Engineering 3d cellular constructs using inter cellular polymeric linker

ENGINEERING 3D CELLULAR CONSTRUCTS USING INTER-CELLULAR POLYMERIC LINKER ONG SIEW MIN B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences & Engineering National University of Singapore 2008 ACKNOWLEDGEMENTS This PhD odyssey was completed with the help of many wonderful people around me. First and foremost, my family members, who gave enormous support although they had no idea what I was doing. Equally noble is my fiancé, Randall, who showed me utmost understanding and encouragement despite groping in the PhD tunnel himself. I am grateful to my supervisor, A/P Hanry Yu, for his mentorship and guidance; members of my thesis advisory committee, Dr Tan Choon Hong and Dr Wang Nai-dy, for their advice and encouragement. Special thanks to comrades Ms He Lijuan, Mr Zhao Deqiang, Ms Nguyen Thi Thuy Linh and Ms Tee Yee Han who started linking cells with me in the early days, and seniors (now Drs) Toh Yi-Chin, Susanne Ng and Khong Yuet Mei, who gave invaluable scientific suggestions and moral support. I am thankful to great friends Ms Zhang Shufang, Ms Zhang Chi and Mr Xia Lei, who drove away the occasional gloomy clouds simply with their very presence. It has been great fun to work with Dr Danny van Noort and Mr Talha Arooz, who had no lack of crazy scientific (or non-scientific) ideas to cheer me up. My thanks to everyone else who was ever part of our lab for the last years and had contributed in other ways. Finally, I would like to thank A*STAR, BMRC, IBN and NGS, NUS for the financial and administrative support. ii CONTENTS LIST OF PUBLICATIONS .iv SUMMARY v LIST OF TABLES vi LIST OF FIGURES .vii LIST OF SYMBOLS AND ABBREVIATIONS xiv 1. INTRODUCTION . 2. BACKGROUND AND SIGNIFICANCE 2.1 3D cell culture .3 2.1.1 Importance of 3D cell culture 2.1.2 Strategies for 3D cell culture . 2.2 Micro-scale cell culture 14 2.2.1 Advantages of micro-scale cell culture . 14 2.2.2 Microtechnology for engineering micro-scale cell culture systems 14 2.2.3 Microfluidics as an attractive micro-scale cell culture system . 15 2.3 3D micro-scale cell culture 22 2.3.1 3D micro-well cell culture systems . 23 2.3.2 3D microfluidic cell culture systems . 23 2.4 Limitations of current 3D cell culture strategies 25 3. OBJECTIVES AND SPECIFIC AIMS 26 3.1 Specific Aim 1: To evaluate the inter-cellular polymeric linker for engineering a 3D cell-dense culture model 26 3.2 Specific Aim 2: To assess linker-engineered aggregates for features of a mature 3D culture .29 3.3 Specific Aim 3: To engineer reproducible 3D cell-dense microconstructs using a combination of inter-cellular linker and microfabricated structures 31 4. MATERIALS AND METHODS . 34 4.1 Evaluation of inter-cellular polymeric linker for engineering a 3D celldense culture model .34 4.1.1 Synthesis and characterisation of inter-cellular linker 34 4.1.2 Forming cellular aggregates using inter-cellular linker 37 4.1.3 Morphological observation of cells in cellular aggregates . 38 i 4.2 Biological characterisation of linker-engineered aggregates 42 4.2.1 Structural characterisation of linker-engineered aggregates and naturally-formed spheroids . 42 4.2.2 Functional assessment of linker-engineered aggregates and naturally-formed spheroids . 46 4.2.3 Drug response of cells in cellular aggregates . 47 4.3 Engineering 3D cell-dense micro-constructs in a microfluidic system 49 4.3.1 Design, fabrication and operation of the microfluidic system . 49 4.3.2 Characterisation of cell-dense micro-constructs . 50 4.3.3 Functional assessment of cell-dense micro-constructs . 52 4.3.4 Drug penetration studies on cell-dense micro-constructs . 53 4.4 Cell Culture .55 5. RESULTS . 56 5.1 Evaluation of inter-cellular polymeric linker for engineering a 3D celldense culture model .56 5.1.1 Synthesis and characterisation of inter-cellular linker 56 5.1.2 Forming cellular aggregates using inter-cellular linker 58 5.1.3 Culture of cellular aggregates 63 5.2 Biological characterisation of linker-engineered aggregates 71 5.2.1 Structural characterisation of linker-engineered aggregates 71 5.2.2 Functional assessment of linker-engineered aggregates . 83 5.2.3 Drug response of mature linker-engineered aggregates . 88 5.3 Engineering 3D cell-dense micro-constructs in a microfluidic system 92 5.3.1 Design and operation of the microfluidic system . 92 5.3.2 Perfusion culture of 3D cell-dense micro-constructs . 96 5.3.3 Functional assessment of cell-dense micro-constructs .101 5.3.4 Utilisation of the 3D microfluidic cell culture system for drug penetration studies .104 6. DISCUSSIONS 107 6.1 Evaluation of inter-cellular polymeric linker for engineering a 3D celldense culture model .107 6.1.1 Chemistry between inter-cellular linker and cell surface .107 6.1.2 Morphological changes of cellular aggregates during culture .109 6.1.3 Summary for Specific Aim 110 6.2 Biological characterisation of linker-engineered aggregates 112 6.2.1 Aggregate-to-spheroid transformation 112 6.2.2 Application of linker-engineered aggregates 115 6.2.3 Summary for Specific Aim 118 ii 6.3 Engineering 3D cell-dense micro-constructs in a microfluidic system 119 6.3.1 Establishing the 3D microfluidic cell culture system .119 6.3.2 Application of the 3D microfluidic cell culture system .121 6.3.3 Summary for Specific Aim 124 6.4 Inter-cellular polymeric linker as a novel biomaterial for engineering 3D cell-dense constructs 125 7. CONCLUSION 126 8. RECOMMENDATIONS FOR FUTURE RESEARCH 127 REFERENCES 131 APPENDIX A IMAGE ANALYSIS 140 APPENDIX B REVERSIBLE SEALING OF MICROFLUIDIC CHANNEL 141 iii LIST OF PUBLICATIONS 1. Ong SM, Zhang C, Toh YC, Kim SH, Foo HL, Tan CH, van Noort D, Park S, Yu H. A gel-free 3D microfluidic cell culture system. Biomaterials 2008; 29: 3237 – 3244. 2. Ong SM, He L, Thuy Linh NT, Tee YH, Arooz T, Tang G, Tan CH, Yu H. Transient inter-cellular polymeric linker. Biomaterials 2007; 28: 3656 – 3667. 3. Zhao D, Ong SM, Yue Z, Jiang Z, Toh YC, Majad K, Shi J, Tan CH, Chen JP, Yu H. Dendrimer hydrazides as multivalent transient inter-cellular linkers. Biomaterials 2008; 29: 3693 – 3702. 4. van Noort D, Ong SM, Zhang C, Zhang S, Arooz T, Yu H. Stem cells in microfluidics. Biotechnology Progress 2008; Invited review article, submitted. 5. Zhang S, Xia L, Kang CH, Xiao G, Ong SM, Toh YC, Leo HL, van Noort D, Kan SH, Yu H. Microfabricated silicon nitride membranes for hepatocyte sandwich culture. Biomaterials 2008; 29: 3993-4002. PATENT Ong SM, Yu H. Engineering 3D micro-scale cellular constructs with transient intercellular linker and micropillar array for maximal mass transport properties. United States (October 2007, 60/960,743), granted. CONFERENCES 1. Siew Min Ong, et al., “Transient Inter-cellular Polymeric Linker for Engineering 3D Cell-Dense Tissues,” presented at International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, January 5-6, 2008, Singapore (Oral) 2. Siew-Min Ong, et al., “Transient Inter-cellular Polymeric Linker for Cell-Dense 3D Culture,” presented at the CELLutions SUMMIT, August 20-23, 2007, Boston, MA (Poster) 3. Siew-Min Ong, et al., “Transient Inter-cellular Polymeric Linker for 3D Cell Culture,” presented at the SBE's 3rd International Conference on Bioengineering and Nanotechnology, August 12-15, 2007, Biopolis, Singapore (Poster) 4. Siew-Min Ong, et al., “Engineering 3-D Cellular Constructs using Inter-cellular Polymeric Linker,” presented at 7th Asian Symposium on Biomedical Materials, August 20-23, 2006, Korea (Oral) iv SUMMARY Three-dimensional cell cultures are indispensible as they mimic the cell-cell and cell-matrix interactions in vivo. To date, biomaterials like scaffolds and hydrogels have been used for engineering 3D cellular or tissue constructs. However, the introduction of such bulk biomaterials might not be suitable for engineering constructs of cell-dense and matrix-poor tissues of the internal organs. To engineer 3D cellular and tissue constructs with minimal biomaterials, we developed an intercellular polymeric linker to facilitate cell-cell interaction and cellular aggregation. The cells in the cellular aggregates proliferated, grew compact and maintained 3D cell morphology while the inter-cellular linker disappeared from the cell surfaces in days. Structural characterisation and functional assessment showed that the cellular aggregates remodeled during culture to exhibit features of a mature 3D spheroid with formation of adherens junctions, secretion of extra-cellular matrix, development of gradients of cell proliferation and oxygen concentration from the periphery to the centre, good cellular functions and resistance to drug penetration. We further incorporated the use of the inter-cellular linker into a 3D microfluidics system to take advantage of the micron-scale and fluidic properties for applications, and at the same time we created a novel method for seeding and culturing mammalian cells three-dimensionally in microfluidic systems without hydrogels. The 3D microfluidic cellular constructs exhibited higher cellular functions than 2D-cultured cells and demonstrated potential for real-time optical-based monitoring of cellular events with good spatial and temporal resolution. The inter-cellular linker hence allows the formation of functional 3D cell-dense constructs for tissue engineering applications. v LIST OF TABLES Table Summary of maturity of C3A linker-engineered aggregates determined via various structural and functional characterisation methods 87 Table Optimised operation parameters for seeding cells into the 3D-µFCCS. 95 vi LIST OF FIGURES Page Figure (A) Polarised light image of electrospun collagen nanofibres as fibrous scaffold.29 (B) SEM image of a tetraethoxysilane (TEOS) polydimethylsiloxane (PDMS) sponge-like scaffold.30 (C) SEM image of polycaprolactone (PCL) scaffold structure produced by fused deposition modeling (FDM).25 Images are reproduced with permission from publishers Figure (A) Schematic representation of principle of organ printing technology: placing of cell aggregates layer by layer in solidifying thermo-reversible gel with sequential cell aggregate fusion and morphing into 3D tube. (B) Ten aggregates (containing ~ 5000 cells) before fusion. (C) Final disc-like configuration after fusion.37 Images are reproduced with permission from publisher. Figure Tissue reconstruction by assembly of cell sheets. (A) By transplanting single cell sheets directly to host tissues, skin, cornea, periodontal ligament, and bladder can be reconstructed. (B) Using homotypic layering of cell sheets, 3D myocardial tissues can be created. (C) With heterotypic stratification of cell sheets, laminar structures such as those in the liver or kidney can be fabricated.12 Figure is reproduced with permission from publisher. 10 Figure A photograph of hanging-drops. Drops of cell suspensions were dispensed onto a tray and the tray inverted. .11 Figure Rotational culture methods to culture or form spheroids from single cell suspensions, adapted.47 (A) Roller-tube method. (B) Spinner flask method. (C) Gyratory shaker method. .12 Figure The induction of cell aggregation by cell surface engineering. Schematic representation of the aggregation concept, where biotinylated cells in suspension are cross-linked by addition of avidin.66 Image is reproduced with permission from publisher. 13 Figure Capability of microtechnology to exert spatial resolution for cell culture.Brightfield images of single hMSC plated onto micro-patterned small (left column) or large (right column) fibronectin islands.79 Scale bar = 50mm Bright-field images of hepatocytes (darker cells) and 3T3 fibroblasts co-cultured on micro-regions with high spatial resolution.81 Images are reproduced with permission from publishers. 15 Figure An overview of the possible uses for a multiplexed microfluidic cell culture system. Inputs can be used for control of the stem cell microenvironment and drug testing. The gradient generator creates a concentration profile of the input factors. The cell culture chamber is a perfusion system in which the cells are seeded. Analysis involves vii monitoring the output of the device, which can be imaged with the aid of biomarkers, analysed using inline sensors or standard lab equipment. 21 Figure Human microvascular endothelial (HMEC-1) cells immediately after seeding in the networks. Staining of CD31 (an endothelial-specific marker) in a branching microfluidic network to recreate a micro-vascular network.127 Images are reproduced with permission from publisher. 22 Figure 10 A high density cell suspension was placed on the microwell arrays and allowed to settle within the wells.133 Figure is reproduced with permission from publisher. .23 Figure 11 Encapsulation of cells in hydrogels in microfluidics using various strategies. (A) Schematic of the micro-patterning of agarose microfluidic devices with (right) and without (left) encapsulated cells to allow fluid perfusion in the centre (white region).131 (B) and (C): Low cell density of cellular constructs obtained using hydrodynamic focussing of cell-laden PuraMatrix hydrogel141 (B) and PEG hydrogel143 (C) in the centre to allow fluid perfusion on the sides. Figures are reproduced with permission from publishers .24 Figure 12 Schematic diagram of the synthesis of PEI-hy. (a) Reaction of PEI with 2iminothiolane results in conjugation of thiols onto the primary amines on PEI. (b) Reaction with EMCH conjugates hydrazides onto the thiols, yielding PEI-hy .57 Figure 13 Schematic diagram of the synthesis of neutral hydrazide. (c) Condensation of methyl malonate with methyl acrylate results in a neutral polymer with ester groups. (d) Reaction with hydrazine hydrate converts the ester groups to hydrazides, yielding neutral hydrazide 58 Figure 14 Cellular aggregation requires both chemically modified cells and positively-charged PEI-hy. a) Unmodified cells in PEI, and b) unmodified cells in PEI-hy are not aggregated. c) Modified cells in PEI form small aggregates. d) Modified cells in PEI-hy form large aggregates. e) Modified cells in neutral hydrazide are not aggregated. HepG2 cells were modified with 1.0 mM NaIO4 and suspended in 0.10 mM of PEI or PEIhy or neutral hydrazide. Scale bar: 60 mm .59 Figure 15 Biocompatibility test of PEI-hy. Percentage viability of HepG2 cells after incubation with PEI-hy at different concentrations .60 Figure 16 Biocompatibility test of NaIO4. Percentage viability of HepG2 cells after incubation with NaIO4 at different concentrations measured with the Trypan Blue exclusion assay and fluorescence viability staining. .62 Figure 17 Different cell types were successfully aggregated with 0.01 mM PEI-hy and 0.50 mM NaIO4, including (a) Primary rat bone marrow stem cells (b) primary porcine hepatocytes and c) HeLa and (d) U251 cell lines. Scale bar: 50 mm. .62 Figure 18 Proliferation of HepG2 cells in aggregates during a 7-day culture. (A) Confocal images of cellular aggregates at various time- points indicate viii 7. CONCLUSION This thesis has documented the development of a transient inter-cellular polymeric linker (PEI-hy) that can rapidly and effectively induce aggregation of live cells to effect cell-dense 3D culture using minimal biomaterials. The cells in the cellular aggregates can proliferate and preserve the 3D structural integrity independently from the inter-cellular linker. Structural characterisation and functional assessment showed that the cellular aggregates remodelled during culture to exhibit features of a mature 3D spheroid with formation of adherens junctions, secretion of extracellular matrix, development of gradients of cell proliferation and oxygen concentration from the periphery to the centre, good cellular functions and drug resistance. Cellular aggregates also displayed drug penetration resistance like mature spheroids. We further incorporated the use of the inter-cellular linker into a microfluidics system to take advantage of the micro-fabricated structures and fluidic properties for reproducibility of cellular construct and culture microenvironment which are crucial for applications. At the same time, the intercellular linker offers a novel gel-free method for seeding and culturing mammalian cells three-dimensionally in microfluidic systems, exhibiting numerous advantages over hydrogel-based systems. The linker-engineered cellular constructs cultured in the microfluidic system showed good cell viability, preserved the 3D cell morphology, and exhibited good cell functionality and differentiation capability. The microfluidic cell culture system was also showed to be a useful platform for optical-based studies with good spatial and temporal resolution. 126 8. RECOMMENDATIONS FOR FUTURE RESEARCH Using inter-cellular linker to facilitate heterotypic cell-cell interaction In this work, the inter-cellular linker was used only for aggregating homotypic cells. Facilitating heterotypic cell-cell interaction in vitro to mimic tissue structures in vivo would be the next step towards engineering functional tissues. For instance, the islets of Langerhans in the pancreas have a distinct architecture whereby nonbeta cells surrounds a mass of beta cells in the centre.208 These homotypic and heterotypic cell-cell interactions are necessary for responding to different nutrient stimuli.209, 210 In engineering tumour models, tumour cells can be co-cultured with other cell types to study interactions between the tumour cells and other cells. For example, tumour cells can be cultured with immune cells to study infiltration of immune cells into tumours;211 or with endothelial cells to study angiogenesis;212 or with fibroblasts to study tumour cell invasion.213 The inter-cellular linker can be used to facilitate these heterotypic cell-cell interactions in vitro to mimic the in vivo situations. Inter-cellular linker with different functional groups In this work, sialic acid residues on cell surface glycoprotein were chemically modified with sodium periodiate to yield aldehyde functional groups, which served as molecular handles for the hydrazides on the inter-cellular linker to react with. This two-step process can be further reduced into one, if functional groups that react with cell surface receptors can be conjugated onto the inter-cellular linker. For instance, galactose groups can be conjugated onto PEI to form PEI-galactose 127 inter-cellular linker, which can be used to aggregate hepatoma cells which display the asialoglycoprotein receptor (ASGPR) for galactose ligands on cell surfaces.214 Three-dimensional patterning of cells This method to form 3D cell-dense cultures with inter-cellular linker does not exert spatial control of the cells to form constructs of desired shapes and structures. One way to that is to use external physical forces to organise the cells into a structure, and then “lock” the cells in the specific organisation with inter-cellular linker. Dielectrophoretic forces for cell manipulation has been used to fabricate tissues of varying microstructures.139, 215, 216 Another technique which uses computer-aided jet-based patterning of cells to form cellular structures, also known as “organ printing”,37, 198 can also be of use. Both techniques are carried out with cell suspension in liquid hydrogels, which are subsequently solidified to lock the cells in place after patterning. With inter-cellular linker, patterning and cell culture would not be confined to take place within hydrogels, which potentially opens up more avenues for innovation. Studying gene expression of linker-engineered aggregates It has been widely reported that cells cultured in 3D in vitro exhibited phenotypes that are different from those in conventional 2D monolayer cultures, and may mirror their in vivo counterpart more closely.2, 146, 217 These claims were validated by assessing the three-dimensionally cultured cells for their gene expression profiles,15 their expression of various differentiated markers (e.g., albumin production in hepatocytes or alkaline phosphatase activity in osteoblasts),14, 218, 219 128 as well as the spatial localisation of structural proteins like F-actin, E-cadherin, and vinculin.16, 220 In this thesis project, we have developed an inter-cellular polymeric linker that enables the development of 3D in vitro cell-dense culture models in both the macro-scale / static (e.g. petri dishes) and the micron-scale / dynamic (e.g. microfluidic systems) environments. It is therefore of interest to investigate the differences in gene expression profiles between cells cultured in 2D and 3D, and between cells cultured in static and dynamic 3D environments. The gene expression profiles of cells cultured in the in vitro model can also be compared to that of cells in vivo, to determine how closely the 3D in vitro model mimics the in vivo situation. Utilisation of the 3D microfluidic cell culture system for high content screening The transparent nature microfluidic cell culture system can be further exploited to monitor various dynamic cellular events in live cells simultaneously, in other words, for high content screening.221 The constant cell culture microenvironment provided by perfusion is also useful for minimising cellular responses to environment changes when performing live-cell experiments. High content screening has revealed valuable functional information by taking into account the intracellular localisation, transport and temporal and spatial control of the activity of biomolecules including DNA, RNA and proteins.222, 223 This allows us the ability to discern inter-relationships of processes affecting cellular organelles.224 For instance, during the introduction of drugs to the microfluidic cellular construct, besides monitoring for cell viability, we can simultaneously monitor other apoptosis markers like mitochondrial membrane potential (MMP), nuclear 129 morphology, membrane permeability; or cellular oxidative stress markers like reactive oxygen species (ROS) or reduced glutathione (GSH).225 130 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. A. Abbott, Nature, 2003, 424, 870-872. F. Pampaloni, E. G. Reynaud and E. H. Stelzer, Nat Rev Mol Cell Biol, 2007, 8, 839-845. E. Alsberg, K. W. Anderson, A. Albeiruti, J. A. 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Lin, Electrophoresis, 2007, 28, 11461153. 139 APPENDIX A Figure 57 IMAGE ANALYSIS Segmentation of images (a) into nuclei staining (b) and protein staining (c) with image analysis software, ImagePro Plus. To quantify the histological staining, the digital images were segmented into blue (counter-staining of nuclei with hematoxylin) and brown pixels (protein of interest). The area selected to be quantified was highlighted in grey (images on the right), and numbered or outlined in green by the software. The number of brown pixels and number of blue objects (nuclei) were then quantified. The level of protein expression was expressed as number of brown pixels divided by number of nuclei, in arbitrary units. 140 APPENDIX B REVERSIBLE SEALING OF MICROFLUIDIC CHANNEL ` Figure 58 (a) Clamps used for reversible sealing of the microfluidic channel for SEM processing of the microfluidic cellular construct. The clamps were custommade with polycarbonate, designed with holes for tubing and cell reservoir (top piece (b)), and a window in the centre for viewing under an inverted microscope (bottom piece (c)). A polyethylene (PE) film was placed between the PDMS channel and a cover slip. The whole set-up (PDMS + PE film + cover slip) was then clamped and tightened with the screws. After days of culture, the cellular construct was fixed and the clamps removed. The cover slip was removed and the PE film peeled off carefully to expose the cellular construct for SEM processing. 141 [...]... types presents an initial step to construct 3D basic building blocks in vitro.10, 11 This thesis project aims to engineer 3D cell-dense constructs for in vitro studies by using an inter -cellular polymeric linker that facilitates aggregation of cells Being low molecular weight molecules, (Mw ~ 2000 Da) the inter -cellular linker allows the formation of cellular constructs with a high ratio of cell to biomaterial... culture medium with dissolved inter -cellular linker and seeded into the microfluidic channel with a withdrawal flow at the outlet .94 Figure 44 (a) Transmission image of the C3A cellular construct in the 3D- µFCCS after seeding Scale bar: 20 mm (b) Confocal image of C3A cells aggregated with fluorescent inter -cellular linker, showing cells in the construct supported in 3D by neighbouring cells Scale... up-regulated in 3D spheroids.21 Generally, cells cultured in 3D maintain a gene expression profile that is closer to tissues in vivo,15 showing that 3D cell cultures better mirror the in vivo environment to yield relevant cellular responses 4 2.1.2 Strategies for 3D cell culture To grow cells in 3D, cells have to be supported in a structure that mimics the ECM in vivo Classical 3D tissue engineering approaches... clinical opportunities in tissue engineering strategies that require a strict structural support, 8 shape-supporting matrices could be less-suited for the engineering of cell-dense and ECM-poor tissues of the internal organs, e.g liver, pancreas, or even tumour models.10, 38 Therefore engineering tissue constructs with minimal biomaterials and even as scaffold-free cellular constructs would be important... constructs with a high ratio of cell to biomaterial volume, a feat unattainable when using conventional bulk biomaterials 1 like scaffolds and hydrogels The cellular aggregates formed with the inter -cellular linker were then structurally characterised and functionally assessed for growth maturation Finally, as there is growing interest to miniaturise in vitro models for achieving more controllable and reproducible... their macro-scale counterparts,13, 14 we incorporated the use of the inter -cellular linker into a microfluidics system to engineer 3D cell-dense micro -constructs for applications At the same time we created a novel method for seeding and culturing mammalian cells threedimensionally and at high density in microfluidic systems without using hydrogels To provide a background for these studies, a literature... pseudo -3D surfaces of the scaffolds To culture cells in 3D, hydrogels of various kinds have been employed to encapsulate cells.6-8 However, these bulk biomaterials may not be appropriate for engineering constructs of cell-dense and matrix-poor tissues of the internal organs, or even tumour models.9-11 Another approach to engineer cellular and tissue constructs with minimal or reduced use of biomaterials... other cells without the use of biomaterials is another 3D culture method that has found various applications.10 The various strategies for 3D cell cultures are described below (1) Scaffolds for 3D support i) Cell seeding in porous 3D matrices Porous 3D matrices, also commonly called scaffolds, have been developed with particular applications in tissue engineering These matrices can have stochastic architectures... Day 2, (d) Day 3, (e) Day 5, (f) Day 7 Inset: Image of a control cellular aggregate acquired on Day 7 where cells had been fixed immediately after aggregation on Day 0 Scale bar: 50 mm 70 Figure 26 Quantification of the amount of fluorescent inter -cellular polymeric linker remaining on cell surfaces over time with software ImagePro Plus using images from Figure 25 70 Figure 27 Growth kinetics... few days to weeks Hence surface modification of cells has emerged as a means to accelerate cell aggregation Engineering 3D cellular aggregates can be conceptually accomplished by modifying cell surfaces to generate reactive handles and gluing cells by a multi-arm inter- 12 cellular molecular linker reacting with the reactive handles on cell surfaces Cell surfaces can be modified genetically, via enzymatic . of inter -cellular polymeric linker for engineering a 3D cell- dense culture model 56 5.1.1 Synthesis and characterisation of inter -cellular linker 56 5.1.2 Forming cellular aggregates using inter -cellular. linker for engineering a 3D cell- dense culture model 34 4.1.1 Synthesis and characterisation of inter -cellular linker 34 4.1.2 Forming cellular aggregates using inter -cellular linker 37 4.1.3. reproducible 3D cell-dense micro- constructs using a combination of inter -cellular linker and micro- fabricated structures 31 4. MATERIALS AND METHODS 34 4.1 Evaluation of inter -cellular polymeric linker