Design of an integrated microfluidic system for culturing patterned tissue Allyson Fry1, Bryan Gorman1, Jonathan Lin2, William Wong1 Advisors: John Wikswo1, Dmitry Markov1, Lisa McCawley3, Phil Samson1 Departments of 1Biomedical Engineering, 2Electrical Engineering, and 3Cancer Biology at Vanderbilt University. Introduction Microfluidic pH Control Characterization of Tissue Culture Model The primary limitation of growing thick tissues in vitro is lack of proper vascular supply for nutrient and metabolite transport. Here, we report progress in developing micro-bioreactors with the ultimate goal of guiding the formation of externally perfused capillary beds in vitro. This project involves engineering an environment in which microvascular endothelial cells can differentiate and form tubular, capillary-like structures.  Currently available micro-bioreactor designs [1-3] do not provide physiologic perfusion over large areas, while allowing manipulation of the local microenvironment. While in situ bioreactors represent the future of tissue engineering and rehabilitation, our approach is directed towards massively parallel microenvironments for basic research in cellular and systems biology. We continue work begun last year to develop components of the bioreactor with the goal of bringing perfused microenvironments to a new level of sophistication and practical laboratory utility. The metabolic processes of living cells cause significant changes in extracellular pH levels. Left alone, such acidification renders the microenvironment inside the bioreactor inhospitable, adversely affecting cell growth. A system to monitor and compensate for significant deviations in pH was thus developed. It consists of a pH sensor placed at the bioreactor exit and two syringe pumps with low- and high-pH RPMI media. The control program constantly monitors pH levels and adjusts the mixing ratios of the media from both pumps to maintain optimal cell growth conditions. In contrast to using a buffered perfusate, this process allows us to monitor the acidification rate of the cells and thus assess the metabolic status of the tissue culture. The organotypic culture used in this project is adapted from a model developed by Manuela Martins-Green [4]. Our cultures contain two layers of human dermal fibroblasts embedded in a collagen matrix and separated by a layer of human microvascular endothelial cells. This culture is constructed in a transwell chamber in a normal 12-well culture plate. The culture is fed from both top and bottom as shown in the diagram below. In this tissue culture model the, molecular cross-talk between two types of cells results in cell rearrangement and formation of microvessels, which can be visualized using standard staining techniques. a Project Goals a a H&E The long-term goals of the VIIBRE bioreactor project: Develop an in vitro microvascular network that will: Create a realistic extracellular matrix Allow evaluation of angiogenesis mechanisms Provide environment for endothelial cell differentiation Facilitate formation of stable tubular structures Support a flow of perfusate Allow study of transendothelial migration Our team addressed the following four problems: Development and fabrication of several classes of microfabricated capillary scaffolds Development of a microfluidic pH control system Histological characterization of an angiogenic tissue culture model 4. Integration of scaffolds into tissue culture a a CD-31 Diagram of a transwell culture plate used to promote formation of microvessels in the intermediate layer, thus making it an ideal platform for scaffold-guided vessel development and study of angiogenesis. Social Impact a a In order to study the functions and mechanisms of a system, scientists commonly use animal models. However this practice has several shortcomings. First, use of animals in research is fraught with ethical implications concerning the value of animal life. Second, results can never fully predict the response of human tissue to treatment. Finally, an animal system is relatively difficult to manipulate. Our project overcomes these shortcomings by creating a tissue microenvironment completely based upon human cells, which can also be instrumented to a greater degree possible than in animal models. Diagram of the perfusion system providing nutrients to the cells and maintaining pH and oxygen (future) levels aSMA Physical constraints for pH control Cells Media needed (ul/cell/hr) Flow rates (ul/hr) Volume of bioreactor (ul) Desired pH range 45,000 4e-4 5-20 1-10 7.2-7.6 Bioreactor Design a a Vimentin The function of our bioreactor is to add another spatial dimension to transwell plate cocultures. The design consists of parallel nutrient supply networks perfusing layers of collagen seeded with fibroblasts. Nano-pore filters support the collagen and allow nutrient/waste exchange. A scaffold seeded with endothelial cells is placed between the collagen layers. The inspiration for the scaffold design is the fractal pattern of vasculature occurring in nature. Cells grown in fractal flows are exposed to similar nutrient, waste, and shear stress levels. Once the endothelial cells in the capillary perfusion system have grown to confluence, the system can support internal flow. Proportional Differential Integral (PID) control scheme Developed tissue cultures stained with H&E and three antibodies. Arrows indicate microvessels. For reference, the filter is 10 mm thick. Modeling and experimental results The bioreactor design concept The design and dimensions of the capillary scaffold For experimental tests of the pH control system, a third pump was used to inject a low pH solution into the bioreactor in order to perturb the pH and imitate the presence of living cells. Additionally, a numerical simulation was constructed in order to verify the experimental results. Scaffold Fabrication and Tissue Culture Integration H & E stained sections of transwell cultures showing healthy fibroblasts as well as microvessel structures in two adjacent sections separated by 15 mm. A microvessel sectioned semi-longitudinally. For reference, the filter is 10 mm thick. H&E Stain. We were able to fabricate the following three different types of tissue culture scaffold: The PDMS ‘Egg-crate’ scaffold Advantage: supports arterial and venous branches in rigid network Disadvantages: difficult to construct, not biodegradable The patterned Matrigel scaffold Advantage: biodegradable Disadvtantage: poor mechanical features The SU-8 scaffold Advantages: good mechanical properties, easy fabrication Disadvantage: not biodegradable We then integrated each type of scaffold into an organotypic tissue culture model and analyzed the results. Control signal and resulting changes in pump rates during the same experiment Real – time pH control following onset of low pH perturbation Simulated pH response Accomplishments: We have grown and characterized an angiogenic tissue culture model by employing several histological labeling methods. 400 Real - time experimental pH response 300 Pump rate uL/hr Pump Rate of High pH Buffer Conclusion 200 In conclusion, we have accomplished the following: We microfabricated tissue culture scaffolds using three different biomaterials. We integrated each type of scaffold into a known tissue culture model. We developed LabVIEW software which integrates a sensor and syringe pumps for real-time pH control. We experimentally verified the performance of a real-time pH control program in a microfluidic environment, We compared our pH control experiments with numerical simulations, We implemented a multi-cell angiogenic tissue culture model, and We have applied various histological staining methods to characterize the developed tissues We have met all four specific project goals outlined in the introduction. Desired pH range: 7.2 – 7.6 Pump Rate of Low pH Buffer 100 The photolithographic mask used to define the structures of the scaffold and supporting fluidics Control Signal (P+I+D) Accomplishments: We have demonstrated computer control of pH in a microfluidic environment. Experimental results are in excellent agreement with numerical simulations. Acknowledgements Cost and Market Analysis We would like to thank Jason Greene, who contributed suggestions for device design and testing. This project is a continuation of a senior design project started in 2004-2005 by Barnett, Garrett, Harvill, Mayer, and McClintock. This project was made possible by use of resources provided by the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) directed by Dr. John Wikswo. Dr. Paul King is the course instructor for BME 272. The predicted market for this product is cellular and systems biology researchers and drug companies desiring to perform massively parallel assays on realistic in vitro human models. Although the pumps, sensors, and computer hardware cost over $8,000, the plastic microfluidic chips are disposable and can be batch-produced off a single mold for less than $1 each. The minimal cost of the microfluidics and supporting framework is estimated at $500. Thus, the system represents an excellent ratio of cost to high-throughput research benefit. Raw Material Expenses of Project Microfabrication $1,325.26 Tissue Culture $3,880.00 Control Systems $8,880.00 Total $14,047.39 References SU-8 mold of capillary scaffold A free-standing PDMS scaffold on glass The PDMS scaffold in cross-section Accomplishments: We have fabricated three different types of tissue culture scaffold and integrated them into a tissue culture model. Leclerc et al.  Biotechnol. Prog. 20, 750-755, 2004, . Borenstein J et al. Biomedical Microdevices 4, 167-175, 2002. 3. Shin M et al. Biomedical Microdevices 6, 269-278, 2004.5 4. Martins-Green M, Li QJ, and Yao M. FASEB 2005; 19:222-224.