ETH Z1418/eth... · DISS. ETH NO. 18943 . LIVING CELL ADHESION MEASURED BY FORCE SPECTROSCOPY . a dissertation submitted to . ETH ZURICH . for the degree of . Doctor of Sciences (Dr

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Doctoral Thesis

Living cell adhesion measured by force spectroscopy

Author(s): Weder, Gilles

Publication Date: 2010

Permanent Link: https://doi.org/10.3929/ethz-a-006092558

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DISS. ETH NO. 18943

LIVING CELL ADHESION MEASURED BY FORCE SPECTROSCOPY

a dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences (Dr. sc. ETH Zurich)

presented by

Gilles WEDER

Master of Science in Biology, University of Neuchâtel, Switzerland, 2006

born on June 13, 1983

citizen of Au (SG)

accepted on the recommendation of

Prof. Dr. Janos Vörös, examiner

Dr. Martha Liley, co-examiner

Dr. Harry Heinzelmann, co-examiner

Dr. Mathis Riehle, co-examiner

2010

Table of contents

iii

SUMMARY VII

RÉSUMÉ XI

CHAPTER 1 INTRODUCTION 1

1.1. CELL ADHESION 1 1.1.1. CONCEPT 1 1.1.2. AT THE MOLECULAR LEVEL 1 1.1.3. AT THE CELL LEVEL 3 1.1.4. APPLICATIONS 5 1.2. CURRENT TECHNIQUES FOR CELL ADHESION STUDIES 6 1.2.1. LIGHT MICROSCOPE-BASED TECHNIQUES 6 1.2.2. MECHANICAL APPROACHES FOR CELL POPULATION STUDIES 7 1.2.3. MECHANICAL APPROACHES FOR SINGLE CELL STUDIES 8 1.3. ATOMIC FORCE MICROSCOPY FOR CELL ADHESION STUDIES 14 1.3.1. PRINCIPLE 14 1.3.2. FORCE SPECTROSCOPY MODE 15 1.3.3. SINGLE BIO-MOLECULE FORCE SPECTROSCOPY 17 1.3.4. SINGLE CELL FORCE SPECTROSCOPY (SCFS) 20 1.4. SCFS CONFIGURATIONS 23 1.5. DISSERTATION OBJECTIVES 25 1.6. ORGANIZATION OF THE DISSERTATION 26

CHAPTER 2 MATERIALS AND METHODS 29

2.1. SUBSTRATE PREPARATION 30 2.1.1. CLEANING PROTOCOL 30 2.1.2. SURFACE MODIFICATION WITH POLYELECTROLYTES 30 2.1.3. PREPARATION OF PNIPAM-GRAFTED SURFACES 31 2.1.4. PREPARATION OF PATTERNED PNIPAM-GRAFTED SURFACES 31 2.1.5. MICRO-FABRICATION OF LOW ASPECT-RATIO PILLARS IN QUARTZ 31 2.1.6. MICRO-FABRICATION OF HIGH ASPECT-RATIO PILLARS IN QUARTZ 32 2.1.7. STERILIZATION PROTOCOL 33 2.2. CELL CULTURE 33 2.2.1. SAOS-2 OSTEOSARCOMA CELLS 33 2.2.2. 3T3 MOUSE FIBROBLASTS 33 2.2.3. PRIMARY EMBRYONIC MOUSE FIBROBLASTS 34 2.2.4. CELL CYCLE SYNCHRONIZATION 34 2.2.5. CELL PROLIFERATION ASSAY 34 2.2.6. CELL SEEDING 35 2.3. INSTRUMENTATION 35 2.3.1. ATOMIC FORCE MICROSCOPE (AFM) 35 2.3.2. FLUORESCENCE ACTIVATED CELL SORTER (FACS) 37 2.3.3. CONFOCAL MICROSCOPE 38 2.3.4. SCANNING ELECTRON MICROSCOPE (SEM) 38

Table of contents

iv

2.4. FORCE-DISTANCE CURVES 39 2.4.1. CANTILEVERS FOR SHORT-TERM ADHESION 39 2.4.2. CELL CAPTURE FOR SHORT-TERM ADHESION 39 2.4.3. SHORT-TERM ADHESION MEASUREMENTS 40 2.4.4. CANTILEVERS FOR LONG-TERM ADHESION 40 2.4.5. LONG-TERM ADHESION MEASUREMENTS 41 2.4.6. CANTILEVERS FOR INDENTATION 41 2.4.7. ELASTICITY MEASUREMENTS 42 2.5. AFM IMAGING 42 2.6. CELL PREPARATION 42 2.6.1. FACS 42 2.6.2. CONFOCAL MICROSCOPY 42 2.6.3. SEM 43 2.7. DATA PROCESSING AND STATISTICAL ANALYSIS 43 2.7.1. FACS 43 2.7.2. ANALYSIS OF FORCE-DISTANCE CURVES 43 2.7.3. STATISTICAL ANALYSIS 45

CHAPTER 3 CELL ADHESION DURING THE CELL CYCLE 47

3.1. INTRODUCTION 48 3.1.1. THE CELL CYCLE 48 3.1.2. ADHESION OF MITOTIC CELLS 49 3.1.3. SHORT-TERM ADHESION MEASUREMENTS 49 3.2. CELL CYCLE SYNCHRONIZATION 51 3.2.1. G1, S AND G2M 51 3.2.2. NATIVE MITOTIC CELLS 52 3.3. SHORT-TERM ADHESION 52 3.3.1. PRESENCE OF ADHESION PROTEINS THROUGHOUT THE CELL CYCLE 52 3.3.2. POSITION OF THE UNBINDING EVENTS 55 3.3.3. ADHESION PER PROTEIN COMPLEX IN THE DIFFERENT CELL CYCLE PHASES 55 3.3.4. MAXIMUM FORCE OF DETACHMENT 56 3.3.5. WORK OF DETACHMENT 57 3.3.6. HIGHER STIFFNESS OF MITOTIC CELLS 58 3.4. DISCUSSION 60 3.4.1. CELL ADHESION BETWEEN M-PHASE AND INTERPHASE 60 3.4.2. STATISTICS 61 3.4.3. THE USE OF TRYPSIN 62 3.4.4. THE SHORT CONTACT TIME 62 3.5. CONCLUSION 62

CHAPTER 4 CANTILEVER FUNCTIONALIZATION FOR CELL DETACHMENT 65

4.1. INTRODUCTION 66 4.1.1. LONG-TERM ADHESION 66 4.1.2. CANTILEVER FUNCTIONALIZATION FOR CELL DETACHMENT 67 4.2. DETACHMENT OF ADHERENT CELLS 68 4.2.1. PERCENTAGE OF CELL DETACHMENT 68 4.2.2. MAXIMUM FORCE OF DETACHMENT 70

Table of contents

v

4.2.3. WORK OF DETACHMENT 70 4.3. DISCUSSION 72 4.3.1. SELECTION OF THE CANTILEVER FUNCTIONALIZATION 72 4.3.2. LIMITATION OF THE CONTACT TIME 73 4.3.3. THE EFFECT OF CANTILEVER FUNCTIONALIZATION ON CELL ADHESION 73 4.4. CONCLUSION 74

CHAPTER 5 CELL ADHESION ON FUNCTIONALIZED SURFACES FOR CELL SHEET ENGINEERING 77

5.1. INTRODUCTION 78 5.1.1. TISSUE ENGINEERING 78 5.1.2. CELL SHEET ENGINEERING 79 5.2. LONG-TERM ADHESION TO GLASS AT 37 °C AND 27 °C 80 5.3. CELL ADHESION ON POLYELECTROLYTES AT 37 °C 84 5.3.1. ADHESION FORCES 84 5.3.2. CHARACTERIZATION OF PLL/HA SUBSTRATES 85 5.3.3. DISCUSSION OF POLYELECTROLYTE COATED SURFACES 86 5.4. CELL ADHESION ON PNIPAM AT 37 °C & 27 °C 87 5.4.1. PRINCIPLE 87 5.4.2. ADHESION FORCES 87 5.4.3. DISCUSSION OF PNIPAM FUNCTIONALIZED SURFACES 89 5.5. CONTROLLING CELL ADHESION USING A PATTERNED PNIPAM COATING 89 5.5.1. PRINCIPLE 89 5.5.2. ADHESION FORCES 90 5.5.3. DISCUSSION OF PNIPAM PATTERNED SURFACES 92 5.6. DISCUSSION 93 5.6.1. DETACHMENT OF ADHERENT CELLS 93 5.6.2. LIMITATION OF THE SAMPLE SIZE 94 5.7. CONCLUSION 94

CHAPTER 6 EFFECT OF SUB-MICRON TOPOGRAPHY ON CELL ADHESION 97

6.1. INTRODUCTION 98 6.1.1. TOPOGRAPHY AND CELL ADHESION 98 6.1.2. TOPOGRAPHY AND CELL TYPE 98 6.1.3. DESCRIPTION OF THE SUB-MICRON TOPOGRAPHY 99 6.2. LONG-TERM ADHESION TO FLAT QUARTZ 100 6.3. EFFECT OF SUB-MICRON TOPOGRAPHY 101 6.4. COMPARISON WITH PRIMARY MOUSE FIBROBLASTS 103 6.4.1. CELL ADHESION 104 6.4.2. CELL PROLIFERATION 104 6.4.3. CELL MORPHOLOGY 105 6.5. DISCUSSION 107 6.5.1. DETACHMENT OF ADHERENT CELLS 107 6.5.2. CELL ADHESION TO QUARTZ AND GLASS 107 6.6. LIMITATION OF THE CELL TYPE 108 6.7. CONCLUSION 109

Table of contents

vi

CHAPTER 7 CONCLUSIONS & OUTLOOK 111

ACKNOWLEDGEMENTS 115

CURRICULUM VITAE 117

REFERENCES 121

Summary

vii

Summary

Cell adhesion is the binding of a cell to another cell, extracellular matrix or a surface, using cell

adhesion molecules. Many of these interactions involve transmembrane integrin receptors.

Integrins cluster to provide dynamic links between extracellular and intracellular environments,

by bi-directional signaling and stabilization of the focal adhesion points. The interactions involve

complex couplings between cell biochemistry, structural mechanics, and surface bonding.

Adhesive interactions play a major role in the development of multicellular organisms, by

guiding and anchoring cells into their appropriate locations. Adhesion is also important in the

maintenance of the body: changes in the expression or function of cell adhesion molecules are

implicated in all steps of tumor progression. Tumor cells are able to loosen their attachment to

leave their original location and become lodged at distant tissues (metastasis). Implantology

research and tissue engineering are directly focused on cell-surface interactions, since events

leading to integration of an implant into bone and to long performance of the device take place at

the interface formed between tissue and implant. Modifying the surface of an implant either by

providing chemical and/or topographical cues encourages bone cell attachment. Cell adhesion is

one way to increase osteointergration and to stabilize the implant. Tissue engineering aims to

replace or restore the anatomic structure and function of damaged or missing tissue. Cell

adhesion is a crucial parameter in the development of biodegradable scaffolds and cell sheet

engineering techniques.

Summary

viii

It is not easy to measure and quantify cell adhesion. Cell adhesion has been investigated using

many techniques. The combination of cell biology with force spectroscopy provides a powerful

tool for exploring the complexity of cell adhesion. The main objective of this work is to use force

spectroscopy to quantify the long-term global adhesion between cells and surfaces and their

response to modified surfaces. Atomic force microscopy-based force spectroscopy is capable of

resolving individual cell binding events as well as global cell adhesion of living cells under

physiological condition. It was used to study and quantify the adhesion of living cells to their

growing substrate.

In order to determine if cell adhesion has to be studied independently of cell cycle or not, cell

adhesion at different phases was measured. The adhesion of osteosarcoma cells to a glass surface

was measured at different phases of the cell cycle. The cells were synchronized in three phases of

the cell cycle: G1, S and G2M. Cells in these phases were compared with unsynchronized and

native mitotic cells. Individual cells were attached to an atomic force microscope cantilever,

brought into brief contact with the glass surface and, then pulled off again. The force-distance

curves obtained allowed the work and maximum force of detachment as well as the number,

amplitude, and position of discrete unbinding steps to be determined. The properties of the

binding proteins present at the cell surface remained similar throughout the cell cycle, including

mitosis. Therefore, next cell detachment experiments were allowed to be studied independently

of cell cycle.

Long-term cell adhesion involves living adherent cells. The challenge was to find a method of

attachment, between cells and atomic force microscope cantilevers, that allows their detachment

from the surface. Fibronectin-coated cantilevers were used to detach individual immortalized

fibroblasts from their growing substrate. The detachment of living adherent cells by force

spectroscopy was tested on a number of chemically functionalized surfaces in order to validate

the technique. The forces involved in the adhesion of fibroblasts were quantified. The cells were

grown on glass surfaces as well as on surfaces used for cell sheet engineering: glass surfaces

coated with polyelectrolyte multilayers (poly-L-lysine and hyaluronic acid) and thermally-

responsive poly(N-isopropylacrylamide) brushes. Large differences in cellular adhesion were

observed on polyelectrolyte coatings, depending on the number of polyelectrolyte bilayers. On

poly(N-isopropylacrylamide)-grafted surfaces, changes of more than an order of magnitude were

observed in cell adhesion above and below the lower critical solution temperature. Glass surfaces

Summary

ix

patterned with periodic poly(N-isopropylacrylamide) microdomains were also investigated. In

this last case, it was shown that cellular adhesion could be reduced while keeping cellular

morphology unchanged.

Finally, in order to investigate the potential and limitations of this technique, two important

experimental parameters were altered: the topography and the cell type. Immortalized and

primary fibroblasts were studied on flat and topographically structured quartz surfaces. Using a

fibronectin-coated AFM cantilever, it was possible to detach a large proportion of the

immortalized cells from the quartz surfaces. Their adhesion to the quartz surface, and the effects

of topography on this adhesion, could be quantified. In contrast, few primary cells were detached

under the same experimental conditions. A qualitative analysis of their behavior showed that

immortalized fibroblasts adhered less strongly than primary fibroblasts to at least one quartz

surface.

The potential and limitations of single cell force spectroscopy in the study of the adhesive

properties of cells are discussed. Quantitative data of this nature should open the way for more

rigorous investigation and comparison of the influence of different parameters on cell/substrate

adhesion.

Summary

x

Résumé

xi

Résumé

L’adhérence cellulaire correspond à la liaison d’une cellule à une autre cellule, à la matrice

extracellulaire ou à une surface, en utilisant des molécules spécifiques d’adhésion. La plupart de

ces interactions impliquent des récepteurs transmembranaires de type intégrines. Les intégrines se

regroupent et s’organisent afin d’établir des liens dynamiques entre le milieu extracellulaire et

intracellulaire, pour la signalisation moléculaire et la stabilisation des points d’adhésion focaux.

Les interactions sont le résultat de couplages complexes entre la biochimie cellulaire, la structure

mécanique et les liaisons de surface.

Les interactions d’adhésion jouent un rôle majeur dans le développement des organismes

pluricellulaires, en guidant et fixant les cellules dans leur emplacement approprié. L’adhésion est

également importante dans l’homéostasie du corps ; des changements dans l’expression et la

fonction des molécules d’adhésion cellulaire sont impliqués dans toutes les étapes de la

progression de tumeurs. Les cellules tumorales sont capables de se détacher et de quitter leur

emplacement d’origine afin de coloniser d’autres tissus (métastases). La recherche sur les

implants et le génie tissulaire sont directement intéressés par les interactions cellule-surface, car

la réussite d’une bonne intégration d’un implant dans l’os et l’efficacité à long terme se joue à

l’interface du tissue et de l’implant. Des modifications chimiques et/ou topographiques de la

surface d’un implant peuvent favoriser l’attachement des cellules osseuses. L’adhésion cellulaire

est responsable de l’ostéo-intégration de l’implant. L’adhésion cellulaire est aussi un paramètre

Résumé

xii

crucial dans le développement des prothèses biodégradables et des feuillets cellulaires.

L’adhésion cellulaire n’est pas facile à mesurer et quantifier. L’adhésion cellulaire a été

investiguée par de nombreuses techniques. La combinaison de la biologie et de la spectroscopie

de force offre un outil puissant pour l’exploration de la complexité de l’adhésion cellulaire.

L’objectif principal de ce travail est d’utiliser la spectroscopie de force pour quantifier les forces

globales d’adhésion à long terme entres des cellules et des surfaces, ainsi que leur réponse à des

surfaces modifiées. La spectroscopie de force issue de la microscopie à force atomique est

capable de mesurer aussi bien des liaisons d’adhésion individuelles que l’adhésion globale de

cellules vivantes en condition physiologique. Elle a été utilisée pour étudier et quantifier

l’adhésion de cellules vivantes à leur substrat de croissance.

L’adhésion cellulaire a été mesurée à différentes phases du cycle cellulaire afin de déterminer si

elle pouvait être étudiée indépendamment, ou non, du cycle cellulaire. L’adhésion entre des

cellules provenant d’un ostéo-sarcome et une surface en verre a été mesurée à différente phases

du cycle cellulaire. Les cellules ont été synchronisées dans trois phases du cycle cellulaire : G1, S

et G2M. Dans ces phases, les cellules ont été comparées avec des cellules non synchronisées et

des cellules naturellement en mitose. Des cellules individuelles ont été attachées à un levier de

microscope à force atomique, mises en bref contact avec la surface en verre et, ensuite retirées à

nouveau. Les courbes de force obtenues en fonction de la distance ont permis de déterminer le

travail et la force maximale de détachement aussi bien que le nombre, l’amplitude et la position

des étapes de détachement. Les propriétés des protéines d’adhésion présentes à la surface de la

cellule sont restées similaires au travers du cycle cellulaire entier, y compris en mitose. Par

conséquent, les mesures suivantes de détachement cellulaire ont été étudiées indépendamment du

cycle cellulaire.

L’adhésion cellulaire à long terme implique des cellules adhérentes vivantes. Le défi a été de

trouver une méthode d’attachement, entre les cellules et les leviers de microscope à force

atomique, capable de supporter leur détachement de la surface. Des leviers recouverts de

fibronectine ont été utilisés pour détacher des fibroblastes individuels immortalisés de leur

surface de croissance. Le détachement de cellules adhérentes vivantes, en utilisant la

spectroscopie de force, a été testé sur de nombreuses surfaces fonctionnalisées chimiquement afin

de valider la technique. Les forces impliquées dans l’adhésion des fibroblastes ont été quantifiées.

Résumé

xiii

La croissance des cellules a été réalisée sur des surfaces en verre ainsi que sur des surfaces

utilisées pour le génie tissulaire : des surfaces en verre fonctionnalisées avec des multi-couches

de polyélectrolytes (poly-L-lysine et acide hyaluronique) et avec le polymère thermo-sensible

poly(N-isopropylacrylamide). De grandes différences en terme d’adhésion cellulaire ont pu être

observées sur les revêtements de polyélectrolytes, en fonction du nombre de bicouches de

polyélectrolytes. Concernant les surfaces fonctionnalisées avec le poly(N-isopropylacrylamide),

des changements de plus d’un ordre de magnitude ont pu être observés au niveau de l’adhésion

cellulaire au-dessus et au-dessous de la température critique inférieure de solution. Des surfaces

en verre modifiées avec des micro-domaines de poly(N-isopropylacrylamide) ont également été

investiguées. Dans ce dernier cas, il a été montré que l’adhésion cellulaire pouvait être réduite

tout en gardant une morphologie cellulaire identique.

Finalement, deux paramètres expérimentaux importants ont été modifiés, la topographie et le type

cellulaire, afin d’investir le potentiel et les limites de cette technique. Des cellules immortalisées

et primaires ont été étudiées sur des surfaces en quartz plates et avec une topographie. Grâce à

l’utilisation d’un levier fonctionnalisé avec de la fibronectine il a été possible de détacher une

grande proportion des cellules immortalisées des surfaces en quartz. Leur adhésion aux surfaces

en quartz, et les effets de la topographie sur cette adhésion, ont pu être quantifiés. En revanche,

seules quelques cellules primaires ont pu être détachées en utilisant les mêmes conditions

expérimentales. Une analyse qualitative de leur comportement a montré que les fibroblastes

immortalisés adhéraient moins fortement que les fibroblastes primaires sur au moins une surface

en quartz.

L’aspect du potentiel et des limites de la spectroscopie de force avec des cellules individuelles

pour l’étude des propriétés d’adhésion est discuté. Des données quantitatives de ce genre

devraient ouvrir le chemin pour des investigations et comparaisons plus rigoureuses de

l’influence de différents paramètres sur l’adhésion entre une cellule et son substrat.

Résumé

xiv

Chapter1 Introduction

1

Chapter 1

Introduction

1.1. Cell adhesion

1.1.1. Concept

One prerequisite for the evolution of multicellular organisms was the development of

mechanisms by which cells could adhere to one another. Cell adhesion is the binding of a cell to

another cell, extracellular matrix or a surface using cell adhesion molecules (CAMs). Many cell

adhesion processes occur in two steps. First, single cell adhesion proteins bind to their respective

ligands at the cell surface and then the interaction is consolidated by the recruitment of

hierarchically assembled protein complexes called focal adhesions (FAs).

1.1.2. At the molecular level

Cell-cell and cell-substrate interactions are mediated through several different families of

receptors. In addition to targeting cell adhesion to specific extracellular matrix proteins and

ligands on adjacent cells, these receptors influence also many processes in cell regulation, tissue


2

homeostasis and morphogenesis. Most of the CAMs belong to five families or superfamilies1:

integrins, immunoglobulins (Ig), cadherins, selectins and homing receptors. These proteins are

typically transmembrane receptors that interact either with similar CAMs (homophilic binding)

or with different CAMs or the extracellular matrix (heterophilic binding). Integrins are

heterophilic molecules that mediate both cell-cell and cell-substratum adhesion2. The

immunoglobulin superfamily is involved in cell-cell adhesion via homo- and heterophilic

interactions. Cadherins are calcium-dependant homophilic cell-cell adhesion proteins3. Selectins

(or LEC-CAMs) mediate white blood cell/endothelial cell adhesion4 and finally homing receptors

target lymphocytes to specific lymphoid tissues. The CAM family that plays a key role in

mediating cell-surface or cell-matrix interactions is the integrin family5.

Integrins are a family of cell adhesion receptors that bind to extracellular matrix ligands, cell-

surface ligands and soluble ligands. They are transmembrane αβ heterodimers and at least

eighteen α and eight β subunits are known in humans, generating twenty-four heterodimers6. The

RGD sequence (arginine-glycine-aspartic acid) in fibronectin was originally identified as an

integrin-binding motifs7. This sequence and related sequences in extracellular matrix molecules

do act as integrin-binding motif. However, integrins also recognize many non-RGD sequences as

ligands8. As far as ligand specificity is concerned, mammalian integrins can bind to, among

others, fibronectin, collagen, laminine, vitronectin, tenascin, thrombospondin, osteopontin or

fibrilin. Some integrins are limited to certain cell types or tissues and other integrins are widely

distributed. Normally, each cell expresses several types of integrins.

Integrins mediate the attachment of the cell to its surroundings and play a role in cell signalling.

They couple the extracellular matrix outside the cell to the cytoskeleton inside the cell. Integrins

are the transducers that directly transmit mechanical stress from the extracellular matrix to the

cytoskeleton and inversely9. This linkage is not only important to stabilize cell adhesion and

morphology but is also involved in cell signalling. The signals received through the integrins

have been shown to be related to cell growth, proliferation, differentiation, viability and

apoptosis10. Table 1.1 shows that the deletion of individual genes coding for integrin subunits, by

gene knockout in mice, plays a critical role in their morphogenesis6. Almost all organs can be

affected.


3

Integrin subunit Viability Phenotype

α3 perinatal lethal kidney, lung and skin defects11

α4 embryonic lethal placental and heart defects12

α5 embryonic lethal mesodermal and vascular defects13

α6 perinatal lethal epidermal detachment and neurogenesis defects14

α7 viable muscular dystrophy15

α8 perinatal lethal kidney defect16

αE viable skin inflammation17

β1 embryonic lethal failure to gastrulate18

β3 viable platelet defect19

β4 perinatal lethal epidermal detachment20

β6 viable inflammation of skin and lungs21

β7 viable gut-associated lymphocyte defects22

Table 1.1 Phenotypes of deletions of integrin subunits in the mice6.

1.1.3. At the cell level

FAs serve as stabilizers of the attachment of the cell to its surrounding and as regulators of cell

signalling. Primary adhesion is dominated by single molecule binding23. These connections of the

integrins with the extracellular matrix or a surface induce intracellular signalling cascades leading

to the formation of hierarchically assembled protein complexes called focal adhesions or focal

contacts24,25. FAs usually measure a few square microns across and are located along the ventral

plasma membrane of adherent cultured cells. Recently, it was shown that layilin, a

transmembrane protein, also interacts with the extracellular matrix notably with hyaluronic

acid26,27.

On their cytoplasmic side, FAs are composed of numerous proteins with and without enzymatic

activity that play the role of stabilizer elements, transducers, modulators and adaptors. They are

also associated with the bundles of actin microfilaments of the cytoskeleton. The main


4

components of FAs are cytoskeletal proteins such as vinculin, paxilin, tensin, α-actinin, actopaxin

or talin that show no enzymatic activity. Kinases, phosphatases, modulators of small GTPases

and other enzymes are also very important. Many of these components may be expressed in a cell

type specific manner. Figure 1.1 illustrates the complexity of focal adhesions connecting the

extracellular matrix via integrins to the actin fibres of the cytoskeleton28.

Fig. 1.1. Illustration of the components of focal adhesions28. On the extracellular face, integrins engage extracellular

matrix macromolecules and on the cytoplasmic face, integrins coordinate the assembly of focal adhesions.

When cells let go of a surface, they don’t let go of all their integrins29. A substantial proportion of

the integrins is left behind on the substrate as the cell detaches while another fraction is collected

into vesicles30. It has been shown that cells use the endocytic protein clathrin to reabsorb the

integrin receptors that attach them to surfaces, instead of simply leaving the molecules behind31.

Cells may use the reabsorbed integrins to build new FAs but this has not yet been demonstrated.


5

1.1.4. Applications

Both cell-cell and cell matrix adhesions are important in multicellular organisms during

morphogenesis and in a variety of basic processes such as proliferation, differentiation, motility,

cellular trafficking, tissue architecture or apoptosis32. Cell adhesion plays an essential role in the

homeostasis of healthy tissues. Unfortunately, cell adhesion can be disrupted and cause a great

variety of diseases.

Some cancers involve mutations in genes for adhesion proteins that result in abnormal cell-cell

interactions. Indeed, changes in the expression or function of cell adhesion molecules have been

implicated in all steps of tumour progression, including the detachment of tumour cells from the

primary site, their intravasation into the blood stream, their extravasation into distant target

organs, and the formation of secondary lesions33. Some people have "blistering diseases" that

result from inherited molecular defects in genes for adhesion proteins. These diseases involve the

formation of a bubble of fluid beneath a thin layer of dead skin. The blisters may start in the

deeper layers of the skin and cover widespread areas. Cell adhesion proteins are also important

for interactions that allow viruses and bacteria to cause damage to humans. Many

pharmaceuticals target the adhesion proteins of viruses and bacteria. Cell adhesion proteins hold

synapses together and the regulation of synaptic adhesion is involved in learning and memory. In

Alzheimer’s disease, there is abnormal regulation of synaptic cell adhesion. The study of cell-

surface interactions allows the indirect study of disturbed cell-cell and cell-matrix interactions in

such diseases.

In contrast, implantology research and tissue engineering are directly focussed on cell-surface

interactions. A goal in the design of bone and dental implants is the development of devices that

induce controlled, guided and rapid healing34. Thus, a better understanding of events at the

interface and of the effects that biomaterials have on bone and bone cells is needed. In tissue

engineering, cells are placed on or within matrices in order to develop functional substitutes for

damaged tissues35.

Cell adhesion has widespread applications. It has been shown that the expression of adhesion

molecules on the surface of cells may change in various diseases. In conclusion, cell adhesion is

fundamental in cancer progression, implantology research, wound healing and against infection

by pathogens.


6

1.2. Current techniques for cell adhesion studies Cell adhesion has been investigated using many techniques. Microscope-based techniques

provide qualitative cell adhesion measurements whereas mechanical approaches quantify semi-

quantitatively or quantitatively cell adhesion. The techniques are not described in chronological

order according to their appearance.

1.2.1. Light microscope-based techniques

Light microscopy was used since the earliest examination of cellular structures. Modern light

microscopy provides a variety of imaging techniques and also the capability to perturb biological

structures in living cells in order to study their functions36,37.

Interference reflection microscopy (IRM) has been used to study the adhesion of cells in contact

with planar substrates38. The technique provides information on the separation of the plasma

membrane of the substrate. The interference between the reflected light waves from the substrate-

medium interface and the plasma membrane-medium interface generate an image with high

contrast. IRM allows the visualization of cell-substratum adhesion in living tissue culture cells

continuously for long periods of time without the use of fluorescent markers39.

The use of fluorochrome-conjugated antibodies and green fluorescent protein (GFP)-fusion

proteins produces high-resolution images of the distribution of binding proteins in cells40. Cells

expressing GFP-fusion proteins have been used to determine the compositional dynamics of the

cell adhesion structures. However, the comparison of the relative distributions of the labeled

components in function of the time is not accurate. This problem was partially solved by a

calculation of the pixel by pixel ratio of two fluorescent images41,28. It is particularly suitable for

colocalization studies of proteins forming distinct and defined structures such as focal adhesions.

This spatiotemporal information is important for structure-function studies in cell adhesion.

However, relative change in protein concentration can be estimated but the absolute

concentration of the specific adhesion protein cannot be calculated.

Microscope-based-techniques can also be used for the detection of close protein-protein

interactions using fluorescence resonance energy transfer (FRET)42,43,44. This is a process

involving the radiationless transfer of energy from a donor fluorophore to an appropriately

positioned acceptor fluorophore. FRET can occur when the emission spectrum of a donor


7

fluorophore significantly overlaps the absorption spectrum of an acceptor and when they are in

favourable mutual orientation. This technique allows the determination of molecular associations

of adhesive components such as the details of the fibronectin fibril assembly process43.

Alternatively, photobleaching techniques have been used to measure the transport of molecules in

living cells or at their surface45,46. The fluorescence of defined regions of the cell was destroyed

and the recovery of fluorescence into those regions reflected the type of transport such as

diffusion or flow. It was used to study the trafficking of cytoskeletal proteins47. The exchange of

α-actinin between the cytoplasmic pool and the focal adhesions was observed. The advantage of

the light-directed technique is the temporal control in the molecular perturbation when compared

to genetic approaches.

In summary, light microscopy-based techniques can be used in situ to study molecular

interactions involved in cell adhesion and dynamics. Specific molecular perturbation in cellular

structures can also be induced. However, cell adhesion is never measured quantitatively.

Although light microscope-based techniques are limited to qualitative images, they are essential

for the implementation of mechanical approaches to measure cell adhesion.

1.2.2. Mechanical approaches for cell population studies

The mechanical behaviour of a living cell cannot be characterized simply in terms of fixed

properties, as the cell structure is a dynamic system that adapts to its local mechanochemical

environment48. An large number of experimental tools have been developed to measure and apply

forces and displacements to probe entire cells as well as individual molecules. Some tools allow

the detection of forces down to the pN regime and displacements down to the Å regime.

In the past, studies were focussed on the mechanical response or mechanical manipulation of

entire cell populations49. A direct manipulation of the substrate to which cells adhere provided a

means of mechanical manipulation50. This approach was used in an attempt to impose cyclic

deformation representative of in vivo conditions for cell adhesion studies. Strains were imposed

and measured via low-resolution displacement sensors and global forces were calculated from

substrate stiffness.

An alternative of providing mechanical stimuli to a cell population through substrate

manipulation was done by controlling the chemical composition. Responses of cells to


8

mechanical properties of the adhesion substrate were examined by varying the concentration of

one component or the degree of crosslinking in polymeric gels51. The forces imposed by the cells

were not quantified.

Techniques based on force-application measurements have also been developed in order to

detach the cells from their growing substrate. Cells were allowed to adhere on substrates that

were placed in a centrifuge52 (Fig. 1.2A). The centrifugal (normal) force allowed the detachment

of cells and the absolute strength of adhesion varied in function of the cell type. The centrifugal

assay was restricted to comparative cell adhesion measurements. The ratio of cells between the

top and the bottom of the chamber of centrifugation was quantified at a particular spin speed but

no information was obtained at the single cell level.

Fig. 1.2. Schematic of a centrifugal assay52 for cell adhesion studies.

1.2.3. Mechanical approaches for single cell studies

Numerous mechanical approaches for single cells have been used to study the interaction

between cells and substrates. Extensive uses of flexible substrates were used for the detection and

the measurement of traction forces exerted by cells including embedded particle tracking,

micropatterned substrates and microfabricated substrates53.

Marker beads were embedded within a polymeric substrate in order to characterize the traction

forces by two-dimensional displacements of the beads54 (Fig. 1.3A). The displacement of the

beads was measured optically and the corresponding force was calculated via the experimentally


9

determination of the stiffness of the substrate. Bead-displacement values were translated into a

traction force map using specialized algorithms. In addition, polyacrylamide gels with variable

degrees of cross-linking allowed the modification of the rigidity of the substrate55,56 (Fig. 1.3B).

Micropatterned substrates were fabricated to allow pointwise control of cell adhesion and traction

measurement through displacement of the pattern features57,51,58. Alternatively, micro-fabricated

arrays containing force-sensing elements were developed59,60,61 (Fig. 1.3C). Silicon substrates

were prepared with flexible cantilevers of known bending stiffness with small pads to grow the

cells. The forces exerted by cells on these pads were directly computed by the deflection of the

cantilevers. Otherwise, silicon elastomeric substrates, that were micropatterned to give a regular

array of surface indentations, were also used to measure the surface distortion of the micropattern

caused by cells62.

Fig. 1.3. (A) Schematic of the embedded particle tracking assay reporting cell tractions as displacements of beads

firmly anchored in the film54. (B) Polyacrylamide-based traction assay after mapping the magnitude of stress in

different colors (dyn/cm2)55. (C) Schematic of a force sensitive substrate with an attached cell on the pads of the

flexible cantilevers61.

Traction forces analysis represents a powerful tool that can easily be combined with light

microscope-based techniques to allow experimentation at high spatial and temporal resolution.

There are a number of measurements that are resulting in increasingly accurate estimates of

traction forces in order to correlate the stresses with the adhesive structures. Basically, traction

forces are generated by actomyosin interactions and actin polymerization whereas adhesion


10

forces by focal adhesions63. Cell traction forces and adhesion forces can be correlated. However,

none of these approaches quantifies the adhesion forces, or only in a semi-quantitative way via

traction forces. Finally, the resolution of the forces and displacements of these techniques (10-9 N

and 10-7 m) limits the measurements to the single cell level, rather than to the single molecule.

The micropipette aspiration technique was originally developed to study time-dependent

deformation of living individual cells subjected to extracellular pressure. Cells were allowed to

spread for varying amounts of time on glass surfaces and then their free extremity was aspirated

into a micropipette at given pressure levels64 (Fig. 1.4A). Applied aspiration pressure ranged

from 0.1-1000 Pa, with resolution of 0.1 Pa65,66. Displacement of the cell membrane was tracked

by light microscopy with a claimed resolution of ± 25 nm. Then dual pipette assays were used to

pull cells apart and quantify the strength of cadherin-dependent cell-cell adhesion67,68. Cells were

manipulated in suspension, an approach that eliminates matrix-mediated signalling and bypasses

the initiation of intercellular adhesion through filopodial activities occurring during adhesion on a

substrate. Alternatively, one of the two cells could adhere on a surface. The weak point of this

technique is the indirect measurement of the adhesion force via the measurement of the aspiration

pressure and the cell deformation in the micropipette. Computational models of the cell

deformation differ in their interpretation and the cell deformation can also interfere in the

adhesion properties. Although the instrumentation is not commercially available, the technique

has been used successfully for cell-cell interactions.

In contrast to static cell adhesion assays, hydrodynamic shear stress was used to analyze the

immobilization and release of non-adherent cells on surfaces (Fig. 1.4B). Parallel and radial flow

chambers as well as capillaries were used to control the hydrodynamic stress level. The problem

is the development of hydrodynamic shear methods in which there is a clear dose-response

relationship between the number and strength of the adhesion bonds and the measured

hydrodynamic parameter69. The application of a hydrodynamic flow is very useful in analysing

catch bond interactions70,71. A catch bond is a bond that becomes stronger or longer lived when

subjected to tensile mechanical force. This adhesion assay was used to mimic the conditions in

the blood vessel under which leucocytes adhere to vascular endothelial cells72,73. The indirect

computation of the adhesion forces via the hydrodynamic forces involves laborious mathematical

models. Moreover, this technique is limited to short-term adhesion. However, it is useful to

simulate what occurs in blood or lymph vessels.


11

Fig. 1.4. Schematic of (A) a micropipette aspiration and (B) a hydrodynamic flow assay74 used for cell adhesion

studies.

Optical tweezers employ light to trap and manipulate cells (Fig. 1.5A). The basic physical

principle underlying optical tweezers is the radiation pressure exerted by light when colliding

with matter75. For transparent materials, light passes through the material but changes direction

upon entering and leaving the material by refraction. A fraction of the light momentum is

imparted onto the refracted material. When a laser beam is focussed tightly by a strong lens, the

effective radiation pressure pulls refractive objects towards the focus and holds it there: an optical

trap. If the laser focus is moved through space, the trapped object is forced to follow. When the

refractive object is pulled away from the laser focus by an external force, the object will scatter

the trapping light, yielding a slight deflection of the ongoing laser light. This allows for three-

dimensional tracking of the position or the force on the trapped object to sub-nanometer or sub-

picoNewton accuracy. Several experimental approaches developed to investigate single cell

mechanics are based on the controlled displacement of dielectric objects that are either attached

to the cell membrane or placed inside the cell. Optical tweezers have been used to study, for

example, short-term adhesion of cells to fibronectin76,77. One drawback is the potential damage to

the cell via radiation. Photo-damage may influence the results because such irradiation levels

were observed to induce a loss of filopodia and a general inhibition of motility78. Moreover,

optical tweezers are limited to the detection of forces in the pN regime and cannot be therefore

employed for the investigation of the global cell adhesion.


12

A similar approach to the optical trap is the magnetic trap. Magnetic tweezers that use a magnetic

field gradient can exert and measure forces on magnetic particles coupled to cells (Fig. 1.5B).

The coupling of the magnetic beads to cells was achieved by phagocytosis or covalent

linkage79,80. Magnetic tweezers have been used to measure integrin interactions in initial cellular

adhesion processes81. Two advantages of this approach over optical tweezers are that out-of-plane

rotations and thus torque can be applied, and the elimination of possible damage to the cell via

radiation. A limitation of this technique is that the magnetic susceptibility of the magnetic beads

varies widely, so that these experiments can be difficult to calibrate accurately. Magnetic

tweezers provide great sensitivity but the maximal vertical force of 200 pN that can be applied

greatly restricts their use in studying global cell adhesion, which requires forces in the nN regime.

Optical and magnetic tweezers are unique tools for force microscopy at the cellular level and the

non-contact nature of both techniques removes complicating surface interactions. The low force

regime available with optical and magnetic tweezers means that the nN regime (and higher) can

only be investigated using contact probe methods.

Fig. 1.5. (A) Schematic of optical tweezers48 and (B) magnetic tweezers81 used for cell adhesion studies.

Thus, other techniques such as the microneedle technique were developed to apply and quantify

large forces (in the nN regime) for global cell adhesion investigations. The instrument applies a

mechanical force normal to the cell/substrate interface via a cantilevered probe. The position of

the probe is monitored within an inverted optical microscope via a position-sensitive photodiode


13

and the force is calculated from this deflection. Fine glass needles were employed to quantify the

forces needed to displace single adherent cells82. This cytodetachment technique is restricted to

horizontal displacements: only shear stress can be applied instead of normal stress for cell

detachment. Therefore, there is no direct measurement of the normal (vertical) adhesion and the

risk to perforate the cell is not negligible.

Fig. 1.6. (A) Fine glass microneedle82 for cell adhesion studies.

In summary, several methods have been developed for measuring the forces that cells exert on

their substrate, mainly involving the use of deformable substrates. These methods provide

quantitative evaluations of the forces, and some combine this with visualization of the adhesion

sites83,84. However, their capacity to compare the forces applied at the focal adhesions of a cell is

limited. The micropipette assay can be used for simultaneous measurement of separation force

between two cells and detection of fluorescent proteins involved in adhesion. However, the

separation force is not monitored in function of the distance of separation because the position of

the micropipette is controlled by a micromanipulator. Force-distance curves cannot be obtained.

Moreover, this instrumentation is not commercially available and therefore, it is difficult to

establish controls that demonstrate the specificity of the molecular interaction being studied.

Hydrodynamic flow assays are especially useful for cells in suspension such as blood cells.

Optical and magnetic tweezers are very sensitive but they are restricted to the detection of low

forces (pN regime) while global cell adhesion requires the detection of high forces (nN regime).

Finally, the microneedle technique doesn’t have a direct measurement of the vertical adhesion.


14

1.3. Atomic force microscopy for cell adhesion studies One of the most promising techniques for the quantification of the adhesion forces is force

spectroscopy, an AFM-based method (Atomic Force Microscopy). The AFM was originally

developed for high-resolution imaging but it has also become a powerful tool to manipulate

biomolecules and cells in a very wide force range. The AFM allows direct force and

displacement measurements from the single cell down to the single molecule level. Force

spectroscopy can be performed in numerous commercially available instruments.

1.3.1. Principle

The atomic force microscope85 is a scanning probe microscope that was developed in 1986. The

AFM can be used to study non-conducting materials, such as insulators and semi-conductors, as

well as electrical conductors. The AFM can operate under ultra high vacuum, ambient air

conditions or in liquid, which is highly useful for biological systems.

The AFM uses a flexible cantilever as a spring to measure the force between the tip of the

cantilever and the sample. The basic principle is that the local attractive or repulsive force

between the tip and the sample is converted into a deflection of the cantilever (Fig. 1.7A). The

deflection of the cantilever is detected via a laser beam that is reflected from the back of the

cantilever onto a detector. A small change in the deflection angle of the cantilever is converted

into a large deflection in the position of the reflected spot. Most AFMs use a photo-sensitive

detector (PSD) that is composed of four quadrants, so that the laser spot deflection can be

determined precisely in two directions.

Originally, the AFM was developed for high-resolution imaging. The detection system measures

the cantilever deflection when the tip is moved over a surface by a scanning system. Different

imaging modes are available: constant height imaging consists of measuring the deflection signal

without changing the height of the cantilever. This is not the most common solution because the

force applied by the cantilever depends on the deflection, so higher parts of the sample will

experience higher applied forces. In contrast, it is much more common to use constant force

imaging. In this mode, a feedback loop monitors the cantilever response and adjusts the

cantilever (or sample) height to take account of the changes in surface height. Thus, all parts of

the probed sample should experience the same force.


15

Several forces typically contribute to the deflection of an AFM cantilever, the most common

being relatively weak, attractive van der Waals forces as well as electrostatic repulsive forces.

The general dependence of these forces upon the distance between the tip and the sample is

shown schematically in figure 1.7B. When the tip is less than a few Angströms from the sample

surface, the interatomic force between the cantilever and the sample is predominantly repulsive,

due to the overlap of electron clouds associated with atoms in the tip with those at the sample

surface. In contrast to this contact regime, there is the non-contact regime where the tip is

somewhere between ten to a hundred Angströms from the sample surface. In this case, the

interatomic force between the tip and the sample is attractive as a result of long-range attractive

van der Waals interactions.

Fig. 1.7. Schematic diagram of (A) an atomic force microscope and (B) the tip-sample separation forces86.

1.3.2. Force spectroscopy mode

AFM is also a powerful tool for sensitive force measurements87. Basic force spectroscopy curves

are obtained with a cantilever in air approaching a hard and incompressible surface (such as

glass) (Fig. 1.8). Initially, the cantilever approaches the surface and the forces are too small to

give a measurable deflection. At some point, the attractive forces overcome the cantilever spring

constant and the tip jumps into contact with the surface. Then, the tip remains on the surface and

the separation between the piezo and the sample decreases further causing a deflection of the

cantilever. During the retraction of the cantilever from the surface, the tip remains in contact with


16

the surface due to adhesion. At some point, the force from the cantilever will be sufficient to

overcome the adhesion and the tip will break free.

Fig. 1.8. Schematic force curve on a hard surface with (A) approach and (B) retraction curves.

For a soft and compressible biological sample in liquid, the approach force curve shows a gradual

increase in force without the sharp onset of the interactions seen in air (Fig. 1.9A). It is often

difficult to define a single point where the tip and sample come into "contact" since the initial

compression of the surface causes a very little deflection of the cantilever88,89.

Forces curves are obtained directly from the simple relationship between the force and the

cantilever deflection, Hooke’s law:

F = -k dc, where F: force k: spring constant of the cantilever dc: deflection of the cantilever

The interpretation of a force curve relies almost entirely on established force laws. These force

laws describe force as a function of the probe-sample separation distance rather than as a function

of the z-piezo position. Thus, the force curves must be transformed into descriptions of force as a

function of distance. However, current AFMs do not have an independent measure of the probe-

sample distance. Instead, the transformation to distance is achieved by subtracting the cantilever

deflection from the z-piezo movement. A corrected force curve is called a force–distance curve

(Fig. 1.9B).


17

Fig. 1.9. Schematics of (A) an uncorrected force curve and (B) a corrected force curve (force-distance curve)90. At

the beginning of the curve (i), the tip is far from the sample and there is no interaction and no cantilever deflection.

The approach brings the tip closer to the sample, and long- and short-range tip-sample interactions cause the

cantilever to deflect (ii). When the tip contacts the surface, the stage movement and cantilever deflection become

coupled (iii). The retracting curve displays hysteresis due to a variety of tip-sample interactions.

1.3.3. Single bio-molecule force spectroscopy

Force spectroscopy can be used to examine rupture forces of molecular bonds and ligand-receptor

interactions, unfolding and folding of various proteins in a stepwise process, extraction of a

protein from a membrane, DNA mechanics or polysaccharide stetching91,92,93,94,91,95. Such an

approach to measuring forces is unique in that components of a single unbinding or unfolding

event can be directly monitored. The flexibility of the AFM is such that force spectroscopy can

be conducted on samples ranging from individual membrane proteins to complex and

heterogeneous whole cells in their native environment. At the cell level, the bond rupture events

represent a combination of the unbinding of ligand-receptor complexes and the disruption of non-

specific binding events.

Adhesion forces between individual ligand-receptor pairs were already quantified more than

fifteen years ago using force spectroscopy on biotin and avidin96. This ligand-receptor pair was

chosen as a model system because of its high affinity and the availability of thermodynamic and

structural data on it. The bond rupture forces were reported for fixed pulling velocities and spring

constants. Further details about the interaction potential of the biotin-avidin pair were revealed by


18

dynamic force spectroscopy97. Similar experiments were reported on the protein A-IgG bond98,99.

The analysis and interpretation of these experiments can be complex, since bond dissociation is a

non-equilibrium dynamical process100. Bonds in cell adhesion are therefore being continually

created, loaded over some period of time and then failing. They will fail under any level of

pulling force if held for sufficient time101. The kinetics of the anchoring process during the

adsorption of fibrinogen molecules on silica surfaces was for example explored102.

In single molecule unfolding experiments, the force applied to a single protein acts as a

denaturant leading to complete unfolding of its three-dimensional structure103. In an early study,

Rief and co-workers applied single molecule force spectroscopy to the giant muscle protein titin

which consists of repeats of globular immunoglobulin and fibronectin domains104,105. The

continuous mechanical extension of the protein resulted in the subsequent unfolding of the

globular domains allowing the unfolding force and pathway of each domain to be detected.

Similarly, individual bacteriorhodopsin molecules were unfolded and extracted from purple

membrane patches from Halobaterium106,107,108. Force spectroscopy was also employed to unfold

single sodium-proton antiporters of Escherichia membrane patches. Some fundamental

differences have to be considered when the forced unfolding by mechanical manipulation is

compared with the classical experiment in which a protein is unfolded by means of chemical

denaturants or by exposure to heat109. As mentioned for receptor-ligand pairs, the unfolding force

of proteins depends also on the force loading rate110. Secondly, mechanically forced unfolding

may proceed via a different path along the protein’s configurational energy landscape than the

thermal process111.

DNA molecule mechanics has also been investigated. Sequence-dependent mechanical properties

of DNA have been measured by stretching individual DNA double strands attached between a

surface and an AFM tip112 (Fig. 1.10). The forces needed to open the double strands showed

variations between 10 and 20 pN on a length scale of 10 bases.


19

Fig. 1.10. Complementary DNA oligonucleotides are chemically attached to an AFM tip and a glass surface. As the

tip is brought in contact with the surface a double strand forms, which can subsequently be unfolded upon retraction

of the tip113.

Single molecule force spectroscopy has also been used to study DNA-protein interactions114.

Overstreched structures of DNA were shown to be stabilized by a protein. Alternatively, direct

force measurements were performed to mechanically unbind a transcriptional regulator protein

that binds to promoter regions in a gene cluster115,116.

The AFM has been used to stretch individual molecules of polysaccharides. Deflections in the

force extension curves for certain polysaccharide molecules were shown to be due to conversion

of the polysaccharide rings in the chain from the energetically favoured “chair” conformation to

the longer “boat” conformation117,118. Dextran molecules were also manipulated and probed by

vertical stretching revealing details of the molecular basis of the mechanical properties of the

polysaccharide that could not have been obtained otherwise. At low forces, the deformation of

dextran was found to be dominated by entropic forces and at elevated forces the strand elongation

was governed by a twist of bond angles119.


20

Force spectroscopy has rapidly evolved and is now on the verge of becoming a standard

technique for the structural and functional investigations of bio-molecules120,121. It allows the

characterization of interactions that stabilize functional membrane proteins as well as those that

destabilize them, leading to malfunction and misfolding. This new tool has more recently been

used to investigate bio-molecules in their native environment using entire living cells as probes:

single cell force spectroscopy.

1.3.4. Single cell force spectroscopy (SCFS)

Living single cell force spectroscopy allows the adhesion of cells and single adhesion proteins to

be studied under near-physiological conditions122. The advantage of using living cells rather than

single proteins is that the receptors probed are under physiological conditions and that they can

interact with the cytoskeleton or other components of the cell. It also ensures the correct

orientation, surface density and posttranslational modification of the proteins.

The first single cell force spectroscopy experiment to investigate cell adhesion in vivo at the

single molecule level was reported by Benoit and co-workers123 in 2000. It should be noted that

previously force spectroscopy had been widely used for cell mechanics measurements such as

estimations of Young’s moduli124,125 (more details in section 2.7.2). Benoit’s measurements were

focussed on the interaction of the glycoprotein contact site A (csA) between two eukaryotic cells

of Dictyostelium. CsA is expressed in aggregating cells of Dictyostelium which are engaged in

the development of a multicellular organism. Using a light microscope, a Dictyostelium cell was

captured on the end of a tipless AFM cantilever. The end of the cantilever was functionalized

with a lectin to give a firm attachment of the cell to the cantilever. A target cell was positioned

underneath the cantilever-mounted cell and was approached until a repulsive contact force was

established. This contact force was held constant for a predefined time interval to allow the

establishment of cell adhesion. Upon retraction of the cantilever, the force was recorded as a

function of the distance that the cantilever was moved until contact between the cells were broken

(Fig. 1.11). A de-adhesion force of 23 pN was quantified which may be due to discrete molecular

entities.


21

Fig. 1.11. Force spectroscopy of adhesion between individual Dictyostelium cells123.

The use of living cells as probes to study receptor-ligand interactions has been carried out using

many cell types. Several studies have focussed on the characterization of integrin interactions

with their ligands on a surface. The unbinding forces of integrins with fibronectin126,127,

collagen128, V-CAM1129,130 (V: vascular), I-CAM1131,132,133 (I: intercellular) and I-CAM2134 have

all been determined. The integrin-fibronectin adhesion forces obtained range from 35 to 80 pN.

The wide range of values obtained might indicate that the strength of the receptor-ligand

interaction depends on the experimental conditions. Indeed, it has been reported that α5β1-

integrin-fibronectin adhesion force on K562 cells varied depending on the force-loading rate

from 40 to 140 pN135. The unfolding and detachment of fibronectin from surface may also

interfere. Other possible influences on the forces measured include non-specific binding of

fibronectin to the cell, extraction of integrins from the cell membrane, temperature, or changes in

cell properties such as membrane fluidity that could result in altered integrin mobility. However,

there is close agreement about the scale of forces required to rupture the bond between integrins

and their ligands. Unfortunately, experimental conditions have not been standardized and are

often not sufficiently documented to allow rigorous comparisons between experiments.

Several CAMs mediate only cell-cell adhesion such as cadherins and selectins. It has proved

difficult to characterize individual cell-cell adhesion events given the heterogeneous and complex

nature of cell surfaces. AFM-based force spectroscopy is capable of resolving such individual

cell-cell binding events but is limited in these kind of applications due to insufficient effective

pulling distances136. Long range force spectroscopy has been made possible by the development


22

of two AFM instruments by Asylum Research (USA) and JPK Instruments (Germany) with

extended vertical ranges of 40 and 100 µm respectively. Using these instruments, homophilic and

heterophilic binding capabilities of E-cadherin and N-cadherin have been compared137. Individual

E- and N-cadherin displayed different biochemical and biophysical properties. The kinetic of

cadherin pairwise bonds formation was also probed138. Similarly, it has been shown that the

visually distinct rolling patterns of leukocytes over selectin substrates are a consequence of the

kinetic and mechanical properties of the participating adhesive groups139.

The use of living cells to study global cell adhesion has been poorly investigated. It is

fundamental to distinguish affinity-based from avidity-based studies. The affinity describes the

strength of a single bond between an individual receptor on the surface of a cell and its ligand on

another cell or a surface. The avidity describes the global adhesion between two cells or between

a cell and a surface. The affinity of all the individual receptors to their ligands control the overall

adhesiveness (avidity) and for that reason, it is also important to investigate the global cell

adhesion. The interpretation of global cell adhesion is much more complex because avidity

depends on a multitude of biochemical and biophysical parameters. There is certainly the

possibility to extrapolate multiple-bond avidity to evaluate single-bond affinity but the

assumptions are not realistic. Avidity may depend on the density and affinity of the receptors for

their ligands as well as on their spatial arrangement and orientation. The surface area with the

probed cell, the duration of contact, the local geometry and stiffness of the plasma membrane,

and the rate of surface diffusion of cell receptors play also important roles140.

Short-term global cell adhesion has been used to compare the global cell adhesion properties of

wild-type embryonic cells from mutant embryonic cells, that carry a loss-of-function mutation in

a gene involved in gastrulation movements141,142. These findings suggested that the Wnt11 gene

is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin during

gastrulation.

Finally, long-term global cell adhesion has never been successfully reported in the literature. It

has been partially explored in a PhD thesis143. Fibroblasts were allowed to adhere to substrates

and then detached with a functionalized cantilever. The fraction of the probed cells that were

successfully detached was not sufficient (less than forty percent) to quantify significantly the

cellular adhesion forces.


23

1.4. SCFS configurations Cell-surface interactions can be investigated using SCFS in several configurations144,145,94,91.

Figure 1.12 summarizes the feasible SCFS configurations. Firstly, the contact time of the

interaction between a cell and a surface can be either short (I-IV) or long (V-VIII). In this thesis,

short-term adhesion is defined as contact times from seconds to minutes whereas long-term

adhesion is defined as contact times from hours to days. Therefore, short-term adhesion

measurements involve cells showing a round morphology in contrast to long-term adhesion

where cells have time to spread and to react to the surface. Secondly, the interaction surface that

is studied can be either the substrate or the cantilever. Finally, the cell can either stay on its initial

surface or be captured and transferred to the opposite surface. The study of cell-surface

interactions can be categorized in eight configurations:

(I) Short-term adhesion measurements have been widely investigated for receptor-

ligand interactions using living cells as probes with the cell on the

cantilever129,135,128,134,132,130,133. To date, global short-term adhesion has been poorly

quantified141,142.

(II) The transfer of a cell from the surface to the cantilever is used to perform short-

term adhesion measurements with the cell on the cantilever (configuration I).

(III) Short-term adhesion measurements have also been widely investigated for

receptor-ligand interactions using living cells as probes with the cell on the

substrate126,131,127.

(IV) The transfer of a cell from the cantilever to the substrate can happen during a

prolonged contact time between the cell and the substrate.

(V) In contrast, long-term adhesion measurements have been very little explored.

Long-term measurements with adherent cells on the substrate were restricted to the

analysis of the mechanical properties of cells124,125.


24

Fig.

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2. S

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atio

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25

(VI) The detachment of living single adherent cells has never been successfully

reported in the literature. It was partially explored in a PhD thesis but only a small

fraction of the probed cells were successfully detached from their growing

substrate143. This fraction was not sufficient to quantify the cellular adhesion

forces.

(VII-VIII) Finally, the direct growth of cells on the cantilevers for long-term adhesion in

order to quantify their adhesion forces has not been reported.

1.5. Dissertation objectives The main objective of this work is to use force spectroscopy to quantify the long-term global

adhesion between cells and surfaces and their response to modified surfaces. Long-term adhesion

measurements are important to allow the cells to spread and their adhesion proteins to be renewed

in order to react to their growing substrate.

Many techniques have been proposed for the quantification of cell adhesion. AFM-based force

spectroscopy has been shown to be a versatile tool allowing the quantification of receptor-ligand

interactions in living cells.

The quantification of long-term cell adhesion involves adherent cells. The first objective of this

work is to determine if the cell cycle influences cell adhesion or not. Cells in interphase all show

the same morphology under the microscope and therefore cannot be selected as a function of their

progression through the distinct phases of the cell cycle.

The quantification of long-term adhesion requires the detachment of adherent cells from their

culture substrate. The challenge is to find a method of attachment, between cells and atomic force

microscope cantilevers, that allows their detachment from the surface. The establishment of the

attachment has to be quick in order to avoid cell adhesion modifications.

The next step is to validate the technique for the detection of the effects of surface modification

on cell adhesion. The application of the technique in tissue engineering and implantology is

particularly interesting because both research fields are directly focused on cell-surface


26

interactions. The objective is to determine if long-term adhesion measurements might be useful in

the development of surfaces for tissue engineering. Long-term cell adhesion forces are quantified

on chemically modified surfaces used in cell sheet engineering in order to correlate the results

with the qualitative effects already reported.

In order to investigate the sensitivity and limitations of the detachment of adherent cells by force

spectroscopy, different experimental parameters can be altered. Surface topography is easily

tunable and its effects on cell adhesion have been widely studied. Another objective therefore is

to analyze the effects of topographical surface modifications on cell adhesion and to evaluate the

possible contribution to bone implant applications. Here cell type is especially important since

both immortalized and primary cells have been studied and very different results have been found

for different cell lines and primary cells. An objective therefore is to compare the detachment of

different cell types in order to determine the potential of the technique in this respect.

1.6. Organization of the dissertation

The dissertation starts with one chapter describing the materials and the experimental techniques

used in the next four chapters focussed on cell adhesion measurements.

Chapter 3 describes the short-term adhesion of Saos-2 human osteosarcoma cells to a glass

surface during the cell cycle. This first experimental chapter examines the effects of the different

phases of the cell cycle on cell adhesion in order to determine if cell adhesion has to be studied

independently of cell cycle or not. The cells were synchronized in three phases of the cell cycle,

trypsinized to release them from the culture surface and captured on a cantilever. Cell adhesion

was compared with unsynchronized cells. Short-term adhesion was used to study the adhesion

proteins already exposed at the cell surface in each phase of the cell cycle without influence of

the glass surface.

Chapter 4 focuses on long-term adhesion measurements with adherent cells. The challenge is to

find a method of attachment, between cells and atomic force microscope cantilevers, that allows

their detachment from the surface. Long-term adhesion on a glass surface was used and several

cantilever functionalizations were tested. Saos-2 osteoblasts were replaced by immortalized 3T3

and primary mouse fibroblasts.


27

Chapter 5 presents the quantification of 3T3 cell adhesion to chemically modified glass surfaces

used for cell sheet engineering. This chapter studies the effects of surfaces modifications on cell

adhesion in order to validate the technique. The cells were grown on different polyelectrolyte

multilayers and on poly(N-isopropylacrylamide) films before their detachment.

Chapter 6 presents the quantification of 3T3 and primary fibroblasts to topographically modified

quartz surfaces in order to investigate the sensitivity, the potential and limitations of the

technique. Two cell types were grown on different topographies and their properties of

detachment were compared.

Finally, the last chapter recaps the objectives in their context and summarizes the main

conclusions of the dissertation with an outlook for future work.


28

Chapter 2 Materials and Methods

29

Chapter 2

Materials and Methods

The materials and experimental techniques used in this thesis are described in this chapter. In

some cases more detailed explanations about the specific experiments are included in the

individual chapters.


30

2.1. Substrate preparation The substrates used were either glass or quartz surfaces.

2.1.1. Cleaning protocol

Glass coverslips (Milian, Switzerland) and quartz wafers (Si-Mat, Germany) were cleaned in

Piranha solution (H2SO4/H2O2) (4: 1)v/v for 10 min at 120 °C. The substrates were rinsed in

flowing water (MilliQ 185 plus, Millipore AG, Switzerland) and dried in a nitrogen flow. Piranha

solution reacts violently with all organics and should be handled with care.

2.1.2. Surface modification with polyelectrolytes*

The cleaned glass coverslips were treated with an oxygen plasma for 2 min. Poly-L-lysine

hydrobromide (PLL, Mw ~15 - 30 kDa, Sigma, Switzerland) and hyaluronic acid sodium salt

from bovine vitreous humor (HA, Mw ~300 kDa, Sigma, Switzerland) were used at a

concentration of 0.5 mg/ml in buffer solution. The buffer solution was 10 mM 4-(2-

hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) with 150 mM NaCl, pH adjusted to 7.4

with NaOH.

The polyelectrolyte multilayers were built up on the substrates by spray deposition, using a

custom-made automated spraying system. Four parallel Paasche VLS airbrushes (Paasche

Airbrush Co., IL, USA) connected to a compressor with pressure control allowed for the spraying

of each polyelectrolyte or buffer solution at a constant pressure of 1 bar, with one airbrush for

each different solution. A Lego Mindstorms NXT programmable robotics kit (Lego Group,

Denmark) was used to fabricate a spraying device with programmable spraying time, pause time

and sequence order. The cleaned substrates were placed on a sample holder allowing for drainage

of the solutions, fixed at a distance of 19 ± 2 cm from the airbrush aperture and tilted at 45°. Each

solution was sprayed according to the following sequence: 5 s polyelectrolyte spraying, 15 s

pause, 5 s buffer spraying, 5 s pause. In this way, the deposition of each bilayer required one

minute. The substrates were rinsed and conserved in the buffer solution.

______________________________________________________________________________*The surface modification with polyelectrolytes was done by O. Guillaume-Gentil at ETH Zurich (Switzerland).


31

2.1.3. Preparation of PNIPAM-grafted surfaces

A thin layer of chromium (2 nm) and another of gold (20 nm) were deposited on the cleaned

glass coverslips by evaporation (Auto 306 Turbo, Edwards, France). A self-assembled monolayer

of 11-mercaptoundecanoic acid (SAM-COOH) was prepared on the gold surface by immersion of

the coverslips in a 2 mM ethanolic solution of 11-mercaptoundecanoic acid (Sigma, MO, USA)

overnight. Finally, the samples were sonicated for 30 s in ethanol and dried in a nitrogen stream.

Amine-terminated poly(N-isopropylacrylamide) (NH2-PNIPAM) with an average molecular

weight of 41 000 g/mol (Polymer Source, Canada was grafted under melt onto anhydride reactive

SAMs (SAM-ANH). The activated anhydride groups were obtained by immersion of the SAM-

COOH in a solution of dimethylformamide containing 0.1 M of trifluoroacetic acid (Sigma, MO,

USA) and 0.2 M of triethylamine (Sigma, MO, USA) for 20 min followed by several washing

steps in dimethylformamide. A PNIPAM film was spin-coated from a 1% w/v ethanolic solution

onto the reactive SAMs at 3000 rpm for 20 s. The substrates were then placed in an oven and

annealed at 150 °C for 5 h. The PNIPAM-grafted surfaces were finally rinsed in ethanol for 1 h

and in deionized water overnight to eliminate the excess of polymer.

2.1.4. Preparation of patterned PNIPAM-grafted surfaces

Patterned PNIPAM-grafted surfaces were obtained using the same method except that the

uniform layer of gold was replaced by a pattern of squares of gold. The squares, 3 x 3 µm, with

an inter-space of 6 µm were obtained by classical photolithography and lift-off. The height of the

gold structures was 20 nm.

2.1.5. Micro-fabrication of low aspect-ratio pillars in quartz*

Low aspect-ratio (270nm high) quartz pillars were fabricated using a modified procedure

previously reported for silicon146. In a first step, a polymer blend thin film was deposited on the

four inch quartz wafers. Poly(methyl methacrylate) (PMMA, Mw=106 kDa, Polymer Standard

Services, Germany) and polystyrene (PS, Mw=101 kDa, Polymer Standard Services, Germany)

were dissolved in dioxane at a concentration of 15 mg/mL to give stock solutions. These were

mixed to obtain a PS/PMMA ratio of (30: 70)w/w. The polymer blend solution was then spin-

______________________________________________________________________________*The microfabrication of pillars in quartz was done by N. Blondiaux & M. Klein at CSEM Neuchâtel (Switzerland).


32

coated onto the substrate under a controlled atmosphere (T °C=21 °C and RH=35 %) using a two

step spin-coating procedure: 2 s at 500 rpm followed by 60 s at 2000 rpm. The resulting thin film

was structured in two phases, one of which (PS) was removed by rinsing the sample in

cyclohexane to reveal the quartz surface underlying the PS areas.

In the second step a durable etch mask was fabricated from the thin polymer film. The polymer-

coated surface was first exposed to an oxygen plasma (O2, 50 sccm, 0.05 torr, 15 W) for 90 s

using an RIE plasma (Plasmalab 80plus, Oxford Instruments, United-Kingdom) to remove any

residual PS layer and expose the quartz substrate. A 10 nm thick chromium layer was then

deposited on the sample using a thermal evaporator (Lab 600H, Leybold Optics, Germany). The

remaining PMMA phase and its chromium layer were removed by sonication in acetone.

The last step was the etching of the quartz, which was carried out in a deep reactive ion etcher

(DRIE) (AMS 200, Alcatel, France) using fluorine chemistry. After etching, the samples were

dipped for 5 min in a chromium etchant CR-7, then cleaned in SC-1 solution

(H2O/NH4OH/H2O2) (5: 1: 1)v/v for 10 min at 70 °C and finally exposed to an oxygen plasma

(RIE, O2, 100 sccm, 0.2 torr, 100 W) for 1 h.

Prior to cell seeding, the quartz surfaces were activated with an oxygen plasma (Harrick plasma,

NY, USA) for 10 min and then incubated in a solution of 0.01 % poly-L-lysine (MW = 150-300

kDa, Sigma, MO, USA) for 30 min to promote cell attachment.

2.1.6. Micro-fabrication of high aspect-ratio pillars in quartz*

The above procedure could be used to fabricate quartz pillars up to 270 nm high. However,

further etching resulted in mask failure. For this reason, an alternative procedure was developed

for higher aspect-ratio structures. A 200 nm layer of amorphous silicon (a-Si) was first deposited

on the quartz by means of LPCVD (Centrotherm Furnace, Germany). The chromium etch mask

was then fabricated on top of the a-Si layer as described above. The chromium layer was used as

an intermediate etch mask that was transferred into the a-Si layer using DRIE. The good etch

selectivity and the relatively thick (200 nm) a-Si mask, allowed the fabrication of deeper

structures in the quartz. After fabrication, the cleaning procedure was the same as for 270

______________________________________________________________________________*The microfabrication of pillars in quartz was done by N. Blondiaux & M. Klein at CSEM Neuchâtel (Switzerland).


33

nm pillars except that the samples were also exposed to KOH after the chromium etchant step in

order to remove the a-Si layer.

2.1.7. Sterilization protocol

The surfaces were sterilized with ethanol before their use in cell culture experiments. The

surfaces were successively immerged in 70% ethanol for 10 s, absolute ethanol for 10 s and

rinsed thrice in sterile deionised water.

2.2. Cell culture Three cell types were used including immortalized human osteoblasts, immortalized mouse

fibroblasts and primary mouse fibroblasts.

2.2.1. Saos-2 osteosarcoma cells

The human osteosarcoma cell line, Saos-2, was obtained from American Type Culture Collection

(Manassas, VA, USA) and was maintained in continuous culture in McCoy’s 5A medium

supplemented with 10% heat-inactivated standardized fetal bovine serum (Biochrom AG,

Germany), 50 units/ml of penicillin (Sigma, MO, USA), 50 µg/ml of streptomycin (Sigma, MO,

USA) and 1.5 mM of L-glutamine (Sigma, MO, USA) at 37°C in a humidified 5% CO2

atmosphere.

2.2.2. 3T3 mouse fibroblasts

The mouse fibroblast cell line, 3T3, was obtained from Marinpharm GmbH (Luckenwalde,

Germany) and was maintained in continuous culture in Dulbecco’s Modified Eagle Medium

(DMEM) supplemented with 10% heat-inactivated standardized fetal bovine serum (FBS,

Biochrom AG, Germany), 50 units/ml of penicillin (Sigma, MO, USA), 50 µg/ml of streptomycin

(Sigma, MO, USA), 1.5 mM of L-glutamine (Sigma, MO, USA) and 1% of non-essential amino

acids (Bioconcept, Switzerland) at 37 °C in a humidified 5% CO2 atmosphere.


34

2.2.3. Primary embryonic mouse fibroblasts

Primary mouse embryonic fibroblasts (PMEF) were isolated from embryos derived from C57

mothers between 12 and 14 days of gestation. Embryos were cut into small pieces and incubated

with a solution of 2.5 g/l trypsin and 0.38 g/l of ethylenediaminetetraacetic acid (EDTA) for 20

min at 37 °C under agitation after removing extracellular tissue, head and liver. Trypsin was

inactivated by addition of DMEM containing 10% FCS and cells were purified by successive

steps of filtration and centrifugation. Cells were maintained in DMEM supplemented with 10%

heat-inactivated standardized fetal bovine serum (FBS, Biochrom AG, Germany), 50 units/ml of

penicillin (Sigma, MO, USA), 50 µg/ml of streptomycin (Sigma, MO, USA) at 37 °C in a

humidified 5% CO2 atmosphere.

2.2.4. Cell cycle synchronization

Saos-2 cells were grown to about 60% confluency. Cell cycle arrest in late G1 phase was induced

using mimosine (Sigma, MO, USA) synchronization. Mimosine was added to a final

concentration of 0.4 mM and the cells were incubated for 48 h.

Cell cycle arrest in late S phase was induced using thymidine (Sigma, MO, USA)

synchronization. Thymidine was added to a final concentration of 4 mM and the cells were

incubated for 48 h. Cells were then washed twice in PBS and incubated with regular medium for

8 h.

Cell cycle arrest in G2M phase was induced using nocodazole (Sigma, MO, USA)

synchronization. Nocodazole-treated cells enter mitosis where they are blocked since they cannot

form metaphase spindles. Nocodazole was added to a final concentration of 0.6 µg/ml and the

cells were incubated for 48 h. Cells were then purified by gentle shaking and by pipetting them

off the dish.

Native mitotic cells were also studied. These were obtained by agitating unsynchronized cultures

of cells to detach mitotic cells from the polystyrene culture surface and then pipetting off the

resulting cells147,148.

2.2.5. Cell proliferation assay

Cells were incubated for 18 h in complete medium before adding Alamar Blue (dilution 1: 10,


35

Invitrogen, CA, USA) for 6 h. Aliquots were transferred to a spectrofluorimeter (Infinite M200,

Tecan, Switzerland) and the fluorescence intensity (λex= 530 nm and λem= 590 nm) was

measured. The remaining Alamar Blue was removed and replaced with fresh complete medium

for similar measurements at 2, 3 and 4 days.

2.2.6. Cell seeding

Cells were detached from the culture dishes by incubating with a solution of 2.5 g/l trypsin and

0.38 g/l of EDTA for 5 min. The dissociated cells were seeded on the surfaces following the

regular conditions of cultivation. Cell detachment experiments were performed in regular

medium supplemented with 25mM HEPES at 37 °C in a temperature-controlled chamber.

G2M synchronized and native mitotic cells were already partially or completely detached from

the culture dishes but were also exposed to trypsin under identical conditions.

2.3. Instrumentation

2.3.1. Atomic force microscope (AFM)

The atomic force microscope (AFM) is one of the family of scanning probe microscopes and was

invented in 198685. It is widely used in biological applications. The AFM uses a flexible

cantilever as a type of spring to measure the force between the tip and the sample. The attractive

or repulsive force between the tip and the sample is converted into a deflection of the cantilever.

This cantilever deflection is detected with a laser beam that is reflected from the back of the

cantilever onto a detector. A small change in the deflection angle of the cantilever is converted to

a measurable large deflection in the position of the reflected spot on the photodiode-detector.

Piezoelectric elements are used to control the position of the cantilever.

AFM is particularly suited for biological applications because the samples can be probed in

physiological conditions. There is no need for staining or coating. The AFM is best known for its

high-resolution imaging capabilities but it is also a powerful tool for sensitive measurements in

force spectroscopy mode. In this mode, the base of the cantilever is moved in the vertical

direction towards the surface using the piezo and then retracted again.


36

A Nanowizard® II BioAFM (JPK Instruments, Germany) was used .It has been developed for life

science research reaching from cell biology and biophysics to surface science and biomedicine.

The Nanowizard® was mounted on an Axiovert 200 inverted optical microscope (Carl Zeiss,

Germany) (Fig. 2.1). A CellHesion® module expended the vertical travel range of the

Nanowizard® to 100 µm by integrating a precise sample lift mechanism into the AFM stage. An

incubation chamber, the BioCell™ (JPK Instruments, Germany), maintained the sample at the

desired temperature (Fig. 2.2).

The atomic force microscope was mainly used in force spectroscopy mode to study cell/surface

interactions at the both cell and molecule levels.

Fig. 2.1. Pictureof the Nanowizard® II life science setup mounted on an inverted optical microscope. The stage and

AFM head are shown sketched separately. The micropositioners to move the sample, represented by a red glass

slide, are surrounded in red (A) and the micropositioners to move the AFM head are surrounded in blue (B).


37

Fig. 2.2. (A) Pictureof the Nanowizard® II life science setup used in this work and, (B) the BioCell™ as a add-on for

the temperature-dependent study of living cells in their native environment.

2.3.2. Fluorescence activated cell sorter (FACS)

Flow cytometry is a technology that simultaneously measures and analyzes multiple physical

characteristics of single cells flowing in a fluid stream through a beam of light. Size, granularity

and fluorescence intensity are measured. These characteristics are determined using an

optoelectronic system that records how the cells scatter the incident laser light and emit

fluorescence.

A flow cytometer is composed of three systems including fluidics, optics and electronics. The

fluidics system transports the cells in a stream to the laser beam for exposure. The optics system

consists of lasers to illuminate the cells in the sample stream and optical filters to direct the

resulting light signals to the appropriate detectors. The electronics system converts the detected

light signals into electronic signals that are processed by the computer (Fig. 2.3). Some

instruments are equipped with an electronics system capable of sorting the cells as a function of

their characteristics.


38

Fig. 2.3. Scattered and emitted light signals are converted to electronic pulses that are processed by the computer.

A FACSAria cell sorter (Becton Dickinson, CA, USA) was used to determine the relative cellular

DNA content during the various phases of the cell cycle using the fluorescent dye propidium

iodide. The incorporated amount of dye was proportional to the amount of DNA. Three distinct

phases were determined including the G1, S and G2M phases. G2 and M phases have an identical

DNA content and cannot be distinguished.

2.3.3. Confocal microscope

Confocal microscopy offers several advantages over conventional optical microscopy: the depth

of field is controlled and all the out of focus structures are suppressed at image formation. The

confocal approach uses spatial filtering to eliminate out-of-focus light in specimens that are

thicker than the focus plane. The confocal microscope offers also the ability to collect serial

optical sections from thick specimens. The cells were imaged with the confocal Leica TCS SP5

(Leica Microsystems, Germany).

2.3.4. Scanning electron microscope (SEM)

A scanning electron microscope (SEM) is a microscope that uses electrons instead of light to

form an image. The image is produced in vacuum by probing the specimen with a high-energy

focused electron beam. The incident electron beam loses some energy at each point on the

specimen that is converted into emission of secondary electrons, back-scattered electrons,

characteristic X-rays, light and heat. The display maps the varying intensity of any of these


39

signals to generate the image.

The samples were imaged with the XL-30 ESEM (Royal Philips, Netherlands) using secondary

electrons since they are the most sensitive to topography.

2.4. Force-distance curves Throughout this work “short-term adhesion” was defined as short cell-substrate contact time

(tcontact = 1 s). The cell was captured on the cantilever before the measurement. In contrast, “long-

term adhesion” was used for long cell-substrate contact time (tcontact = 24 h). In this case, the cell

was grown on the substrate and then detached with the cantilever.

2.4.1. Cantilevers for short-term adhesion

AFM cantilevers were functionalized with concanavalin A using a protocol adapted from

Wojcikiewicz et al149. 500µm long tipless silicon cantilevers (Arrow TL1, Nanoworld,

Switzerland) with a nominal spring constant of 0.03 N/m were used for all short-term adhesion

measurements. Cantilevers were cleaned in an oxygen plasma (Harrick plasma, NY, USA) for 5

min, and then incubated overnight at 37°C in a solution of 0.6 mg/ml biotinamidocaproyl-labeled

bovine serum albumin (Sigma, MO, USA) in 100 mM NaHCO3, pH 8.6. The cantilevers were

then rinsed twice in PBS, incubated in a solution of 0.6 mg/ml streptavidin (Sigma, MO, USA) in

PBS at pH 7.3 for 30 min. After two further rinses in PBS, they were finally incubated in a

solution of 0.6 mg/ml biotin-labeled concanavalin A (Sigma, MO, USA) in PBS for 60 min.

Cantilevers were calibrated in water before each measurement using the thermal fluctuation

method.

2.4.2. Cell capture for short-term adhesion

Using the AFM in force capture mode, the extremity of a concanavalin A-decorated cantilever

was precisely positioned above one cell. An approach step brought cell and cantilever into

contact for 2 s. This was immediately followed by a retraction step to remove the cell from the

surface. The cell was then left undisturbed on the cantilever for 15 min to ensure strong adhesion

between cell and cantilever.


40

2.4.3. Short-term adhesion measurements

Force distance curves were acquired with an approaching and retracting speed of 5 µm/s using

the maximum z-range of 100 µm. The cell was approached towards the surface until a repulsive

force of 900 pN was reached. It was left in contact with the surface for 1 s before the retract step

was started. There was a pause of 60 s at the maximal retract distance between each measurement

(Fig. 2.4).

Fig. 2.4. Schematic illustrating a short-term adhesion measurement with a contact time of 1 s between the cell and

the substrate.

For each cell the first and last force distance curves were compared to ensure that there were no

gross differences due to cell damage. To ensure that the cell-surface contact areas were similar

for all measurements, the largest and smallest cells of each population – in total no more than

20% of the population – were excluded from measurements.

2.4.4. Cantilevers for long-term adhesion

AFM cantilevers were functionalized with fibronectin to promote cell adhesion143. 130 µm long

silicon tipless cantilevers (NSC12, Mikromasch, Estonia) with a rectangular shape and a nominal

spring constant of 4.5 N/m were used for all long-term adhesion measurements. Cantilevers were

cleaned in piranha solution at 120°C for 10 min and activated with an oxygen plasma (Harrick

Plasma, NY, USA) for 3 min. The cantilevers were then incubated in a solution containing 94 %

acidic methanol (1 mM acetic acid in methanol), 5 % water and 1 % (3-aminopropyl)-

triethoxysilane (Sigma, MO, USA) for 30 min. The cantilevers were extensively rinsed in

methanol and dried before incubation in a solution of 1 % glutaraldehyde for 1 min. They were

finally rinsed in water and incubated in a solution of 10 µg/ml fibronectin (Sigma, MO, USA) for

1 h. The cantilevers were kept in the solution of fibronectin. Cantilevers were calibrated in water

before each measurement using the thermal fluctuation method150.


41

2.4.5. Long-term adhesion measurements

The extremity of a fibronectin-decorated cantilever was precisely positioned above the targeted

adherent cell. To ensure that the cell-cantilever contact area was similar for all measurements,

representative cells of comparable size were selected for each condition. The cantilever was

approached towards the cell until a repulsive force of 1.5 nN was reached. It was left in contact

with the cell in constant height for 30 min before the retract step was started. Force distance

curves were acquired with an approaching and retracting speed of 0.5 µm/s using the maximum

z-range of 100 µm. A new cell sample and a new cantilever were used before each measurement

(Fig. 2.5).

Fig. 2.5. Schematic illustrating a long-term adhesion measurement on an adherent cell with a contact time of 30 min

between the cantilever and the cell.

2.4.6. Cantilevers for indentation

Cell elasticity measurements were performed with the same cantilevers as for short-term

adhesion but without functionalization. Arrow TL1 tipless cantilevers (Nanoworld, Switzerland)

were send to Novascan to be modified by gluing glass spheres (radius = 5 µm, Novascan, IA,

USA) to the end of each cantilever to give well-defined indenters151 (Fig. 2.6).

Fig. 2.6. TL-1 tipless cantilever modified by Novascan with a 10 µm sphere attached (SiO2).


42

2.4.7. Elasticity measurements

Force distance curves were acquired with an approach speed of 2.5 µm/s to minimize

hydrodynamic effects. A maximum applied force of 200 pN resulted in a maximal indentation of

300 nm for the softest samples. There was a pause of 60 s at the maximal retract distance between

each measurement. Measurements were performed only on thicker regions of the cell.

2.5. AFM imaging* Polyelectrolyte layers were imaged on a Nanowizard I BioAFM (JPK Instruments, Germany)

using CSC38/noAl cantilevers (Mikromasch, Estonia) in intermittent-contact (fluid) mode.

2.6. Cell preparation

2.6.1. FACS

Cells were harvested by trypsinization, washed in PBS, collected by centrifugation and fixed in

cooled 70% ethanol for 1h. Cells were collected by centrifugation once more and suspended in

PBS containing 40 µg/ml propidium iodide (Sigma, MO, USA), 0.015% v/v Nonidet P40

substitute (Sigma, MO, USA) and 100 µg/ml RNase A (Sigma, MO, USA). The DNA content

was analyzed after at least 30 min incubation on ice.

2.6.2. Confocal microscopy

Cells were washed with cold PBS and fixed for 20 min in PBS containing 4% paraformaldehyde.

They were then rinsed twice with PBS and incubated for 10 min in a solution of 0.1 M glycine in

PBS. After two further rinses in PBS, the cells were incubated in a blocking solution composed

of 0.05 % saponine and 0.2 % bovine serum albumin in PBS, for 20 min. Cells were stained for

actin filaments with AlexaFluor 488 Phalloidin (dilution 1: 40, Sigma, MO, USA) for 2 h at room

temperature under agitation. The cells were finally washed twice in blocking solution, in PBS and

in water. A mounting medium was used to preserve fluorescence.

______________________________________________________________________________*The AFM images were obtained by O. Guillaume-Gentil at ETH Zurich (Switzerland).


43

2.6.3. SEM

Cells were rinsed with PBS and fixed in a solution of 2.5 % glutaraldehyde in 0.2 M cacodylate

buffer, pH 7.4 overnight. After this incubation, the cells were dehydrated in a series of

ethanol/water mixtures: 20 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 100 %

ethanol (5 min each incubation), followed by drying in air. The cells were observed after

sputtering with gold.

2.7. Data processing and statistical analysis

2.7.1. FACS

For each cell population, 15’000 cells were analyzed. Each synchronization experiment was

repeated at least 5 times. Data acquisition was performed with the FACSDiva software (Becton

Dickinson, CA, USA) and further analysis of the data was carried out with the WinMDI software

(J. Trotter, Scripps Research Institute, CA, USA).

2.7.2. Analysis of force-distance curves

After completion of a force-distance cycle, the measured changes in piezo z-position and the

corresponding deflection signal in newtons were saved by the Scanning Probe Microscopy

software (JPK Instruments, Germany)152. Force-distance curves were inspected before they were

included in the data analysis. They were discarded if the baseline was too short to ensure that the

cell was completely detached from the surface during cantilever retraction. The baseline was

linear in all curves analyzed. Acoustic noise, mechanical interference, microscopic objects or

other cells floating in the medium may cause unwanted cantilever deflection, which results in

undulating baselines.

For short-term adhesion, data analysis was carried out using ORIGIN 7.5 (OriginLab, MA,

USA). The force-distance curves were complex, usually containing a maximal force peak and

several force steps either preceded by a force plateau or not. The parameters analyzed were the

maximum force of detachment; the number, position and amplitude of the discrete detachment

steps observed along the retraction curve; and the work required to detach the cell from the

substrate. The work of detachment was obtained by integrating the adhesion forces over the


44

distance travelled by the cantilever. For each cell population, 10 force distance curves were

obtained from 10 different cells, giving 100 measurements. Each measurement provided one

maximum force of detachment, one work of detachment and several positions and amplitudes of

the discrete detachment steps.

For long-term adhesion, data analysis was carried out using the Image Processing software (JPK

Instruments, Germany). The force-distance curves were simple and contained one single

maximal force peak. A number of mechanical parameters related to cell detachment were

determined from the force-distance curves including the maximum force, work and distance of

detachment. For each condition, 1 force distance curve was obtained from each of 20 different

cells, giving 20 independent measurements. Each measurement provided one maximum force,

one work and one distance of detachment. The percentage of cells that detached from the surface

was determined by optically examining the end of the AFM cantilever after each measurement.

For elasticity measurements, data analysis was carried out using the Image Processing software

(JPK Instruments, Germany). The software gave the possibility to derive the Young’s modulus

from force curves running through several steps. All operations were applied to the approach

curve. The first step of the processing was to remove any offset or tilt from the curve. The contact

point was calculated automatically. Then the software calculated the indentation depth by taking

the difference between the piezo movement and the cantilever vertical deflection in units of

length. Finally the curve was ready to be fitted to the Hertz model to derive the Young’s

modulus. The formula was adapted for parabolic indenter (Fig. 2.7)153. For each condition, 5

force distance curves were obtained from each of 20 different cells, giving 100 measurements.

The Hertz model assumes linear elastic behaviour as well as homogeneity of the sample.

However, most biological materials are neither homogeneous nor absolutely elastic. The viscous

nature of the cell means that the energy delivered by the indenter is not completely given back by

a cell but dissipates. The variability of the viscous behaviour becomes visible if different

indentation velocities are tested154,155. Each cell type has its own relaxation time. Moreover, the

Hertz model is only valid for small indentations (a maximum of 10% of the cell height) where the

substrate doesn’t influence the measurements and where the geometry of the indentation matches

the geometry of the indenter. The Hertz model is a simple model to have a number in order to

quantify the stiffness of the cells.


45

Fig. 2.7. The Young’s modulus can be fitted using the Hertzian model for parabolic indenter. The model assumes

infinite thickness and linear elasticity of the sample156.

2.7.3. Statistical analysis

The forces, the work and the distances of detachment of the cells were quantified as described in

the previous section. The mean of the values together with its standard error was taken as

characteristic for each mechanical parameter of detachment.

For short-term adhesion, several force distances were obtained from each cell. Therefore, not all

the data were independent: results from any one cell were dependent, while data compared

between cells were independent. For this reason, the median value of each parameter was

determined separately for each cell. The mean of these median values together with its standard

error was then taken as characteristic for each condition.


46

The data obtained were analyzed with the statistical analysis and data analysis software S-PLUS

8.0 (Insightful, Switzerland). The two-sample T-test was used to test whether or not two

population means, m1 and m2, are equal. The samples are assumed to come from Gaussian

(normal) distributions. If it was not the case, logarithmic transformations giving normal

distributions of the mechanical parameters were analyzed. A two-tailed test was used, with a null

hypothesis H0 that is the logical counterpart, mutually exclusive and exhaustive, to an alternative

hypothesis H1. Depending on the outcome of the test, the null hypothesis is either rejected or

retained. Rejection of the null hypothesis logically leads to acceptance of the alternative

hypothesis and retention of the null hypothesis leads to an inability to accept the alternative

hypothesis; H0: m1 – m2 = 0 and H1: m1 – m2 ≠ 0. The Bonferroni correction was used for

multiple comparisons to account for the inflation of the Type I-error.

For all statistical tests, a p-value <0.05 was considered significant. The p-value is the probability

of obtaining a test statistic at least as extreme as the one that is actually observed, assuming that

the null hypothesis is true. The smaller the p-value, the more strongly the test rejects the null

hypothesis, that is, the hypothesis being tested.

Chapter 3 Cell adhesion during the cell cycle

47

Chapter 3

Cell adhesion during the cell cycle‡

In order to determine if cell adhesion has to be studied independently of cell cycle or not, cell

adhesion at different phases was measured. In this chapter, force spectroscopy was used to

measure the adhesion of Saos-2 cells to a glass surface at different phases of the cell cycle.

The cantilevers were functionalized for cell capture in order to perform short-term adhesion

measurements.

The cells were synchronized in 3 phases of the cell cycle: G1, S and G2M. Cells in these phases

were compared with unsynchronized and native mitotic cells. Individual cells were attached to an

AFM cantilever, brought into brief contact with the glass surface and then pulled off again. The

force-distance curves obtained allowed the work and maximum force of detachment as well as

the number, amplitude and position of discrete unbinding steps to be determined.

______________________________________________________________________________‡Part of this chapter was published in Measuring cell adhesion forces during the cell cycle by force spectroscopy.

Weder G., Vörös J., Giazzon M., Matthey N., Heinzelmann H. and Liley M. Biointerphases, 2009, 4(2), 27-34.


48

3.1. Introduction

3.1.1. The cell cycle

The eukaryotic cell cycle is divided into four phases157. The two most dramatic events are when

the nucleus divides, a process called mitosis, and when the cell splits into two cells, a process

called cytokinesis. These two processes constitute the M phase (M = mitosis) of the cell cycle.

The period between the M phase is called interphase, during which the cell grows in size. The

interphase is divided into the remaining three phases of the cell cycle: G1, S and G2. During the S

phase (S = synthesis), the cell replicates its DNA. The G1 phase (G = gap) is the interval between

the completion of the M phase and the beginning of the S phase. The G2 phase is the interval

between the end of the S phase and the beginning of the M phase.

Fig. 3.1. (A) The four successive phases of a standard eukaryotic cell cycle. (B) The relative amount of DNA per cell

during the cell cycle.

Cell cycle can be analyzed by fluorescence labeling of the DNA of cells in suspension and using

a fluorescence activated cell sorter (FACS). G1 cells have one copy of DNA and have therefore

1X fluorescence intensity. Cells in G2M phase have two copies of DNA and have therefore 2X

fluorescence intensity. Since the cells in S phase synthesize DNA, they have fluorescence values

between the 1X and 2X populations.


49

3.1.2. Adhesion of mitotic cells

For most adherent cells in vitro, dramatic changes in cell morphology take place during the cell

cycle158. For most of the cell cycle, the cells are spread over the surface of the substrate, then, at

the beginning of mitosis, they round up and lose most of their contact to the substrate in

preparation for cytokinesis. Mitotic rounding up is associated with changes in the cytoskeleton,

plasma membrane and cell volume, while adhesion of the cell to the substrate is greatly

reduced159. However, the underlying mechanisms of these changes are poorly understood.

Adherent cells attach to their surroundings via focal adhesions. These large protein complexes

consist of a variety of cytoskeletal and cytoskeletal-associated proteins providing the primary

stabilizing force for the attachment of cultured cells as well as initiation sites for actin stress

fibres160,161. There have been many studies of the role of individual adhesion proteins in the

rounding up of mitotic cells162. These studies show that various phosphorylation events of

cytoskeletal163 and focal adhesion proteins164,165,166,167,168,169 contribute to the pre-mitotic

disassembly of focal adhesions170, the deconstruction of the actin cytoskeleton of the

interphase171 and the construction of a new mitotic cytoskeleton172. However, despite this

dissociation and deconstruction, the mitotic cell remains attached to the substrate. There is direct

contact between the plasma membrane and the substrate173. In addition, there are a number of

retraction fibres, thin actin filaments, that anchor the cell to the substrate via focal adhesions.

Thus some adhesive elements remain during mitosis.

Not only the cytoskeleton, but also the plasma membrane is remodelled during mitosis. Rounding

up of the cell involves a reduction of the total area of plasma membrane, which has been recently

found to take place via normal endocytosis of the plasma membrane and a reduction in the

recycling of internalized membranes174.

3.1.3. Short-term adhesion measurements

A single cell was attached to an AFM cantilever, brought briefly into contact with a glass

substrate, and then pulled off the substrate175 (Fig. 3.2A). Measurement of the cantilever

deflection during this process allowed the forces between cell and surface to be quantified at each

moment and represented in force distance curves (Fig. 3.2B). Several different mechanical

parameters were extracted from these curves152 including the maximum force of detachment, the


50

displacement needed to completely remove the cell from the substrate and the work of

detachment. Not only global cellular parameters were studied: it was also possible to identify and

analyze individual unbinding events, discrete steps in the force distance curve that occur on the

release of individual proteins or protein complexes from the surface176.

Fig. 3.2.(A) Schematic of a single-cell force spectroscopy measurement, and (B) a force-distance curve acquired

during approach (I-II in blue) and retracting steps (III-IV in orange). In the initial phase of the approach there is no

contact between the cell and the surface (I). Then the cell is pressed onto the surface until a pre-set maximal force is

attained. During this phase the elastic response of the cell can be observed (II). The position of the cantilever is held

constant for a given contact time. Information on different mechanical parameters can be obtained from the

retraction: the work of detachment, the number, amplitude and position of the unbinding events corresponding to

single proteins or protein complexes and, finally, the maximal force needed to detach the cell from the surface (III).

In the last step, there is no physical contact between cell and surface (IV).

With this approach, the results obtained reflected the composition and organization of the plasma

membrane, for example, concerning the presence, activity and organization of adhesion proteins.

However, the short contact time – one second - between cell and substrate means that no

information could be gained about how the cell responds to the surface on a longer time scale,

thus excluding the effects of, for example, changes in protein expression.


51

3.2. Cell cycle synchronization

3.2.1. G1, S and G2M

Saos-2 cells were synchronized in 3 phases of the cell cycle, G1, S and G2M. Unsynchronized cell

populations showed the expected distribution of 56% of cells in the G1 phase, 21% in the S phase

and 23% in the G2M phase. Good synchronization was obtained in both the G1 phase with 78% of

cells in G1 and in the G2M phase with 80% of cells in G2M. S phase synchronization was limited

to 62% due to differences in resumption of the cell cycle for individual cells after release from

the thymidine synchronization (Fig. 3.3). A more specific synchronizing agent to synchronize the

cells in the intermediate S phase of DNA replication was not found.

Fig. 3.3.DNA content analysis of the cell cycle measured by flow cytometry of SaOs-2 cells in the presence of

synchronizing agents: (A) unsynchronized, (B) mimosine as a G1 synchronizing agent, (S) thymidine as a S

synchronizing agent and, (D) nocodazole as a G2M synchronizing agent.


52

3.2.2. Native mitotic cells

When studying synchronized cells, it is important to be aware that the synchronizing agent may

affect several properties of the cell; nocodazole, for example, inhibits the polymerization of the

microtubules. In order to exclude the possibility that any differences observed between G2M cells

and unsynchronized cells were an unexpected artifact due to the use of nocodazole, native mitotic

cells were also investigated. Native mitotic cells were obtained from unsynchronized cell cultures

and were separated from cells in other phases of the cell cycle thanks to their reduced adhesion to

the culture surface. Gentle agitation of the medium or culture support resulted in their release

from the surface into the culture medium while other cells remained firmly attached.

3.3. Short-term adhesion

3.3.1. Presence of adhesion proteins throughout the cell cycle

First experiments carried out with the Saos-2 cells were used to select the experimental

parameters for the short-term adhesion measurements. An applied force of 900 pN was chosen

and a contact time of 1 s was selected to avoid very strong cell-surface interactions that might

result in a failure to totally detach the cell from the surface even at a retraction distance of 100

µm.

Using these experimental parameters, typical force distance curves obtained for each of the

synchronized cell populations are shown in figure 3.4. Qualitatively, the curves look similar, with

discrete unbinding events indicating the presence of adhesion-protein complexes at the cell

membrane in each phase of the cell cycle. A statistical analysis of these events was carried out,

comparing the number of events per curve, the amplitude of the events and the position of the

events along the retraction curve for the three synchronized cell populations and for a population

of unsynchronized cells.


53

Fig. 3.4. Typical force distance curves showing the detachment of G1, S and G2M synchronized human osteoblasts

from a glass surface (contact for 1 s with an applied force of 900 pN). The step-like features in the curves

correspond to the release of adhesion proteins from the glass and can be observed in all three phases of the cell

cycle.

Histograms showing the number of unbinding events are shown in figure 3.5. In all cell

populations, there were between 4 and 23 unbinding events per curve, reflecting the number of

adhesion protein complexes that bound to the surface. Comparing the mean numbers of

unbinding events for the different populations, the values obtained are 8.9 and 12 in interphase

(G1 and S), while in the two M-phase populations (G2M and native mitotic) the means are 11.3

and 10.9. For the unsynchronized cells, which are expected to be approximately 80% in

interphase (since made up of 56% G1 and 21% S), 12.6 is obtained. The number of unbinding

events in interphase and metaphase are broadly similar: there is no large decrease in unbinding

events that might be associated with a loss of adhesion in M-phase (Table 3.1).


54

Fig. 3.5. Histograms showing the number of unbinding events of human osteoblasts on a glass surface after an

applied force of 900pN for 1s. The number of unbinding events was measured for unsynchronized cells and

synchronized cells in G1, S and G2M phases. Between 4 and 23 unbinding events were observed in each force

distance curve independently of the cell cycle phase.

Mean (Median) ±SEUnsynchronized 12.6 0.5G1 phase 8.9 0.4S phase 12 1G2M phase 11.3 0.5Native mitotic 10.9 0.8

Number of unbinding events

Table 3.1. Comparison of the number of unbinding events for 5 conditions: unsynchronized cells, G1, S, G2M

synchronized cells and native mitotic cells (SE: standard error).


55

3.3.2. Position of the unbinding events

The positions (distance from the surface) of the unbinding events in the force distance curves

were also analyzed. Clear differences were observed between the G2M phase and the other

populations, while differences between unsynchronized, G1 and S phases were not significant. A

statistical analysis of the number of events before and after 30 µm displacement of the cantilever

is shown in table 3.2.

● For unsynchronized cells, 78% of unbinding events occur within the first 30 µm and 22% in

the remaining distance. Similar values were obtained for cells in G1 and S.

● For cells in G2M, 93% of unbinding events occur within the first 30 µm and 7% in the

remaining 70 µm of travel.

● For native mitotic cells, 95% of unbinding events occur within the first 30 µm and 5% in the

remaining 70 µm of travel.

Clearly, cells in G1 and S can be stretched further before the cell is released from the surface than

cells in G2M or native mitotic cells.

≤ 30µm (%) > 30µm (%)Unsynchronized 78 22G1 phase 80 20S phase 79 21G2M phase 93 7Native mitotic 95 5

Position of unbinding events

Table 3.2. Comparison of the distribution of the position of unbinding events between 5 conditions: unsynchronized

cells and G1, S, G2M synchronized cells and native mitotic cells.

3.3.3. Adhesion per protein complex in the different cell cycle phases

The amplitudes of the unbinding events are very similar in all the different conditions, with mean

values between 84 and 108 pN (Table 3.3). The size of these events suggests that they correspond

to the rupture or release of either individual proteins or small protein complexes from the glass

surface.


56

Comparing the values obtained for interphase (G1 and S) with M-phase (G2M and native mitotic),

86 and 84 pN respectively for the interphase populations and 108 and 96 pN for the M-phase

populations are observed. In contrast, the unsynchronized population, which is expected to be

approximately 80% in interphase, gives a value of 104 pN.

The similarity of the unbinding forces obtained for all phases of the cell cycle is perhaps

surprising: there are numerous reports of the phosphorylation of individual proteins associated

with the cytoskeleton and with focal adhesions, which might lead one to expect differences (i.e. a

reduction) in the binding ability of the proteins exposed at the cell membrane.

Mean (Median) (pN) ±SEUnsynchronized 104 17G1 phase 86 6S phase 84 13G2M phase 108 5Native mitotic 96 6

Amplitude of unbinding events

Table 3.3. Comparison of the amplitude of the unbinding events for 5 conditions: unsynchronized cells, G1, S, G2M


3.3.4. Maximum force of detachment

The values obtained for the means of the maximum forces of detachment are similar for all

phases of the cell cycle: the values, between 0.88 and 1.2 nN, are shown in table 3.4. Comparing

interphase and M-phase, 0.88 and 0.99 nN for G1 and S populations and 1.2 nN for both G2M and

native mitotic cells were obtained. The unsynchronized population gives a value of 1.2 nN. The

maximum force of detachment is not strictly an independent parameter as it is related to the

number of unbinding events and their amplitude. It is, however, not simply the product of the

number of unbinding events and their amplitude due to contraction and/or relaxation of the cell

during the measurement, particularly the first few microns of retraction. The values obtained for

the unsynchronized cell population are compatible with those obtained for each of the phases

individually. The only statistically significant differences are between the distributions of the G1-

phase and mitotic cells.


57

Mean (Median) (nN) ±SE

Unsynchronized 1.2 0.1

G1 phase 0.88 0.03

S phase 0.99 0.09

G2M phase 1.2 0.06

Native mitotic 1.2 0.06

Maximum force of detachment

Table 3.4. Comparison of the maximum force of detachment for 5 conditions: unsynchronized cells, G1, S, G2M


3.3.5. Work of detachment

The work of detachment – similarly to the maximum force of detachment – is not strictly an

independent parameter as it is related to the amplitude and number of unbinding events and their

position during retraction of the cantilever. The mean works of detachment are similar for the

unsynchronized and synchronized cells with values between 1.9 and 0.94 x 10-14 J (Table 3.5). In

interphase, the values obtained are 1.4 and 1.7 x 10-14 J for G1 and S populations. In M-phase the

values are 1.1 and 0.94 x 10-14 J for G2M and native mitotic. 1.9 x 10-14 J was obtained for

unsynchronized cells. The only statistically significant differences are between the distributions

of the unsynchronized and mitotic cells.

Mean (Median) (J) ±SE

Unsynchronized 1.9 x 10-14 0.2 x 10-14

G1 phase 1.4 x 10-14 0.2 x 10-14

S phase 1.7 x 10-14 0.2 x 10-14

G2M phase 1.1 x 10-14 0.1 x 10-14

Native mitotic 0.94 x 10-14 0.2 x 10-14

Work of detachment

Table 3.5. Comparison of the work of detachment for 5 conditions: unsynchronized cells, G1, S, G2M synchronized

cells and native mitotic cells (SE: standard error).


58

3.3.6. Higher stiffness of mitotic cells

The analysis of the positions of the unbinding events suggested that G2M and native mitotic cells

were stiffer than cells in the other phases. The increased stiffness of mitotic cells has been

reported by other authors177,124 and is associated with a reorganization of the cytoskeleton (Fig.

3.6).

Fig. 3.6. Comparison of unsynchronized (A) and G2M synchronized cells (B) by optical (I) and confocal microscopy

(II) showing the actin fibres (labeled with AlexaFluor 488 Phalloidin). The cell rounding and reorganization of the

cytoskeletal actin fibres observed in B are characteristic of cytokinesis.

This was verified for Saos-2 cells using force spectroscopy to obtain an estimate of the Young’s

modulus of the cells. For these measurements, the cells were adherent on a glass surface and a

round glass sphere attached to an AFM cantilever was brought into contact with them. Given the

size of the glass sphere and the forces applied during the indentation process it was possible to

calculate a value for the Young’s modulus using a Hertzian model (section 2.7.2). The

indentation is the difference between the piezo movement and the cantilever vertical deflection in

units of length (Fig. 3.7).


59

Fig. 3.7. Force indentation curves derived from (A) unsynchronized and (B) G2M synchronized Saos-2 cells. The

applied force of 200 pN resulted in an indentation of 210 nm for the unsynchronized cells and of 90 nm for the G2M

synchronized cells.

Values for the Young’s modulus of individual G2M synchronized cells and individual adherent

unsynchronized cells are shown in figure 3.8. Typically, the Young’s moduli of the G2M

synchronized cells (mean value 1.4 kPa) are higher than those of the unsynchronized cells (mean

value 0.4 kPa) confirming the increase of the cell stiffness in mitosis. The data obtained are

within reported values for the Young’s modulus of living cells, (between 0.1 and 10 kPa)178,179. It

is difficult to make close comparisons with literature values, given the range of experimental

parameters that influence the values obtained (e.g. geometry of the indenter, depth of indentation,

speed of measurement). However, studies on osteosarcoma cell lines and on primary human bone

cells including osteoblasts give very similar Young’s moduli: between 1 and 2 kPa for MG-63

cells180, from 0.7 to 2.6 kPa for primary human bone cells and MG-6335, and from 2.1 to 8.8 kPa

for Saos-2 cells181.


60

Fig. 3.8. Histogram showing the values of the Young’s modulus obtained for individual unsynchronized cells and

cells synchronized in G2M phase. Vertical lines indicate the mean values and the error bar is the standard error. No

native mitotic cells were chosen for measurement in the unsynchronized cell population.

3.4. Discussion

3.4.1. Cell adhesion between M-phase and interphase

The experiments described here show that binding proteins and/or protein complexes at the cell

membrane remain similar both in number and in their binding properties during all the phases of

the cell cycle. Indeed, while some differences are observed in the surface binding properties of

the cells, the variations are, in all cases, less than a factor of two and the lowest values are not

associated with the M phase.

A detailed statistical analysis of the data was used to determine the probability that each

measured parameter of each group of cells is the same (the p-value). Table 3.6 shows that the

statistical differences (see white dots on table 3.6) existing between two cell populations are not

associated with the M phase. However, the statistical analysis cannot determine whether or not

the ten cells selected for analysis are representative of the entire cell population under this

condition. It is almost certainly this limitation that results in some unexpected data points (see

white dots). Despite the difficulty to draw a conclusion for small differences between cell

populations, cell adhesion related to M-phase was not different from those related to interphase.


61

Unsynchro G1 phase S phase G2M phase Unsynchro G1 phase S phase G2M phaseUnsynchroG1 phase ○ ●S phase ● ○ ● ●G2M phase ● ○ ● ● ○ ○Native mitotic ● ● ● ● ● ● ○ ●

Unsynchro G1 phase S phase G2M phase Unsynchro G1 phase S phase G2M phaseUnsynchroG1 phase ● ●S phase ● ● ● ●G2M phase ● ○ ● ○ ● ●Native mitotic ● ○ ● ● ○ ● ● ●

Amplitude of unbinding events

Work of detachment

Number of unbinding events

Maximum force of detachment

Table 3.6. Comparison of the number and amplitude of unbinding events, and the maximum force and work of

detachment for 5 conditions: unsynchronized cells, G1, S, G2M synchronized cells and native mitotic cells. Data were

compared using the two sample T-test with Bonferroni correction (p-value 0.05). The black dots indicate that the cell

populations are not statistically significantly different (p-value ≥0.05) and the white dots indicate that the cell

populations are statistically significantly different (p-value <0.05).

3.4.2. Statistics

Force spectroscopy is inherently a single cell technique: values for different mechanical

properties are determined individually for each cell of each population studied. Single cell

analyses allow us to study not only the average properties of a sample (an ensemble of cells), but

also the variation and heterogeneity within the sample. While this gives access to qualitatively

new information, it also comes with its disadvantages. In our case, the statistical analysis was

made significantly more complicated: for each cell condition ten cells were selected for study –

this relatively small number was chosen because of the lengthy preparation required for

measurements on each individual cell - and ten force distance measurements were carried out per

cell. This method gave a dataset that was a mix of dependent and independent results. In general,

the analysis required that the data for each cell (dependent results) were analyzed first to obtain a

value characteristic of that cell. The values for each cell in the sample were then analyzed as

independent data points to give a value that was representative of the sample as a whole.


62

3.4.3. The use of trypsin

A significant limitation of this study is the use of trypsin to detach the cells from the culture

surface before fixing them to the cantilever. Trypsinization clearly damages at least some of the

binding proteins exposed at the cell surface and so may well influence results in a study of these

proteins. The effects have tried to be minimized by using a short, reproducible trypsinization on

all samples and allowing the cells to recover for 30 minutes before measurement. Studies by

other authors have shown that, at least in some cases, trypsinization leaves the binding functions

of surface proteins largely intact128,130. SDS-PAGE analyses also showed no effect of the

trypsinization on either α2 integrin or β1 integrins28. While trypsinization may limit the work to

the observation of relatively large effects, it is believed to have no influence on the conclusions of

this study.

3.4.4. The short contact time

Finally, the short contact time between cell and surface during this study limits the analysis to a

study of proteins and protein complexes that are already present at the cell membrane and to the

gross mechanical properties of the cell. There is insufficient time for the cell to react significantly

to the presence of the surface, for example, by changes in the cytoskeleton or in protein

expression. A more complete study of cell surface interactions requires much longer contact

times between cell and surface. However, increased contact times between SaOs-2 cells and glass

result in increased cell/surface adhesion. The results are either a rupture of the cell as cantilever

and surface are separated, or alternatively, the cell does not rupture, but remains attached to both

surface and cantilever even at separations of 100 microns.

3.5. Conclusion Single cell force spectroscopy was used to study the adhesive and mechanical properties of SaOs-

2 cells in culture. The study focused in particular on the changes in these properties between

interphase, when cells are well spread and firmly attached to culture surface and M-phase, when

the cells round up and are much more loosely attached to the surface. The differences in

cell/surface attachment are striking: in M-phase SaOs-2 cells may be removed by shaking culture

vessels while in interphase attempts to pull SaOs-2 cells off the surface may result in cell rupture.


63

Because of the short time of contact between cell and surface, the investigation is relevant to cell

surface properties and cell stiffness. The experiments described here show that binding proteins

and/or protein complexes at the cell membrane remain similar both in number and in their

binding properties (amplitude of the unbinding events) during interphase and M phase. While

some differences can be observed in the mean values obtained in the different phases, the

differences are, in all cases, less than a factor of 2 and the extreme values are not associated with

the G2M or native mitotic (M) phase. No large reduction was observed either in number or in

amplitude of the unbinding events that might contribute to the loss of adhesion of M-phase cells.

This may be surprising given the numerous studies showing the phosphorylation of proteins

associated with focal adhesions during mitosis.

A second clear conclusion can be drawn from the position of the unbinding events. As the cell

was stretched during detachment from the surface, unbinding events took place at a smaller

extension for the mitotic/G2M cells than for cells in other phases. These cells cannot be stretched

as far before they are released from the surface. This is due to an increased stiffness of the cell

caused by a reorganization of the cytoskeleton during mitosis as also observed by other

researchers in the field.

The maximum force of detachment and the work of detachment were also studied for all the cells.

These two parameters are not strictly independent as they are related to the number and amplitude

of unbinding events and to their position. Here, again the differences observed between M phase

and interphase were relatively small compared to the differences between cellular adhesion

during the cell cycle.


64

Chapter 4 Cantilever functionalization for cell detachment

65

Chapter 4

Cantilever functionalization for cell

detachment

Long-term cell adhesion involves adherent cells. The challenge is to establish an attachment

between cells and AFM cantilevers strong enough to allow their detachment. In this chapter,

AFM cantilevers were functionalized for long-term adhesion measurements in order to detach

living adherent 3T3 fibroblasts.

The cantilevers were prepared with 4 different functionalizations: concanavalin A, fibronectin,

poly-L-lysine and zirconium. 3T3 mouse fibroblasts were allowed to grow for one day on a glass

surface. Then the cantilevers were pressed onto the adherent fibroblasts for a given contact time

and withdrawn. The functionalization showing the highest percentage of cell detachment was

selected for the rest of this work.


66

4.1. Introduction

4.1.1. Long-term adhesion

The use of force spectroscopy permits the study of adhesion of living cells to be studied. In this

thesis, “short-term adhesion” is defined as short cell-substrate contact times, from seconds to

minutes, whereas “long-term adhesion” is defined as long cell-substrate contact times, from hours

to days. Long-term adhesion allows the cells to form strong adhesion complexes that involves a

large number of receptors bindings cooperatively. Long-term adhesion measurements

characterize the cell-surface interaction because the cells can spread, react to the surface and their

adhesion proteins can be renewed. That is the reason why the main objective of this work is to

detach living single adherent cells in order to quantify their global adhesion forces.

Other authors have reported that single cell force spectroscopy is currently restricted to short

contact times that range from milliseconds to about twenty minutes152. This restriction was

observed using single cells captured on an AFM cantilever and the main limitation was the loss

of the cell from the cantilever at prolonged cell/surface contact times i.e. exceeding one hour

(Fig. 4.1). A single cell was captured on a coated AFM cantilever and allowed to settle on the

cantilever for approximately fifteen minutes away from the surface. Then the cell was pressed

onto the substrate for more than one hour. At long contact times, the adhesion between the

cantilever and the cell was weaker than that between the substrate and the cell. Using

concanavalin A to immobilize the cell on the cantilever, forces of up to 50 nN were measured

before the cells detached from the cantilever. This force was, however, dependent on cell type. In

addition, the authors noticed that the almost unavoidable thermal and mechanical drifts in AFM

complicate those experiments that exceeded thirty minutes.

Fig. 4.1. Schematics illustrating a cell captured on an AFM cantilever for (A) a short cell-substrate contact time and

(B) a long cell-substrate contact time exceeding one hour: the interaction cell-cantilever is weaker than the

interaction cell substrate and there is a loss of the cell from the cantilever.


67

One possible solution is to first seed the cell on the surface and then to detach it with

functionalized AFM cantilever (Fig. 4.2). In this way, the cell can be incubated for the desired

time on a specific surface under physiological condition without mechanical stress by the

cantilever on it. In this approach, the challenge is to establish a quick adhesion between cells and

AFM cantilevers capable of supporting their detachment. The establishment of the connection

cell-cantilever has to be fast. This is to avoid a disturbance of the adhesion properties of the cell

on the specific surface, and to limit problems related to the thermal and mechanical drifts at the

system. Another advantage of seeding cells on the surface is the elimination of the use of trypsin.

During the long contact time between cell and substrate, cellular adhesion proteins are renewed.

Fig. 4.2. Schematic illustrating a long-term adhesion measurement. The cell is first incubated on the surface for a

long cell-substrate contact time and then detached with an AFM functionalized cantilever. The interaction cell-

cantilever is stronger than the interaction cell-substrate and the cell is detached from the surface.

4.1.2. Cantilever functionalization for cell detachment

In order to successfully detach cells, the adhesion between cell and cantilever has to be stronger

that between cell and substrate. A wide variety of molecules exposed at the cell surface such as

sugars, proteins or lipids are candidates to establish this strong adhesion.

Several cantilever functionalizations have been used by other authors for cell capture. Lectin-

decorated cantilevers, especially with concanavalin A182,136,183,128 and wheat germ

agglutinin123,175, have been widely used to capture several cell types. Lectins are sugar-binding

proteins that interact with the glycoproteins exposed at the cell surface. Cantilevers have also

been decorated with fibronectin that interacts with integrins that are the major adhesion receptors

that mediate cell-substrate adhesion184,143. Alternatively, a charge-based approach has been

reported to allow capture of cells on a cantilever array (several cantilevers in parallel)185. The


68

cantilevers were functionalized with poly-L-lysine and then the cells were added in a low

conductivity medium. The biotin-streptavidin system has been used to capture cells on

cantilevers140,137. Cell surface molecules were biotinylated while the cantilever was decorated

with streptavidin. Finally, antibody-antigen interactions have been used for specific cell capture.

Jurkat cells, an immortalized line of T lymphocytes, were coupled to AFM cantilevers using an

anti-leucosialin functionalization129. All these cantilever functionalizations have been shown to

be efficient for cell capture in order to perform short-term adhesion measurements. In contrast,

cantilever functionalization for long-term adhesion with cell detachment is much more

demanding due to the necessity for a stronger attachment of the cell to the substrate.

In order to determine which cantilever functionalization was the most suitable for cell

detachment, three different cantilever functionalizations, commonly used for cell capture, were

systematically tested. The cantilever functionalizations targetted various molecules exposed at the

cell surface. Concanavalin A was selected to bind sugars, fibronectin to bind proteins and poly-L-

lysine to interact with charges.

In addition, a zirconium cantilever functionalization was tested to interact with the lipids of the

cell membrane. Indeed, tetravalent metal ions such as Zr4+ have been shown to interact strongly

with phosphates and phosphonates186. Recently, the affinity between the metal ion and

phosphates/phosphonates was exploited in the bioanalytical field187: capillary columns188 and

porous wafers189 were coated with zirconium phosphonate for phosphopeptide enrichment for

mass spectroscopy. Zirconium coatings on AFM cantilevers have never been used for cell capture

or cell detachment.

4.2. Detachment of adherent cells

4.2.1. Percentage of cell detachment

Single adherent cells were detached from glass surfaces using different AFM cantilever

functionalizations. 3T3 mouse fibroblasts were grown for 24 hours on a flat glass surface. The

glass sample was placed in a temperature-controlled fluidic chamber and a single adherent cell

was selected. The functionalized-cantilever was positioned above the cell and gently pressed on it


69

until a force of 1.5 nN was attained (Fig 4.3A). The cantilever was left in contact for 30 minutes

to allow the cell to interact and bind actively with the functionalized cantilever. Finally, the

cantilever was withdrawn until the cell detached either from the surface or from the cantilever.

For each condition, one force distance curve was obtained from each of twenty different cells.

The cantilevers were coated with concanavalin A, fibronectin, poly-L-lysine or zirconium.

During this process, the forces exerted on the cell were continuously recorded and the values

were represented in a force distance curve (Fig 4.3B). The first qualitative information that was

obtained was the percentage of cells detached from the glass surface. The maximum force,

corresponding to the maximum deflection of the cantilever, and the work of detachment were

quantified for the cells successfully detached from the surface.

Fig. 4.3. (A) Schematic of the detachment of a single adherent cell and, (B) a corresponding force-distance curve.

The cantilever is pressed onto the adherent cell until a preset maximal force of 1.5 nN is attained. The position of the

cantilever is held constant for 30 min before it is withdrawn. A force-distance curve is recorded indicating the

maximum force and work of detachment. On both panels, orange defines the approach and turquoise defines the

retraction.


70

The percentages of cell detachment from the glass surface were between zero and seventy-five

percent for all cantilever functionalizations. Fifteen percent of cells were detached using the

concanavalin A-functionalized cantilevers, seventy-five percent with the fibronectin, thirty-five

percent with the poly-L-lysine and zero percent with the zirconium. Native cantilevers without

functionalization didn’t allow the detachment of any cells.

Concanavalin AFibronectinPoly-L-lysineZirconiumNative silicon

Cantilever functionalization

00

Percentage of cell detachment (%)

157535

Table 4.1 Comparison of the percentages of detachment of 3T3 cells detached from glass surfaces using four

cantilever functionalizations: concanavalin A, fibronectin, poly-L-lysine and zirconium.

4.2.2. Maximum force of detachment

The maximum forces of detachment of the cells detached with the different functionalized

cantilevers were not statistically different. The maximum forces were quantified only for the cells

that detached from the glass surface. The mean values for the maximum forces of detachment

were 170 nN with the concanavalin A, 230 nN with the fibronectin and 190 nN with the poly-L-

lysine (Fig. 4.4).

4.2.3. Work of detachment

The works of detachment – similarly to the maximum forces of detachment – were similar. The

mean values for the work of detachment were 2.5 pJ with the concanavalin A, 3.3 pJ with the

fibronectin and 2 pJ with the poly-L-lysine (Fig. 4.5). The works of detachment showed no

statistically significant differences.


71

Fig. 4.4. Histogram showing the distribution of the maximum forces of detachment of 3T3 cells that detached from

glass surface using cantilevers functionalized with concanavalin A, fibronectin and poly-L-lysine.

Fig. 4.5. Histogram showing the distribution of the works of detachment of 3T3 cells that detached from glass

surface using cantilevers functionalized with concanavalin A, fibronectin and poly-L-lysine.


72

4.3. Discussion

4.3.1. Selection of the cantilever functionalization

The results described in the previous sections demonstrate that it is possible to detach living 3T3

fibroblasts from the surface on which they are growing after 24 hours of culture using

functionalized AFM cantilevers. A functionalization of the AFM cantilever is necessary to

achieve a strong adhesion between cell and cantilever.

A functionalization of fibronectin on the cantilever and 30 minutes contact between cell and

cantilever provided a sufficiently strong cell/cantilever adhesion to detach seventy-five percent of

3T3 cells from glass surfaces. In contrast, a cantilever functionalization of concanavalin A

allowed the detachment of only fifteen percent of cells. A cantilever functionalization of poly-L-

lysine allowed an intermediate percentage of thirty-five percent. The fibronectin was found to be

the best cantilever functionalization for the detachment of adherent 3T3 fibroblasts and was

selected for the rest of this work.

The selection of the cantilever functionalization depends on both the cell type and the SCFS

configuration. For example, fibronectin does not induce spreading in HeLa cells190 and their

formation of focal adhesions is not fibronectin-dependent191. However, while the cantilever

functionalization is crucial to promote the attachment of the cell to the cantilever, it may also

influence the adhesion of the cell to the substrate. For this reason, an interaction with the sugars

exposed at the cell surface, instead of the adhesion proteins, may be advantageous for some

SCFS configurations. A functionalization of concanavalin A was selected in the previous chapter

for cell capture and short-term adhesion measurements. The interaction between the cell and the

cantilever was strong enough to avoid a loss of the cell from the cantilever and probably has only

a limited effect in terms of the reorganization of the cell adhesion proteins. Alternatively, a

functionalization of poly-L-lysine might be more efficient to interact with another cell type. In

this case, the preparation of poly-L-lysine-functionalized cantilevers would be simpler and faster.

The cantilevers coated with a thin layer of zirconium (10 nm) didn’t allow the detachment of any

cells. The interaction of the cantilever with the lipids of the cell membrane was probably not

possible due to the presence of other molecules at the cell surface, notably sugars. However, there

are literature reports of three-dimensional vesicle matrices created using multivalent zirconium


73

ion linkers between the liposomes186. For this reason, the approach seems promising for the

manipulation of liposomes.

4.3.2. Limitation of the contact time

Good results were obtained using a fibronectin functionalization and a cell contact time of thirty

minutes. During these thirty minutes of contact, cell/cantilever adhesion increases. This is

presumably due to a reorganization of both the focal adhesion proteins exposed at the cell surface

and the cytoskeleton. This reorganization is one limitation of this functionalization because it will

inevitably modify the adhesion of the cell to the substrate. Thus longer cell/cantilever contact

times will result in stronger cell/cantilever interactions but will increasingly modify the

cell/substrate adhesion measured. For this reason, the preparatory cell/cantilever contact time was

kept to the minimum necessary to successfully detach a reasonable proportion of cells from the

culture surface.

4.3.3. The effect of cantilever functionalization on cell adhesion

During the thirty minutes of contact between the cell and the AFM cantilever, the

functionalization of the cantilever may also influence the adhesion of the cell to the substrate. For

this reason, the detachment forces from glass surfaces were compared using different cantilever

functionalizations to observe possible changes to cell adhesion caused by fibronectin.

The quantification of the global cell adhesion between the 3T3 cells and the glass surface was

similar for all measurements independently of the cantilever functionalization (Table 4.2). The

mean maximum forces of detachment were between 180 and 230 nN with no statistically

significant differences for the different fonctionalizations. The mean works of detachment were

between 2 and 3.3 pJ with no statistically significant differences. While some differences are

observed in the maximum forces and works of detachment, the variations are less than a factor of

two and the lowest values are not associated with the fibronectin. Any effects of the cantilever

functionalization on cell-surface interaction after 30 minutes contact were too small to be

detected.


74

Mean ±SE Mean ±SE

Concanavalin A 180 55 2.5 0.8Fibronectin 230 47 3.3 0.7Poly-L-lysine 190 31 2 0.5Zirconium ‒ ‒ ‒ ‒Native silicon ‒ ‒ ‒ ‒

Cantilever functionalizationMaximum force of detachment (nN) Work of detachment (pJ)

Table 4.2 Comparison of the maximum forces and works of detachment of 3T3 cells detached from glass surface

using four cantilever functionalizations: concanavalin A, fibronectin, poly-L-lysine and zirconium.

The acquisition of a sufficiently large data set for a statistically significant analysis of cell/surface

adhesion forces was difficult under these experimental conditions. Force distance curves from

twenty separate cells were used for each condition. Only three cells detached from the surface

using cantilevers functionalized with concanavalin A, so only three values (15% of cell

detachment) were available for analysis. The standard error was taken as characteristic of the

variance in order to consider the sample size because it estimates the standard deviation of the

sample mean of a population mean. The three values obtained for the two mechanical parameters

of detachment using concanavalin A-functionalized cantilevers are probably not representative of

a population mean. However, the results seem to indicate similar detachment forces from the

glass surface for the three cells that were detached.

4.4. Conclusion Force spectroscopy can be used to detach single adherent mouse fibroblasts from glass surfaces.

The necessary strong adhesion between cell and cantilever can be obtained by functionalizing the

cantilever with fibronectin. Fibronectin is recognized by integrins that mediate cell-surface

interactions and therefore promotes cell attachment. Another advantage is that the integrins

transmit the traction forces directly to the cytoskeleton157.

Measurements of the maximum forces and works of 3T3 cells detached from their growing

substrate were used to investigate the influence of the cantilever functionalization on the adhesion

of cells to surfaces. Fibroblast adhesion was shown to be similar on glass surfaces independently


75

of the cantilever functionalization. However, incomplete sample detachments led to less precise

characterizations of cell detachment forces.

The contact time between cell and cantilever was minimized to thirty minutes to avoid a major

reorganization of the focal adhesion proteins binding the cells on their growing substrate.

Fibronectin-functionalized cantilevers and a contact time of thirty minutes were used to detach

adherent cells forn the rest of this work.


76

Chapter 5 Cell adhesion on functionalized surfaces for cell sheet engineering

77

Chapter 5

Cell adhesion on functionalized surfaces

for cell sheet engineering‡

In this chapter, the detachment of living adherent cells by force spectroscopy was tested on a

number of chemically functionalized surfaces in order to validate the technique. The objective is

to determine if long-term adhesion measurements are useful in the development of surfaces for

tissue engineering. The adhesion of living fibroblasts was studied twenty-four hours after seeding.

The cantilevers were functionalized for cell detachment in long-term adhesion measurements.

The cells were grown on glass surfaces as well as on surfaces used for cell sheet engineering:

glass surfaces coated with polyelectrolyte multilayers (of poly-L-lysine and hyaluronic acid) and

PNIPAM films. Individual adherent cells were detached from their culture substrate using an

AFM cantilever coated with fibronectin. The maximum forces of detachment of each cell were

measured and taken as characteristic of the cellular adhesion.

______________________________________________________________________________‡Part of this chapter was included in Quantification and characterization of single cell adhesion on functionalized

surfaces for cell sheet engineering. Weder G., Guillaume-Gentil O., Matthey N., Montagne F., Heinzelmann H.,

Vörös J. and Liley M. (submitted to Biomaterials).


78

5.1. Introduction

5.1.1. Tissue engineering

Tissue engineering35 is a multidisciplinary field involving the development of biological

substitutes and/or the fostering of tissue remodelling with the purpose of repairing or enhancing

tissue function192. Tissue engineering includes biomaterials193 that are designed to direct cells in

the formation of functional tissue by providing both physical and chemical cues. Finally,

engineering design aspects are very important for two-dimensional cell expansion and three

dimensional tissue growth.

Cells are often seeded into an artificial structure capable of supporting three-dimensional tissue

formation. These structures, typically called scaffolds194, serve as a synthetic extracellular matrix.

They often mimic the in vivo environment and allow the cells to influence their own

microenvironment. Common scaffolds use natural biopolymers or synthetic polymers. The most

commonly natural biopolymers include demineralised bone matrix195, agarose196, collagen197,

hyaluronan198 and alginate199. Synthetic polymers use mainly polyglycolic acid, polylactic acid

and their copolymers that are hydrolytically degradable polymers200,201,202. As an alternative, a

variety of hydrogels, a class of highly hydrated polymer materials, are also employed as scaffold

materials203,204.

These artificial scaffolds have been used in various tissue engineering applications205 such as

fractures repairing206,207. Ideally, they should not only promote cell adhesion, growth, and

revascularization but also be biodegradable without toxic and inflammatory responses. The

synchronization of the rate of new tissue formation with the rate of scaffold degradation has not

been totally solved208,209,210. In order to avoid some of these limitations of tissue reconstruction

using biodegradable scaffolds, Okano et al. developed “cell sheet engineering”211. This concept is

based on the use of sheets of cells for tissue reconstruction instead of individual cells.


79

5.1.2. Cell sheet engineering

Cell sheet engineering is based on the control of cell adhesion, so that intact sheets of confluent

cells may be recovered with minimal damage to cell structure and function. Clinical applications

include the repair of ocular trauma by corneal epithelial cell sheet transplantation212,213. Similarly,

multilayered cell sheets have been used in cardiac tissue regeneration in rats214.

Various polymers and copolymers of poly(N-isopropylacrylamide) (PNIPAM) have been used as

coatings to control cell-surface adhesion215,216,217,211,218. These thermally-actuated polymers show

a phase transition between a collapsed state above their lower critical solution temperature

(LCST) and an extended, highly hydrated state below their LCST. Above the LCST, which is

typically around 32 °C, cells adhere, spread and proliferate on a PNIPAM-coated surface, while

below the LCST, cells detach spontaneously from the surface. These coatings have been

successfully applied to produce many types of cell sheets including oral mucosal epithelial212,

aortic endothelial219, urothelial220 as well as periodontal ligament cells221, cardiomyocytes222 and

keratinocytes223.

Polyelectrolyte multilayer films are also being developed, as an alternative to PNIPAM coatings,

to control the adhesion of several cell types224,225,226,227 including human mesenchymal stem

cells228. Polyelectrolyte layers are built up by adsorbing polyions onto oppositely charged

surfaces via electrostatic interactions229,230. Multilayered polyelectrolyte films have been shown

to desorb from conductive substrates on the application of an electrical potential231,232. The first

step in the preparation of cell sheets consist of growing cells to confluence onto polyelectrolyte

thin films. The cell sheets then detach either spontaneously or under electrical control233.

Electrical desorption has been shown to be effective for several cell types. In contrast, it has been

postulated that the spontaneous detachment requires weak adhesion to the substrate and high

cell/cell cohesion.

An extensive literature on these two polymer-based coatings documents their properties.

However, to date, there have been no quantitative investigations of their influence on cell

adhesion.


80

5.2. Long-term adhesion to glass at 37 °C and 27 °C Direct cell adhesion measurements were carried out by detaching single adherent fibroblasts from

glass surfaces using an atomic force microscope and a temperature-controlled fluidic chamber.

3T3 cells were grown for 24 hours on a glass surface. The glass sample was placed in the fluidic

chamber and a single adherent cell was selected. To ensure that the interaction between cell and

surface was similar for all measurements, cells of comparable size were selected for each

condition. The extremity of a fibronectin-decorated cantilever was precisely positioned above the

targeted cell (Fig. 5.1A). The cantilever was gently pressed onto the single adherent cell with a

predefined contact force of 1.5 nN for 30 min at constant height to allow the cell to bind to the

cantilever. Finally the cantilever was withdrawn at 0.5 µm/s until the cell detached either from

the surface or from the cantilever. The viability of the cell after detachment was assessed by

visually observing its active re-spreading on the cantilever (Fig. 5.1B). For each condition,

twenty different cells were probed. Each time a freshly prepared cantilever and new sample of

cells were used.

The force exerted on the cell during this process was continuously recorded. These forces are

typically displayed as a function of the distance between cantilever tip and surface in a force

distance curve (Fig. 5.1C). The maximum force corresponds to the maximal deflection of the

cantilever. There were two possible outcomes of the cell detachment experiments; detachment of

the cell from the glass surface occurred when the adhesion between cell and cantilever was

stronger than that between cell and substrate. In contrast, when the adhesion between cell and

glass surface was stronger, the cell detached from the cantilever. The percentage of cell

detachment gave qualitative information about the cell/surface interaction. The maximum force

of detachment was evaluated separately for experiments where the cell detached from the surface

and those where it did not.


81

Fig. 5.1. (A) Schematic of the detachment of a single adherent cell by force spectroscopy. In the initial phase of the

approach there is no contact between the cantilever and the adherent cell (A I-II). The cantilever is pressed onto the

adherent cell until a preset maximal force of 1.5 nN is attained (A III). The position of the cantilever is held constant

for 30 min before it is withdrawn at 0.5 µm/s (A IV). Finally, the cell is either on the cantilever if the cell detaches

from the surface (A V, possibility 1), or on the surface if the interaction between cell and surface is stronger than the

interaction between cell and cantilever (A VI, possibility 2). (B) The living detached cell with the last protrusion of

anchoring (B I) is observed after 3 min (B II) and 30 min (B III) to control the re-spreading of the cell on the

cantilever. (C) During the separation of the cell from the surface (or from the cantilever) a force-distance curve is

recorded indicating the maximum force and distance of detachment. The hatched area shows the work of

detachment. On both panels, red defines the approach and blue defines the retraction.

Detachment experiments on glass were carried out at both 37 °C and 27 °C. Two temperatures

were necessary in order to have a direct comparison with measurements on PNIPAM coatings

above and below the LCST and to exclude any possible effects of a physiological response of the

cells to the change in temperature.

In the detachment experiments carried out at 37 °C, seventy-five percent of the cells were

detached from the glass surface whereas twenty-five percent of the cells were left on the surface.

No qualitative differences were observed between force-distance curves where cells detached and

those where the cells remained on the glass surface (Fig. 5.2).


82

Fig. 5.2. Typical force-distance curves showing the detachments of single adherent fibroblasts either from a glass

surface (green and blue) or from the cantilever (red and black) at 37 °C using a fibronectin-decorated cantilever.

The mean values of the maximum forces of detachment were 230 nN for the cells detached from

the surface and 150 nN for the cells detached from the cantilever (Fig. 3 and Table 1).

Surprisingly, no statistically significant differences were observed between cells detaching from

the glass surface or from the cantilever (two sample T-test, p-value 0.05).

The maximum forces of detachment of the cells exposed to a temperature of 27 °C for 1 h were

not statistically different from those of the cells continuously incubated at 37 °C. The mean

maximum force of detachment from glass at 27 °C was 200 nN (Table 1), while the mean

maximum force of detachment from the cantilever was 170 nN. The maximum forces of

detachment from the glass surface and from the cantilever were not statistically different. Eighty

percent of the cells detached from the surface and twenty percent from the cantilever. Any

possible effect of temperature was too small to be observed in this study.


83

Fig. 5.3. Histogram showing the distribution of the maximum forces of detachment of single adherent 3T3 cells

either from the glass surface (red) or from the cantilever (blue) at 37 °C.

Mean (nN) ±SE

Glass 37°C Glass 27°C

From surface From surface

From surface 230 47 0.8205

From cantilever 150 20 0.4145

From surface 200 27


Glass 37°C

Glass 27°C

Two sample T-test

Table 5.1 Comparison of the maximum forces of detachment of mouse fibroblasts on glass at 37 °C and 27 °C (SE:

standard error). Data were evaluated using the two sample T-test and the limit of significance was fixed at a p-value

of 0.05. The values ≥0.05 indicate that the conditions are not significantly different.


84

5.3. Cell adhesion on polyelectrolytes at 37 °C

5.3.1. Adhesion forces

Multilayer polyelectrolyte coatings were prepared on glass by alternately spraying dilute

solutions of the polycation poly(L-lysine) (PLL) and the polyanion hyaluronic acid (HA). Two

coating types were tested: coatings with one layer of each polyelectrolyte (PLL/HA)1 and

coatings with nine layers of each polyelectrolyte (PLL/HA)9.

Detachment experiments were carried out on adherent 3T3 cells on the polyelectrolyte surfaces at

37 °C as described for glass. The results of the cell detachments from the surface were similar for

(PLL/HA)1 to those obtained on glass: sixty percent of the cells detached from (PLL/HA)1. The

mean value of the maximum force of detachment was 200 nN (Fig. 5.4 and table 5.2). The two-

sample T-test showed no statistically significant differences between cell adhesion on glass or

(PLL/HA)1. Additionally, no statistically significant differences were observed between the mean

values of the maximum forces of detachment from the (PLL/HA)1 surface or from the cantilever.

Fig. 5.4. Histogram showing the distribution of the maximum forces of detachment of single adherent 3T3 cells on

polyelectrolytes films at 37 °C: (PLL/HA)1 in red and (PLL/HA)9 in blue. The cells detach either from the surface

(dark color) or from the cantilever (light color). All the cells detach from the surface on (PLL/HA)9.


85

In contrast, the results obtained on (PLL/HA)9 were very different: All the adherent cells were

detached from the surface. The mean maximum force of detachment was 100 nN – approximately

half of the value observed on glass or on (PLL/HA)1 (Fig. 5.4 and table 5.2).

Mean (nN) ±SE

Glass 37°C (PLL/HA)1From surface From surface

From surface 200 22 0.9824


(PLL/HA)9 From surface 100 6 0.0003 0.0000

Two sample T-test

(PLL/HA)1

Table 5.2 Comparison of the maximum forces of detachment of mouse fibroblasts on glass at 37 °C and 27 °C (SE:

standard error). Data were evaluated using the two sample T-test and the limit of significance was fixed at a p-value

of 0.05. Values ≥0.05 indicate that the conditions are not significantly different.

5.3.2. Characterization of PLL/HA substrates*

The buildup of (PLL/HA)n multilayers is characterized by the initial formation of isolated

polyelectrolyte complexes that grow by addition of new polyelectrolyte layers and by mutual

coalescence of the complexes234. The growth and coalescence of these complexes finally leads to

the formation of continuous films, and the number of layer pair depositions required to reach a

continuous coverage of the substrate depends highly on the working conditions. Figure 5 shows

the morphologies of the (PLL/HA) coatings used in this study, which both consisted of randomly

distributed polyelectrolyte complexes. The deposition of (PLL/HA)1 yielded nano-droplets

covering less than 1% of the substrate, with heights ranging from 5 to 20 nm. (Fig. 5.5B) The

surface modification with (PLL/HA)9 resulted in polyelectrolyte complexes covering more than

50% of the substrate, with droplet heights around 100 nm in average. (Fig. 5.5C)

______________________________________________________________________________*AFM characterization of the PLL/HA layers was carried out by O. Guillaume-Gentil at ETH Zurich (Switzerland).


86

Fig. 5.5. Characterization of PLL/HA modified substrates by atomic force microscopy. (A) Unmodified glass

substrate. (B) (PLL/HA)1 on glass. (C) (PLL/HA)9 on glass. The graphs show one representative profile for each

AFM images.

5.3.3. Discussion of polyelectrolyte coated surfaces

Cell adhesion on (PLL/HA) multilayers has been shown to be relatively low233,235. In general,

polyelectrolyte multilayers based on biopolymers or polypeptides like PLL/HA are considered

soft, highly hydrated materials, and it has been shown that their cell adhesive properties relate

mainly to their hydration degree and viscoelastic properties, regardless of serum protein

adsorption236. As such, one can consider that (PLL/HA)1 coatings do not significantly affect the

hydration and stiffness of the glass substrate due to the low amount of adsorbed polyelectrolytes

and the low surface coverage. However, (PLL/HA)9 coatings yielded bigger complexes and

higher surface coverage, significantly affecting the substrate properties, and consequently its cell

adhesive properties.


87

5.4. Cell adhesion on PNIPAM at 37 °C & 27 °C

5.4.1. Principle

The second surface chemistry investigated was a reactive PNIPAM layer. This polymer exhibits a

lower critical solution temperature (LCST) in aqueous solution at approximately 32 °C. Above

the LCST, the PNIPAM layer is present in a compact relatively hydrophobic form, while below

the LCST the layer changes to an expanded, swollen and hydrophilic state.

The PNIPAM layers were produced by grafting PNIPAM chains to a thin layer of gold on glass

substrates using a self-assembled monolayer (SAM-COOH) of thiols (HS(CH2)11CO2H) as an

intermediate coupling agent. The amine-terminated PNIPAM was spin-coated onto the reactive

SAM of thiols and the substrate was then annealed above the glass transition temperature to

enable the amine groups to react with the SAM-COOH.


Cell detachment experiments were carried out on adherent 3T3 cells incubated for 24 hours at 37

°C on the PNIPAM-functionalized surfaces as described for the glass and polyelectrolyte

substrates. The results obtained for PNIPAM at 37 °C were similar to those obtained on glass and

on (PLL/HA)1. Sixty percent of the cells detached from PNIPAM at 37 °C. The mean value of

the maximum force of detachment was 270 nN (Fig. 5.6 and table 5.3). No statistically

significant differences were observed in the maximum forces of detachment for the cells detached

from the PNIPAM surface or from the cantilever.

Cell detachment experiments were also carried out at 27 °C. At the start of the experiment the

temperature was reduced to 27 °C for 20 minutes during which time the PNIPAM layer changed

from a compact to an extended configuration215. After twenty minutes, the cells showed a

retracted morphology but were still firmly attached to the surface (Fig. 5.7). The temperature was

maintained at 27 °C for the rest of the detachment experiment, which was carried out as

described for glass and polyelectrolytes. Cell adhesion was dramatically reduced at 27 °C. All the

cells were released from the surface. The mean maximum force of detachment was 10 nN (Table

5.3).


88


PNIPAM at 37 °C in red and at 27 °C in blue. The cells detach either from the surface (dark color) or from the

cantilever (light color). All the cells detach from the PNIPAM surface at 27 °C.

Fig. 5.7. Schematic and optical images showing the morphological response of single adherent 3T3 cells on thermo-

responsive 100%-PNIPAM layers (A) at 37 °C and, (B )after cooling the sample at 27 °C for 20 min. 100%-PNIPAM

induces a retraction of the cell at 27 °C.


89

Mean (nN) ±SE

PNIPAM 100% 37°C Glass 37°C Glass 27°CFrom surface From surface From surface

From surface 270 50 0.3829 0.2368


PNIPAM 100% 27°C From surface 10 3 <0.0001 <0.0001 <0.0001

PNIPAM 100% 37°C

Two sample T-test

Table 5.3 Comparison of the maximum forces of detachment of mouse fibroblasts on 100%-PNIPAM and glass at 37

°C and 27 °C (SE: standard error). Data were evaluated using the two sample T-test and the limit of significance

was fixed at a p-value of 0.05. Values ≥0.05 indicate that the conditions are not sig nificantly different whereas

values <0.05 indicate that the conditions are significantly different.

5.4.3. Discussion of PNIPAM functionalized surfaces

The study of PNIPAM layers showed a remarkable switching behavior between the adhesive

state at 37 °C and the non-adhesive state at 27 °C. While this behavior has been extensively

studied and investigated in past years, this is the first study to quantify these changes in adhesion.

The mean maximum force of detachment at 27 °C was around 10 nN indicating a reduction in

cell adhesion of more than an order of magnitude between the adhesive and non-adhesive states.

5.5. Controlling cell adhesion using a patterned

PNIPAM coating

5.5.1. Principle

A patterned PNIPAM coating was used to control cell adhesion by locally detaching the cell from

the surface on lowering the temperature at 27 °C. The patterned film was obtained using the

grafting method previously described except that the uniform layer of gold was replaced by a

patterned gold layer. The gold layer was composed of squares of 3 x 3 µm, and covered

approximately 30% of the glass substrate (Fig. 5.8).


90

Fig. 5.8. Single adherent 3T3 cell grown on PNIPAM-patterned glass and cooled to 27 °C for 1h showing the actin

fibres (labeled with AlexaFluor 488 Phalloidin) by confocal microscopy. In average, 29 squares of PNIPAM (in

black) are in contact with each cell.


Cell adhesion measurements on PNIPAM-patterned glass surfaces were performed using the

experimental procedure described previously. The results obtained for PNIPAM-patterned glass

at 37 °C were similar to those obtained on plain glass. Eighty-five percent of cells detached from

PNIPAM-patterned glass at 37 °C. The mean value of the maximum force of detachment was 240

nN (Fig. 5.9 and table 5.4).

The results obtained at 27 °C were different from those obtained at 37 °C. Cooling the sample to

27 °C for 1 h did not alter the morphology of the cells (Fig. 5.10). Ninety percent of cells

detached from PNIPAM-patterned glass at 27 °C. The mean value of the maximum force of

detachment was 110 nN (Table 5.4). A statistical comparison of the maximum forces of

detachment showed a clear difference (p-value 0.0007).


91


PNIPAM-patterned glass at 37 °C in red and at 27 °C in blue. The cells detach either from the surface (dark color)

or from the cantilever (light color).

Fig. 5.10. Schematic and optical images showing the morphological response of single adherent 3T3 cells on

thermo-responsive 33%-PNIPAM layers (A) at 37 °C and, (B )after cooling the sample at 27 °C for 20 min. 33%-

PNIPAM does not induce any major morphological modifications of the cell at 27 °C.


92

Mean (nN) ±SE

PNIPAM 33% 37°C Glass 37°C Glass 27°CFrom surface From surface From surface

PNIPAM 33% 37°C From surface 240* 35 0.6419 0.4546

PNIPAM 33% 27°C From surface 110** 19 0.0007 0.0035 0.0030

Two sample T-test

Table 5.4 Comparison of the maximum forces of detachment of mouse fibroblasts on 33%-PNIPAM and glass at 37

°C and 27 °C (SE: standard error). Data were evaluated using the two sample T-test and the limit of significance

was fixed at a p-value of 0.05. Values ≥0.05 indicate that the conditions are not sig nificantly different whereas

values <0.05 indicate that the conditions are significantly different. *This value was calculated for the 17 cells that detached from the surface (85% of cell detachment)

**This value was calculated for the 18 cells that detached from the surface (90% of cell detachment)

5.5.3. Discussion of PNIPAM patterned surfaces

A patterned glass/PNIPAM surface has been also studied. The glass adhesive substrate was

covered with an array of small gold squares functionalized with PNIPAM. The array was chosen

so that each adherent cell covered on average 29 squares and one third of the glass surface was

covered with the gold-PNIPAM coating. A reduction in temperature to 27 °C should therefore

result in a local de-adhesion of the cell at each of the 3 x 3 micron squares of PNIPAM under the

cell, while leaving the gross morphology of the cell and, to a large extent, the cytoskeleton

unchanged. Ideally this would reduce the maximum detachment force of the cell by one third, so

that highly adherent cells could be detached using fibronectin-coated AFM cantilevers.

The results reported here show that the 3T3 cells used in this study proliferate well on the

patterned PNIPAM/glass substrates and have similar morphologies to those grown on plain glass

surfaces. In addition, at 37 °C, cellular adhesion to the PNIPAM/glass surfaces is

indistinguishable from that on plain glass. When the temperature is reduced to 27 °C, there is no

observable change in cellular morphology, but there is a clear and significant reduction in mean

detachment forces from 240 nN to 110 nN.


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5.6. Discussion

5.6.1. Detachment of adherent cells

The maximum force needed to detach a cell from the culture surface was measured separately for

each individual cell and was taken as characteristic of the adhesion of the cell. On most surfaces,

a very broad distribution of results was obtained, indicating a large heterogeneity in the

attachment of the cells. Despite the wide distribution of individual values, statistically significant

results were obtained. One notable exception was the (PLL/HA)9 surface which had a strikingly

narrow distribution of cell adhesions.

The results for cells detaching from the culture surface or from the cantilever were evaluated

independently. Surprisingly, similar distributions of adhesion strengths were observed between

cell/culture surface and the cell/cantilever for most of the culture surfaces. This can clearly be

seen from the percentage detachments of the cells from the culture surfaces (60-85% of the cells

detached). Only on the two substrates with much lower maximum detachment forces – the

(PLL/HA)9 and the PNIPAM 100% (27 °C) surfaces – was it possible to detach all the cells from

the culture surfaces. These results suggest that the adhesion between cell and cantilever also

varies widely. Thus the exact percentage of cells detaching from the culture surface and the mean

maximum force obtained for a population of cells depends, for most of the surfaces studied here,

on the interplay of the two distributions: cell/surface adhesion and cell/cantilever adhesion.

Moreover, small changes in experimental parameters such as the cell/cantilever contact force or

the retraction speed, may have a strong influence on the percentage of cells that detach from the

quartz surface.

It was possible to quantify cell adhesion on glass, PNIPAM and (PLL/HA) polyelectrolyte

surfaces as well as on mixed glass/PNIPAM surfaces at 37 °C and 27 °C. Very similar mean

maximum detachment forces (200 – 270 nN) were observed on glass (at both 27 °C and 37 °C),

PNIPAM (100% and 33%) at 37 °C and on (PLL/HA)1 surfaces: all substrates on which the 3T3

cells studied proliferate well. On (PLL/HA)9 a very different cellular adhesion was observed,

with a much lower maximum detachment force of 100nN. This reduction in cellular adhesion

associated with an increase in the number of PLL/HA layers has been documented qualitatively

in the literature237,235. It should therefore be possible to correlate cell detachment forces with the


94

number of (PLL/HA) layers as well as with initial cellular proliferation and the later detachment

of confluent cell sheets.

5.6.2. Limitation of the sample size

The acquisition of a sufficiently large data set for a statistically significant analysis of cell

behavior is one of the major limitations of the technique. Single cell force spectroscopy is

currently a laborious technique because each cell must be individually bound to a clean freshly-

mounted AFM cantilever. Similarly each cell/substrate sample must be tested at the same time

after cell seeding (24 hours in this case). Force distance curves from twenty separate cells were

used for each experimental condition. In our hands, data from two separate cells could be

acquired per day per atomic force microscope under ideal circumstances.

A significant reduction in the standard errors associated with the means of the different force

distributions would require the number of measurements to be increased by a factor of at least

four. In future studies, an increased cell/cantilever adhesion – giving detachment of all cells from

all surfaces - might allow a more precise characterization of cell detachment forces: a slightly

larger sample size would be available and any skew in the distributions due to incomplete sample

detachment would be eliminated.

5.7. Conclusion Cell adhesion was quantified on a number of different chemically functionalized surfaces: plain

glass, glass coated with polyelectrolyte layers (PLL and HA), homogeneous PNIPAM films and

patterned surfaces with areas of PNIPAM and others of plain glass. On several surfaces,

measurements of cell adhesion were performed at both 37 °C and 27 °C. For most of the cell

culture surfaces very similar values were obtained for the maximum forces of detachment; for

glass at both 37 °C and 27 °C, 100% or patterned PNIPAM surfaces at 37 °C and for (PLL/HA)1

surfaces the mean maximum detachment forces were between 200 and 270 nN. The

measurements of cell adhesion on glass showed that a reduction of the temperature from 37 °C to

27 °C did not induce significant changes in cell adhesion due to physiological changes in the cell.

Very different results were observed on (PLL/HA)9 layers, where mean maximum detachment

forces were 100 nN. This reduced level of cell adhesion is clearly sufficient to allow good initial


95

cell proliferation followed, according to literature reports, by detachment of confluent cell sheets.

Reducing the temperature of the homogeneous PNIPAM layers to below the LCST reduced the

cell adhesion by a factor of more than one order of magnitude. This effect has been extensively

studied and reported in the literature. However, this is the first time, to our knowledge, that the

effect has been measured quantitatively.

Finally, preliminary results on patterned glass/PNIPAM surfaces, showed that these substrates

can be used to reduce cell surface adhesion – in this case by a factor of two - while keeping cell

morphology unchanged. It is hoped that these substrates may be used to investigate the adhesion

of highly adherent cells to surfaces in a semi-quantitative way.

Single cell force spectroscopy was used to study the effects of surface modifications on cell

adhesion. These effects of modified surfaces on cell adhesion were quantified. Force

spectroscopy allows unrivalled spatial and temporal control of cells, as well as highly quantitative

force actuation and measurement under physiological condition. Force spectroscopy is a suitable

tool for the development of surfaces in cell sheet engineering.


96

Chapter 6 Effect of sub-micron topography on cell adhesion

97

Chapter 6

Effect of sub-micron topography on cell

adhesion‡

In this chapter, the potential and limitations of force spectroscopy to detach adherent cells were

explored by altering the cell type and the topography of the surface. The adhesion of living

immortalized and primary fibroblasts to their culture substrate was studied twenty-four hours

after seeding.

The cantilevers were functionalized for cell detachment in long-term adhesion measurements.

Both primary fibroblasts and a continuous cell line were studied on flat and topographically

structured quartz surfaces. Individual cells were detached using an AFM cantilever, pressed onto

the cell for thirty minutes and then withdrawn. The force-distance curves obtained allowed the

maximum force, work and distance of detachment to be determined.

______________________________________________________________________________‡Part of this chapter was published in Use of force spectroscopy to investigate the adhesion of living adherent cells.

Weder G., Blondiaux N., Giazzon M., Matthey N., Klein M., Pugin R., Heinzelmann H. and Liley M. Langmuir,

2010, Article ASAP DOI: 10.1021/la904526u.


98

6.1. Introduction

6.1.1. Topography and cell adhesion

Adherence-dependent cells are profoundly influenced by the topography of the substrate to which

they adhere. Cellular responses such as adhesion, morphology, alignment, guidance, gene

expression and proliferation are all influenced by topography238,239,240,241,242,243. The effects may

differ widely depending on the dimensions and form of the topography244.

Brunette et al. divided cells into “rugophobic” cells, such as fibroblasts, that prefer smooth

surfaces over rough ones, and “rugophilic” cells, such as osteoblasts245,246. In the last ten years, it

has been shown that the rugophobia/rugophilia classification depends both on the dimensions of

the topography and on the cell type. A large and sometimes contradictory literature on this topic

seems to indicate a number of other poorly-controlled factors that influence cell response to

topography.

Taking the example of fibroblasts, many studies have shown that surface nanotopography affects

fibroblast response, with both increased247,244 and decreased248 adhesion observed. Nanometric

islands have been reported both to speed up initial cell adhesion249 and to have no influence at all

when compared with planar samples250. Numerous studies have shown a reduction in cellular

adhesion and a decrease in spreading when fibroblasts are grown on sub-micron topographies251

such as islands252, columns253 or pits254. In contrast, microtopography has been used to transform

non-adhesive into adhesive substrates for fibroblasts255 by increasing cellular adhesion.

While the effects of nano-sized topography on fibroblast adhesion remain unclear, most authors

report that sub-micron topographies inhibit fibroblast adhesion and result in cell rounding.

6.1.2. Topography and cell type

There are certainly several reasons why studies of cell/surface adhesion are so confused. In many

studies, surface chemistry is poorly controlled, and experimental parameters vary widely between

different research groups. However, the effects of topography on cell adhesion may also differ

widely depending on the cell type256. Immortalized and primary cells from the same tissue origin

have been shown both to exhibit distinct cell morphologies and mechanical properties179. The

effects of surface topography on cell adhesion can be very different for different continuous cell


99

lines and different from those of primary cells257. Although the effects of surface topography on

cell adhesion were demonstrated to depend on the cell type, there is a lack of suitable methods to

quantify cell/surface interactions.

Here, force spectroscopy is used to directly measure the adhesion of two cell types to their culture

substrates. The adhesion of immortalized and primary fibroblasts to polylysine-treated quartz

surfaces after 24 hours of growth in situ was first quantified. It was then compared with the

adhesion on identical quartz surfaces that have been structured with a sub-micron pillar motif.

6.1.3. Description of the sub-micron topography

Two different sub-micron topographies on quartz surfaces were tested. Each consisted of quartz

pillars with an average diameter of 400 nm and an approximate surface coverage of 30 %. In one

case the pillars were 270 nm high and in the other 520 nm high. Both topographies are shown in

figure 6.1. The wide distribution of pillar diameters is due to the use of a polymer blend mask in

the fabrication process. Differences in the shape of the pillars are due to differences in the

fabrication process and particularly the lift-off step. After fabrication the samples were

thoroughly cleaned to ensure a controlled and reproducible surface chemistry. All traces of

chromium and amorphous silicon used as etch masks were removed using a chrome etchant and

potassium hydroxide solution. Possible fluorine traces from the dry etching step were removed by

an oxygen plasma.

Fig. 6.1. Scanning electron microscopy pictures of sub-micron pillars in quartz with a height of 270 nm (A), 520 nm

(B) and a flat control surface (C).


100

6.2. Long-term adhesion to flat quartz Cell adhesion was measured directly by detaching single adherent 3T3 fibroblasts from quartz

surfaces coated with poly-L-lysine. The forces exerted on the cell during this process were

continuously recorded and the values were represented in a force-distance curve. Each force-

distance curve consisted of one peak independently of whether or not the cell detached from the

quartz surface. Three mechanical parameters were extracted from each curve i.e. the maximum

force, the work and distance of detachment.

The percentage of cells detached from the surface gives the first qualitative information about the

relative adhesion forces. Seventy percent of cells detached from the flat quartz. The maximum

force of detachment was evaluated separately for experiments where the cell was detached from

the surface and those where it did not. No significant statistical differences were observed

between cells detaching from the quartz surface and from the cantilever (two sample T-test, p-

value 0.05) (Fig. 6.2). The mean values of the maximum forces of detachment were 420 nN for

the cells detached from the surface and 310 nN for the cells detached from the cantilever (Table

6.1B)

Fig. 6.2. Histogram showing the mean values of the maximum forces of detachment obtained for the detachments

from the surface (grey) and from the cantilever (black). Vertical lines indicate the means and the error bar is the

standard deviation.


101

The mean values for the work of detachment were 7.9 pJ for the cells detached from the surface

and 6.9 pJ for the cells detached from the cantilever (Table 6.1C). Again no significant statistical

differences were observed between the two populations.

The distance of retraction from the surface required to totally detach the cell was also analyzed.

The mean values of the distance of detachment were 33 µm for the detachments from the

substrate and 38 µm for the detachments from the cantilever (Table 6.1D). The two-sample T-test

showed no significant difference between the two populations.

6.3. Effect of sub-micron topography The percentage of cells detached from the quartz surfaces was greatly increased on both pillar

topographies. The percentage of cell detachment was 95 % on the 270 nm pillars and 100 % on

the 520 nm pillars. The maximum forces of detachment were dramatically reduced on the

microstructures, with mean values of 210 nN on the 270 nm pillars and 180 nN on the 520 nm

pillars (Table 6.1B). Wide dispersions in the force values were observed on both flat and

structured surfaces reflecting the heterogeneity of the cell adhesion properties within the same

population (Fig. 6.3).

Similarly, the work of detachment was reduced on the pillars. The mean values on the 270 nm

and 520 nm pillars were 2.6 pJ and 2.7 pJ (Fig. 6.4 and Table 6.1C). The analysis of the distance

of detachment showed that shorter distances were required to totally detach the cells from the

pillars. The values were 22 µm on both 270 nm and 520 nm pillars (Table 6.1D). No statistically

significant differences were observed between the 270nm and 520nm pillars.


102

Fig. 6.3. Histogram showing the distribution of the maximum forces of detachment of single adherent 3T3 cells

grown for 24 h on quartz surfaces: Flat (grey), 270 nm pillars (white) and 520 nm pillars (black).

Fig. 6.4. Histogram showing the distribution of the works of detachment of single adherent 3T3 cells grown for 24 h

on quartz surfaces: Flat (grey), 270 nm pillars (white) and 520 nm pillars (black).


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Mean ±SEQuartz flat 70 Quartz flat270 nm pillars 95 From the surface 420 37520 nm pillars 100 From the cantilever 310 50

270 nm pillars* 210 36520 nm pillars 180 29

Mean ±SE Mean ±SEQuartz flat Quartz flatFrom the surface 7.9 1 From the surface 33 3From the cantilever 6.9 1.7 From the cantilever 38 8

270 nm pillars* 2.6 0.7 270 nm pillars* 22 2520 nm pillars 2.7 0.7 520 nm pillars 22 2

A. Percentage of cell detachment (%)

C. Work of detachment (pJ)

B. Maximum force of detachment (nN)

D. Distance of detachment (µm)

Table 6.1 Comparison of the percentages of cell detachment and the mean values of the maximum force of

detachment, the work of detachment, and the distance of detachment of 3T3 cells on three quartz surfaces: flat, 270

nm pillars and 520 nm pillars (SE: standard error). *These values were calculated for the 19 cells that detached

from the quartz surface (95 % of cell detachment).

6.4. Comparison with primary mouse fibroblasts A key point in the study of cell/surface interactions and the role of topography is the choice of

cell type. Both immortalized cell lines and primary cells have been studied and very different

results have been found for different cell lines and primary cells. For this reason, the study was

extended to the more biologically-relevant, but harder to study, primary fibroblasts. The primary

fibroblasts tested were obtained from mouse embryos. As 3T3 cells showed no differences in

adhesion when the height of the submicrometer pillars was changed from 270 nm to 520 nm, the

primary fibroblasts were detached only from the 520 nm pillars.


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6.4.1. Cell adhesion

It was found that – with the experimental parameters used for 3T3 cells – very few primary cells

detached from the flat quartz surface: 90 % of the cells were left on the surface. On the 520 nm

pillars the percentage of detachment increased to 30 %.

Since so few cells detached, the analysis of the force distance curves essentially characterizes the

interaction between cell and cantilever. The mean values of the maximum force of detachment

were 190 nN on the flat quartz and 210 nN on the 520 nm pillars (Table 6.2B). The mean values

obtained for the work of detachment were 1.7 pJ on the flat surface and 3.8 pJ on the 520 nm

pillars (Table 6.2C). The differences between the values obtained on flat quartz and on the pillars

were not statistically significant (two-sample T-test, p-value 0.05) as can be expected from the

fact that they mostly reflect the cell/cantilever interactions. Finally, the mean values for the

distance of detachment were between 13 and 16 µm showing that the primary cells were less

stretched than the 3T3 cells during detachment (Table 6.2D).

Mean ±SEQuartz flat 10 Quartz flat 190 41520 nm pillars 30 520 nm pillars 210 58

Mean ±SE Mean ±SEQuartz flat 1.7 0.5 Quartz flat 13 2520 nm pillars 3.8 1.4 520 nm pillars 16 3

B. Maximum force of detachment (nN)

D. Distance of detachment (µm)

A. Percentage of cell detachment (%)

C. Work of detachment (pJ)

Table 6.2 Comparison of the percentages of cell detachment and the mean values of the maximum force of

detachment, the work of detachment, and the distance of detachment of PMEF cells on two quartz surfaces: flat and

520 nm pillars (SE: standard error).

6.4.2. Cell proliferation

Cell proliferation on the test substrates was investigated to confirm the viability of the cells on

the flat and structured surfaces. The Alamar Blue assay incorporating a non-toxic fluorometric

growth indicator based on detection of metabolic activity was used. The quartz surfaces were

particularly attractive for the 3T3 cells showing an optimal proliferation activity that was not


105

modified by the topography (Fig. 6.5A). In contrast, the primary cells were less viable than the

3T3 cells, with a slow and constant proliferation that was reduced on the pillars (Fig. 6.5B).

Fig. 6.5. Alamar Blue assay results from 3T3 mouse fibroblasts (A), and primary mouse embryonic fibroblasts (B).

They were cultured on structured and flat quartz for 4 days and each value represents the mean of 6 samples.

6.4.3. Cell morphology

Differences in morphology were observed between the immortalized and primary fibroblasts and

on the different quartz surfaces. F-actin staining (phalloidin) and scanning electron microscopy

were used to compare cell morphologies.

3T3 cells grown on flat quartz had a spread morphology with a mean diameter of 50 µm (Fig.

6.6). In contrast, a spherical morphology was observed on both pillar structures with a mean

diameter of 30 µm. The fixing and dehydration of the cells considerably limited the extent to

which the images may be interpreted, particularly the position of the cell membrane on the pillar

structures.

The primary cells had a highly spread and flattened morphology on flat quartz and were

significantly larger at around 150 µm in diameter (Fig. 6.7). The 520 nm pillars dramatically

modified their morphology resulting in a spindle-shaped, flattened morphology with a

considerable reduction in spreading.


106

Fig. 6.6. Comparison of 3T3 cells by confocal microscopy (ACE) showing the actin fibres (labeled with AlexaFluor

488 Phalloidin) and scanning electron microscopy (BDF) on different quartz surfaces: flat (AB), 270 nm pillars

(CD) and 520 nm pillars (EF).

Fig. 6.7. Comparison of PMEF cells by confocal microcopy (AC) showing the actin fibres (labeled with AlexaFluor

488 Phalloidin) and scanning electron microscopy (BD) on two quartz surfaces: flat (AB) and 520 nm pillars (CD).


107

6.5. Discussion

6.5.1. Detachment of adherent cells

The force distance curves obtained during cell detachment were analyzed to obtain three

characteristic mechanical parameters: the maximum force applied during the detachment; the

work necessary for cell detachment; the distance of retraction at which the cell detached from the

surface. Of the three, the maximum force was the most intuitively accessible and seemed most

characteristic of the different experimental conditions used in this study. The rest of the

discussion is focussed on this parameter.

The maximum forces applied in the detachment experiments on 3T3 cells on quartz (Fig. 6.2)

reveal, firstly, that the adhesion between cell and substrate varies widely. In order to investigate

the sensitivity of this technique to changes in cell/surface adhesion, two important experimental

parameters were altered: the topography of the quartz surface and the cell type. Changing the

quartz substrate from a flat surface to one covered with sub-micron pillars resulted in a

significant reduction in cell adhesion. The fraction of cells detached from the substrate went from

70% to 95-100% while the mean maximum force applied to the cell during detachment was

reduced by a factor of approximately two. These results are in accordance with most literature

reports on the effect of topography on fibroblast adhesion252,253,251,254,258,259,260.

6.5.2. Cell adhesion to quartz and glass

Adhesion strength for 3T3 cells detaching from quartz surfaces coated with poly-L-lysine or from

glass surfaces were different. While the percentages of cell detachment were similar on both

surfaces (70%-75%), the maximum forces of detachment were different. The mean maximum

forces of detachment were 420 nN from flat quartz surfaces coated with poly-L-lysine and 230

nN from plain glass surfaces. The difference of the maximum forces of detachment is probably

due to the presence of poly-L-lysine on quartz. In contrast, the explanation of the similar

percentages of detachment remains unclear. The fibronectin-based functionalization of the AFM

cantilevers is capable of supporting at least a mean maximum force of detachment of 420 nN.

The cantilever functionalization was also capable of detaching all the cells from the quartz

surfaces with sub-micron pillars. Therefore, there is an explanation that is independent of the

efficiency of the cantilever functionalization. The cells might have exhibited a different


108

morphology modifying their area of contact with the AFM cantilever but in this case, the

percentages of cell detachment should have been different. Alternatively, poly-L-lysine may

active cell adhesion not only on the surface but also on the cantilever, resulting in higher

maximum forces of detachment and similar percentages of cell detachment than from glass.

6.6. Limitation of the cell type When the 3T3 cells were replaced by PEMF cells, only 10% of the cells detached from flat

quartz, while 30% detached from the sub-micron pillars on quartz. The absolute numbers of cells

detached from the surfaces are so low that no meaningful quantitative analysis of cell/substrate

adhesion is possible. The force distance curves obtained with PEMF cells do, however, give

information about the cell/cantilever interactions. The mean maximum forces obtained on PEMF

cells were approximately 200 nN on both flat and structured quartz. In contrast the mean

maximum forces obtained for the detachment of the 3T3 cells from the cantilever were around

300 nN. A statistical analysis finds the probability that these distributions are the same to be 5%

(p=0.05), the limit of statistical significance chosen for this study. It is likely, that a reduction in

adhesion to the cantilever contributes to the failure to detach PEMF from the quartz surface.

The differences in cell/cantilever adhesion may be linked to inherent differences in the adhesive

properties of 3T3 and PEMF cell surfaces, or to the speed of reaction of the different cell types. It

is likely, however, that the very different morphologies of 3T3 and PEMF cells also play a role.

3T3 cells on quartz are relatively thick – at least at the cell nucleus – when compared to the

extremely flat, spread form of the PEMFs. The raised cell nucleus allows a relatively large area

of contact between cell and AFM cantilever (which is tilted at 10° to the horizontal). In contrast,

it is very difficult to obtain a large contact area between PEMF and cantilever.

Despite the failure to detach the PEMF, it is possible to draw some qualitative conclusions about

their adhesion to the quartz surfaces. Firstly, the PEMF adhere more strongly to the flat quartz

surfaces than the structured quartz surfaces. Secondly, a comparison between the detachment of

3T3s from the 520 nm pillars (100% detachment with a mean maximum force of 180 nN) and

PEMF from the same structure (30% detachment with a mean maximum force of 210 nN) shows

clearly that the PEMF adhere more strongly to the structured quartz surfaces than the 3T3s.


109

The failure to detach a significant number of PEMF cells highlights a key issue with this

approach: if the number of cells detached from the substrate is close to 100% then the values

obtained for the detachment parameters (force, work and distance) may be considered

representative of the cell’s adhesion, irrespective of the forces between cell and cantilever.

However, as the percentage of cells detached from the substrate decreases, the accuracy of the

values obtained also decreases: a bias is introduced into the distribution because only the least

adherent cells can be measured.

6.7. Conclusion Force spectroscopy can be used to detach single adherent fibroblasts from flat and structured

quartz surfaces by traction normal to the surface. Measurements of the percentage of cells

detached from the substrate surface and the maximum force necessary for detachment were used

to investigate different factors influencing the adhesion of fibroblasts to surfaces. 3T3 mouse

fibroblast adhesion was shown to be higher on flat quartz surfaces and lower on quartz surfaces

with a structure of sub-micron pillars. The rugophobia of fibroblasts on sub-micron topography

has been reported by other authors252,253,251,254,258,259,260 but in this work the adhesion forces were

quantified. No differences in adhesion were observed when the height of the sub-micron pillars

was changed from 270nm to 520nm.

Primary embryonic mouse fibroblasts were also studied and were found to be rugophobic.

However, only very few PEMFs could be detached from the surface using the experimental

parameters optimized for 3T3s. For this reason it was not possible to quantify the PEMF adhesion

to the surface. Qualitatively, it is clear that PEMF adhesion is lower on structured quartz than on

flat quartz. The PEMF also adhere more strongly at least one of the quartz surfaces than the 3T3s.

Cell/cantilever adhesion seems lower for PEMF than for 3T3. A less favourable cell morphology

resulting in smaller contact area between cell and cantilever may contribute to this effect. This

reduced cell/cantilever adhesion for the PEMFs seems to be a major factor in the different

percentages of cell detachment. The different cell/cantilever adhesions obtained for different cell

types is a significant limitation of the technique because it renders comparisons between cell

types difficult. The experimental parameters of cell detachment have to be similar and optimized

for both cell types when adhesion forces have to be compared.


110

Chapter 7 Conclusions & Outlook

111

Chapter 7

Conclusions & Outlook

The main objective of this work was to use force spectroscopy to quantify the long-term global

adhesion between cells and surfaces and their response to modified surfaces. This objective was

achieved. Force spectroscopy was used to quantify the global adhesion forces of individual

adherent cells. Adherent cells were detached from glass surfaces modified with polyelectrolytes

and a thermo-responsive polymer, as well as from quartz surfaces with a structure of sub-micron

pillars. The cells were detached after twenty-four hours in culture using fibronectin-decorated

AFM cantilevers that were pressed onto the adherent cells for thirty minutes. The mechanical

parameters acquired during detachment were used to quantify the cell adhesion forces.

The first step was to verify if cell adhesion has to be studied independently of cell cycle or not.

Short-term adhesion was used to demonstrate that binding proteins at the cell membrane remain

similar both in number and in their binding properties during interphase and mitosis. The

rounding up and greatly reduced adhesion of cells during mitosis was associated with an

increased stiffness of the cell caused by a cytoskeletal reorganization. Mitosis was not paired with

large differences in the binding proteins at the cell membrane but with cytoskeletal changes. In

this way, it was demonstrated that cell adhesion can be studied independently of cell cycle as


112

long as mitotic cells are not chosen due to their different morphology. However, the

demonstration was performed using short-term adhesion with osteoblasts captured on the AFM

cantilever. It is hoped that results are similar for the adherent fibroblasts studied in the rest of the

thesis. A more powerful demonstration, verifying this, would use adherent fibroblasts,

synchronized in the different phases of the cell cycle, and detached from their culture substrate.

Long-term cell adhesion was quantified on glass surfaces coated with polyelectrolyte bilayers.

Large differences in cellular adhesion were observed depending on the number of polyelectrolyte

bilayers. The reduced level of cell adhesion associated with an increased number of bilayers was,

however, sufficient to allow good initial cell attachment and proliferation. Wide dispersions in

the force values were observed within the same condition reflecting the heterogeneity of the cell

population. However, the statistical analysis of the values indicated highly significant differences

between the conditions.

The measurements of cell adhesion on glass showed that a reduction of the temperature from 37

°C to 27 °C did not induce significant changes in cell adhesion due to physiological changes in

the cell. This result allows global cell adhesion measurements, not exceeding one hour, to be

performed without an accurate control of the temperature (between 27 °C and 37 °C).

Long-term cell adhesion was quantified on thermally-responsive PNIPAM brushes. Cell adhesion

was dramatically reduced by lowering the temperature to below the LCST. The observation of

cells between the adhesive and non-adhesive states indicates only if cells are detached or not. In

contrast, the quantification of the adhesion forces details the PNIPAM’s effect on cell adhesion in

both states. Glass surfaces patterned with periodic PNIPAM micro-domains were used to reduce

cell surface adhesion while keeping cell morphology unchanged. This approach to reduce cell

adhesion and to keep cell morphology could be interesting in the stimulation of single adherent

cells that are detachable. The stimulation could be performed at 37 °C and the detachment at 27

°C for further single cell analysis.

Long-term cell adhesion was shown to be higher on flat quartz surfaces than on quartz surfaces

with a structure of sub-micron pillars. This was already reported by other authors but here the

adhesion forces were quantified. In contrast to cell adhesion, cell proliferation was not affected

by the sub-micrometer pillars. No differences in adhesion were observed when the height of the

sub-micrometer pillars was changed. Three-dimensional positioning of cells was not clearly


113

determined by microscopic analysis due to artifacts coming from the cell fixation. The micro-

fabrication of higher quartz pillars in order to eliminate any contact of the cells with the surface

between the pillars was not possible.

Finally, an immortalized cell line was compared with primary cells in long-term adhesion

experiments. The failure to detach the primary cells revealed the importance of the cell type. This

allowed the potential and limitations of the technique in measuring cell/surface interactions to be

discussed in more detail. The differences of detachment between the two cell types may be linked

to several independent reasons including the contact area with the AFM cantilever and how

quickly the cells adhere to the cantilever. The cell surface-to-volume ratio is important because in

cases where the cell adhesion forces are greater than the cell cohesion forces, detachment of such

cells is not possible. Primary cells may break in case of strong connection with the AFM

cantilever.

As with most new techniques, single cell force spectroscopy needs to mature. The modification

of experimental parameters during this study highlighted some biological and physical

limitations. A key issue with the technique is the adhesion of the cells to the AFM cantilever used

to detach them. The cantilever functionalization is crucial to promote cell attachment. The contact

time cell/cantilever has to be minimized to limit the inevitable reorganization of the adhesion

cell/substrate. The connection could be addressed by a mechanical approach instead of

functionalizing the cantilever. A mechanical connection would suppress the reorganization of the

adhesion of the cell to the substrate and would reduce the time of the measurements. Hollow

force-controlled AFM cantilevers were used for local dispensing of a solution into individual

cells261. The adaptation of these micro-channeled cantilevers for aspiration instead of injection

could be used to capture single cells. The concept should be first validated for cell capture of

non-adherent cells.

Another significant limitation was the acquisition of a sufficiently large data set for a statistically

significant analysis of cell behavior. Adhesion measurements that use single cells are time

consuming because only one cell can be characterized at a time. Therefore, it would be useful to

adjust the size of the representative sample depending on the magnitude of the effect which is

sought. The detection of the influence of one polyelectrolyte bilayer on cell adhesion may require

fifty or hundred measurements whereas ten measurements could have been sufficient for one and


114

nine bilayers. Since the cell dynamics limits the speed of individual experiments, the throughput

has to be increased by parallelizing the AFM experiments. Performing experiments in parallel

also offers the possibility of testing various parameters under the same experimental conditions.

Parallelizing the AFM experiments requires the use of a cantilever array. One of the major

challenges is the development of a readout system for cantilever arrays.

A large proportion of cells were detached from glass and quartz surfaces. Surface modifications

with polyelectrolytes, PNIPAM and sub-micron topography allowed the detachment of all the

adherent cells. In this work, cell adhesion measurements were focused on surfaces dedicated for

cell sheet engineering and bone implant applications. Many effects on cell adhesion were already

reported but this was the first time that global cell adhesion was measured quantitatively.

Single cell force spectroscopy is a powerful tool for the study of long-term adhesion. Under

suitable experimental conditions, force spectroscopy allows quantification of adhesion forces

between adherent cells and their culture substrates. Quantitative data of this nature should open

the way for more rigorous investigation and a comparison of the influence of different parameters

on cell/substrate adhesion. Force spectroscopy is highly complementary to molecular biology

approaches and to existing qualitative techniques already used in cell adhesion studies.

Acknowledgements

115

Acknowledgements

It is my great pleasure to thank all the people who supported me during my project, scientifically

as well as personally (2006-2010). I would like to thank them either in English or in French.

I would like to thank Janos Vörös for giving me the opportunity to perform my PhD thesis in his

group and for being my official supervisor as I carried out my PhD thesis outside the ETH

domain. - Janos, you always considered me as a full member of your group and you gave me so

many incredible and great ideas.

I would like also to thank Mathis Riehle who agreed to come from Glasgow to be part of my

board of co-referees. - Mathis, I thank you very much for coming in Switzerland and for the time

you spent for me.

Je tiens à remercier sincèrement Martha Liley d’avoir été ma superviseure principale. - Martha,

ces quelques mots seront insuffisants pour t’adresser toute ma reconnaissance. Je te remercie

pour m’avoir engagé dans ton groupe, pour m’avoir guidé techniquement et pour m’avoir

supervisé durant ce projet de quatre ans. Ces éléments font partis du cahier des charges d’un bon

superviseur et tu les as accomplis avec brio. Par contre, ton côté humain est unique. J’ai apprécié

quotidiennement ta porte toujours ouverte, ton écoute attentive, ta compréhension des problèmes

personnels, ta tolérance et ta bonne humeur. Ces valeurs ne s’apprennent pas dans les livres

scientifiques. Tu m’as démontré qu’elles étaient compatibles avec un travail appliqué et je m’en

souviendrai toute ma vie, merci infiniment.

Acknowledgements

116

Je remercie Marta Giazzon et Nadège Matthey qui sont mes 2 amours du boulot ! - Marta,

Nadège, je vous remercie du fond du cœur pour votre soutien continu et pour votre contribution

dans ce travail. Votre bonne humeur légendaire reste inoubliable.

Je remercie chaleureusement Harry Heinzelmann d’avoir été mon co-superviseur. Harry, je te

remercie pour m’avoir engagé dans ta division et pour ta disponibilité en toutes circonstances. Je

pense que ce n’est pas un hasard si tu es à la tête d’une division regroupant tant de personnes

intéressantes.

Un grand merci à tous les collègues de mon groupe : Nadège Matthey, Joanna Bitterli, Mélanie

Favre, Marta Giazzon, Silvia Angeloni, Martha Liley, Sher Ahmed, Jérôme Maris-Polesel,

Fabrizio Spano et André Meister.

Première ligne : Martha Liley, Marta Giazzon, Nadège Matthey, Harry Heinzelmann, Janos Vörös et Jérôme Maris-

Polesel. Deuxième ligne : André Meister, Silvia Angeloni, Fabrizio Spano, Joanna Bitterli, Mélanie Favre et Sher

Ahmed.

Un grand merci à tous les autres collègues qui ont contribué de près ou de loin à ce travail,

notamment à Orane Guillaume-Gentil, Mona Klein, Patricia Fernandes, Marta Bally, Jérôme

Maris-Polesel, Nicolas Blondiaux, Franck Montagne et Raphaël Pugin.

Les mots ne sont pas assez forts pour remercier Delphine Hügli, la femme de ma vie, et mes

parents, Sylvia Weder et Michel Miserez, qui ont été là inconditionnellement pour moi.

Finalement, je remercie les amis qui ont eu une oreille particulièrement attentive à mes aventures

professionnelles, notamment Cédric Jecker, Nicolas Forster, Thibaud Rossel, Grégory Franc et

les autres.

Curriculum Vitae

117

Curriculum Vitae

Gilles WEDER

Vivier 20 Ι 2016 Cortaillod Ι Switzerland

[email protected]

13th June 1983 Ι Swiss

Education

2006 – 2010 Doctoral student at the Swiss Center for Electronics and Microtechnique (CSEM

SA), Division Nanotechnology & Life Sciences, Neuchâtel, Switzerland. PhD

performed in collaboration with the Laboratory of Biosensors and Bioelectronics,

Institute for Biomedical Engineering, Department of Information Technology and

Electrical Engineering, ETH Zürich, Switzerland.

2005 – 2006 Master of Science part 2, University of Neuchâtel, Switzerland - Cum Laude

2004 – 2005 Master of Science part 1, University of Lausanne, Switzerland

2001 – 2004 Bachelor of Science, University of Neuchâtel, Switzerland - Cum Laude

1998 – 2001 Baccalaureate of Science, Lycée Denis-de-Rougemont, Neuchâtel, Switzerland

Curriculum Vitae

118

Publications

2010 G. Weder, N. Matthey, H. Heinzelmann, J. Vörös, and M. Liley. Cantilever

Functionalization for Cell Detachment. In Preparation.

2010 G. Weder, O. Guillaume-Gentil, N. Matthey, F. Montagne, H. Heinzelmann, J.

Vörös, and M. Liley. Quantification and Characterization of Single Cell Adhesion

on Functionalized Surfaces for Cell Sheet Engineering. Submitted to Biomaterials.

2010 G. Weder, N. Blondiaux, M. Giazzon, N. Matthey, M. Klein, R. Pugin, H.

Heinzelmann, J. Vörös, and M. Liley. Use of Force Spectroscopy to Investigate the

Adhesion of Living Adherent Cells. Langmuir. Article ASAP DOI:

10.1021/la904526u.

2009 G. Weder, J. Vörös, M. Giazzon, N. Matthey, H. Heinzelmann and M. Liley.

Measuring Cell Adhesion Forces during the Cell Cycle by Force Spectroscopy.

Biointerphases. Vol. 4(2): 27 – 34.

2009 G. Guibert, T. Rossel, G. Weder, B. Betschart, C. Meunier and S. Mikhailov.

Surface Treatment of Polymers by Ion Beam Irradiation to Control the Human

Osteoblast Adhesion: Fluence and Current Density. AIP Proceedings. Vol. 1099:

511 – 515.

2008 R. Pugin, N. Blondiaux, A. M. Popa, E. Scolan, Hoogerwerf A., T. Overstolz, M.

Giazzon, G. Weder and H. Heinzelmann. Surface Nanostructuration: From

Coatings to MEMS Fabrication. NSTI Proceedings. Vol. 1: 431 – 434.

2006 M. Vukicevik, G. Weder, A. Boillat, A. Boesch and S. Kellenberger. Trypsin

Cleaves Acid-Sensing Ion Channel 1a in a Domain that is Critical for Channel

Gating. The Journal of Biological Chemistry. Vol. 281: 714 – 722.

Curriculum Vitae

119

Oral presentations

2009 G. Weder et al. Measuring Cell Adhesion Forces during the Cell Cycle by Force

Spectroscopy. European Society of Biomaterials, Lausanne, Switzerland.


Spectroscopy. European Society of Biomechanics, Luzern, Switzerland.

2007 G. Weder et al. Atomic Force Microscopy: Applications in Biology and Materials

Sciences. EU Project Newbone, Public Conference, Lugano, Switzerland.

Poster presentations

2009 G. Weder et al. Microstructured Non-Metallic Bone Implants: In Vitro & In Vivo

Evaluation. Bioforum, Neuchâtel, Switzerland.


Spectroscopy. Bioforum, Neuchâtel, Switzerland.


Spectroscopy. Single Cell Analysis Workshop, Zürich, Switzerland.


Spectroscopy. World Biomaterials Congress, Amsterdam, Netherlands.

2007 G. Weder et al. Measuring Cell Adhesion Forces of the Cell Cycle by Force

Spectroscopy. Swiss Society for biomedical Engineering, Neuchâtel, Switzerland.


Spectroscopy. European Cell and Materials VIII: bone tissue engineering, Davos,

Switzerland.

2007 G. Weder et al. Measuring Cell Adhesion Forces on Nanostructured Surfaces by

Force Spectroscopy. Gorden Research Conference Cell Contact & Adhesion, Il

Ciocco, Italy.


Spectroscopy. Swiss Society for Biomaterials, Neuchâtel, Switzerland.

Curriculum Vitae

120

2007 G. Weder et al. Measuring Cell Adhesion Forces on Nanostructured Surfaces by

Force Spectroscopy. Swiss User Group Surfaces and Interfaces, Fribourg,

Switzerland.

2006 G. Weder et al. Topographical and Chemical Modifications of Polymer Surfaces

and their Influence on Human Cell Behaviour. Swiss User Group Surfaces and

Interfaces, Fribourg, Switzerland.

Prize

2009 First Baxter Prize for the Interuniversity Doctoral Program in Organismal

Biology. Microstructured Non-Metallic Bone Implants: In Vitro & In Vivo

Evaluation . Bioforum, Neuchâtel, Switzerland.

References

121

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ETH Z1418/eth... · DISS. ETH NO. 18943 . LIVING CELL ADHESION MEASURED BY FORCE SPECTROSCOPY . a dissertation submitted to . ETH ZURICH . for the degree of . Doctor of Sciences (Dr