Download pdf - Inverted Colloidal Crystals as Three-Dimensional Cell Scaffolds

Inverted colloidal crystals as three-dimensional microenvironments forcellular co-cultures

Jungwoo Lee,a Sachin Shanbhagb and Nicholas A. Kotov*abc

Received 24th April 2006, Accepted 4th July 2006

First published as an Advance Article on the web 25th July 2006

DOI: 10.1039/b605797g

Cellular scaffolds made on the basis of inverted colloidal crystals (ICC) provide a unique system

for investigation of cell–cell interactions and their mathematical description due to highly

controllable and ordered 3D geometry. Here, we describe three new steps in the development of

ICC cell scaffolds. First, it was demonstrated that layer-by-layer (LBL) assembly with clay/PDDA

multilayers can be used to modify the surface of ICC scaffolds and to enhance cell adhesion.

Second, a complex cellular system made from adherent and non-adherent cells co-existing was

created. Third, the movement of non-adherent cells inside the scaffold was simulated. It was

found that floating cells are partially entrapped in spherical chambers and spend most of their

time in the close vicinity of the matrix and cells adhering to the walls of the ICC. Using this

approach one can efficiently simulate differentiation niches for different components of

hematopoietic systems, such as T-, B- and stem cells.

Introduction

Colloidal crystals represent an exceptionally dynamic area of

research capitalizing on the unique spatial organization and

diffraction characteristics of sub-micron scale lattices.1–4 These

structures are designed, having in mind, primarily, applica-

tions in optics,5 sensors6 and catalysis.7 Inverted colloidal

crystals (ICC) also open an interesting opportunity for a rather

unexpected, but tremendously important area of science

related to cell communication. We recently introduced the

use of micron-scale ICC systems for three-dimensional (3D)

cell cultures, which mimic the microenvironment of three-

dimensionally organized native tissues.8–10 Unlike other

disordered cell supports, the ICC geometry affords systematic

study of cell signaling in 3D, which has been proven to be

fundamentally essential for proper development of tissues.11–16

Adequate understanding and proper methods of control of

cell signaling are particularly important for stem cell research.

For instance, the rate and direction of the differentiation of

stem cells are strongly affected by their 3D microenvironment

and soluble signaling molecules.17–22 Recent studies have

shown that a 3D culture environment significantly promotes

the efficiency of stem cell differentiation.23,24 Intense cell–cell

and cell–matrix interactions have been distinguished as key

factors that determine the fate of individual cells by serving

as important communication channels.23,25,26 In order to

reproduce the complexity and dynamics of cellular environ-

ments, various scaffold fabrication techniques have been

developed.27–31 However, the geometry of these scaffolds

mainly depends on the process, and usually they have a poorly

ordered or chaotic structure. Recently, rapid prototyping and

3D deposition techniques, assisted by computer-aided design

and complex robotic equipment, were developed to construct

more controlled 3D architectures.27,32 These techniques allow

researchers to design 3D scaffolds with desired properties such

as porosity, interconnectivity and pore size. Nevertheless,

besides being heavily equipment-dependent, they suffer from

limited material selection and inadequate resolution. From a

manufacturing standpoint, the fabrication procedure of ICC

scaffolds is simple and flexible. Any precursor solution capable

of undergoing a liquid-to-solid transition may potentially be

used as a scaffolding material. An ordered structure, with a

high degree of uniformity, can be achieved without the need

for complex computer design programs and facilities.

Beyond that, several unique characteristics of ICC used as

cell scaffolds make them particularly convenient for the use

with stem cell cultures,8–10,33 which can help uncover methods

for successful tissue engineering from them. In this respect,

ICC systems possess high surface areas with a void fraction of

76% and a regularly spaced network of pores which provides a

mechanically strong, well-connected open porous geometry.33

These features enhance cell seeding efficiency, transport of

nutrients and metabolites, and the rapid and uniform

distribution of soluble signaling molecules. The exceptionally

uniform and three-dimensionally ordered structure of ICC

scaffolds enables the development of computational models to

systematically study the effect of signaling molecules, cell–cell

and cell–matrix interactions, and other processes.34 Until now,

only single cell culture studies have been reported for ICC

scaffolds.8–10,33 Considering that this system could be a

convenient discovery tool for research on cell–cell interactions,

achieving the next level of complexity, i.e. the construction of

a system with two or more different cell types co-populating

the ICC matrix, appears to be the most essential step in

this area.

aDepartment of Biomedical Engineering, University of Michigan, 3074H.H. Dow Building, 2300 Hayward Street, Ann Arbor, MI 48109, USAbDepartment of Chemical Engineering, University of Michigan, 3074H.H. Dow Building, 2300 Hayward Street. Ann Arbor, MI 48109, USAcDepartment of Material Science and Engineering. University ofMichigan, 3074 H.H. Dow Building, 2300 Hayward Street, Ann Arbor,MI 48109, USA. E-mail: [email protected]; Fax: +1-734-764-7453;Tel: +1-734-763-8768

PAPER www.rsc.org/materials | Journal of Materials Chemistry

3558 | J. Mater. Chem., 2006, 16, 3558–3564 This journal is � The Royal Society of Chemistry 2006

In this paper, we introduce a model system combining two

types of cells co-existing in an ICC scaffold, which paves the

way for future systematic studies of cell evolution mechanisms.

Since these interactions are of particular importance for the

development of hematopoietic stem cells, the cell cultures

were chosen having in mind the recreation of the 3D

microenvironment of bone marrow and thymus differentiation

niches.26,35,36 The selection of particular model cell cultures

was also aided by the fact that the characteristic geometry of

the ICC scaffolds resembles that of bone marrow and thymus

niches (i.e. stromal cells cover the surface and well intersticed

sinus cavities). Human thymus epithelial cells (Hs202.Th)

and human monocytes (HL-60) were used as anchorage-

dependent feeder cells and suspension cells mimicking pro-

genitors, respectively. Before using hematopoietic stem cells

in our 3D culture system, we tried to use the HL-60 cell line

because it is easier to deal with and has been proven a unique

in-vitro model system for studying the cellular and molecular

events involved in the proliferation and differentiation of

normal and leukemic cells.37 Cell–cell interactions within

ICC scaffolds were evidenced by simplified Brownian

Dynamics (BD) simulations taking advantage of the unique

3D morphology.

Experimental

Colloidal crystal construction

An aqueous suspension of polystyrene (PS) spheres with a

diameter of 100 mm (Duke Scientific, 3 6 104 particles per

milliliter and 1.4% size distribution) was changed with

isopropanol solution before use. A 0.5 ml plastic centrifuga-

tion tube was glued on the plastic dish and the top of the

centrifuge tube was cut and connected with a long Pasteur

glass pipette. The complex unit was attached to the bottom

of a glass beaker, and the glass beaker was placed on an

ultrasonic bath (VWR). Two drops of the solution were

released through a long Pasteur glass pipette at 15 minute

intervals (25 intervals total) under gentle agitation generated

by the ultrasonic bath. To reduce thermal motions of the

spheres, the bath temperature was maintained below 20 uC.

After the dropping was finished, the isopropanol was

evaporated off overnight at 60 uC.

Scaffold fabrication

Prepared colloidal crystals were heat treated at 120 uC for

4 hours, which caused partial melting of the beads’ surface. As

a result, the PS microspheres fused together and the free

standing colloidal crystals were easily extracted from the mold.

As scaffolding materials, a poly(acrylamide) hydrogel com-

posed of a 30 wt% acrylamide (Sigma) precursor containing

5 wt% of N,N-methylenebisacrylamide (NMBA) cross-linker

was used. The precursor was infiltrated into the colloidal

crystal by centrifugation at 5800 rpm for 10 min. An initiator,

1 wt% of potassium peroxide solution, and an accelerator,

N,N,N9,N9-tetramethylethylenediamine (TEMED), were

added. Polymerization occurred in a glass vial. After the

polymerization was complete, the colloidal crystal containing

the hydrogel part was cut out and soaked in tetrahydrofuran

(THF) for 24 hours to remove PS beads. Finally the ICC

hydrogel scaffolds were equilibrated in deionized water.

Fluorescent ICC hydrogel scaffolds were prepared by adding

0.05 wt% of fluorescent monomer, Polyfluor 511 (Polyscience

Inc.), to the hydrogel precursor solution.

Layer-by-layer surface coating

ICC hydrogel scaffold surfaces were coated with the sequential

deposition of positively charged 0.5 wt% poly(diallyldimethyl-

ammonium chloride) (PDDA, Sigma, MW = 200 000) solution

for 15 min, and a negatively charged 0.5 wt% clay platelet

(average 1 nm thick and 70–150 nm in diameter, Southern

Clay Products) dispersion for 15 min. Each adsorption

step was followed by rinsing in deionized water for 15 min,

and all processes were performed under a gentle flow

generated by a stirrer. Cyclic repetition of the polymer

adsorption/rinsing/clay adsorption/rinsing process was carried

out 10 times.38

Mechanical property testing

Compressive moduli of hydrated ICC scaffolds, where the

surface was coated with 10 layers of clay/PDDA, were

measured at a constant strain rate (10 mm s21) using a

mechanical properties tester and a 1.1 lb load cell

(TestReciurces Inc., MN).

Dynamic co-culture

Human thymus cell line Hs202.Th (CRL-7163) and human

premyeloblast cell line HL-60 (CCL-240) were purchased

from ATCC (Manassas, VA). Hs202Th cells were grown in

Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented

with 10% Fetal Bovine Serum (FBS) (GIBCO, CA). HL-60

cells were cultured in Iscove’s Modified Dulbecco’s Medium

(IMDM) containing 20% FBS. The cells were maintained at

37 uC with 5% CO2 and the medium was changed twice a week

until they reached a confluent or desired population on T-75

culture flasks.

Co-culture was carried out in 10 ml rotary cell culture

vessels (RCCS-4D, Synthecon Inc.). Scaffolds were sterilized

by soaking in 70% EtOH for one hour followed by washing

in phosphate buffered saline (PBS) for 15 min twice. 2 6105 Hs202.Th cells were placed in a culture vessel, which

subsequently was filled with the medium. The rotation speed

was set at 12 rpm for the first 12 hours and later it was

decreased to 8 rpm, the normal speed. The medium was

replaced once every three days. On day six, both Hs202.Th and

HL-60 were stained with fluorescent dyes followed by a five

day co-culture period. The HL-60 cells were stained with 5 mM

chloromethyl derivatives fluorescent dye (CMRA, Molecular

Probes) diluted in PBS buffer following the protocol provided

by the vendor. Hs202.Th cells on the scaffold were stained with

carboxyfluorescein diacetate succinimidyl ester (CFDA-SE,

Molecular Probes). The culture medium was replaced with

10 ml of 5 mM CFDA-SE diluted in PBS buffer, and the

culture was incubated at 37 uC for 20 min. After that, the

medium was changed to IMDM supplemented with 20% FBS,

and pre-stained 1 6 106 HL-60 cells were seeded.

This journal is � The Royal Society of Chemistry 2006 J. Mater. Chem., 2006, 16, 3558–3564 | 3559

Cell culture characterization

Scaffolds were carefully removed from the culture vessel

together with the medium and moved to a glass bottom culture

dish (MatTek Corporation). The scaffolds were kept immersed

in the medium during the process. Co-cultured scaffolds were

investigated utilizing a Leica SP2 confocal microscope with

106 and 206 objective lenses. Thymic epithelial cells stained

with CFDA-SE were imaged using a 470 nm filter while

excited with 457 nm laser light (green). CMRA stained

monocytes were excited at 543 nm and fluorescent lights were

detected at 576 nm (red). Cross-sectional images were obtained

in the same manner by taking pictures at 1 mm depth

increments down to 160 mm.

Scanning Electron Microscope (SEM) observations were

performed with a Philips XL30 SEM at 5.0 KV. Before

imaging, hydrogel scaffold samples were first fixed in 2%

cacodylate-buffered glutaraldehyde for 2 hours and washed

three times in 0.1 M cacodylate buffer for 30 min. Fixed

hydrogel scaffolds were dehydrated through a series of ethanol

solutions concentrations of 50, 70, 90, 95, and 100% for 10 min.

Dehydrated samples were freeze dried overnight utilizing a

Labconco FreeZone (Labconco), and then were coated with

gold for 180 s using a sputter coater (Desktop 2, Denton

Vacuum Inc.). Cross-section images of the internal architec-

ture were obtained after cutting the sample with a razor blade.

Results and discussion

ICC scaffold construction

The diameter of the spheres commonly used as colloidal

crystals is around 100–1000 nm for the purpose of matching

the optical band-gap in the visible region. Various methods

such as electrophoretic deposition,39 solvent evaporation,40

dipping,41 agitation42,43 and most recently spin coating44 have

been developed to construct highly ordered colloidal crystal

structures. In order to utilize the unique geometry of the

inverted colloidal crystals as a scaffold, the sphere size has to

be increased to the 10–1000 mm range. However, it is difficult

to obtain the same degree of order with micron scale beads

using the above methods of synthesis, mainly due to their

larger volume and heavier mass. Fortunately, micron-sized

beads offer two advantages over nanosize spheres. First, the

agitation of beads by shear force works more effectively

because of their larger volume. Previously, we reported the

construction of colloidal crystals by employing a gentle

agitation method.9 The second advantage is a faster sedimen-

tation rate due to their greater mass. However, the sedimenta-

tion rate was often too fast for them to self-assemble into a

closely-packed ordered array. The opposite problem, viz., how

to retard the sedimentation rate, was solved by introducing a

Pasture glass pipette before the beads entered into the mold.

The pipette extended the sedimentation distance and worked

as a thin funnel, which caused a bottleneck effect for

precipitating beads (Fig. 1a).

Once beads precipitated at the bottom of the mold, gentle

agitation generated by an ultrasonic bath assisted the move-

ment of beads and positioned them at the lowest energy spots.

This led to a highly packed and ordered array of spheres.

When the bottom area was covered with beads, their rugged

surface served as a template for the formation of the second

layer. Since structural defects accumulated from the bottom

area, incomplete layers and less ordered arrays were usually

observed on the top area.

The sedimentation rate was controlled further by adjusting

the concentration of beads in the solution and the time interval

between injections. For example, decreasing the amount of

beads and increasing the interval period provided more time

for the repositioning of precipitated beads. The use of

isopropanol guaranteed that the agitation was not too violent

to destroy the whole structure, while its buoyancy made it

easier for the PS beads to rearrange. Generated colloidal

crystal structures were 8 mm diameter and 1–1.5 mm in

thickness and SEM investigations revealed a highly ordered

hexagonally close-packed structure (Fig. 1b–d).

Following sedimentation, the colloidal crystals were heat

treated which resulted in partial melting of the spheres.29 This

step allowed the beads to stick together and on subsequent

cooling (re-solidification), junctions were created between the

spheres setting the structure in place. The resulting free

standing colloidal crystals were strong enough to be easily

handled and removed from the mold. The formation of the

junctions later prevented breakage of the crystal lattice during

the infiltration of scaffolding material and ensured the

connectivity between spheres and continuity of the chain of

pores in the final scaffold. The channel diameter was

determined at this stage, because the size of the melted

area depended on the annealing temperature. Increasing

Fig. 1 (a) Schematic diagram of the experimental setup used for

assembling micron-range polystyrene beads in 3D ordered structure.

(b–d) SEM images of the colloidal crystal structure made from 100 mm

polystyrene beads, showing the bottom (d), and internal structure at

different magnifications (b, c). Internal images were taken after cutting

the colloidal crystal with a razor blade. The small white spots on each

PS sphere in (b) are contact points between beads, which later become

channels.


temperature enlarged the melting spot, but it caused shrinkage

of the colloidal crystal. Usually it led to the cracking of the

crystal structure and/or incomplete precursor solution infiltra-

tion. For 100 mm diameter PS beads, heat-treatment at 120 uCfor 4 hours gave the optimal result.

The diameter of pores was dictated by the details of the co-

culture system. Although monocytes and trypsinized thymic

epithelial cells have similar dimensions, epithelial cells stretch

out after attachment to the surface. Based on 2D charac-

terization, the size of the elongated thymic epithelial cells was

around 80–160 mm. Kotov et al. studied the pore size effect of

ICC scaffolds on a 3D cell culture utilizing three different sizes

of beads: 10 mm, 75 mm and 160 mm. The 75 mm pore diameter

favored bone marrow stromal cells nesting, while the 10 mm

pore size was too small for even a single cell, and 160 mm

diameter pores were too large to effectively entrap cells.10

Also, Zinger et al. investigated osteoblast-like cell cultures

on well-defined 2D cavities which were analogous to ICC

scaffolds, and found that 100 mm cavities favored osteoblast

attachment and growth.45 For the entrapment and transport of

suspension cells, the channel diameter, which is determined by

the size of the microspheres, was the most important para-

meter. PS beads which had a 100 mm diameter made 25–30 mm

diameter channels after annealing at 120 uC. The diameter of

the suspension cells (approximately 15–20 mm) was small

enough to enable them to pass through the channels.

As a scaffolding material, we selected poly(acrylamide)

hydrogel. Hydrogel is a broadly used scaffolding material

because of its biocompatibility, mechanical strength, and

transparency.46,47 The transparency of the hydrogel makes it

easier to monitor cell migration and growth deep inside the

scaffold using optical microscopy. Recently the observation of

cell growth at a depth greater than 250 mm 9 and real time cell

migration via a channel33 were reported. In addition, the

hydrogel exhibited another feature that facilitated its use in

ICC work. At low viscosity of the precursor solution, it

completely infiltrated to the colloidal crystal, and the whole

structure of the crystal template was transferred intact (Fig. 2).

The monomer concentration was set low enough to prevent

incomplete infiltration due to increased viscosity, and simulta-

neously to prevent deformation of the geometry during solvent

extraction.

The compressive moduli of hydrated and LBL coated ICC

scaffolds were 189.4 ¡ 5.89 KPa (Fig. 3). Compared to the

mechanical strengths of other porous hydrogel substrates, it

showed stronger mechanical stability.33,48 This was mainly

due to the higher content of polymer and the highly ordered

structure of the hydrogel ICC scaffolds. The achieved

compressive modulus was within the range of normal articular

cartilage.49 This degree of mechanical property was adequate

to construct artificial supports of targeted soft tissues.

Layer-by-layer surface modification

Hydrogel matrices rarely support adherent cell adhesion

without surface modification, because acrylamide polymer

chains do not have cell adhesion receptors, and the hydrophilic

nature of the hydrogel inhibits adsorption of cell binding

proteins on the gel surface.47 To render the surface bioactive,

we selected a layer-by-layer (LBL) deposition technique

instead of the commonly used covalent coupling of specific

peptide sequences such as RGD or an entire ECM protein to

the polymer.47 The driving forces for LBL coating are the

electrostatic, van der Waals, and hydrogen bonding interac-

tions between oppositely charged polyelectrolytes dissolved in

aqueous solution.50 This unique feature of the LBL technique

allows a complex porous 3D geometry, such as the intricate

and convoluted ICC surface, to be coated as long as fluid

transport in and out of the sample is not severely constrained.

It has been reported that 2D polyelectrolyte multilayers

Fig. 2 SEM images of the hydrogel scaffold after dehydration: (a)

bottom structure image, (b) internal structure image taken after cutting

the scaffold with a razor blade. The dehydration process caused

shrinkage of the ICC hydrogel scaffold, which led to some deformation

of the structure. Confocal images of a fluorescent hydrogel scaffolds:

(c) a 3D reconstruction of serial z-section images taken in 0.5 mm steps

showing the organization of main pores and interconnected channels

of a hydrogel ICC scaffold without shape deformation, and (d) 3D

overlapping images of serial z-sectional images of 160 mm interval with

5 mm step size.

Fig. 3 A compressive stress–strain curve from the mechanical

property test.


supported anchorage dependent cell attachment without using

adhesive proteins.51

In our system, we used clay nanoparticles/poly(diallyl-

dimethylammonium chloride) (PDDA) multilayers.38 The clay

particles are biocompatible and their flat shape effectively

covered the hydrogel surface. Coated clay nanoparticles

created nanoscale roughness, increased charging on the

surface, and created much stiffer films than hydrogel. An

increase of Young’s modulus was shown to be the primary

factor determining the adhesion of cells to materials.52,53 These

synchronous effects promoted cell adhesion.54,55 Ten layers of

PDDA/clay easily changed the surface property from cell

repulsive to cell adhesive, and thymic epithelial cells could

attach to the hydrogel scaffold.

Dynamic co-culture

Human thymic epithelial cells and human premyelote mono-

cytes were co-cultured in a rotary cell culture bioreactor.

Rotary motion induced convective flow, and the scaffold

geometry utilized this flow as a continuous driving force for

the cell movement.56 After five days of co-culture, the hydrogel

ICC scaffolds were imaged through a confocal microscope.

Different emission ranges of fluorescent dyes were used to

stain the thymus cells and monocytes with green and red,

respectively. Thymic epithelial cells attached to the cavity were

observed as green circles, and floating monocytes were imaged

as red spots (Fig. 4). Although many monocytes were diffused

out of the scaffold during the sample preparation, some of

them remained entrapped inside the pores. Confocal cross-

sectional images reveal that suspended cells were distributed

uniformly throughout the scaffold (Fig. 4b).

Co-cultured hydrogel ICC scaffolds were dehydrated and

observed under an SEM. The dehydration process deformed

the structure, which is the reason for the dimensional

differences between the two rows of images in Fig. 4. It was

found that the scaffold exterior was covered densely with

thymic epithelial cells, and their population reduced the

inward movement of other epithelial cells (Fig. 4a, b, d).

Secondly, epithelial cells migrated between pores through

interconnected channels, and some colonies expanded over

several pores (Fig. 4f). Thirdly, a few suspension cells were

trapped inside when they were observed at the interior of the

scaffold in SEM cross-sectional images (Fig. 4e). It suggests

that monocytes travel deep into the ICC scaffolds.

Modeling

To study the interaction of a floating cell with the scaffold

quantitatively, we constructed a simplified Brownian

Dynamics model. The cell was treated as a hard sphere of

radius acell that is suspended in an ICC geometry, composed of

hollow spherical chambers of nominal radius R connected by

channels of radius b.34 To simplify the treatment, we assumed

that the fluid inside the scaffold was quiescent and that the

motion of the cells was purely diffusive. Under the assump-

tions stated above, the motion of the cell results from a balance

between the drag force and the random Brownian force:

fdr/dt = FB (1)

where f = 6pgacell is the hydrodynamic drag exerted by the

solvent of viscosity g on a cell of radius acell, and r is the

position vector of the center of mass of the cell. The Brownian

Fig. 4 Confocal images (a–c) of co-cultured ICC hydrogel scaffolds; thymic epithelial cells (green) and monocytes (red). (a) Bottom area image

shows the surface of the scaffold was densely covered with thymic epithelial cells. Most of the monocytes around the edge of the scaffold were

released. (b) A cross-sectional image after cutting the co-cultured ICC scaffold with a razor blade shows decreasing thymic epithelial cell density

moving into the inside of the ICC scaffold. Monocytes were distributed through the whole ICC scaffold and a similar number of cells were

entrapped at each pore. (c) A lateral section image of 80 mm in depth. SEM images (d–f) of co-cultured hydrogel scaffolds. (d) Cross-sectional image

of the scaffold’s interior. (e) Entrapped monocytes. (f) Thymic epithelial cells covering pores and channels.


force, FB, satisfies the fluctuation–dissipation theorem57 which

necessitates ,FB. = 0, and ,FBFB. = 2kBTfI. Here, I is the

unit tensor, kB = 1.38 6 10223 J K21 is Boltzmann’s constant,

and T is the absolute temperature.

The diffusivity, D, was obtained from the hydrodynamic

drag via the Einstein relation,57 D = kBT/f. In accordance with

microscopy measurements, we took R = 50 mm, b = 12.5 mm,

and acell = 7.5 mm. Thus, f = 6p(1 cP)(7.5 mm) = 1.414 61024 g s21, and D = kBT/f = 2.91 6 1022 mm2 s21. We used

the algorithm outlined by Larson57 to implement the BD

simulation, choosing the simulation time step, dt, so that

!6Ddt # 0.05acell. We employed reflecting boundary condi-

tions to model collisions between the cell and the scaffold.

Grigoriev et al. considered a dimensionless Brownian

particle trapped inside a spherical chamber of volume V.58

They estimated that the time, t*, that it takes for the particle to

escape from a small circular hole of radius b on the surface of

the chamber is given by t* = V/4bD, where D is the diffusivity

of the particle. We adapted the expression for t* to obtain a

crude estimate for the escape time of a Brownian particle of

finite size from an ICC scaffold as:

t*ICC = (p/3ZD)(R 2 acell)3/(b 2 acell) (2)

where Z = 12 is the co-ordination number of the ICC lattice.

From eqn (2), we obtained t*ICC y 5.5 6 105/12 s y 12 hours.

Thus, the ICC geometry provides very suitable geometry

for cell interactions due to partial entrapment of the cells in

the cavity.

We simulated the dynamics of the cell in the ICC scaffold

using BD, and recorded its trajectory from t = 0 to t =

1000 days. Over this period, the cell visited several chambers.

From the simulation, we observed that by the time the cell

vacated a chamber by escaping through the interconnecting

channel to another chamber, it thoroughly, and uniformly,

sampled the whole chamber. In other words, the amount of

time the cell spent in any region of the chamber was

proportional to the volume of that region. Fig. 5a shows a

cross section of a spherical chamber that has been divided into

shells of equal thickness, DR = acell. These shells do not have

the same volume. For illustration, if we approximate the

volume of a shell by DVshell = 4pRi2DR, where Ri is the inner

radius of the shell, we can see that the volume of the outer

shells is greater than that of the inner shells. As mentioned

previously, the center of mass of the cell resides in a shell, in

proportion to the volume of that shell. Thus, it spends a

significant fraction of time (about 41%, see Fig. 5a) in the

outermost shell, where the distance between the surface of the

cell and the inner surface of the chamber is less than or equal

to the radius of the cell. Thus the ICC geometry fosters contacts

between the cell and the matrix surface or between the

suspension and adherent cells in a co-culture.

Conclusions

We have demonstrated a 3D co-culture model system within a

LBL surface modified ICC scaffold in both experimental and

modeling works. Clay/PDDA multilayer successfully formed

on a complex porous ICC scaffold walls, and significantly

improved adhesion of epithelial cells. The unique geometry of

ICC scaffolds can accommodate two different types of cells

within a same chamber. Modeling results indicated that cells

were effectively trapped in the spherical chambers and that

entrapped suspension cells spent a significant fraction of time

in the vicinity of the ICC chamber wall or the epithelial coating

in co-cultures.

Well controlled multiscale structures which can build real-

size organ systems and generate the essential subcellular

morphology are a key factor for the successful investigation

of cell–molecule and cell–cell interactions.12,59 It is obvious

that the full function of the tissues and organs cannot be

recovered without rebuilding the ultrastructure of the tissue

itself. Proposed ICC scaffolds and surface modification

utilizing a LBL technique will be excellent approaches for this

purpose. ICC scaffold structure generates super- and cellular-

scale microenvironments for intense cell contacts with other

types of cell or matrix. On this surface, various insoluble

signaling molecules such as ECM components, membrane

bound receptors and ligands can be incorporated through a

LBL method which can produce a subcellular, nanoscale

resolution environment for cellular receptor–molecular inter-

actions. In particular, this could greatly facilitate the study of B

and T cell development from stem cells which requires under-

standing and controlling precise 3D molecular interactions.

Acknowledgements

This work was supported by a grant from DARPA and

VaxDesign Inc. We thank Dr Michael Solomon and Tesfu

Fig. 5 Radial probability distribution of a finite-sized Brownian

particle of radius 7.5 mm diffusing in a spherical ICC chamber obtained

from BD simulations, when the chamber is divided into shells of the

same (a) thickness, and (b) volume. In (a), the dotted arc and the disc

represent the inner surface of the chamber, and the cell which is

modeled as a hard sphere, respectively. The thickness of each shell is

equal to the radius of the cell, acell. From (b), it can be seen that the cell

spends the same amount of time in each of the equi-volume shells,

whereas in (a), it spends more time in the exterior shells due to their

greater volume.


Solomon (University of Michigan, Ann Arbor) for assistance

with the laser scanning confocal microscope.

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