C H A P T E R 4

Patterned Cocultures for ControllingCell-Cell Interactions

Sun Min Kim, Junji Fukuda, and Ali Khademhosseini

4.1 Introduction

Cell-cell interactions play an important role in the function of many organ systems.In addition to homeostatic function, cell-cell interactions are also vital for regenera-tive processes, as well as for in vitro reconstruction of tissues for tissue replacement[1–8]. In the body, the dynamic interplay between various cell types regulates thefunction of the cells. The lack of such cell-cell interactions is one potential reason forthe loss of functional capability of cell types such as hepatocytes outside the body. Intissue culture, much of the native cell-cell interactions present in the body are lostdue to tissue isolation and digestion, as well as processes such as purification of spe-cific cell populations. To overcome this limitation, cocultures of two (or more) celltypes have been used to better mimic the organization and complexity of the in vivomicroenvironment [9–16].

Traditionally, in vitro cellular interactions were investigated by random seedingof multiple cell types on a tissue culture substrate; however, using this approach, itis difficult to control the degree of homotypic (i.e., contact between same cell types)and heterotypic (i.e., contact between different cell types) cell-cell interactions [17].To overcome these limitations, patterned coculture techniques have been developedbased on advances in microengineering technology, material science, and chemistry[17–25].

Patterned coculture techniques enhance the spatial control of cells in culture,the precise manipulation of homotypic/heterotypic cell-cell interactions, and thefunction of cell types through introduction of support cells that provide the signalsto maintain these cells in culture [17, 18, 20, 22, 24, 26]. Many different types ofpatterned coculture techniques have been investigated, most of which could beplaced into one of four categories. The first approach uses the selective adhesion ofcells to micropatterned substrates or islands [18–20, 27, 28]; the second approachuses the liquid flow in microfluidic channels to pattern cells and proteins on a sub-strate [29–31]; the third approach uses stencil-based patterning for localizing cellsto specific regions on a substrate [17, 21–23, 32, 33]; and, finally, the fourthapproach utilizes micropatterned surfaces that can be switched from cell repulsiveto adhesive by specific stimuli (electrical potential [34–37], temperature [38–44], orlight exposure [45]).

53

In this chapter, we review each patterned coculture method in detail based onthe above-mentioned categories. Also, emerging patterned coculture methods thatfacilitate cell culturing in three-dimensional environments, as well as with con-trolled temporal control, are discussed. Finally, we analyze the future directions ofthe use of patterned cocultures for in vitro systems.

4.2 Random Coculture Systems

Traditionally, cocultures of two or more cell types were generated by randomlyseeding the cells on a substrate [9, 46–50]. These cocultures have enabled the studyof cell-cell interactions. For example, it has been shown that the coculturing of adultrat hepatocytes with liver epithelial cells improved hepatocyte function in vitro asdemonstrated by the expression of liver-specific markers in culture. In thesecocultures, hepatocytes showed distinct morphology and function in vitro.Cocultures have also been applied to many other biological systems to recapture thecomplexity of the cellular microenvironment. Random coculture systems have pre-sented insight into homotypic and heterotypic cell-cell interactions but have beenlimited by the inability to vary local cell seeding density and the degree of cell-cellcontact. To overcome these limitations, micropatterned coculture systems have beenused to enhance the control of spatial localization of multiple cell types relative toeach other and to enable detailed mechanistic studies of the processes that regulatecell-cell interactions.

4.3 Patterned Coculture Systems

The majority of current patterned coculture systems can be generated based on oneof the following techniques: (1) selective adhesion to micropatterned substrates, (2)microfluidic patterning, (3) stencil-based patterning, and (4) micropatterned switch-able surfaces.

4.3.1 Selective Adhesion of Cells to Micropatterned Substrate

In this approach, the cells of interest selectively adhere to specific regions of amicropatterned substrate by the surface characteristics of substrate, including varia-tions in surface charge, chemistry, hydrophilicity, and topology [18, 51–57]. Differ-ent cell types have different levels of adhesiveness to various substrates based ontheir level of expression of various adhesion molecules, such as integrins andcadherins. Based on these differences, it is possible to localize specific cell types tomicropatterned regions on a substrate for culturing multiple cell types. One of thefirst examples of the use of this approach was demonstrated by Bhatia et al. [18, 19,28] through the use of a micropatterning technique for generating two-dimensional,anisotropic surfaces used to localize different cell types in specific region.Figure 4.1(a) shows the schematic of the process of generating such micropatterncocultures. In this approach, micropatterned cell-adhesive biomolecules (e.g.,

54 Patterned Cocultures for Controlling Cell-Cell Interactions

collagen) mediated the adhesion of the first-type cell. For example, collagenmicropatterns mediated the selective adhesion of hepatocytes compared to thenoncoated regions. Finally, secondary cells were seeded and adhered to the unmodi-fied region of the substrate by nonspecific, serum-mediated attachment. Using thistechnique, homotypic and heterotypic cell-cell interactions of rat hepatocytes andfibroblasts in maintaining liver functions were examined to yield important biologi-cal insight into the role of cell-cell contact [Figure 4.1(b)].

This micropatterning technique can efficiently localize cells within patternedcocultures. Furthermore, this approach can be easily implemented since it requiresonly access to existing techniques, including photolithography, soft lithography,and microcontact printing [58–67]. However, despite these advantages, thetechnique is limited by several issues [20]. First, this technique depends on therelative cell-cell and cell-substrate adhesiveness of each cell type. For example,first seeded cells must weakly adhere to the unmodified region but strongly attachto the patterned region. Also, secondary cells should adhere to the unmodifiedregion of the substrate rather than the primary cells. Furthermore, the seedingorder of cell, as well as the choice of the matrix materials, is limited using thesecultures.

4.3 Patterned Coculture Systems 55

(a)

(b)

Patterned cell adhesivetwo-molecules

Seedcell A

Seedcell B

Cell A Cell B

Removeunattached cell A

Figure 4.1 Micropatterning using selective adhesion of cells to patterned substrate: (a) A sche-matic diagram of the procedure to generate micropattern cocultures. Cell-adhesive biomoleculeswere patterned by typical photolithography process. Then, type A cells were spread and attachedto the wafer, and unattached cells were washed out. Finally, type B cell were attached to anunmodified region of the wafer for coculturing. (b) A micrograph of cocultures of hepatocytes(200 µm lanes of collagen-derivatized glass) with fibroblasts (500 µm glass lanes). The photographwas taken forty-eight hours after hepatocyte seeding and twenty-four hours after fibroblast seed-ing. (Source: [18], reprinted with permission from John Wiley and Sons.)

4.3.2 Microfluidic Patterning for Cocultures

Fluid flow in microfluidic channels can also be used for patterning the substratewith cells and biomolecules [29–31]. In this approach microfluidic channels can beused to deliver cells and soluble factors to specific regions of a substrate. Forexample, Takayama et al. [30] presented the patterning of two different cell typesby using the multiple flow streams in capillary channels fabricated withpoly(dimethylsiloxane) (PDMS). Within these microchannels, the fluid flow is inthe low Reynolds number regime; thus, two or more laminar flow streams flowparallel to each other due to low convective mixing [25, 68–71]. This feature ofmultiple laminar flows enables the spatial patterning of cells and theirmicroenvironments within microchannels. In this process, the width of each cellpattern can easily be controlled by adjusting the relative volumetric flow rate of cellsuspensions and sheath flow.

This patterning technique has distinctive advantages for versatile applications,which include a straightforward fabrication process and the ability to generatepatterns inside microfluidic channels [30]. However, this method patterns cellsonly on parallel stripes, not on the various shapes that can be achieved by otherpatterning methods. Moreover, this method can be applied only to a few metaboli-cally slow cell types because the delivery of oxygen and nutrient is arrested, whilethe energy-consuming processes of cell anchorage and spreading occur [23, 43].

An alternate approach is to patterning multiple cell types within individualreversibly sealed microchannels can be used to localize cells to specific regions on asubstrate. In this approach, an array channel containing different cell types can beused to localize the cells in specific regions [26, 72]. For example, Khademhosseini etal. [26] presented multiphenotype cell patterning within an array of reversibly sealedmicrofluidic channels. Microfluidic channels deliver various fluids or cells onto spe-cific locations on a substrate, and microwells on the substrate capture and immobi-lize cells within low shear stress regions. Both of the microfluidic channels andmicrowells were fabricated by the soft lithography of PDMS polymer. Thisapproach utilizes three distinct concepts: (1) the capture of cells inside themicrowells that contain low shear stress regions, (2) reversible sealing between thetop layer of microchannels and microwell-patterned substrate; and (3) alternativeorthogonal placing of microchannel arrays to deliver a unique set of fluids or celltypes (see Figure 4.2).

This approach can reduce the time for cell attachment on the substrate andallow the top layer of microchannel to be realigned and moved without disturbingthe cells because cell are docked inside microwells. This approach also enablescoculture of multiple cell types by sequences of fluid flow of different cell types andhas potential applications in high-throughput screening of drug and optimization ofcell-soluble signal interactions for biological research.

4.3.3 Stencil-Based Patterning

In this approach, elastomeric stencil can be used to pattern cells to specific regions ofa substrate [17, 21–23]. Elastomeric stencils with microengineered holes can bereversibly sealed on a substrate to promote patterned deposition of biomolecules orcells directly on a substrate.

56 Patterned Cocultures for Controlling Cell-Cell Interactions

For example, Ostuni et al. [21] presented a stencil-based lift-off approach togenerate patterned cell arrays [73, 74]. In this process, PDMS polymer was spin-coated onto a microstructured silicon wafer containing photoresist posts togenerate a PDMS membrane. Subsequently, the cured membrane was removedfrom the master and brought into conformal contact with a substrate. To generatecell patterns, cell-adhesion-promoting molecules (fibronectin or gelatin) wereabsorbed into the patterned holes in the membranes, and then membrane wasremoved from the substrate to generate a pattern of adhesion-promoting molecules.After coating the rest of the substrate with cell-repulsive molecules (bovine serumalbumin) to inhibit cell adhesion, cells were only patterned on the adhesiveregions. In an alternative approach, adhesive cells such as fibroblasts can be directly

4.3 Patterned Coculture Systems 57

(a)

(b)

Microwell patternedsubstrate

Seal with primary arrayof microchannelson microwells

Flow each cell type inthe microwells

Each cell type docksin the microwells

Orthogonally attach thesecondary array ofmicrochannels

Multiphenotype cellsare placed in eachmicrochannel

Remove themicrofluidic mold

Figure 4.2 Micropatterned cell patterning using microfluidic channels: (a) A schematic ofmultiphenotype cell patterning by utilizing reversible sealing of microfluidic channel arrays ontomicrowell-patterned substrate. Each cell type flows through an independent microchannel anddocks inside microwells. The PDMS microfluidic mold can be removed and replaced with anothermold, which is placed orthogonally to generate multiphenotype cell arrays inside eachmicrochannel. (b) A fluorescent image of patterned AML12 (red) and NIH-3T3 (green) cells insidemicrowells. (Source: [26], reprinted with permission from the Royal Society of Chemistry.)

patterned by adhering to the substrate through the holes in the membranes.Patterning of multiple cell types can also be accomplished by sequentially patterningcell-adhesion promoters and cells using this technique (see Figure 4.3).

Folch et al. [22, 23] presented two techniques for the fabrication of elastomericmembranes for cell patterning. First, elastomeric membranes were fabricated byinjecting or suctioning uncured PDMS prepolymer into the microchamber, whichwas composed with photoresist posts on a wafer and a thin adhesive film as a toproof. After the PDMS prepolymer was cured, the adhesive film was carefullyremoved, and the PDMS membrane was peeled off from the wafer. Then, the PDMSmembrane was sealed onto the substrate for cell culturing. Second, the elastomericmembrane was fabricated by using a compression-molding process. PDMSprepolymer was poured on a photoresist mold, and pressure was uniformly appliedby plate stack. After the PDMS curing was complete, the next steps were performedlikewise. Cell patterning and cocultures of multiple cell types were performedfollowing similar procedures to those previously discussed.

58 Patterned Cocultures for Controlling Cell-Cell Interactions

(a)

(b)

Elastomeric membrane Fibronectin (FN) Cell A

Cell D

Seed cell AExpose to FN solution

Seed cell BPeel off membrane andexpose to FN solution

Figure 4.3 Micropatterned cell cocultures using elastomeric membrane: (a) A schematic ofgeneric procedure of patterned cocultures using elastomeric membrane. At first,cell-adhesion-promoting molecules (fibronectin, or FN) adhered to the substrate through pat-terned holes in the membrane, and then type A cells adhered to FN. Subsequently, the membranewas removed from the substrate to generate a pattern, and the substrate was exposed to FN solu-tion again for seeding second-type cells. Finally, type B cells were seeded on the substrate and cellswere only attached to the unpatterned area. (b) A fluorescent image of cocultured mES cells (red)with AML12 cells (green) using parylene membrane. (Source: [32].)

These patterning methods using PDMS membranes are applicable to a broadrange of substrates that can absorb adhesion-promoting proteins and can makeconformal contact with PDMS membranes [21]. PDMS membranes also can be rep-licated many times from the same master mold because the replication proceduredoes not break the mold [23]. Furthermore, three-dimensional structures formed byPDMS could make it possible to generate a three-dimensional cell-culturemicroenvironment [21, 72]. However, the attachment of PDMS membranes overthe substrates and the peeling off of membranes after cell seeding can cause troublein large-scale applications [43].

Recently, microfabricated parylene membranes, which have been used formicropatterning methods to pattern cells, proteins, and antibodies [33, 75, 76],were used for static and dynamic cocultures of multiple cell types, which can manip-ulate the spatial and temporal cell-cell interactions in tissue culture by changing thecell adhesiveness to parylene membrane surfaces [32]. In this coculture system, thetop surface of the parylene membrane was pretreated with hyaluronic acid (HA) tolessen nonspecific cell adhesion and then placed onto a PDMS substrate. First-typecells were seeded and only adhered to the substrate through the holes in the mem-brane. Collagen was then deposited on the parylene membrane to change the sur-face properties of parylene to cell adhesive. Subsequently, second-type cells wereseeded on the membrane to form a patterned coculture. To seed the third-type cells,second-type cells were removed by peeling off the parylene membrane from thesubstrate. This method enables the spatial and temporal regulation of multiple cellcultures for studying the complexity of cell-cell interactions in in vitro cell cultures.

Parylene membranes have several advantages over PDMS membranes. Parylenemembranes can be easily removed or attached to a surface without tearing due totheir mechanical robustness compared to PDMS [77, 78] and can form a reversiblebinding with hydrophobic surfaces. Thus, parylene membranes could be used formultiple patterning processes. Moreover, more cells adhere to parylene comparedto PDMS [32]. However, the parylene membrane fabrication procedure requiresmany steps and much special equipment compared to the PDMS membrane fabrica-tion procedure [33].

4.3.4 Micropatterning Using Surfaces Switchable from Cell Repulsive toAdhesive

This approach is based on using the surfaces that can be turned from beingcell repulsive to adhesive by specific stimuli: electrical potential, temperature, andlight exposure. Recently, layer-by-layer assembly of biopolymers [79–82] has alsobeen employed for cell patterning by switching the cell adhesion of the substrate[24, 67, 83].

Yousaf et al. [35] developed an electroactive mask that enables two differentcell types to be patterned on a single substrate. This mask was fabricated with aself-assembled monolayer of alkanethiolates on gold, which can be switched fromcell adhesive to repulsive.

Figure 4.4(a) shows the schematic strategy of patterning two different celltypes using switchable monolayers. At first, the monolayer was patterned bymicrocontact printing [56, 63, 84], and extracellular matrix (ECM) proteins

4.3 Patterned Coculture Systems 59

(fibronectin) were attached to the regions where no monolayer was patterned. Thisfibronectin pattern resulted in the attachment of first-type cells to the fibro-nectin-coated region. Then, external stimulus (electric potential) was applied to theentire substrate, and the monolayer was changed to cell adhesive. Finally, second-type cells were attached to this activated layer. This technique does not requireextensive or invasive manipulation of the substrate, so this technique is suited forcell-culturing systems where physical manipulations are not applicable [35]. Thistechnique can control the receptor-ligand interactions between cell and substrates,as well as the properties of substrate [85–87].

Yamato et al. [42, 43, 88] presented a coculture technique utilizing a thermo-responsive polymer whose cell-polymer adhesion is changed by temperature. A

60 Patterned Cocultures for Controlling Cell-Cell Interactions

(a)

(b)

Switchable polymer monolayer, inert state Cell A

Extracellular matrix proteins

Depositcell A

Depositcell B

Activation byexternal stimulti

Activated monolayerCell B

Figure 4.4 Micropatterned cell cocultures using switchable substrates via external stimuli: (a) Ageneric procedure for patterning two different cell types using switchable substrates. At first, themonolayer was patterned, and fibronectins were attached to the regions where no monolayer waspatterned. This fibronectin pattern resulted in the attachment of first-type cells to thefibronectin-coated region. Then, external stimulus (electrical, thermal, or ultraviolet light) wasapplied to the entire substrate, and the monolayer was changed to cell adhesive. Finally, sec-ond-type cells were attached to this activated layer. (b) A micrograph of cocultured ES cells (brightarea) with fibroblasts (dark area) using layer-by-layer deposition. (Source: [24], reprinted with per-mission from Elsevier.)

thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) was covalently graftedas a thin layer onto tissue culture grade polystyrene (TCPS) dishes by electron beamradiation through a pattering mask [38–41, 88, 89]. Above the lower critical solu-tion temperature (LCST, 32°C) of PIPAAm polymer, PIPAAm is dehydrated andcell adhesive. Under its LCST of 32°C, PIPAAm polymer is hydrated, and cellattachment is highly suppressed [40, 90]. The cell-patterning procedure usingthermoresponsive PIPAAm polymer is quite simple and versatile. The first cell typewas seeded over the PIPAAm-patterned TCPS dishes at 20°C and attached only tothe non-PIPPAm region of TCPS dishes. Then, the second cell type was seededabove LCST and only attached to the PIPAAm-patterned region. This patterningtechnique possibly can be utilized for coculturing three or more cell types by varyingthe LCST of the PIPAAm polymer, both over successive temperature regimes andspatially across the culture surface, simultaneously using sequential masks andcopolymerization. PIPAAm copolymerized with other monomers permits wideaccess to copolymer-grafted culture surfaces with variable hydration temperatures[91, 92]. Recently, Edahiro et. al. [45] improved this method with thermo- andphotoresponsive surfaces. Culture surfaces were coated with a polymer materialcomposed of PIPAAm having spiropyran chromophores as side chains. These sur-faces can be switched from being cell repulsive to adhesive by being exposed to dif-ferent wave lengths of light, as well as by temperature changing. Since lightexposure can be applied to the small area of culture surfaces, this approach canimprove the regional control of cell adhesion by reducing the size of patterns com-pared to the previous thermoresponsive method.

Khademhosseini et al. [24, 67] developed a method for patterning cellcocultures using layer-by-layer deposition of ionic biomolecules. Hyaluronic acid(HA), which is a biocompatible and biodegradable material [93, 94], was patternedon glass slides by capillary force lithography [95, 96], followed by fibronectinadsorption onto the non-HA-patterned region. Then, the first-type cells were seededand only attached to the fibronectin-coated region. Subsequent ionic adsorption ofpoly-L-lysine (PLL) to the HA pattern was used to change HA surfaces from cellrepulsive to cell adhesive. Finally, the second-type cells were seeded and attached tothe PLL pattern. Coculturing of ES cells with fibroblasts and hepatocytes withfibroblasts was successfully performed by utilizing this method [Figure 4.4(b)] [24].This coculturing method utilizes the switchable but does not require electroactive orthermoresponsive surfaces that may be difficult to fabricate. Both HA and PLL arecommercially available and do not require any chemistry or complex techniques forimmobilization.

4.3 Other Approaches

Recently, various micropatterned coculture approaches not in one of fourcategories have been developed to better mimic the organization and complexity ofin vivo microenvironments using microfabrication technique. Although the pat-terned coculture methods discussed above provide the spatial control of cellsand the manipulation of homotypic/heterotypic cell-cell interactions in culture,they do not replicate dynamic and three-dimensional aspects of the in vivoenvironment.

4.3 Patterned Coculture Systems 61

The dynamics of cell-cell interactions as regulated by embryonic morphogenesisand mechanical factors are a key regulator of cell fate decisions. Khademhosseini et.al. [32] presented the dynamic coculture system using microfabricated parylenemembranes as discussed previously (see Section 4.3.4) This system enables thecocultures of multiple cell types, which can manipulate the spatial and temporalcell-cell interactions in tissue cultures by changing the cell adhesiveness to parylenemembrane surfaces. Hui and Bhatia [97] have demonstrated the dynamic cocultureof various cell types by using a microfabricated interdigitating system. This systemutilized a silicon platform to bring cells in close proximity to each other in a dynamicmanner and manipulate the complexity of cell-cell interactions in a spatially andtemporally regulated way.

A three-dimensional structure containing various cell types is required to recon-struct an organ system to function as it does in vivo. Ito et. al. [98] utilized magneticforce to precisely place magnetically labeled cells onto target cells and promoteheterotypic cell-cell adhesion to form a three-dimensional structure. Magnetitecationic liposomes carrying a positive surface charge accumulated in endothelialcells to improve adsorption. Subsequently, endothelial cells specifically accumulatedonto hepatocyte monolayers at sites where a magnet was positioned, then adheredto form a heterotypic layered construct with tight and close contact. Tsang et. al.[99] fabricated a three-dimensional hepatic tissue construct embeddinghepatocytes in poly(ethylene glycol) (PEG) hydrogel structures using a multilayerphoto- patterning platform [Figure 4.5(a)]. They perfused these three-dimensionaltissue structures in a continuous flow bioreactor and demonstrated improvedviability and liver-specific function of the encapsulated hepatocytes over unpat-terned controls. Albrecht et. al. [100] also presented a method for the rapidformation of three-dimensional cellular structures within a photopolymerizablePEG hydrogel using dielectrophoretic forces. In this method, cells weremicropatterned via dielectrophoretic forces, and each single hydrogel layer wasincorporated into multilayer constructs for cocultures. Mikos et. al. [101] suggestedlight-based process for creating bilayered hydrogel structures. At first, one layer ofpartially gelled liquid polymer solution is placed in the mold, and then a secondlayer of liquid polymer solution is added to the first layer. The whole construct isthen fully crosslinked by light to form a bilayered hydrogel. Different cell types canbe embedded in each layer for cocultures. This method also can be extended tocreate a multilayered hydrogel construct. Yeh et al. [102] also presented ahydrogel-based cell-coculturing method in three-dimensional constructions.

Figure 4.5(b) shows a fluorescence image of hydrogel arrangement and assem-bly. Cells were suspended in a HA hydrogel prepolymer solution and molded using aPDMS stamp. Hydrogels were then formed via exposing the prepolymer to ultravio-let light. Two different cell types encapsulated in separate HA hydrogels were subse-quently arranged in a checkerboard pattern for cocultures.

These coculture systems have been developed to better mimic the organizationand complexity of in vivo tissue structure in dynamic and three-dimensional aspects.However, these approaches are still under active investigation in the area of materi-als, chemistry, and pattering techniques.

62 Patterned Cocultures for Controlling Cell-Cell Interactions

4.4 Conclusion

Understanding cell-cell interactions is important for tissue engineering, which stud-ies in vitro reconstructing of tissue architecture for tissue replacement. The

4.4 Conclusion 63

(a)

Glass slide

Mask

Methacyrlated glass

Silicone spacer

Teflon base

Patterning process

Apparatus assembly

Invert

Perfuse

Prepolymer/cells

Additive photopatterning

Prepolymer/cells

Figure 4.5 (a) Schematic procedure for fabricating three-dimensional living tissues by additivephotopatterning of cellular hydrogels. The chamber was filled with cell-laden prepolymer solution,and exposure to light through a photomask causes photocrosslinking in pattern. The height of thechamber is then increased, and the process repeated, resulting in a multilayer cell-ladenthree-dimensional tissue construct with microscale features. (Source: [99], reprinted with permis-sion from The Federation of American Societies for Experimental Biology.) (b) A fluorescenceimage of hydrogel arrangement and assembly. Two different cell types (red and green) encapsu-lated in separate HA hydrogels were arranged in a checkerboard pattern for cocultures. (Source:[102], reprinted with permission from Elsevier.)

coculturing of two or more cell types has been used to establish a more biomimeticenvironment. In particular, micropatterning coculture methods have been devel-oped and challenged to enhance coculture environment control through spatiallocalization of multiple cells. Each method has its own advantages, as well as limita-tions due to the lack of suitable materials and the complex fabrication procedure, asdiscussed. Micropatterning methods have usually been performed on static andtwo-dimensional culture systems; however, cells in tissue and natural organs are indynamic and three-dimensional environments. Recently, some methods have beendeveloped to fabricate dynamic coculture systems using specific patterning methodand three-dimensional coculture constructions using hydrogel, but these systems arestill under investigation in the area of biomaterials and patterning techniques.

References

[1] Michalopoulos, G. K., and DeFrances, M. C., “Liver regeneration,” Science, Vol. 276, No.5309, 1997, pp. 60–66.

[2] Slavkin, H. C., Snead, M. L., Zeichnerdavid, M., Jaskoll, T. F., and Smith, B. T., “Conceptsof epithelial-mesenchymal interactions during development—tooth and lungorganogenesis,” J. Cell. Biochem., Vol. 26, No. 2, 1984, pp. 117–125.

[3] Davies, P. F., “Vascular Cell-Interactions with Special Reference to the Pathogenesis ofAtherosclerosis,” Laboratory Investigation, Vol. 55, No. 1, 1986, pp. 5–24.

64 Patterned Cocultures for Controlling Cell-Cell Interactions

(b)

Figure 4.5 (b)

[4] Aufderheide, E., Chiquetehrismann, R., and Ekblom, P., “Epithelial mesenchymal interac-tions in the developing kidney lead to expression of tenascin in the mesenchyme,” J. Cell.Biol., Vol. 105, No. 1, 1987, pp. 599–608.

[5] Simonassmann, P., Bouziges, F., Arnold, C., Haffen, K., and Kedinger, M., “Epithelialmesenchymal interactions in the production of basement-membrane components in thegut,” Development, Vol. 102, No. 2, 1988, pp. 339–347.

[6] Brown, L. F., Yeo, K. T., Berse, B., Yeo, T. K., Senger, D. R., Dvorak, H. F., andVandewater, L., “Expression of vascular-permeability factor (vascular endothelialgrowth-factor) by epidermal-keratinocytes during wound-healing,” J. Exp. Med., Vol.176, No. 5, 1992, pp. 1375–1379.

[7] Nishida, M., Springhorn, J. P., Kelly, R. A., and Smith, T. W., “Cell-cell signaling betweenadult-rat ventricular myocytes and cardiac microvascular endothelial-cells in heterotypicprimary culture,” J. Clin. Invest., Vol. 91, No. 5, 1993, pp. 1934–1941.

[8] Grinnell, A. D., “Dynamics of nerve-muscle interaction in developing and matureneuromuscular-junctions,” Physiological Reviews, Vol. 75, No. 4, 1995, pp. 789–834.

[9] Guguenguillouzo, C., Clement, B., Baffet, G., Beaumont, C., Morelchany, E., Glaise, D.,and Guillouzo, A., “Maintenance and reversibility of active albumin secretion by adult-rathepatocytes co-cultured with another liver epithelial-cell type,” Exp. Cell Res., Vol. 143,No. 1, 1983, pp. 47–54.

[10] Guillouzo, A., Delers, F., Clement, B., Bernard, N., and Engler, R., “Long-term productionof acute-phase proteins by adult-rat hepatocytes co-cultured with another liver-cell type inserum-free medium,” Biochem. Biophys. Res. Comm., Vol. 120, No. 2, 1984, pp.311–317.

[11] Lebreton, J. P., Daveau, M., Hiron, M., Fontaine, M., Biou, D., Gilbert, D., andGuguenguillouzo, C., “Long-term biosynthesis of complement component C-3 andalpha-1 acid glycoprotein by adult-rat hepatocytes in a coculture system with an epithelialliver cell-type,” Biochem. J., Vol. 235, No. 2, 1986, pp. 421–427.

[12] Jongen, W. M. F., Sijtsma, S. R., Zwijsen, R. M. L., and Temmink, J. H. M., “Acocultivation system consisting of primary chick-embryo hepatocytes and V79chinese-hamster cells as a model for metabolic cooperation studies,” Carcinogenesis, Vol.8, No. 6, 1987, pp. 767–772.

[13] Conner, J., Valletcollom, I., Daveau, M., Delers, F., Hiron, M., Lebreton, J. P., andGuillouzo, A., “Acute-phase-response induction in rat hepatocytes cocultured withrat-liver epithelial-cells,” Biochem. J., Vol. 266, No. 3, 1990, pp. 683–688.

[14] Donato, M. T., Gomezlechon, M. J., and Castell, J. V., “Drug-metabolizing-enzymes in rathepatocytes cocultured with cell-lines,” In Vitro Cellular and Developmental Biology,Vol. 26, No. 11, 1990, pp. 1057–1062.

[15] Matsuo, R., Ukida, M., Nishikawa, Y., Omori, N., and Tsuji, T., “The role of Kupffer cellsin complement activation in D-galactosamine lipopolysaccharide-induced hepatic-injuryof rats,” Acta Medica Okayama, Vol. 46, No. 5, 1992, pp. 345–354.

[16] Rojkind, M., Novikoff, P. M., Greenwel, P., Rubin, J., Rojasvalencia, L., Decarvalho, A.C., Stockert, R., Spray, D., Hertzberg, E. L., and Wolkoff, A. W., “Characterization andfunctional-studies on rat-liver fat-storing cell-line and freshly isolated hepatocyte coculturesystem,” Am. J. Path., Vol. 146, No. 6, 1995, pp. 1508–1520.

[17] Folch, A., and Toner, M., “Microengineering of cellular interactions,” Ann. Rev. Biomed.Eng., Vol. 2, 2000, pp. 227–256.

[18] Bhatia, S. N., Yarmush, M. L., and Toner, M., “Controlling cell interactions bymicropatterning in co-cultures: Hepatocytes and 3T3 fibroblasts,” J. Biomed. Mater. Res.,Vol. 34, No. 2, 1997, pp. 189–199.

[19] Bhatia, S. N., Balis, U. J., Yarmush, M. L., and Toner, M., “Probing heterotypic cell inter-actions: Hepatocyte function in microfabricated co-cultures,” J. Biomaterials Sci-ence-Polymer Edition, Vol. 9, No. 11, 1998, pp. 1137–1160.

References 65

[20] Bhatia, S. N., Balis, U. J., Yarmush, M. L., and Toner, M., “Effect of cell-cell interactions inpreservation of cellular phenotype: Cocultivation of hepatocytes and nonparenchymalcells,” Faseb J., Vol. 13, No. 14, 1999, pp. 1883–1900.

[21] Ostuni, E., Kane, R., Chen, C. S., Ingber, D. E., and Whitesides, G. M., “Patterning mam-malian cells using elastomeric membranes,” Langmuir, Vol. 16, No. 20, 2000, pp.7811–7819.

[22] Folch, A., and Toner, M., “Cellular micropatterns on biocompatible materials,” Biotech.Progress, Vol. 14, No. 3, 1998, pp. 388–392.

[23] Folch, A., Jo, B. H., Hurtado, O., Beebe, D. J., and Toner, M., “Microfabricatedelastomeric stencils for micropatterning cell cultures,” J. Biomed. Mater. Res., Vol. 52, No.2, 2000, pp. 346–353.

[24] Khademhosseini, A., Suh, K. Y., Yang, J. M., Eng, G., Yeh, J., Levenberg, S., and Langer,R., “Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cellco-cultures,” Biomaterials, Vol. 25, No. 17, 2004, pp. 3583–3592.

[25] Khademhosseini, A., Langer, R., Borenstein, J., and Vacanti, J. P., “Microscale technolo-gies for tissue engineering and biology,” Proc. Natl. Acad. Sci. USA, Vol. 103, No. 8, 2006,pp. 2480–2487.

[26] Khademhosseini, A., Yeh, J., Eng, G., Karp, J., Kaji, H., Borenstein, J., Farokhzad, O. C.,and Langer, R., “Cell docking inside microwells within reversibly sealed microfluidic chan-nels for fabricating multiphenotype cell arrays,” Lab Chip, Vol. 5, No. 12, 2005, pp.1380–1386.

[27] Bhatia, S. N., Toner, M., Tompkins, R. G., and Yarmush, M. L., “Selective adhesion ofhepatocytes on patterned surfaces,” Annals NY Acad. Sci., Vol. 745, 1994, pp. 187–209.

[28] Bhatia, S. N., Balis, U. J., Yarmush, M. L., and Toner, M., “Microfabrication ofhepatocyte/fibroblast co-cultures: Role of homotypic cell interactions,” Biotech. Progress,Vol. 14, No. 3, 1998, pp. 378–387.

[29] Delamarche, E., Bernard, A., Schmid, H., Michel, B., and Biebuyck, H., “Patterned deliv-ery of immunoglobulins to surfaces using microfluidic networks,” Science, Vol. 276, No.5313, 1997, pp. 779–781.

[30] Takayama, S., McDonald, J. C., Ostuni, E., Liang, M. N., Kenis, P. J. A., Ismagilov, R. F.,and Whitesides, G. M., “Patterning cells and their environments using multiple laminarfluid flows in capillary networks,” Proc. Natl. Acad. Sci. USA, Vol. 96, No. 10, 1999, pp.5545–5548.

[31] Takayama, S., Ostuni, E., LeDuc, P., Naruse, K., Ingber, D. E., and Whitesides, G. M.,“Laminar flows—subcellular positioning of small molecules,” Nature, Vol. 411, No. 6841,2001, pp. 1016–1016.

[32] Wright, D., Rajalingam, B., Selvarasah, S., Dokmeci, M. R., and Khademhosseini, A.,“Generation of static and dynamic patterned co-cultures using microfabricated parylene-Cstencils,” Lab on a chip, Vol. 7, No. 10, 2007, pp 1272–1279.

[33] Wright, D., Rajalingam, B., Karp, J. M., Selvarasah, S., Ling, Y., Yeh, J., Langer, R.,Dokmeci, M. R., and Khademhosseini, A., “Reusuable, reversibly sealable parylene mem-branes for cell and protein patterning,” Journal of Biomedical Materials Research, Part A,2007, DOI: 10.1002/jbm.a.31281, in print.

[34] Mrksich, M., “A surface chemistry approach to studying cell adhesion,” Chem. Soc. Rev.,Vol. 29, No. 4, 2000, pp. 267–273.

[35] Yousaf, M. N., Houseman, B. T., and Mrksich, M., “Using electroactive substrates to pat-tern the attachment of two different cell populations,” Proc. Natl. Acad. Sci. USA, Vol. 98,No. 11, 2001, pp. 5992–5996.

[36] Jiang, X. Y., Ferrigno, R., Mrksich, M., and Whitesides, G. M., “Electrochemicaldesorption of self-assembled monolayers noninvasively releases patterned cells from geo-metrical confinements,” J. Am. Chem. Soc., Vol. 125, No. 9, 2003, pp. 2366–2367.

66 References

[37] Yeo, W. S., Yousaf, M. N., and Mrksich, M., “Dynamic interfaces between cells and sur-faces: Electroactive substrates that sequentially release and attach cells,” J. Am. Chem.Soc., Vol. 125, No. 49, 2003, pp. 14994–14995.

[38] Yamada, N., Okano, T., Sakai, H., Karikusa, F., Sawasaki, Y., and Sakurai, Y.,“Thermoresponsive polymeric surfaces—control of attachment and detachment of cul-tured-cells,” Makromolekulare Chemie—Rapid Communications, Vol. 11, No. 11, 1990,pp. 571–576.

[39] Okano, T., Yamada, N., Sakai, H., and Sakurai, Y., “A novel recovery-system for cul-tured-cells using plasma-treated polystyrene dishes grafted withpoly(N-isopropylacrylamide),” J. Biomed. Mater. Res., Vol. 27, No. 10, 1993, pp.1243–1251.

[40] Yamato, M., Okuhara, M., Karikusa, F., Kikuchi, A., Sakurai, Y., and Okano, T., “Signaltransduction and cytoskeletal reorganization are required for cell detachment from cellculture surfaces grafted with a temperature-responsive polymer,” J. Biomed. Mater. Res.,Vol. 44, No. 1, 1999, pp. 44–52.

[41] Yamato, M., Konno, C., Kushida, A., Hirose, M., Utsumi, M., Kikuchi, A., and Okano,T., “Release of adsorbed fibronectin from temperature-responsive culture surfacesrequires cellular activity,” Biomaterials, Vol. 21, No. 10, 2000, pp. 981–986.

[42] Hirose, M., Yamato, M., Kwon, O. H., Harimoto, M., Kushida, A., Shimizu, T., Kikuchi,A., and Okano, T., “Temperature-responsive surface for novel co-culture systems ofhepatocytes with endothelial cells: 2-D patterned and double layered co-cultures,” YonseiMed. J., Vol. 41, No. 6, 2000, pp. 803–813.

[43] Yamato, M., Konno, C., Utsumi, M., Kikuchi, A., and Okano, T., “Thermally responsivepolymer-grafted surfaces facilitate patterned cell seeding and co-culture,” Biomaterials,Vol. 23, No. 2, 2002, pp. 561–567.

[44] Tsuda, Y., Kikuchi, A., Yamato, M., Nakao, A., Sakurai, Y., Umezu, M., and Okano, T.,“The use of patterned dual thermoresponsive surfaces for the collective recovery asco-cultured cell sheets,” Biomaterials, Vol. 26, No. 14, 2005, pp. 1885–1893.

[45] Edahiro, J., Sumaru, K., Tada, Y., Ohi, K., Takagi, T., Kameda, M., Shinbo, T., Kanamori,T., and Yoshimi, Y., “In situ control of cell adhesion using photoresponsive culture sur-face,” Biomacromolecules, Vol. 6, No. 2, 2005, pp. 970–974.

[46] Shimaoka, S., Nakamura, T., and Ichihara, A., “Stimulation of growth of primary culturedadult-rat hepatocytes without growth-factors by coculture with nonparenchymalliver-cells,” Exp. Cell Res., Vol. 172, No. 1, 1987, pp. 228–242.

[47] Goulet, F., Normand, C., and Morin, O., “Cellular interactions promote tissue-specificfunction, biomatrix deposition and junctional communication of primary cul-tured-hepatocytes,” Hepatology, Vol. 8, No. 5, 1988, pp. 1010–1018.

[48] Lawrence, M. B., McIntire, L. V., and Eskin, S. G., “Effect of flow on polymorphonuclearleukocyte endothelial-cell adhesion,” Blood, Vol. 70, No. 5, 1987, pp. 1284–1290.

[49] Lawrence, M. B., Smith, C. W., Eskin, S. G., and McIntire, L. V., “Effect of venousshear-stress on Cd18-mediated neutrophil adhesion to cultured endothelium,” Blood, Vol.75, No. 1, 1990, pp. 227–237.

[50] Schrode, W., Mecke, D., and Gebhardt, R., “Induction of glutamine-synthetase inperiportal hepatocytes by cocultivation with a liver epithelial-cell line,” Eur. J. Cell. Biol.,Vol. 53, No. 1, 1990, pp. 35–41.

[51] Hammarback, J. A., McCarthy, J. B., Palm, S. L., Furcht, L. T., and Letourneau, P. C.,“Growth cone guidance by substrate-bound laminin pathways is correlated with neu-ron-to-pathway adhesivity,” Dev. Biol., Vol. 126, No. 1, 1988, pp. 29–39.

[52] Stenger, D. A., Georger, J. H., Dulcey, C. S., Hickman, J. J., Rudolph, A. S., Nielsen, T. B.,McCort, S. M., and Calvert, J. M., “Coplanar molecular assemblies of aminoalkylsilaneand perfluorinated alkylsilane—characterization and geometric definition of mamma-lian-cell adhesion and growth,” J. Am. Chem. Soc., Vol. 114, No. 22, 1992, pp.8435–8442.

References 67

[53] Lee, J.-S., Kaibara, M., Iwaki, M., Sasabe, H., Suzuki, Y., and Kusakabe, M., “Selectiveadhesion and proliferation of cells on ion-implanted polymer domains,” Biomaterials, Vol.14, No. 12, 1993, pp. 958–960.

[54] Soekarno, A., Lom, B., and Hockberger, P. E., “Pathfinding by neuroblastoma cells in cul-ture is directed by preferential adhesion to positively charged surfaces,” NeuroImage, Vol.1, No. 2, 1993, pp. 129–144.

[55] Singhvi, R., Stephanopoulos, G., and Wang, D. I. C., “Effects of substratum morphologyon cell physiology—review,” Biotechnol. Bioeng., Vol. 43, No. 8, 1994, pp. 764–771.

[56] Singhvi, R., Kumar, A., Lopez, G. P., Stephanopoulos, G. N., Wang, D. I. C., Whitesides,G. M., and Ingber, D. E., “Engineering cell-shape and function,” Science, Vol. 264, No.5159, 1994, pp. 696–698.

[57] Oakley, C., and Brunette, D. M., “Topographic compensation—guidance and directedlocomotion of fibroblasts on grooved micromachined substrata in the absence ofmicrotubules,” Cell Motility and the Cytoskeleton, Vol. 31, No. 1, 1995, pp. 45–58.

[58] Kumar, A., and Whitesides, G. M., “Features of gold having micrometer to centimeterdimensions can be formed through a combination of stamping with an elastomeric stampand an alkanethiol ink followed by chemical etching,” Appl. Phys. Lett., Vol. 63, No. 14,1993, pp. 2002–2004.

[59] Duffy, D. C., McDonald, J. C., Schueller, O. J. A., and Whitesides, G. M., “Rapidprototyping of microfluidic systems in poly(dimethylsiloxane),” Analytical Chemistry,Vol. 70, No. 23, 1998, pp. 4974–4984.

[60] Anderson, J. R., Chiu, D. T., Jackman, R. J., Cherniavskaya, O., McDonald, J. C., Wu, H.K., Whitesides, S. H., and Whitesides, G. M., “Fabrication of topologically complexthree-dimensional microfluidic systems in PDMS by rapid prototyping,” Analytical Chem-istry, Vol. 72, No. 14, 2000, pp. 3158–3164.

[61] McDonald, J. C., Duffy, D. C., Anderson, J. R., Chiu, D. T., Wu, H. K., Schueller, O. J. A.,and Whitesides, G. M., “Fabrication of microfluidic systems in poly(dimethylsiloxane),”Electrophoresis, Vol. 21, No. 1, 2000, pp. 27–40.

[62] McDonald, J. C., Metallo, S. J., and Whitesides, G. M., “Fabrication of a configurable, sin-gle-use microfluidic device,” Analytical Chemistry, Vol. 73, No. 23, 2001, pp. 5645–5650.

[63] Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X. Y., and Ingber, D. E., “Soft lithogra-phy in biology and biochemistry,” Ann. Rev. Biomed. Eng., Vol. 3, 2001, pp. 335–373.

[64] McDonald, J. C., Chabinyc, M. L., Metallo, S. J., Anderson, J. R., Stroock, A. D., andWhitesides, G. M., “Prototyping of microfluidic devices in poly(dimethylsiloxane) usingsolid-object printing,” Analytical Chemistry, Vol. 74, No. 7, 2002, pp. 1537–1545.

[65] Khademhosseini, A., Yeh, J., Jon, S., Eng, G., Suh, K. Y., Burdick, J. A., and Langer, R.,“Molded polyethylene glycol microstructures for capturing cells within microfluidic chan-nels,” Lab Chip, Vol. 4, No. 5, 2004, pp. 425–430.

[66] Khademhosseini, A., Suh, K. Y., Jon, S., Eng, G., Yeh, J., Chen, G. J., and Langer, R., “Asoft lithographic approach to fabricate patterned microfluidic channels,” Analytical Chem-istry, Vol. 76, No. 13, 2004, pp. 3675–3681.

[67] Fukuda, J., Khademhosseini, A., Yeh, J., Eng, G., Cheng, J. J., Farokhzad, O. C., andLanger, R., “Micropatterned cell co-cultures using layer-by-layer deposition ofextracellular matrix components,” Biomaterials, Vol. 27, No. 8, 2006, pp. 1479–1486.

[68] Brody, J. P., Yager, P., Goldstein, R. E., and Austin, R. H., “Biotechnology at low Reynoldsnumbers,” Biophys. J., Vol. 71, No. 6, 1996, pp. 3430–3441.

[69] Weigl, B. H., and Yager, P., “Microfluidics—microfluidic diffusion-based separation anddetection,” Science, Vol. 283, No. 5400, 1999, pp. 346–347.

[70] Kenis, P. J. A., Ismagilov, R. F., and Whitesides, G. M., “Microfabrication inside capillariesusing multiphase laminar flow patterning,” Science, Vol. 285, No. 5424, 1999, pp. 83–85.

[71] Takayama, S., Ostuni, E., Qian, X. P., McDonald, J. C., Jiang, X. Y., LeDuc, P., Wu, M.H., Ingber, D. E., and Whitesides, G. M., “Topographical micropatterning of

68 References

poly(dimethylsiloxane) using laminar flows of liquids in capillaries,” Adv. Mater., Vol. 13,No. 8, 2001, pp. 570–574.

[72] Chiu, D. T., Jeon, N. L., Huang, S., Kane, R. S., Wargo, C. J., Choi, I. S., Ingber, D. E., andWhitesides, G. M., “Patterned deposition of cells and proteins onto surfaces by usingthree-dimensional microfluidic systems,” Proc. Natl. Acad. Sci. USA, Vol. 97, No. 6,2000, pp. 2408–2413.

[73] Jackman, R. J., Duffy, D. C., Cherniavskaya, O., and Whitesides, G. M., “Usingelastomeric membranes as dry resists and for dry lift-off,” Langmuir, Vol. 15, No. 8, 1999,pp. 2973–2984.

[74] Duffy, D. C., Jackman, R. J., Vaeth, K. M., Jensen, K. F., and Whitesides, G. M., “Pat-terning electroluminescent materials with feature sizes as small as 5 mu m usingelastomeric membranes as masks for dry lift-off,” Adv. Mater., Vol. 11, No. 7, 1999, pp.546–552.

[75] Ilic, B., and Craighead, H. G., “Topographical patterning of chemically sensitive biologi-cal materials using a polymer-based dry lift-off,” Biomed. Microdevices, Vol. 2, No. 4,2000, pp. 317–322.

[76] Pal, R., Sung, K. E., and Burns, M. A., “Microstencils for the patterning of nontraditionalmaterials,” Langmuir, Vol. 22, No. 12, 2006, pp. 5392–5397.

[77] Noh, H. S., Moon, K. S., Cannon, A., Hesketh, P. J., and Wong, C. P., “Wafer bondingusing microwave heating of parylene intermediate layers,” J. Micromechanics andMicroengineering, Vol. 14, No. 4, 2004, pp. 625–631.

[78] Lotters, J. C., Olthuis, W., Veltink, P. H., and Bergveld, P., “The mechanical properties ofthe rubber elastic polymer polydimethylsiloxane for sensor applications,” J.Micromechanics and Microengineering, Vol. 7, No. 3, 1997, pp. 145–147.

[79] Decher, G., “Fuzzy nanoassemblies: Toward layered polymeric multicomposites,” Sci-ence, Vol. 277, No. 5330, 1997, pp. 1232–1237.

[80] Vazquez, E., Dewitt, D. M., Hammond, P. T., and Lynn, D. M., “Construction of hydro-lytically-degradable thin films via layer-by-layer deposition of degradablepolyelectrolytes,” J. Am. Chem. Soc., Vol. 124, No. 47, 2002, pp. 13992–13993.

[81] Thierry, B., Winnik, F. M., Merhi, Y., and Tabrizian, M., “Nanocoatings onto arteries vialayer-by-layer deposition: Toward the in vivo repair of damaged blood vessels,” J. Am.Chem. Soc., Vol. 125, No. 25, 2003, pp. 7494–7495.

[82] Kidambi, S., Chan, C., and Lee, I. S., “Selective depositions on polyelectrolyte multilayers:Self-assembled monolayers of m-dPEG acid as molecular template,” J. Am. Chem. Soc.,Vol. 126, No. 14, 2004, pp. 4697–4703.

[83] Kidambi, S., Lee, I., and Chan, C., “Controlling primary hepatocyte adhesion and spread-ing on protein-free polyelectrolyte multilayer films,” J. Am. Chem. Soc., Vol. 126, No. 50,2004, pp. 16286–16287.

[84] Mrksich, M., Dike, L. E., Tien, J., Ingber, D. E., and Whitesides, G. M., “Usingmicrocontact printing to pattern the attachment of mammalian cells to self-assembledmonolayers of alkanethiolates on transparent films of gold and silver,” Exp. Cell Res., Vol.235, No. 2, 1997, pp. 305–313.

[85] Roberts, C., Chen, C. S., Mrksich, M., Martichonok, V., Ingber, D. E., and Whitesides, G.M., “Using mixed self-assembled monolayers presenting RGD and (EG)(3)OH groups tocharacterize long-term attachment of bovine capillary endothelial cells to surfaces,” J. Am.Chem. Soc., Vol. 120, No. 26, 1998, pp. 6548–6555.

[86] Houseman, B. T., and Mrksich, M., “Efficient solid-phase synthesis of peptide-substitutedalkanethiols for the preparation of substrates that support the adhesion of cells,” J.Organic Chem., Vol. 63, No. 21, 1998, pp. 7552–7555.

[87] Houseman, B. T., and Mrksich, M., “The microenvironment of immobilized Arg-Gly-Asppeptides is an important determinant of cell adhesion,” Biomaterials, Vol. 22, No. 9, 2001,pp. 943–955.

References 69

[88] Yamato, M., Kwon, O. H., Hirose, M., Kikuchi, A., and Okano, T., “Novel patterned cellcoculture utilizing thermally responsive grafted polymer surfaces,” J. Biomed. Mater. Res.,Vol. 55, No. 1, 2001, pp. 137–140.

[89] Hirose, M., Kwon, O. H., Yamato, M., Kikuchi, A., and Okano, T., “Creation of designedshape cell sheets that are noninvasively harvested and moved onto another surface,”Biomacromolecules, Vol. 1, No. 3, 2000, pp. 377–381.

[90] Uchida, K., Sakai, K., Ito, E., Kwon, O. H., Kikuchi, A., Yamato, M., and Okano, T.,“Temperature-dependent modulation of blood platelet movement and morphology onpoly(N-isopropylacrylamide)-grafted surfaces,” Biomaterials, Vol. 21, No. 9, 2000, pp.923–929.

[91] Feil, H., Bae, Y. H., Jan, F. J., and Kim, S. W., “Effect of comonomer hydrophilicity andionization on the lower critical solution temperature of N-isopropylacrylamide copoly-mers,” Macromolecules, Vol. 26, No. 10, 1993, pp. 2496–2500.

[92] Takei, Y. G., Aoki, T., Sanui, K., Ogata, N., Okano, T., and Sakurai, Y., “Tempera-ture-responsive bioconjugates .2. molecular design for temperature-modulatedbioseparations,” Bioconjugate Chem., Vol. 4, No. 5, 1993, pp. 341–346.

[93] Vercruysse, K. P., and Prestwich, G. D., “Hyaluronate derivatives in drug delivery,” Criti-cal Reviews in Therapeutic Drug Carrier Systems, Vol. 15, No. 5, 1998, pp. 513–555.

[94] Luo, Y., Kirker, K. R., and Prestwich, G. D., “Cross-linked hyaluronic acid hydrogel films:New biomaterials for drug delivery,” J. Controlled Release, Vol. 69, No. 1, 2000, pp.169–184.

[95] Suh, K. Y., Seong, J., Khademhosseini, A., Laibinis, P. E., and Langer, R., “A simple softlithographic route to fabrication of poly(ethylene glycol) microstructures for protein andcell patterning,” Biomaterials, Vol. 25, No. 3, 2004, pp. 557–563.

[96] Suh, K. Y., Khademhosseini, A., Yang, J. M., Eng, G., and Langer, R., “Soft lithographicpatterning of hyaluronic acid on hydrophilic substrates using molding and printing,” Adv.Mater., Vol. 16, No. 7, 2004, pp. 584–588.

[97] Hui, E. E., and Bhatia, S. N., “Micromechanical control of cell-cell interactions,” Proc.Natl. Acad. Sci. USA, Vol. 2007, pp. 0608660104.

[98] Ito, A., Takizawa, Y., Honda, H., Hata, K. I., Kagami, H., Ueda, M., and Kobayashi, T.,“Tissue engineering using magnetite nanoparticles and magnetic force: Heterotypic layersof cocultured hepatocytes and endothelial cells,” Tissue Eng., Vol. 10, Nos. 5–6, 2004, pp.833–840.

[99] Tsang, V. L., Chen, A. A., Cho, L. M., Jadin, K. D., Sah, R. L., DeLong, S., West, J. L., andBhatia, S. N., “Fabrication of 3-D hepatic tissues by additive photopatterning of cellularhydrogels,” Faseb J., Vol. 21, No. 3, 2007, pp. 790–801.

[100] Albrecht, D. R., Underhill, G. H., Wassermann, T. B., Sah, R. L., and Bhatia, S. N.,“Probing the role of multicellular organization in three-dimensional microenvironments,”Nature Methods, Vol. 3, No. 5, 2006, pp. 369–375.

[101] Mikos, A. G., Herring, S. W., Ochareon, P., Elisseeff, J., Lu, H. H., Kandel, R., Schoen, F.J., Toner, M., Mooney, D., Atala, A., Dyke, M. E. V., Kaplan, D., and Vunjak-Novakovic,G., “Engineering complex tissues,” Tissue Eng., Vol. 12, No. 12, 2006, pp. 3307–3339.

[102] Yeh, J., Ling, Y. B., Karp, J. M., Gantz, J., Chandawarkar, A., Eng, G., Blumling, J.,Langer, R., and Khademhosseini, A., “Micromolding of shape-controlled, harvestablecell-laden hydrogels,” Biomaterials, Vol. 27, No. 31, 2006, pp. 5391–5398.

70 References