Printing of biochemically-patterned slides OK of biochemically-patterned slides1.pdfimmobilized on ECM micro patterns of various shapes (the patterns are depicted by dashed lines)

INNOPSYS Parc Activestre – 31390 CARBONNE – France

Tel : +33 561 971 974 / Fax : +33 561 971 975 / www.innopsys.com

Printingofbiochemically-patternedslideswiththeInnoStamp40®fordeterministiccellimmobilization.

Jean‐christophe CAU1 1 Innopsys, Parc activestre, Carbonne – France – [email protected]

www.innopsys.com

www.biosoftlab.com

Introduction

Mastering the adhesion landscape of living

cells on a surface is emerging as a new tool for

investigating many fundamental mechanisms

of cell biology such as shape control,

differentiation, division, polarity or motility [1‐

12]. The common point between all these

studies is the production of micro‐patterns of

various shapes and dimensions along which

adherent cells are immobilized in a

deterministic way. The control of cell adhesion

through this technology allows the biologists to

design specific scenari and record how various

living cell types (neural, epithelial, tumoral and

stem cells) respond to them. Because this

technology allows reproducing identical

precise patterns along well arranged periodical

arrays, these experimental observations can be

made systematically over large population of

cells thus reaching a high level of

representativeness. The micro‐contact printing

of proteins of the Extra Cellular Matrix (ECM)

on conventional glass slides or coverslips

turned out to be an efficient method for

fabricating these micro‐patterned cell culture

substrates. However, when this technological

process is made manually, it fails to generate

reproducible patterns over large surfaces thus

limiting the actual potential of this new

approach. In order to bring this process to a

standard level in cell biology, we propose to

automate the printing process of ECM proteins

on cell culture substrates. As a matter of fact, a

fine control of the stamping parameters is a

sine qua none condition to be able to print in

one step reproducible patterns of different

shape, size and pitch.

In this note, we present a dedicated study in

which we screen different pattern shapes and

pattern sizes for the deterministic

immobilization of cancer cells. With the

perspective in mind of remote production of

these patterned cell‐culture supports, we have

also investigated the conservation potential

over time of the printed slides before cell

seeding.

We have used a patterned stamp designed

with four different classes of features (disc,

line, square, and triangle) of different sizes

(from 10 to 50µm) and pitches (10 to 100µm).

The samples were seeded with Prostate Cancer

3 (PC3) cells. Then, we have quantified the

spreading of the immobilized cells on these

different patterns and the selectivity of the

immobilization on the adhesive patterns

compared to the antifouling background.

1‐Cellular growth on patternedsamples fabricated with a fullyautomatedmicrocontact printer: theInnoStamp40.

The workflow of this study is based on three

steps (figure 1). The first one is the fabrication

of ECM (Extra Cellular Matrix) protein micro



patterns on glass slides. For that, we used the

automatic microcontact printer named

InnoStamp40. It is based on magnetic‐field

assisted microcontact printing [13]. This

technology allows for:

An automation of the full process of

microcontact printing,

A fine control of the pressure applied

during the printing step,

An alignment of the patterns on the

sample,

A high reproducibility of the process.

Three elements are handled in the

InnoStamp40® (www.innopsys.com):

A PDMS stamp with the micropatterns,

A sample (in this study: a glass slide),

The ink (In this study, it is a 100µg/mL

fibronectin solution with PBS 1X).

This equipment automatically generates

micropatterns of fibronectin on the glass slide

according to the layout of figure 2. Four

different shapes of patterns with five different

sizes (from 10µm to 50µm) and four different

gaps (from 10µm to 100µm) are printed and

arranged into periodic arrays. Each shape array

is repeated at least one time on a stamp.

The second step is the backfilling of the

fibronectin micro patterns with an antifouling

layer (figure 1). For that, we incubated the

printed sample in a 100µg/mL solution of Pll‐g‐

PEG during 1h. After drying, the space between

fibronectin patterns is filled with Pll‐g‐PEG.

The third step is the PC3‐GFP cells seeding

(figure 1). The PC3‐GFP cells are seeded during

3h at 37°C with a concentration of 42.000

cells/cm². Then, they are washed with PBS and

dehydrated with 3 baths of 50%, 75% and 100%

of ethanol.

Figure 1: the workflow of Cellular growth on

patterned samples

The images are acquired with a standard

fluorescence microscope.



Figure 2: layout of the micropatterns. There are 4

different cells with different shapes: disc, line,

square, and triangle. Their size varies between

10µm to 50µm and the gap between the patterns

varies between 10 to 100µm.

2‐Differentshapesofthebiochemicalpatterns.

The cells are selectively captured on the micro‐

patterned slides. We can observe on figure 3

that the PC3‐GFP cells in adhesion with the

surface of the sample decorate the printed

fibronectin patterns. The PC3‐GFP cells which

are selectively immobilized on the molecular

patterns spread on and fill them while the very

few cells immobilized outside the patterns are

circular and do not spread. We quantify the

selectivity of the immobilization as the number

of cells counted inside the printed patterns

divided by the number of cells counted in the

PLL‐g‐PEG background. Table 1 displays the

selectivity observed for the four different

shapes. We find a good selectivity for all the

shapes (higher than 75%). With triangles, the

selectivity is lower than the line patterns. In a

more general manner, we observe that the

higher the spatial confinement produced by

the patterns is, the lower the selectivity is.

Selectivity (inside/outside

%)

Triangle 75 Square 85 Round 91 Line 99

Figure 3: fluorescence images of PC3‐GFP cells

immobilized on ECM micro patterns of various

shapes (the patterns are depicted by dashed lines).

3‐Quantification of the area of a celltrappedontoabiochemicalpattern.

After shape investigation, we studied the

minimal surface needed to trap a single PC3‐

GFP cell and the area occupied by captured

cells. To achieve that, we selected a region near

the interface between two areas (figure 4). In

order to distinguish and well identify the

different areas (one with 50µm and the other

one with 40µm triangle patterns), we have also

printed a dashed‐line pattern composed of

20µm*10µm rectangles (violet arrow).

We first estimated the mean area of the cells

trapped into triangles by dividing the triangle



area by the number of trapped cell. We found

a mean area of 420µm².

Figure 4: A selected region at the interface between

two areas. The upper part is made of 50µm triangles

and the lower part is made of 40µm triangles. At the

interface, rectangles of 20µm*10µm are printed

(yellow dashed line).

We can observe in figure 4 that single cells

decorate the rectangular patterns of the

printed dashed line. However, they do not

follow exactly the contours of the rectangle but

instead they overfill the patterns. The area of

these patterns is 200µm². Compared to the cell

trapped into triangles, we assume that a

200µm² rectangular area is enough to trap

single cells but not enough to make the

contours of the cell match the contours of the

printed features. This is why the PC3‐GFP cells

on this kind of patterns exhibit circular shapes

and not rectangular ones. The interplay

between PC3‐GFP cells and 200µm² rectangles

of fibronectin enables to achieve a

deterministic immobilization of single cells on

the patterns but do not conform the cell shape

to the contours of the pattern. The circular

shape of these cells could also be

demonstrated by the interplay of the

protruding parts of the cells (outside of the

patterns) with the antifouling layer.

Single cells trapped on the 30µm triangles

(figure 5) seem to be shaped by the patterns.

The area of these triangles is 450µm², which is

close to the 420µm² area found previously.

Several cells can sometimes be observed on

these patterns because the pattern area is a bit

larger than 420µm² and also due to cell size

heterogeneity. Put together, these results

indicate that, for patterns of area close to

200µm2, single cells can be deterministically

immobilized but their shape is not constricted

by the shape of the patterns, while for patterns

of area close to 400 µm2, single cells are

selectively immobilized and shaped by the

printed patterns.

Figure 5: fluorescence image of PC3‐GFP cells

immobilized on 30µm printed triangles (scale bar

40µm).

4‐Aggregates of cells near themolecularpatterns.

We have also investigated the interplay

between cells according to their positions into

the patterns.

We observe on figure 6 the cell spreading on

lines of different widths: 10, 20 and 40µm. On

10µm lines (figure 6.A), the PC3‐GFP cells

spread on the entire length of the lines and

create a pattern similar to a cell “rosary”. If

there are more cells across, PC3‐GFP cells

reduce their spreading in order to “cohabit”

within the line. Cells found outside the patterns

exhibit circular shapes and they hang on to the

spread ones. For 20µm and 40µm‐wide lines

(figure 6.B and C), the cells interplay is similar.

However, due to a lower spatial confinement,

PC3‐GFP cells are less stressed and, thus, can

shape the lines with two or three cells across.

On figure 7, PC3‐GFP cells shape discs of 40µm

diameter but some additional cells hang on to



them. We observe the same shape difference

between cells inside (spread) and outside

(circular) the patterns. This effect being visible

for different shape and size patterns, we

assume that it is due to some cell aggregates

present in the seeding solution.


trapped immobilized on 10µm (A), 20µm (B) and

40µm (C) wide printed lines. Scale bar: 40µm.


immobilized on 40µm circular patterns. Scale bar:

40µm.

5‐Study of the patterned slides shelftime

To conclude this study, we have investigated if

these printed slides could be used days after

the manufacturing process. Indeed, in order to

evaluate this technology, it could be interesting

to send to researchers some printed slides,

raising the question: “what is the shelf time of

the printed slide?”. To answer this question,

we compared cell culture on a fresh printed

slide and cell culture operated 12 days after

printing in different conditions of conservation.

The temperature in the conservation phase

was changed (room temperature, 4°C or ‐20°C)

and two media of conservation were tested

(ambient air or PBS1X). We could not notice

any difference across these conditions. This is

why we present the results after 12‐day

conservation at room temperature, ambient

air. We are now using these parameters to

send some samples to interested customers.

C B

A



Figure 8: comparison between a fresh printed slide

(A) and a 12‐day stored patterned glass slide (B).

On figure 8, we can observe no significant

differences between freshly printed slides and

conserved ones. The selectivity is better after

12 days but the number of cells per pattern is

higher. This trend could be explained by a small

variation in the cell concentration of the

seeding solution.

Conclusion

Patterned slides with ECM proteins can be

routinely and reproducibly produced using the

InnoStamp40 microcontact printer. Adherent

cells can be immobilized deterministically on

these patterns. Single‐cell arrays can be

generated. The shape of these individual cells

can be dictated by the shape of the patterns, if

the area of each pattern is smaller than the

mean area of a cell in adhesion on a non‐

spatially confined zone. To complete the study,

we have characterized the conservation of the

patterned slides before cell seeding and have

proven that functional micro‐patterned slides

are compatible with standard shipping.

Acknowledgment

This work was done within the framework of a

joint laboratory named BIOSOFT

(www.biosoftlab.com) between LAAS‐CNRS

and INNOPSYS. Author wants to acknowledge

Julie FONCY, Charline BLATCHE, Emmanuelle

TREVISIOL and Christophe VIEU.

References

[1] McBeath R et al. Cell shape, cytoskeletal

tension, and RhoA regulate stem cell lineage

commitment. Dev Cell. 2004 Apr;6(4):483‐95.

[2] Tseng Q et al. Spatial organization of the

extracellular matrix regulates cell–cell junction

positioning. Proc Natl Acad Sci U S A. 2012 Jan

31;109(5):1506‐11

[3] Gao L et al. Stem cell shape regulates a

chondrogenic versus myogenic fate through

Rac1 and N‐cadherin. Stem Cells. 2010 Mar

31;28(3):564‐72.

[4] Mrksich M et al. Using microcontact printing

to pattern the attachment of mammalian cells

to self‐assembled monolayers of

alkanethiolates on transparent films of gold

and silver. Exp Cell Res. 1997 Sep

15;235(2):305‐13.

[5] Huang S et al. Shape‐dependent control of

cell growth, differentiation, and apoptosis:

switching between attractors in cell regulatory

networks. Exp Cell Res. 2000 Nov 25;261(1):91‐

103.

A

B



[6] Théry M et al. The extracellular matrix

guides the orientation of the cell division axis.

Nat Cell Biol. 2005 Oct;7(10):947‐53.

[7] Théry M. Micropatterning as a tool to

decipher cell morphogenesis and functions. J

Cell Sci. 2010 Dec 15;123(Pt 24):4201‐13.

[8] James J et al. Subcellular curvature at the

perimeter of micropatterned cells influences

lamellipodial distribution and cell polarity. Cell

Motil Cytoskeleton. 2008 Nov;65(11):841‐52.

[9] Dupin I et al. Classical cadherins control

nucleus and centrosome position and cell

polarity. J Cell Biol 185(5):779‐786

[10] Desai AR et al. Cell polarity triggered by

cell‐cell adhesion via E‐cadherin. J Cell Sci. 2009

Apr 1;122(Pt 7):905‐11.

[11] Théry M et al. Cell shape and cell division.

Curr Opin Cell Biol. 2006 Dec;18(6):648‐57.

[12] Parker KK et al. Directional control of

lamellipodia extension by constraining cell

shape and orienting cell tractional forces.

FASEB J. 2002 Aug;16(10):1195‐204.

[13] Brock A et al. Geometric Determinants of

Directional Cell Motility Revealed Using

Microcontact Printing. Langmuir, 2003, 19 (5),

pp 1611–1617

[14] CAU JC et al. Magnetic field assisted

microcontact printing: A new concept of fully

automated and calibrated process.

Microelectronic Engineering, volume 110,

October 2013, Pages 207–214

Documents

Printing of biochemically-patterned slides OK of biochemically-patterned slides1.pdfimmobilized on ECM micro patterns of various shapes (the patterns are depicted by dashed lines)