Engineered, Perfusable, Human Microvascular Networks on a Microfluidic Chip by MC4 MASSA O)' vl jkTlJTE I Jordan Ari Whisler OF TECHNOLOGY NOV 12 2013 B.S. Mechanical Engineering 3.RARESI Washington University in St. Louis, 2009 SUBMITTED TO THE DEPARTMENT OF MECHANICAL ENGINEERING IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MECHANICAL ENGINEERING AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY SEPTEMBER 2013 @Massachusetts Institute of Technology 2013. All rights reserved. Signature of Author: Department of Mechanical Engineering August 26, 2013 Certified by: Roger D. Kamm Professor of Mechanical Engineering Thesis Supervisor Accepted by: David E. Hardt Chairman, Committee for Graduate Students Department of Mechanical Engineering Engineered, Perfusable, Human Microvascular Networks on a Microfluidic Chip by Jordan Ari Whisler Submitted to the Department of Mechanical Engineering on August 26, 2013 in Partial Fulfillment of the Requirements for the Degree of Master of Science in Mechanical Engineering ABSTRACT In this thesis, we developed a reliable platform for engineering perfusable, microvascular networks on-demand using state of the art microfluidics technology. We have demonstrated the utility of this platform for studying cancer metastasis and as a test bed for drug discovery and analysis. In parallel, this platform enabled us to study, in a highly controlled environment, the physiologic processes of angiogenesis and vasculogenesis to further elucidate their underlying mechanisms. In addition to using our platform for real-time observation of physiological processes, we also took advantage of the ability to influence these processes through precise control of the extracellular environment. By manipulating the mechanical and bio-chemical inputs to our system, we controlled the dynamics of microvascular network formation as well as key properties of the network morphology. These findings will aid in the design and engineering of organ specific constructs for tissue engineering and regenerative medicine applications. Finally, we explored the potential use of stem cells for engineering microvascular networks in our system. We found that human mesenchymal stem cells can act as secondary, support cells during microvascular network formation. Thesis Supervisor: Roger D. Kamm Title: Cecil and Ida Green Distinguished Professor of Biological and Mechanical Engineering 2 Acknowledgments I would like to thank my advisor, Professor Roger Kamm, for his scientific insight as well as his motivation and support throughout this project. He was willing to take me in as an inexperienced researcher with little background in biology, and had the foresight and patience to allow me to develop the necessary skills to complete a successful project and make an independent contribution to the scientific community. His rigorous scientific and ethical standards serve as an example for all of his students. A special thanks to Joan and Leslie, in the MechE grad office, for keeping me up to date with all the requirements and deadlines and making sure that this thesis became a reality. You are always looking out for your students, and we greatly appreciate it. I would also like to thank my parents for their continuous support throughout my academic career thus far. They have always encouraged me to pursue my passions and provided me the resources to do so. They have instilled in me the belief that any accomplishment is possible with the right attitude and lots of hard work. Finally, I would like to thank my fiancee, Rena. You have experienced my late nights in the lab and intensely focused periods of writing, and you still want to marry me. In truth, the long hours are bearable only because I know you will be there waiting when I finish. The material in this thesis is based upon work supported by the National Science Foundation Graduate Research Fellowship and the Science and Technology Center Emergent Behaviors of Integrated Cellular Systems (EBICS) Grant No. CBET-0939511. 3 Table of Contents In tro d u ctio n ................................................................................ 7 Chapter 2: Microfluidic Device Fabrication and Designs ........... 9 Device Design ............................................................................................................................................ 9 Device Fabrication ................................................................................................................................... 10 Gel Filling M ethod ................................................................................................................................... 10 M icro-injection .................................................................................................................................... 10 Gel Filling Port ..................................................................................................................................... 11 Com parison ......................................................................................................................................... 12 Chapter 3: Vascularization Strategies ...................................... 14 Angiogenesis ........................................................................................................................................... 14 S e e d in g ................................................................................................................................................ 1 5 Angiogenesis Device ........................................................................................................................... 15 R e s u lts ................................................................................................................................................. 1 7 Alginate Beads ........................................................................................................................................ 18 Bead Form ing M ethods ....................................................................................................................... 19 Results and Discussion ........................................................................................................................ 22 Interm ediate Post Device ........................................................................................................................ 23 Vasculogenesis ........................................................................................................................................ 25 Com parison ............................................................................................................................................. 27 Chapter 4: Control of Microvascular Network Morphology ..2.9. Angiogenesis ........................................................................................................................................... 30 Effects of Extracellular M atrix ............................................................................................................. 30 Growth factors .................................................................................................................................... 34 Interstitial flow .................................................................................................................................... 35 Vasculogenesis ........................................................................................................................................ 38 M aterials and M ethods ....................................................................................................................... 40 R e su lts ................................................................................................................................................. 4 3 4 D isc u ssio n ............................................................................................................................................ 5 0 Chapter 5: Stem Cells for Microvascular Engineering......55 Hum an M esenchym al Stem Cells (hM SCs) ......................................................................................... 55 M ouse Em bryonic Stem Cells (m ESCs).............................................................................................. 57 Conclusion and Future Directions..........................................60 References............................................................................ 61 Appendix 1: Seeding ECs in Fibrin Gel....................................65 Appendix II: P-Values for Figures 17-20................................66 5 List of Figures Figure 1. Two designs implementing the gel filling port system.. ......................................................... 12 Figure 2. Diagram and results using angiogenesis gel filling device. .................................................... 17 Figure 3. Confocal images of perfusable microvascular network formed using angiogenisis method.....18 Figure 4. Schematic diagram of the air-cutting method for producing alginate beads........................ 20 Figure 5. Setup of the two-phase microfluidic alginate bead making system........................................ 21 Figure 6. Alginate bead angiogenesis device. ........................................................................................ 22 Figure 7. Dextran perm eability experim ents. ........................................................................................ 23 Figure 8. Interm ediate post device. ........................................................................................................... 24 Figure 9. Results from interm ediate post device.................................................................................. 25 Figure 10. Vasculogenesis experim ents.................................................................................................. 26 Figure 11. ECM com position com parison. ............................................................................................ 31 Figure 12. PEG gel im m obilization of fibroblasts ..................................................................................... 32 Figure 13. M echanical effects on angiogenesis. .................................................................................... 33 Figure 14. Biochem ical effects on angiogenesis. ................................................................................... 34 Figure 15. Interstitial flow effects on angiogenesis. ............................................................................. 36 Figure 16. Multiculture vasculogenesis device: diagram and results. .................................................. 39 Figure 17 . Fibro blast stabilizatio n.............................................................................................................. 44 Figure 18. Paracrine siganling effects on vasculogenesis network morphology. .................................. 46 Figure 19. Fibrin concentration effects on vasculogenesis network morphology.................................47 Figure 20. EC seeding density effects on vasculogenesis network morphology. .................................. 49 Figure 21. Control of microvascular network morphology: aproach and results...................................53 Figure 22. Human bone marrow derived MSCs cultured with ECs in our microfluidic device..............56 Figure 23. M ouse em bryonic stem cells for angiogenesis ..................................................................... 57 Figure 24. Mouse embryonic stem cells for vasculogenesis................................................................... 58 6 Introduction These are exciting times for the fields of Tissue Engineering and Regenerative Medicine. Recent headlines have reported the successful implantation of engineered tracheas, bladders, and urethras in human patients.-3 There are currently several viable products on the market for cell-based skin grafts used to treat burn and wound victims.4'5 All these tissues can be grown from a patient's own cells, reducing the risk of rejection by the body. To date, however, the success stories have been limited to thin tissues providing basic structural function which do not require a functional vasculature for tissue survival. In order to engineer sustainable thick and complex tissues and organs, it will be necessary to include such vasculature to provide oxygen and nutrients to the cells to meet their metabolic needs upon 6 implantation. This is the goal of microvascular tissue engineering. Tissue engineers are currently pursuing two approaches to supplying engineered tissues with a functioning microvasculature: 1) host induced vascularization and 2) pre-vascularization. The former attempts to minimize the time that implanted cells must survive without a proper nutrient supply by inducing angiogenesis from the host vasculature through a combination of angiogenesis promoting matrix ligands and growth factor delivery.7 The latter attempts to eliminate the time to vascularization by engineering a functional vasculature into the tissue construct before implantation. 8, Successful implementation will likely comprise a combination of the two. The findings of our work advance the causes of both approaches. Our pre- vascularized tissue constructs can potentially be implanted directly into the body for therapeutic applications. Additionally, the platform has enabled us to study the process of 7 vascularization in great detail, providing insight and strategies for more quickly and effectively inducing angiogenesis in-vivo. In this thesis, we present the development and application of a new microfluidic platform for engineering perfusable microvascular networks in a 3D environment. Chapter 2 discusses the technical designs and methods for fabrication of the device and carrying out cell culture experiments. Chapter 3 describes the various forms of vascularization that our device is capable of replicating along with a discussion of the relevant applications for each. Chapter 4 presents our major contribution to the field of microvascular tissue engineering in our efforts to control the process of vascularization using the mechanical and biochemical inputs of our microfluidic system. Chapter 5 describes our studies on the use of stem cells for microvascular tissue engineering and cell therapies. 8 Chapter 2: Microfluidic Device Fabrication and Designs To carry out our work, we have taken advantage of the latest in microfluidic technology for mammalian cell-culture. The use of microfluidics for cell culture provides several advantages over traditional biological assays.'0 The miniaturization of in-vitro lab experiments through the use of microfluidics enables many conditions to be tested while minimizing the volume of expensive reagents used. It allows for precise control of the geometry and the chemical composition of the environment in which cells are cultured. Importantly for our studies, it allows for spatial segregation of cell types while still maintaining close enough proximity for the cells to communicate. It also enables controlled fluid flow to be applied to cells cultured in 3D. Device Design We based the design of our microfluidic device for vascularization upon previous designs from our lab for devices used to study the initial stages of sprouting angiogenesis."- 3 These designs provide a vertical interface between the hydrogel and the medium channel (see Figures 1 & 2 for illustrative diagrams) on which a confluent endothelial monolayer can be formed. It is from this vertical monolayer that the endothelial cells sprout horizontally in to the hydrogel, enabling the process to be visualized in real time using standard microscopy. We needed to make several key adjustments to these previous designs to ensure complete, efficient, and uniform vascularization of the entire gel region. These adjustments, and the findings that motivated them, are further discussed in Chapter 3. 9 Device Fabrication We used standard procedure for generating silicone molds.' Briefly, CAD designs were generated and used to print negative pattern transparency masks. A 100 pm layer of SU-8 photoresist was coated onto a silicon wafer, and the mask was used to photo-polymerize the pattern on to the wafer. The mask was repeatedly used to form microfluidic chips. Briefly, PDMS (Ellsworth Adhesives, WI USA) and curing agent were mixed at a 10:1 ratio and poured onto the wafer. After degassing, the PDMS was baked in an 80 degree oven for two hours. The individual devices were then cut out and a biopsy punch was used to create ports for gel filling and medium channels. Tape was used to remove dust from the surface, and the devices were placed in an autoclave for sterilization. Clean devices and coverslips were plasma treated (Harrick Plasma, CA, USA) and bonded together. Gel Filling Method We used two different techniques for filling the gel chamber of our microfluidic device with hydrogel: (1) micro-injection (2) and gel-filling via a port in the closed device. Each technique presents its own advantages and disadvantages. Ultimately, the gel-filling design was preferred due to its consistent formation of a uniform gel/channel interface and avoidance of leakage. Micro-injection In the micro-injection method developed by Vickerman et. al.' 5 (see this reference for a detailed protocol and parts list), sterile devices are plasma treated and allowed to recover some 10