Engineering soluble microenvironments in microfluidic systems

ENGINEERING SOLUBLE MICROENVIRONMENTS IN MICROFLUIDIC SYSTEMS ZHANG CHI B. Eng. (South China University of Technology) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences & Engineering National University of Singapore 2009 ACKNOWLEDGEMENT The four years of PhD study has so far been the most beautiful chapter in my life. It has not been easy to study away from my own country and without using my mother tongue. It is not only a matter of degree, but also a process of growing up. I owe my thanks to more people than I could remember during this process. First and foremost, my parents who showed their utmost concerns from our weekly phone calls even though they are thousands of miles away from me. Many thanks to my boyfriend, Yang Yang, who is struggling with his own PhD study, who suffered what I suffered and who took every possible initiative to make our life fulfilling. I am grateful to my supervisor, Prof. Hanry Yu, for his guidance. He has greatly shaped my thinking in research. Special thanks to Dr. Danny van Noort, who is a great friend. It was great fun to work with him and he always had ideas to cheer people up. I want to thank my colleagues and seniors, Dr. Toh Yi Chin and Dr. Ong Siew Min, who gave me invaluable suggestions and moral support and who taught me how to seed cells in microfluidic channels when I tried to start my own project. I am grateful to Dr. Chia Ser Mien, without whom, my first paper would not have been published so easily. I owe my thanks to Mr. Zhao Ziqing and Dr. Aida Rahim, who contributed a lot to my project. I am grateful to my friends, Mr. Xia Lei and Ms Zhang Shufang, who have been supportive all these years. Also, I will remember every member in the lab who supported me in one way or another. Last but not least, I want to thank my two cats, Niu Niu and Ka Ka, for keeping me company. Their sleeping on my lap or beside my laptop made the dark nights sweeter. TABLE OF CONTENTS LIST OF PUBLICATIONS .4 SUMMARY LIST OF TABLES .7 LIST OF FIGURES .8 LIST OF SYMBOLS AND ABBREVATIONS .14 CHAPTER Introduction .16 CHAPTER Background and Significance 19 2.1 Constructing a microenvironment for in vitro cell cultures .20 2.1.1 Importance of constructing a microenvironment in vitro .20 2.1.2 Strategies of constructing a microenvironment in vitro 23 2.2 Microscale cell cultures for controllable cellular microenvironment 30 2.2.1 Microtechnology for engineering microscale cell culture systems 30 2.2.2 Microfluidics as a cell culture platform 33 2.2.3 Controlling the soluble microenvironment in microfluidics 36 2.3 Controlled-release technique and the cellular microenvironment .38 2.3.1 Gelatin as carriers for polyion complexation .39 2.3.2 Different gelatin matrics for controlled release 40 2.3.3 GMs as novel controlled release carriers 41 2.4 Limitations of current studies and rationale of thesis research 44 CHAPTER Objectives and Specific Aims .47 3.1 Specific Aim 1: To fabricate, characterize and incorporate GMs into the 3D-μFCCS to ensure the feasibility of this strategy for engineering a soluble microenvironment. .48 3.2 Specific Aim 2: To access the engineered soluble microenvironment in the 3D-μFCCS for functional enhancement of primary rat hepatocytes 50 3.3 Specific Aim 3: To create individually controlled soluble microenvironments in a multi-channel 3D-μFCCS for functional enhancement of multiple cell types 52 CHAPTER Fabrication, Characterization and Incorporation of GMs into the 3D-μFCCS .54 4.1 Introduction .54 4.2 Materials and Methods 57 4.2.1 Device fabrication 57 4.2.2 Fabrication of GMs 57 4.2.3 Characterization of FITC-dextran release profile from the GMs .58 4.2.4 Polyelectrolytes fabrication 59 4.2.5 Seeding GMs in the 3D-μFCCS .59 4.2.6 Cell maintenance 59 4.2.7 Seeding of cells in the 3D-μFCCS .60 4.2.8 Cell viability staining .60 4.2.9 Seeding of GMs and cells in the 3D-μFCCS 61 4.2.10 Characterization of fluorescent intensity distribution in the 3D-μFCCS 61 4.3 Results and discussion .63 4.3.1 Design and fabrication of the modified 3D-μFCCS .63 4.3.2 Reducing the occurrence of air bubbles in the 3D-μFCCS 65 4.3.3 Culturing of various cell types in the 3D-μFCCS 67 4.3.4 Fabrication conditions for GMs 69 4.3.5 Entrapment of GMs in the 3D-µFCCS .72 4.3.6 Seeding of GMs with cells into the 3D-µFCCS .73 4.3.7 Characterization of the soluble microenvironment in the 3D-µFCCS .75 4.4 Conclusion .81 CHAPTER Engineering the Soluble Microenvironment in the 3D-µFCCS for Functional Enhancement of Primary Rat Hepatocytes 82 5.1 Introduction .82 5.2 Materials and Methods 85 5.2.1 Assessment of the size distribution of the GMs 85 5.2.2 Scanning electron microscopy (SEM) 85 5.2.3 Characterization of TGF-β1 release profile from the GMs 85 5.2.4 Estimation of the concentration of in situ controlled-released TGF-β1 in the 3D-μFCCS .86 5.2.5 Primary rat hepatocyte isolation .86 5.2.6 Hepatocyte seeding and culture in the 3D-μFCCS .87 5.2.7 Qualitative assessment of the GM distribution in the 3D-μFCCS 87 5.2.8 Functional assessment of hepatocytes 87 5.2.9 Hepatotoxicity testing in the 3D-μFCCS 88 5.3 Results and discussions .90 5.3.1 Incorporating TGF-β1 loaded GMs into the 3D-μFCCS 90 5.3.2 Determining the optimal TGF-β1 concentration for primary hepatocytes in 3D-µFCCS .94 5.3.3 Assessment of metabolic enzyme activities of hepatocytes in 3D-µFCCS 97 5.3.4 Improved drug sensitivity of hepatocytes with controlled TGF-β1 presentation in 3D-µFCCS .98 5.4 Conclusion .104 CHAPTER Culturing Multiple Cell Types on a Multi-Channel 3D-μFCCS with Compartmentalized Microenvironments .105 6.1 Introduction .105 6.2 Materials and methods .108 6.2.1 Cell maintenance 108 6.2.2 Cell seeding and culture in the multi-channel 3D-μFCCS .108 6.2.3 Characterization of TGF-β1 release profile from the GMs 109 6.2.4 Estimation of the concentration of controlled released TGF-β1 in the multi-channel 3D-μFCCS .109 6.2.5 Functional characterization of the cells 110 6.3 Results and discussion .111 6.3.1 Development of a common, serum-free culture medium .111 6.3.2 Effects of TGF-β1 on cellular functions .112 6.3.3 Perfusion culture of multiple cell types in the multi-channel 3D-µFCCS 115 6.3.4 Controlled-release of TGF-β1 from the GMs .118 6.3.5 Compartmental isolation between different cell types on the multi-channel 3D-μFCCS .120 6.4 Conclusion .124 CHAPTER Conclusion 125 CHAPTER Recommendations for Future Research 127 8.1 Biological validation of cells cultured in the 3D-µFCCS with the engineered soluble microenvironment .127 8.2 Development of an integrated system for physiologically based pharmacokinetic (PBPK) studies 129 8.3 Development of a platform for monitoring the effects of a microenvironment on stem cells .132 CHAPTER References .133 APPENDIX I ImportantProtocols 143 APPENDIX II the Design of the Multi-Channel 3D-µFCCS According to Human Physiology 145 APPENDIX III Imaging-Based Assay for Quantifying the Viability of Hepatocytes .147 LIST OF PUBLICATIONS Zhang C, Zhao Z, Rahim A, van Noort, Yu H. Towards a Human-on-Chip: Culturing multiple cell types on a chip with compartmentalized microenvironments. Lab Chip 2009; 9(22): 3185-3192 Zhang C, Chia SM, Ong SM, Zhang SF, Toh YC, van Noort D, et al. Controlled presentation of TGF-β1 to hepatocyte functions in a 3D-microfluidic cell culture system. Biomaterials 2009; 30(23-24): 3847-3853 van Noort D, Ong SM, Zhang C, Zhang S, Arooz T, Yu H. Stem cells in microfluidics. Biotech Prog 2009;25(1):52-60 Ong SM, Zhang C, Toh YC, Kim SH, Foo HL, Tan CH, et al. A gel-free 3D microfluidic cell culture system. Biomaterials 2008;29(22):3237-44 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(3):302-9 PATENT Zhang C, van Noort D, Yu H. A microfluidic system based localized soluble environment to enhance cell functions by controlled release of growth factors from gelatin microspheres Application Number – PCT/SG2008/000293 Accorded Filling Date – August 2008 SUMMARY Highly functional in vitro cultured cells are of great usefulness in various applications such as cell-based testing, constructing large tissues and pathological research. The key to culturing functional cells in vitro is to recapitulate an in vivo-like cellular microenvironment which allows extensive mechanical support as well as cell-cell, cell-matrix and cell-soluble factor interactions. The development of microfluidic cell culture devices has made possible the controlling cellular microenvironments, phenotypes and behaviors under novel experimentations. Although they are believed to be advantageous over other systems in terms of a more controllable microenvironment, few attempts to engineer a soluble microenvironment for extensive cell-soluble factor interactions within these microfluidic systems are reported. Hence, there is a great need to develop an in vitro model with well-controlled soluble microenvironment to primarily supplement in vivo animal models, thus reducing the cost and ethical issues surrounding animal experimentation. This thesis demonstrates the strategy of engineering soluble microenvironments in microfluidic cell culture systems for various cell types, which significantly enhanced biological functions of either primary cells or cell lines. To engineer the soluble microenvironment, growth factors were loaded to gelatin microspheres (GMs) and the growth factor-releasing GMs were immobilized together with cells in the microfluidic cell culture system to enhance cellular functions. Characterizations have shown that the growth factor-releasing GMs were able to give rise to an isolated and stable soluble microenvironment even under constant fluid perfusion. When multiple cell types were cultured inside one system simultaneously, their individual soluble microenvironment can still be established without interference with each other. Such an in vitro cell culture model could aid or even, in a later stage, replace animal models for studying the systemic effect of a chemical in pharmaceutical or food industry (i.e. the pharmacokinetic and pharmacodynamic characterizations of newly synthesized drugs in the screening process). LIST OF TABLES Table l composition of the growth factors and supplements used in CM Table parameters for cell seeding in 3D-µFCCS Table physiological data for human LIST OF FIGURES Figure schematic of the cellular microenvironment. ECM, soluble factor and some mechanical stress are primarily presented. Cells form extensive cell-cell interactions within the microenvironment. Figure the cellular microenvironment varies with tissue injury. (a) In a normal ECM, cells are anchored to it through multiple receptor-ligand bonds. (b) Tissue injury, which can initially reduce the stiffness of the ECM and the number of ligands, can destabilize receptor-ligand bonds, leading also to a decrease in cellular contractility. (c) Conversely, pathological processes such as tumor formation may lead to enhanced expression of adhesion receptors, an accumulation of ECM and other changes in the microenvironment (for example, hypoxia). Figure schematic representation of the microencapsulation technique. The capsule membrane allows the bi-directional diffusion of nutrients, oxygen and waste, and the secretion of the therapeutic product, but prevents immune cells and antibodies, which might destroy the enclosed cells, from entering the capsule. Figure different types of scaffolds. (A) synthetic scaffold made by a layer-by-layer technique (B) assembly of microscopic Lego-like building blocks into a scaffold (C) a PCL scaffold with surface coating Figure substrates with different patterning. (A) surface coating with proteins (B) surface conjugation with peptides Figure microcontact printing (A) schematic of the microcontact printing on surfaces (B) Endothelial cells plated onto islands of ECM assume the geometry of the stamped region (C) Cells seeded onto bowtie shaped features Figure schematic of generating micropatterns using photolithography technique Figure schematic of microcontact printing using PDMS as a stamp Figure schematic of REM Figure 10 the microscale cell culture analogue (μCCA). Lung cells (L2), liver cells (HepG2/C3A) and fat cells (3T3-L1) were cultured in the μCCA to mimic lung, liver and fat. The geometry of the cell culture compartments 8.3 Development of a platform for monitoring the effects of a microenvironment on stem cells The key focus on stem cell research is to maintain them in vitro and to coax them to differentiation into desired cellular phenotypes.117 Stem cells exist within the complex microenvironment, where extensive cell-cell, cell-matrix and cell-soluble factor interactions are found.19 However, there is still not enough knowledge on the stem cell microenvironment which regulates their self-renewal and differentiation.214, 215 This deficiency of understanding is possibly due to the complexity of the soluble factors in the microenvironment, the lack of available tools for observation113 and the lack to technology to engineer a microenvironment. Our 3D-µFCCS is transparent and allows for imaging and analysis of cellular behaviors. 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Pour 1L of acetone into sterile 1L bottle (x2) 3. Transfer all items into laminar flow hood. 4. Precipitate collagen by pouring 50ml of collagen I solution (Vitrogen 1000) into 1L of acetone. Swirl bottle slightly. 5. Pour excess acetone into a clean bottle and use sterile glass rod to squeeze out remaining acetone from precipitated collagen. Dry inside the hood for 5-10 to allow acetone to evaporate. Remove as much acetone as possible. 6. Pour 500 ml of methanol + 0.1 M HCl into each bottle containing precipitated collagen. 7. Transfer into 4°C cold room and stir (at low speed) for days. Preparation of dialysis tubings 1. Cut dialysis tubing into length of 32cm (x33) 2. Soak in DI water for 1-2 days; run DI water through the tubings for approximately half a day. 3. Transfer dialysis tubings into a 1L sterile bottle and sterilize with 70% ethanol for 30 min-1 hr (max). 4. Rinse with 2L of autoclaved DI water and store dialysis tubing. Day Sterilized items: z 4L Nalgene containers filled with DI water (x11; containers with red labels should contain magnetic stirrer as well) - autoclaved. z 2L beakers (x2) – autoclaved z Funnel (x1) – autoclaved z Freeze drying bottles (x3) – autoclaved z Freeze drying bottle tops (x3) – soaked in 70% ethanol z Filters for freeze drying bottles (x3) – autoclaved 1. Rinse dialysis tubings with container of sterile DI water and transfer tubings into sterile beaker. 143 2. Tie one end of the dialysis tubing and fill it up with methanol-collagen solution using the sterile funnel. Tie the other end of the dialysis tubing to secure it. 3. Place filled dialysis tubing into 4L container with sterile DI water (red label with stirrer). 4. Repeat step 2-3 until all methanol-collagen solutions are transferred into dialysis tubings. 5. Place container into 4°C cold room. 6. Complete changes of water with remaining containers of DI water with the next 24 hours. Check the PH of the water (~7 after changes of water). 7. Pour away excess water into waste bottle. Transfer all the dialysis tubings into a sterile beaker. 8. Untie dialysis tubing and transfer collagen solution into sterile 50 ml tubes. 9. Freeze collagen solution at -30°C. 10. Freeze dry collagen. 11. Collect dried collagen from 50 ml tubings and pool collagen modified from an initial volume of 50 ml collagen. Weigh collagen and indicate on the weight on the tubings. Note: when preparing collagen solution, DO NOT weighs small quantities of collagen as it increases weighing errors. B. Procedures for seeding cells in 3D-µFCCS Table 2: parameters for cell seeding in 3D-µFCCS SINGLE CHANNEL Cell • hepatocytes density = 1-2x106 cells/ml immobilization • Withdrawal flow rate = 0.02 – 0.04 ml/hr Complex coacervation (methylated collagen and terpolymer) Medium perfusion for cell culture Flow rates of polyelectrolytes during holdi complex coacervation time reaction (ml/hr) Terpolymer Methylated (P1) collagen(P2) 0.2-0.25 0.01 0.2-0.25 4-CHANNEL • cell line density (cell lines)= 9-10x106 cells/ml • Total withdrawal flow rate (withdrawal pump) = 0.02-0.04 ml/hr Flow rates of polyelectrolytes during holdi complex coacervation time reaction (ml/hr) Terpolymer Methylated (P1) collagen(P2) 0.4-0.5 0.01 0.4-0.5 Perfusion flow rate = 0.05-0.07 Perfusion flow rate = 0.15-0.25 ml/hr ml/hr 144 APPENDIX II the Design of the Multi-Channel 3D-µFCCS According to Human Physiology Table 3: physiological data for human Body weight (kg) 70 Cardiac output (CO) (L/hr) 347.9 Organ weight % of body weight Liver Kidney Fat Lung 3.14 0.44 23.1 1.15 Organ blood flow % of CO Liver Kidney Fat Lung 25.0 25.0 5.0 10 Calculation of resistances Cell lines are used to construct the artificial organs in the four-channel system. The difference of their size and density is negligible. Thus, provided the constant width of the channels, the ratio of cell mass in each channel is the same as that of the channel length. Ro is incorporated into the system as a resistance to facilitate blood distribution. When the channels are fully packed with cells, the final resistances of the channels should be reversely proportional to their flow distribution (Table 1). Rlu: resistance of the lung channel Rli: resistance of the liver channel Rk: resistance of the kidney channel Rf: resistance of the fat channel Ro: resistance of the other organ channel Rli= Rk, Rf= Rk, Ro= 5/9 Rk Flow distribution through lung is 100% Figure 35: flow distribution resistances However, each channel has an intrinsic resistance due to the ratio of the organs. Thus, 145 by incorporating additional resistances to specific channels, the final resistances can be obtained to mimic flow distribution. The calculation process is shown in Fig. 36. Resistance value of channel, with cells and flow distribution Rli =3.14/0.44Rk = 7.1 Rk; Rli = Rk Rf = 3.465/.044Rk = 7.9 Rk; Rf = Rk Ro =5/9 Rk Rl = 1.15/0.44Rk = 2.61 Rk Total resistance Rk = Rk + 6.1 Rk Rli = 7.1 Rk Rf = 7.9 Rk + 27.6 Rk Ro = 3.9 Rk Rl = 2.61 Rk Figure 36: total resistances (Red highlights indicate the value of the additional resistances) To ensure enough cells for biological characterization, the lower limit of cell number in the microfluidic channel is 5000. The size of a cell is around 10 µm (By empirical observation). Thus, the volume of a cell is no more than 1000µm3 (10µm×10µm×10µm). Based on this assumption, and provided that the width of the channels is consistent (200µm+200µm+200µm), the length of the channels can be calculated: Liver: cm Kidney: 0.14 cm Fat: 1.1 cm Lung: 0.36 cm 146 APPENDIX III Imaging-Based Assay for Quantifying the Viability of Hepatocytes Figure 37: confocal images of staining for necrotic and total cell population in the 3D-µFCCS after 24 hours of treatment with 40 mM APAP (A) The necrotic cell population was stained with 25 μg/ml PI and (B) the total cell population was stained with 5μg/ml Hoechst 33342. (C) Overlay of red and blue channels where blue indicates total cells and co-stained red nuclei indicate necrotic cells. Images are projection of a 100 μm optical stack at μm interval. Scale bars = 50 μm. 147 [...]... microenvironment in vitro In order to create more in vivo-like microenvironments for cell culture, many engineering methods are employed and both natural and synthetic biomaterials are chosen for fabricating various systems This section will focus on reviewing different systems as well as materials for engineering a microenvironment in vitro a) microencapsulation of cells for in vitro culture Microcapsule... traditional way for obtaining better defined surfaces is by precoating the surfaces with a purified ECM protein, followed by blocking remaining cell adsorption sites on the surfaces with a nonadhesive protein such as albumin.31 However, these nonadhesive proteins are prone to degradation.31 Thus, a more stable blocking agent other than nonadhesive proteins is in need A more flexible systems to modify surfaces... fabricated using photolithography The microcontact printing normally provides the patterning of SAMs (e.g alkanethiol) to control the adsorption of subsequent proteins of interest to facilitate the patterning of cells on surfaces.89, 90 Figure 8: schematic of microcontact printing using PDMS as a stamp.90 b) replica molding (REM) Replica molding is the process of duplicating information in the surface... miniaturizations of a wide range of biological systems, 82, 83 whereas others focus on low Reynolds numbers for obtaining controllable laminar flows which are common in the microcirculations in vivo.15 Cells cultured in microscale systems can be easily incorporated into micro-total analysis systems (μ-TAS) that includes cell culture, cell sorting and analysis in one device to probe the biochemical processes... includes fabrication, handling and practical use of the chips Streams of gases or fluidized solids/particles in microscale are also considered a form of microfluidics 64, 91 Microfluidic channels can be obtained by contacting or bonding a substrate surface with channel structures in PDMS and these channels allow fluid delivering.17, 92 The flow inside microfluidic channels are always laminar, meaning... cells in a 3D 25 fashion, enabling the delivery of soluble signals to cells in all directions Scaffolds and hydrogels have been used extensively in engineering 3D in vitro cell culture systems For example, breast epithelial cells encapsulated and cultured within 3D hydrogel formed acinus structures, resembling the morphology found in vivo.57 Fibroblasts in 3D collagen gels have a different shape and a... mostly use poly(dimethylsiloxane) (PDMS), since it is biocompatible, optically transparent, gas permeable and durable.76, 88 The most commonly used patterning methods involving PDMS are microcontact 31 printing and replica molding.76 a) microcontact printing Instead of patterning a surface using photoresist, PDMS can be used as a stamp to pattern the material of interest onto a substrate surface (Fig 8)... hypothesized that stable cell -soluble factor interactions can be established for 17 functional enhancement of cells, with laminar flow within microfluidic devices and continuous release of soluble factors from their carriers Thus, to test the hypothesis, we report in this thesis a strategy to engineer a soluble microenvironment by controlled-releasing of a growth factor, transforming growth factor-beta 1... medium in this case) was the same as that in vivo Figure 11 schematic representation of the microfluidic channels (A) the original microfluidic channel (B) the modified microfluidic channel (C) the connections and connectors needed for the original microfluidic channel (D) the connections and connectors needed for the modified microfluidic channel (E) the uneven flow caused in the original microfluidic. .. and removal of soluble factors.15 Recently, many three-dimensional (3D) microfluidic devices have been developed for enabling cell-cell and cell-matrix interactions during cell culture.16, 17 However, few attempts to engineer a soluble microenvironment for extensive cell -soluble factor interactions within the microfluidic devices are reported Hence, there is a great need to develop an in vitro model . ENGINEERING SOLUBLE MICROENVIRONMENTS IN MICROFLUIDIC SYSTEMS ZHANG CHI B. Eng. (South China University of Technology) . 59 4.2.5 Seeding GMs in the 3D-μFCCS 59 4.2.6 Cell maintenance 59 4.2.7 Seeding of cells in the 3D-μFCCS 60 4.2.8 Cell viability staining 60 4.2.9 Seeding of GMs and cells in the 3D-μFCCS. other systems in terms of a more controllable microenvironment, few attempts to engineer a soluble microenvironment for extensive cell -soluble factor interactions within these microfluidic systems