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. 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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,