pid construction of mechanically confined multi cellular structures using dendrimeric inter cellular linker

RAPID CONSTRUCTION OF MECHANICALLYCONFINED MULTI- CELLULAR STRUCTURES USING DENDRIMERIC INTER- CELLULAR LINKER MO XUEJUN B.Appl.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENT First of all, I would like to express my sincere appreciation to everyone who helps me to make this thesis possible. The special thanks should go to my supervisors, Assoc. Prof Choon- Hong Tan and Prof. Hanry Yu for their valuable advice, patient guidance and inspirational motivation throughout my PhD course. I would like to express my appreciation to Dr. Zhilian Yue on her technical support as well as constructive guidance. I would like to express my sincere appreciation to Prof. Chwee Teck Lim, Assist. Prof Jie Yan, Assist. Prof Chorng Haur Sow, Dr. Lena Wai Yi Lui, Dr. Hongxia Fu, Dr. Siew-min Ong, Dr. Deqiang Zhao, and Dr. Lei Xia for their kind help and helpful advice. I wish to extend my sincere appreciation to my research colleagues: Qiushi Li, Baixue Zheng, Alvin Kang Chiang Huen, Bramasta Nugraha, Ruirui Jia, Deepak Choudhury, Talha Arooz, Jie Zhang, Abhishek Ananthanarayanan, Balakrishnan Chakrapani Narmada, who have offered invaluable help on experiments and useful discussions. I would also like to thank other members of the Cell and Tissue Engineering Laboratory and GEM4 (Global Enterprise for Micro- Mechanics and Molecular Medicine) for technical supports and stimulating scientific discussions. Special thanks go to my husband Chun Wei for endless emotional rescues and moral support. Finally to my families whose support I can never thank enough. CONTENTS LIST OF PUBLICATIONS……………………………………………………… viii SUMMARY……………………………………………………………………… x LIST OF FIGURES…………………………………………………………………xi LIST OF SYMBOLS AND ABBREVIATIONS……………………………… xviii 1. Introduction……………………………………………………………………… 2. Background and significance…………………………………………………… 2.1 Liver tissue engineering……………………………………………………….4 2.2 Liver physiology……………………………………………………………….6 2.3 Mammalian cellular membrane and its lipid domains……………………… .8 2.3.1 Overview of cellular membrane……………………………….8 2.3.2 Lipid domain and its charge………………………………….11 2.4 Dendrimers in bioengineering……………………………………………… 13 2.4.1 Chemistry and Synthesis of multivalent dendrimer molecule………………………………………………… ….13 2.4.2 Biological applications of multivalent dendrimer……………18 2.4.3 Cytotoxicity of dendrimers………………………………… .21 2.5 Cell surface engineering…………………………………………………… 22 2.5.1 Insertion of molecules onto cell membrane surface………….23 2.5.2 Reaction using exogenous enzymes………………………….24 2.5.3 Inhibition of biosynthetic pathways………………………….25 2.5.4 Metabolic engineering……………………………………… 25 2.5.5 Covalent ligation to cell surface chemical groups……………26 i 2.5.6 Application of surface engineered cells…………………… .27 2.6 Importance of 3D cellular culture……………………………………………29 2.6.1 Approaches for engineering 3D cellular culture…………… 30 2.6.1.1 Naturally formed 3D cellular culture……… .30 2.6.1.2 Scaffold approaches………………………….33 2.6.1.3 3D microfluidic cell culture systems…………36 2.6.1.4 Scaffold- free approaches…………………….37 Cell sheet assembly………………………… 37 Organ printing……………………………… 38 Synthetic inter- cellular linker approaches… 39 2.7 Laser assisted cell assembly…………………………………………………41 2.7.1 Laser assisted technology for formation of defined and precise 3D cellular constructs / culture………………………………42 2.8 Limitation of current 3D cell culture technologies………………………… 44 3. Objectives and specific aims…………………………………………………… 46 3.1 Specific aim 1: To functionalize the cellular membrane surface with nonnative functional group……………………………………………………….47 3.2 Specific aim 2: To design a novel dendrimeric inter- cellular linker for engineering 3D multi- cellular constructs……………………………………47 3.3 Specific aim 3: Characterization of 3D multi- cellular constructs engineered by dendrimeric inter- cellular linker…………………………………………… 48 3.4 Specific aim 4: Rapid precision engineering of 3D cellular lego using linkerengineered tissue spheroids as building blocks in micro- fabricated structures using mechanical confinement methods…………………….……………… 49 4. Cell surface functionalisation with non- native functional group…………… 50 ii 4.1 Introduction……………………….………………………………………….50 4.2 Materials and methods……………………………………………………… 51 4.2.1 Synthesis and characterisation of cholesterol- PEG conjuagte with ketone functionality…………………………………… 51 4.2.1.1 Synthesis procedure and compound characterisation .51 4.2.1.2 Synthesis and characterisation of cho- PEG- ketone conjugate…………………………………………… 52 4.2.1.3 Cell number………………………………………… 52 4.2.1.4 Solution preparation………………………………….53 4.2.1.5 Cell viability staining of cells treated with cho- PEGketone conjugate…………………………………… .53 4.2.2 Labelling cellular membrane with cholesterol- PEG conjugate with ketone functionality…………………………………… 53 4.2.2.1 Detection of displayed functional group on cell surface……………………………………………… 54 4.3 Results……………………………………………………………………… 54 4.3.1 Functionalisation of cellular membrane surface with ketone functionality………………………………………………….54 4.3.1.1 Synthesis of cholesterol- PEG conjugate with ketone functionality………………………………………… 54 4.3.2 Labelling cellular membrane with cholesterol- PEG conjugate with ketone functionality…………………………………… 55 4.4 Discussions………………………………………………………………… .60 4.4.1 Cell surface functionalisation with non- native groups………60 4.4.1.1 The chemistry of cell membrane chemoselective iii ligation……………………………………………… 60 4.5 Summary for Specific aim 1………………………………………………….65 5. Design a novel dendrimeric inter- cellular linker for engineering 3D multicellular constructs………………………………………………………………… 65 5.1 Introduction…………………………………………………………………65 5.2 Materials and methods………………………………………………………67 5.2.1 Synthesis and characterisation of dendrimeric inter- cellular linker…………………………………………………………67 5.2.1.1 Synthesis procedures and compound characterisation.67 5.2.1.2 Synthesis and characterisation of oleyl- PEG conjugate…………………………………………… 67 5.2.1.3 Synthesis and characterisation of oleyl- PEG conjugated DAB dendrimer………………………………………68 5.2.2 Forming 3D multi- cellular constructs using the dendrimeric inter- cellular linker………………………………………….68 5.2.2.1 Formation of multi- cellular structures using dendrimeric inter- cellular linker…………………… 68 5.2.2.2 Zeta potential measurement………………………… 69 5.2.2.3 MTS cytotoxicty assay of dendrimeric inter- cellular linker…………………………………………………69 5.2.2.4 Microarray analysis………………………………… 70 iv 5.3 Results……………………………………………………………………… 70 5.3.1 Synthesis and characterisation of the dendrimeric inter- cellular linker…………………………………………………………70 5.3.2 Forming 3D multi- cellular constructs using the dendrimeric inter- cellular linker………………………………………… 74 5.4 Discussions………………………………………………………………… .80 5.4.1 Evaluation of dendrimeric inter- cellular linker for engineering 3D multi- cellular structures…………………………………80 5.4.1.1 Interaction between dendrimeric inter- cellular linker and cell surface………………………………………80 5.5 Summary for Specific aim 2…………………………………………………78 6. Biological characterisation of linker- engineered multi- cellular constructs…83 6.1 Introduction………………………………………………………………… 84 6.2 Materials and methods……………………………………………………… 84 6.2.1 Structural characterisation of linker- engineered multi- cellular constructs…………………………………………………… 84 6.2.2 6.2.1.1 Live- dead assay of multi- cellular structures……… 85 6.2.1.2 DNA quantification assay……………………………85 6.2.1.3 Scanning electron microscopy……………………….85 6.2.1.4 Hydroxyproline assay……………………………… 85 6.2.1.5 Actin staining……………………………………… .86 Functional assessment of linker- engineered multi- cellular constructs…………………………………………………….86 6.2.2.1 Albumin secretion and Cytochrome P450 1A1/2 enzymatic activity……………………………………86 v 6.3 Results……………………………………………………………………… 87 6.3.1 Structural characterisation of linker- engineered multi- cellular constructs…………………………………………………… 88 6.3.2 Functional assessment of linker- engineered multi- cellular constructs…………………………………………………… 92 6.4 Discussions………………………………………………………………… .94 6.4.1 Biological characterisation of linker- engineered multi- cellular constructs…………………………………………………….94 6.4.1.1 Morphological changes of multi- cellular constructs during culture……………………………………… 94 6.4.1.2 Applications of linker- engineered multi- cellular constructs…………………………………………….96 6.5 Summary for Specific aim 3…………………………………………………97 7. Rapid construction of defined multi- cellular structures with dendrimeric inter- cellular linker using optical tweezer……………………………………… 98 7.1 Introduction………………………………………………………………….98 7.2 Materials and methods……………………………………………………….99 7.2.1 Adhesion force measurement between cells with dendrimeric inter- cellular linker using a dual micro- pipette manipulator system……………………………………………………….100 7.2.2 Precise construction of defined multi- cellular constructs….100 7.2.3 Other methods………………………………………………100 7.3 Results………………………………………………………………………100 7.3.1 Rapid construction of defined multi- cellular structures with dendrimeric inter-cellular linker using optical tweezer …….100 vi 7.3.1.1 Design and operation of optical tweezer……………100 7.3.1.2 Adhesion force measurement between cells with dendrimeric inter- cellular linker……………………103 7.3.1.3 Precise construction of defined multi- cellular constructs……………………………………………105 7.4 Discussions………………………………………………………………….107 7.4.1 Adhesion force of oleyl- PEG conjugated DAB dendrimeric linkers on cells………………… ………………………… .107 7.4.2 Formation of defined 3D cellular structures with optical trapping method……………………………………………108 7.4.3 Applications of 3D cellular lego……………………………109 7.5 Summary for Specific Aim 4……………………………………………… 110 8. Conclusion……………………………………………………………………….111 9. Recommendations for future research……………………………………… .115 9.1 Use of dendrimeric inter-cellular linker to facilitate formation of heterocellular cell aggregates………………………………………………………………115 9.2 Using linker engineered tissue spheroids as building blocks for organ printing ………………………………………………………………………………115 9.3 Inter- cellular linker with different functional groups…………………… 116 9.4 Inter- cellular linker with photo cross- linkable and photo- degradable functionality…………………………………………………………………117 10. References…………………………………………………………………… .119 vii LIST OF PUBLICATIONS 1. Mo X, Li Q, Wai YLL, Zheng B, Kang CH, Nugraha B, Yue Z, Jia R, Fu H, Choudhury D, Arooz T, Yan J, Lim CT, Shen S, Tan CH, Yu H. Rapid construction of mechanically- confined multi- cellular structures using dendrimeric inter- cellular linker. Biomaterials 2010; 31(29): 7455-7467. 2. Ananthanarayanan A, Narmada BC, Mo X, McMillian M, Yu H. Purpose- driven biomaterials research in liver- tissue engineering. Trends in Biotechnology 2011; 29 (3): 110-118. 3. Choudhury D, Mo X, Iliescu C, Tan L, Tong WH, Yu H. Exploitation of chemical and physical constraints for 3D microtissue construction in microfluidics. Biomicrofluidics 2011; (2): 022203-1 - 18. 4. Nugraha B, Hong X, Mo X, Tan L, Zhang W, Chan, P-M, Kang CH, Wang Y, Beng LT, Sun W, Choudhury, D, Rubens JM, McMillian M, Silvia J, Dallas S, Tan CH, Yue Z, Yu H. Galactosylated cellulosic sponge for multi- well hepatotoxicity drug testing. Biomaterials 2011; 32 (29): 6982- 6994. 5. Zheng B, Tan L, Mo X, Yu W, Wang Y, Tucker- Kellogg L, Welsch R, So P, Yu H. An anti- hepatofibrotic drug efficacy predictor that correlates and predicts in vivo drug response based on in vitro high- content analysis. Journal of Hepatology 2011 viii [236] Wang S, Nagrath D, Chen PC, Berthiaume F, Yarmush ML. Three-dimensional primary hepatocyte culture in synthetic self-assembling peptide hydrogel. Tissue Eng Part A. 2008;14:227-36. [237] Adelow CA, Frey P. Synthetic hydrogel matrices for guided bladder tissue regeneration. Methods Mol Med. 2007;140:125-40. [238] Mann BK, Gobin AS, Tsai AT, Schmedlen RH, West JL. Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering. Biomaterials. 2001;22:3045-51. [239] Wilson WC, Jr., Boland T. Cell and organ printing 1: protein and cell printers. Anat Rec A Discov Mol Cell Evol Biol. 2003;272:491-6. [240] Mironov V, Boland T, Trusk T, Forgacs G, Markwald RR. Organ printing: computer-aided jet-based 3D tissue engineering. Trends Biotechnol. 2003;21:157-61. [241] Yasuda K, Ping Gong J, Katsuyama Y, Nakayama A, Tanabe Y, Kondo E, et al. Biomechanical properties of high-toughness double network hydrogels. Biomaterials. 2005;26:4468-75. [242] Choi NW, Cabodi M, Held B, Gleghorn JP, Bonassar LJ, Stroock AD. Microfluidic scaffolds for tissue engineering. Nat Mater. 2007;6:908-15. [243] Leclerc ES, Y.; Fujii, T. Cell culture in 3-dimensional microfluidic structure of PDMS (polydimethylsiloxane). Biomed Microdevices. 2003;5:109-14. 148 [244] Toh YC, Zhang C, Zhang J, Khong YM, Chang S, Samper VD, et al. A novel 3D mammalian cell perfusion-culture system in microfluidic channels. Lab Chip. 2007;7:302-9. [245] Tan W, Desai TA. Layer-by-layer microfluidics for biomimetic threedimensional structures. Biomaterials. 2004;25:1355-64. [246] Viravaidya K, Sin A, Shuler ML. Development of a microscale cell culture analog to probe naphthalene toxicity. Biotechnol Prog. 2004;20:316-23. [247] Kim L, Toh YC, Voldman J, Yu H. A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab Chip. 2007;7:681-94. [248] Albrecht DR, Underhill GH, Wassermann TB, Sah RL, Bhatia SN. Probing the role of multicellular organization in three-dimensional microenvironments. Nat Methods. 2006;3:369-75. [249] Ling Y, Rubin J, Deng Y, Huang C, Demirci U, Karp JM, et al. A cell-laden microfluidic hydrogel. Lab Chip. 2007;7:756-62. [250] Kim MS, Yeon JH, Park JK. A microfluidic platform for 3-dimensional cell culture and cell-based assays. Biomed Microdevices. 2007;9:25-34. [251] Toh YC, Ng S, Khong YM, Samper V, Yu H. A configurable three-dimensional microenvironment in a microfluidic channel for primary hepatocyte culture. Assay Drug Dev Technol. 2005;3:169-76. 149 [252] Wong AP, Perez-Castillejos R, Christopher Love J, Whitesides GM. Partitioning microfluidic channels with hydrogel to construct tunable 3-D cellular microenvironments. Biomaterials. 2008;29:1853-61. [253] Frisk T, Rydholm S, Andersson H, Stemme G, Brismar H. A concept for miniaturized 3-D cell culture using an extracellular matrix gel. Electrophoresis. 2005;26:4751-8. [254] Yamato M, Utsumi M, Kushida A, Konno C, Kikuchi A, Okano T. Thermoresponsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng. 2001;7:473-80. [255] Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation. 2004;77:379-85. [256] Shiroyanagi Y, Yamato M, Yamazaki Y, Toma H, Okano T. Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps. BJU Int. 2004;93:1069-75. [257] Hasegawa M, Yamato M, Kikuchi A, Okano T, Ishikawa I. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng. 2005;11:469-78. [258] Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res. 2002;90:e40. 150 [259] Shimizu T, Yamato M, Kikuchi A, Okano T. Cell sheet engineering for myocardial tissue reconstruction. Biomaterials. 2003;24:2309-16. [260] Harimoto M, Yamato M, Hirose M, Takahashi C, Isoi Y, Kikuchi A, et al. Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes. J Biomed Mater Res. 2002;62:464-70. [261] Tsang VL, Bhatia SN. Three-dimensional tissue fabrication. Adv Drug Deliv Rev. 2004;56:1635-47. [262] Jakab KD, B.; Neagu, A.; Kachurin, A.; Forgacs, G. Three-dimensional tissue constructs built by bioprinting. Biorheology. 2006;45:509-135. [263] Jakab K, Norotte C, Damon B, Marga F, Neagu A, Besch-Williford CL, et al. Tissue engineering by self-assembly of cells printed into topologically defined structures. Tissue Eng Part A. 2008;14:413-21. [264] Yang J, Yamato M, Shimizu T, Sekine H, Ohashi K, Kanzaki M, et al. Reconstruction of functional tissues with cell sheet engineering. Biomaterials. 2007;28:5033-43. [265] Greenberg ME, Brackenbury R, Edelman GM. Alteration of neural cell adhesion molecule (N-CAM) expression after neuronal cell transformation by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1984;81:969-73. [266] Charter NW, Mahal LK, Koshland DE, Jr., Bertozzi CR. Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells. Glycobiology. 2000;10:1049-56. 151 [267] Mahal LK, Bertozzi CR. Engineered cell surfaces: fertile ground for molecular landscaping. Chem Biol. 1997;4:415-22. [268] Kellam B, De Bank PA, Shakesheff KM. Chemical modification of mammalian cell surfaces. Chem Soc Rev. 2003;32:327-37. [269] Medof ME, Nagarajan S, Tykocinski ML. Cell-surface engineering with GPIanchored proteins. FASEB J. 1996;10:574-86. [270] Yamada K, Kamihira M, Iijima S. Enhanced cell aggregation and liver functions using polymers modified with a cell-specific ligand in primary hepatocyte cultures. J Biosci Bioeng. 1999;88:557-62. [271] Dai W, Belt J, Saltzman WM. Cell-binding peptides conjugated to poly(ethylene glycol) promote neural cell aggregation. Biotechnology (N Y). 1994;12:797-801. [272] Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S. Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett. 1986;11:288. [273] Block SM, Blair DF, Berg HC. Compliance of bacterial flagella measured with optical tweezers. Nature. 1989;338:514-8. [274] Grier DG. A revolution in optical manipulation. Nature. 2003;424:810-6. [275] Neuman KC, Block SM. Optical trapping. Rev Sci Instrum. 2004;75:2787-809. [276] G. Sinclair PJ, J. Leach, M. J. Padgett, J. Cooper. Defining the trapping limits of holographical optical tweezers Journal of modern optics. 2004;51:409-14. 152 [277] Ashkin A. History of optical trapping and manipulation of small- neutral particles, atoms, and molecules. IEEE Selected Topics in Quantum Electronics. 2000;6:841-56. [278] Neuman KC, Nagy A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods. 2008;5:491-505. [279] Chiou PY, Ohta AT, Wu MC. Massively parallel manipulation of single cells and microparticles using optical images. Nature. 2005;436:370-2. [280] Mirsaidov U, Scrimgeour J, Timp W, Beck K, Mir M, Matsudaira P, et al. Live cell lithography: using optical tweezers to create synthetic tissue. Lab Chip. 2008;8:2174-81. [281] Jordan P, Leach J, Padgett M, Blackburn P, Isaacs N, Goksor M, et al. Creating permanent 3D arrangements of isolated cells using holographic optical tweezers. Lab Chip. 2005;5:1224-8. [282] Neuman KC, Chadd EH, Liou GF, Bergman K, Block SM. Characterization of photodamage to Escherichia coli in optical traps. Biophys J. 1999;77:2856-63. [283] Aguiari P, Leo S, Zavan B, Vindigni V, Rimessi A, Bianchi K, et al. High glucose induces adipogenic differentiation of muscle-derived stem cells. Proc Natl Acad Sci U S A. 2008;105:1226-31. [284] Dittrich PS, Manz A. Lab-on-a-chip: microfluidics in drug discovery. Nat Rev Drug Discov. 2006;5:210-8. 153 [285] Hung PJ, Lee PJ, Sabounchi P, Lin R, Lee LP. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnol Bioeng. 2005;89:1-8. [286] Demel RA, Bruckdorfer KR, van Deenen LL. The effect of sterol structure on the permeability of lipomes to glucose, glycerol and Rb +. Biochim Biophys Acta. 1972;255:321-30. [287] Chapman D. Biological membranes. Academic Press, London. 1973;2:91-144. [288] Dongen S, Nallani, M., Schoffelen, S., Cornelissen, J., Nolte, R., Hest, J. A Block Copolymer for Functionalisation of Polymersome Surfaces. Macromolecular Rapid Communications. 2008;29:321-5. [289] Nauman DA, Bertozzi CR. Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting. Biochim Biophys Acta. 2001;1568:147-54. [290] Chen I, Howarth M, Lin W, Ting AY. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Methods. 2005;2:99-104. [291] Cook BN, Bertozzi CR. Chemical approaches to the investigation of cellular systems. Bioorg Med Chem. 2002;10:829-40. [292] Jacobs CL, Goon S, Yarema KJ, Hinderlich S, Hang HC, Chai DH, et al. Substrate specificity of the sialic acid biosynthetic pathway. Biochemistry. 2001;40:12864-74. [293] Winans KA, Bertozzi, C. R. Inner space exploration: the chemical biologist's guide to the cell. Chemistry and biology. 1998;5:R313-R5. 154 [294] Muir TW. A chemical approach to the construction of multimeric protein assemblies. Structure. 1995;3:649-52. [295] Schnolzer M, Kent SB. Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease. Science. 1992;256:221-5. [296] Liu CF, Rao, C., Tam, J. P. . Orthogonal Ligation of Unprotected Peptide Segments through Pseudoproline Formation for the Synthesis of HIV-1 Protease Analogs. Journal of the American Chemical Society. 1996;118:307-12. [297] Williams MJ, Muir, T. W., Ginsberg, M. H. Kent, S. B. H. Total Chemical Synthesis of a Folded .beta.-Sandwich Protein Domain: An Analog of the Tenth Fibronectin Type Module. Journal of the American Chemical Society. 1994;116:10797-8. [298] Muir TW, Williams MJ, Ginsberg MH, Kent SB. Design and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cellsurface receptor: integrin alpha IIb beta (platelet GPIIb-IIIa). Biochemistry. 1994;33:7701-8. [299] Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A. Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science. 1986;232:162932. [300] Vasan S, Zhang X, Kapurniotu A, Bernhagen J, Teichberg S, Basgen J, et al. An agent cleaving glucose-derived protein crosslinks in vitro and in vivo. Nature. 1996;382:275-8. 155 [301] Jencks WP. Studies on the mechanism of oxime and semicarbazone formation. Journal of the American Chemical Society. 1959;81:475-81. [302] Rideout D. Self-assembling drugs: a new approach to biochemical modulation in cancer chemotherapy. Cancer Invest. 1994;12:189-202; discussion 68-9. [303] Rideout D. Self-assembling cytotoxins. Science. 1986;233:561-3. [304] Rideout D, Calogeropoulou T, Jaworski J, McCarthy M. Synergism through direct covalent bonding between agents: a strategy for rational design of chemotherapeutic combinations. Biopolymers. 1990;29:247-62. [305] Sadamoto R, Niikura K, Ueda T, Monde K, Fukuhara N, Nishimura S. Control of bacteria adhesion by cell-wall engineering. J Am Chem Soc. 2004;126:3755-61. [306] Rose K. Facile synthesis of homogenous artificial proteins. Journal of the American Chemical Society. 1994;116:30-3. [307] Shao J, Tam, J. P. Unprotected Peptides as Building Blocks for the Synthesis of Peptide Dendrimers with Oxime, Hydrazone, and Thiazolidine Linkages. Journal of the American Chemical Society. 1995;117:3893-9. [308] Hussey SL, He, E., Peterson, B. R. . A synthetic membrane- anchored antigen efficiently promotes uptake of antifluorescein antibodies and associated protein A by mammalian cells. Journal of the American Chemical Society. 2001;123:12712-3. [309] Cameron DF, Hushen JJ, Colina L, Mallery J, Willing A, Sanberg PR, et al. Formation and structure of transplantable tissue constructs generated in simulated 156 microgravity from Sertoli cells and neuron precursors. Cell Transplant. 2004;13:75563. [310] Kichler A. Gene transfer with modified polyethylenimines. J Gene Med. 2004;6 Suppl 1:S3-10. [311] Mislick KA, Baldeschwieler JD. Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci U S A. 1996;93:12349-54. [312] Mounkes LC, Zhong W, Cipres-Palacin G, Heath TD, Debs RJ. Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo. J Biol Chem. 1998;273:26164-70. [313] Fujimoto K. Supramolecular approaches for cellular modulation. Supramolecular Design for Biological Applications. Yui, N, Ed; CRC Press: Boca Raton. 2002:343-69. [314] Takahashi TF, K.; Kawaguchi, H. Surface modifications of cell with a nonionic polymer and assessment of the hybrid function. Polym Prep, Japan. 1996;45:572-. [315] Baba T, Rauch C, Xue M, Terada N, Fujii Y, Ueda H, et al. Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly (ethylene glycol)-derivatized cholesterol in the plasma membrane. Traffic. 2001;2:501-12. [316] van Broekhoven CL, Parish CR, Vassiliou G, Altin JG. Engrafting costimulator molecules onto tumor cell surfaces with chelator lipids: a potentially convenient approach in cancer vaccine development. J Immunol. 2000;164:2433-43. 157 [317] van Broekhoven CL, Altin JG. A novel approach for modifying tumor cellderived plasma membrane vesicles to contain encapsulated IL-2 and engrafted costimulatory molecules for use in tumor immunotherapy. Int J Cancer. 2002;98:63-72. [318] Van Broekhoven CL, Altin JG. A novel system for convenient detection of lowaffinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II. Immunol Cell Biol. 2001;79:274-84. [319] Kato K, Itoh C, Yasukouchi T, Nagamune T. Rapid protein anchoring into the membranes of Mammalian cells using oleyl chain and poly(ethylene glycol) derivatives. Biotechnol Prog. 2004;20:897-904. [320] Nyberg SL, Hardin J, Amiot B, Argikar UA, Remmel RP, Rinaldo P. Rapid, large-scale formation of porcine hepatocyte spheroids in a novel spheroid reservoir bioartificial liver. Liver Transpl. 2005;11:901-10. [321] Mironov V, Visconti RP, Kasyanov V, Forgacs G, Drake CJ, Markwald RR. Organ printing: tissue spheroids as building blocks. Biomaterials. 2009;30:2164-74. [322] Furukawa KS, Suenaga H, Toita K, Numata A, Tanaka J, Ushida T, et al. Rapid and large-scale formation of chondrocyte aggregates by rotational culture. Cell Transplant. 2003;12:475-9. [323] Kunz-Schughart LA, Freyer JP, Hofstaedter F, Ebner R. The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J Biomol Screen. 2004;9:273-85. 158 [324] Wu LY, Di Carlo D, Lee LP. Microfluidic self-assembly of tumor spheroids for anticancer drug discovery. Biomed Microdevices. 2008;10:197-202. [325] Bhatia SN, Balis UJ, Yarmush ML, Toner M. Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J. 1999;13:1883-900. [326] Bates RC, Edwards NS, Yates JD. Spheroids and cell survival. Crit Rev Oncol Hematol. 2000;36:61-74. [327] Santini MT, Rainaldi G, Indovina PL. Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids. Crit Rev Oncol Hematol. 2000;36:75-87. [328] Santini MT, Rainaldi G. Three-dimensional spheroid model in tumor biology. Pathobiology. 1999;67:148-57. [329] Walker TM, Rhodes PC, Westmoreland C. The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures. Toxicol In Vitro. 2000;14:475-85. [330] Sutherland RM. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science. 1988;240:177-84. [331] Mueller-Klieser W. Three-dimensional cell cultures: from molecular mechanisms to clinical applications. Am J Physiol. 1997;273:C1109-23. [332] Mueller-Klieser W. Tumor biology and experimental therapeutics. Crit Rev Oncol Hematol. 2000;36:123-39. 159 [333] Dhiman HK, Ray AR, Panda AK. Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen. Biomaterials. 2005;26:979-86. [334] Schmeichel KL, Bissell MJ. Modeling tissue-specific signaling and organ function in three dimensions. J Cell Sci. 2003;116:2377-88. [335] Nelson CM, Bissell MJ. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol. 2006;22:287-309. [336] Kyle AH, Huxham LA, Chiam AS, Sim DH, Minchinton AI. Direct assessment of drug penetration into tissue using a novel application of three-dimensional cell culture. Cancer Res. 2004;64:6304-9. [337] Tannock IF, Lee CM, Tunggal JK, Cowan DS, Egorin MJ. Limited penetration of anticancer drugs through tumor tissue: a potential cause of resistance of solid tumors to chemotherapy. Clin Cancer Res. 2002;8:878-84. [338] Johnson JI, Decker S, Zaharevitz D, Rubinstein LV, Venditti JM, Schepartz S, et al. Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer. 2001;84:1424-31. [339] Bhadriraju K, Chen CS. Engineering cellular microenvironments to improve cell-based drug testing. Drug Discov Today. 2002;7:612-20. [340] Fracasso G, Colombatti M. Effect of therapeutic macromolecules in spheroids. Crit Rev Oncol Hematol. 2000;36:159-78. 160 [341] Boland T, Mironov V, Gutowska A, Roth EA, Markwald RR. Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels. Anat Rec A Discov Mol Cell Evol Biol. 2003;272:497-502. [342] Ho CT, Lin RZ, Chang WY, Chang HY, Liu CH. Rapid heterogeneous liver-cell on-chip patterning via the enhanced field-induced dielectrophoresis trap. Lab Chip. 2006;6:724-34. [343] Du Y, Lo E, Ali S, Khademhosseini A. Directed assembly of cell-laden microgels for fabrication of 3D tissue constructs. Proc Natl Acad Sci U S A. 2008;105:9522-7. [344] Moffitt JR, Chemla YR, Izhaky D, Bustamante C. Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc Natl Acad Sci U S A. 2006;103:9006-11. [345] Odde DJ, Renn MJ. Laser-guided direct writing of living cells. Biotechnol Bioeng. 2000;67:312-8. [346] Suresh S. Biomechanics and biophysics of cancer cells. Acta Biomater. 2007;3:413-38. [347] Hornby JE. Measurements of cell adhesion. II. Quantitative study of the effect of divalent ions on cell adhesion. J Embryol Exp Morphol. 1973;30:511-8. [348] Lim CT, Zhou E.H., Li, A., Vedula, S.R.K., Fu, H.X. Experimental techniques for single cell and single molecule biomechanics. Materials Science and Engineering C. 2006;26:1278-88. 161 [349] Dahl U, Sjodin A, Semb H. Cadherins regulate aggregation of pancreatic betacells in vivo. Development. 1996;122:2895-902. [350] Hauge-Evans AC, Squires PE, Persaud SJ, Jones PM. Pancreatic beta-cell-tobeta-cell interactions are required for integrated responses to nutrient stimuli: enhanced Ca2+ and insulin secretory responses of MIN6 pseudoislets. Diabetes. 1999;48:1402-8. [351] Hamaguchi K, Utsunomiya N, Takaki R, Yoshimatsu H, Sakata T. Cellular interaction between mouse pancreatic alpha-cell and beta-cell lines: possible contactdependent inhibition of insulin secretion. Exp Biol Med (Maywood). 2003;228:122733. [352] Gottfried E, Kunz-Schughart LA, Andreesen R, Kreutz M. Brave little world: spheroids as an in vitro model to study tumor-immune-cell interactions. Cell Cycle. 2006;5:691-5. [353] Oudar O. Spheroids: relation between tumour and endothelial cells. Crit Rev Oncol Hematol. 2000;36:99-106. [354] Kunz-Schughart LA, Kreutz M, Knuechel R. Multicellular spheroids: a threedimensional in vitro culture system to study tumour biology. Int J Exp Pathol. 1998;79:1-23. [355] Lodish HF. Recognition of complex oligosaccharides by the multi-subunit asialoglycoprotein receptor. Trends Biochem Sci. 1991;16:374-7. [356] Langer R, Tirrell DA. Designing materials for biology and medicine. Nature. 2004;428:487-92. 162 [357] Mano JF. Stimuli- responsive polymeric systems for biomedical applications. Adv Eng Mater. 2008;1:515-27. [358] Helmchen F, Denk W. Deep tissue two-photon microscopy. Nat Methods. 2005;2:932-40. [359] Zipfel WRW, R. M.; Webb, W. W. Nat Biotechnol. 2003, 21, 1368). Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol. 2003;21:1368- 76. [360] Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol. 2001;19:316-7. [361] Kloxin AM, Kasko AM, Salinas CN, Anseth KS. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science. 2009;324:59-63. 163 [...]... represents the dendrimeric inter- cellular linker Figure 12 Formation of multi- cellular structures using 0.5 µM oleyl- PEG 75 conjugated DAB dendrimeric linker (A) Cells formed multicellular structures with average diameter of 90 µm within 30 min incubation on an orbitron shaker (B) 88 ± 5 % of the linker treated cells were effectively clustered by centrifugation at 40 rcf for 1 min (C) The multi- cellular. .. histogram The dendrimeric linker can form multi- cellular structures with average of 184 ± 44 cells/ construct Figure 13 Zeta potential measurement of linkers treated cells Zeta 77 potential measurement of cell solutions treated with various concentrations of dendrimeric linkers No further increase in charge or size of the multi- cellular structures were observed xiii beyond 5 µM of dendrimeric linker Figure... successfully pulled off the flexible pipette Figure 22 Rapid construction of optically- trapped defined multi- cellular 106 structures using dendrimeric inter- cellular linker Rapid assembly of (A) ring, sheet and branching rod for linkerengineered cells at 44.1 mW laser power Disruption of rod construct for (B) untreated cells and (C) linker- engineered cells at 775 mW laser power (direction of pulling laser... of biopolymers and their mimetics and in the modification of biopolymers and cells [7] xii Figure 10 Characterisation of dendrimeric inter- cellular linker (A) 71 Structure of DAB- Am 16 (B) Schematic diagram of synthesis of oleyl- PEG conjugated DAB dendrimeric linker (C) 1H NMR spectra of oleyl- PEG conjugate, (D) 1H NMR spectra of oleylPEG conjugated DAB dendrimeric linker in CDCl3 (E) MALDI- TOF... significantly higher in the multi- cellular structures than the 2D monolayer culture Data plotted represent the mean ± s.e.m of 3 independent experiments Figure 19 Schematic representation of single beam optical tweezer system 102 Figure 20 Cellular assembly using dendrimeric inter- cellular linker: 102 Dendrimeric inter- cellular linker with the lipid oleyl- PEG arms can stabilize cell- cell interaction that... anchorage of hydrophobic oleyl groups at the end terminal of the dendrimeric linker into the cell membrane surface We demonstrate rapid assembly of C3A cells into multi- cell structures using the dendrimeric inter- cellular linker Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular. .. for in vitro studies by using a novel dendrimeric inter- cellular linker that can rapidly stabilize cell- cell contacts within minutes The 3D multi- cellular constructs formed with the dendrimeric inter- cellular linker were then structurally characterised and functionally 2 assessed for growth maturation Finally, we mechanically confined the linker- treated cells with the use of the optical- trapping... X, Nugraha B, Zhang C, Toh Y-C, Tan C-H, Wang Y and Yu H., “MicroFabrication Factory of Complex Tissues,” 3rd East Asian Pacific Student Workshop on Nano-Biomedical Engineering 21-22 December 2009, Singapore 2 Mo X, Tan C-H, Yu H., “Rapid construction of mechanically- confined multicellular structures using dendrimeric inter- cellular linker, ” TERMIS North America Meeting 2010, 5-8 December 2010, Orlando,... multi- cellular structures within minutes The multi- cellular structures exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and improved cellular functions over a 7day culture period The linker stabilizes the multi- cellular structures of defined shape and pattern in a gel- free environment by mechanically confining the cells Defined multi- cellular structures such... conjugated DAB dendrimeric linker in CDCl3 (E) MALDI- TOF MS spectra of oleyl- PEG conjugate, (F) MALDI- TOF MS spectra of oleyl- PEG conjugated DAB dendrimeric linker using CHCA as a matrix Figure 11 Schematic representation of cell- polymer network 75 Dendrimeric inter- cellular linker promotes aggregation of C3A cells into 3D multi- cellular aggregates H2 N H2 N HN 2 H2 N N N N N H2 N N N H2 N N H2 . the dendrimeric inter- cellular linker. 75 Figure 12 Formation of multi- cellular structures using 0.5 µM oleyl- PEG conjugated DAB dendrimeric linker. (A) Cells formed multi- cellular structures. 5.4.1 Evaluation of dendrimeric inter- cellular linker for engineering 3D multi- cellular structures ………………………………80 5.4.1.1 Interaction between dendrimeric inter- cellular linker and cell. 2009, Singapore 2. Mo X, Tan C-H, Yu H., “Rapid construction of mechanically- confined multi- cellular structures using dendrimeric inter- cellular linker, ” TERMIS North America Meeting 2010,