1. Three-Dimensional Closed-Loop Microfabricated Bioreactor Michael Hwang, Jenny Lu, Alex Makowski, Advisors: Lisa McCawley 1, Dmitry Markov 2, Phil Samson 2, John Wikswo Vanderbilt University, Department of Biomedical Engineering; Vanderbilt Medical Center, Department of Cancer Biology 1 ; Vanderbilt Institute for Integrated Biosystems Research and Education 2 Microfabricated bioreactors offer significant advantages over traditional cell culture techniques: o constrained μ-scale dimensions o small volumes and cell numbers o physiologically relevant environment Our VIIBRE developed bioreactors are: o fabricated from biologically inert PDMS material o suitable for multicelluar 3D cell structure cultures (e.g. MCF10A) o designed for automated measurements and long-term culture The combination of the above features of the bioreactor allows for tissue morphology formations in a simulated in vivo environment with accurate data collection and feedback-mediated homeostasis. Goals for this Project- Develop a Bioreactor capable of: o supporting long-term cell culture o taking near real time pH measurements o growing 3D multicellular morphological structures o maintaining constant nutrient supply and waste removal BACKGROUND MATERIALS AND METHODS pH Sensors – Iridium Oxide (IrOx) o inexpensive and manufacturable o low sensitivity degradation o fast response time o ease of integration Flow-Through IrOx pH Sensor Microfluidic Bioreactor Instrumentation System o bioreactor o computer controlled Cavro XLP 6000 syringe pumps o PDMS mixing manifold o IrOx sensors working in differential mode o instrumentation amplifier circuit o LabVIEW analysis and control software 3D Culture of MCF10A Human Breast Epithelials o epithelial cells are the origin of 80% of all breast tumors (Knauss) o excellent model system for multicellular 3D structure in our bioreactor o fully develop acinar (hollow sphere) mammospheres after 20 days o relatively inexpensive to purchase and maintain o Approved growth protocols and cells readily available from ViCBC Illustration of Acinar Morphology Formation (from Debnath and Brugge 2005) MATERIALS AND METHODS Bioreactor Components o inert PDMS cell growth chamber contains Matrigel matrix and cells o nanopore filter provides diffusive nutrient exchange and prevents clogging o microfluidic supply network provides media/waste exchange o fluidic ports in acrylic lid interfaces device with pumps o brass clamp provides structural integration RESULTS AND DISCUSSIONS Original Device Design and Fabrication o generated assembly and fabrication protocols  repeatable characteristics o device cross-sections show appropriate feature dimensions and shape o gravity feed tests show viable sealed fluidics and quantifiable flow o volume calculations indicate maximum diffusion volume <12 μL Miniature Flow-Through pH Sensor Design and Fabrication o Ti-Iridium Oxide ph sensors fabricated and fully characterized o average responsivity of >60mV/pH o sensor drift was within ± 2mV o response time ~7sec with 2.0pH change o no significant hysteresis Graph of bioreactor pH measurements during pumping indicates cell health MCF10A Cells Formed Acini in Bioreactor Culture Well Optimized Concentration of Cells for Culture Chamber: ~1500 cells seeded Acinar morphology in bright field image (left) confirmed by Confocal “Z-stack” (right) Microfluidic Instrumentation System Developed LabVIEW modules for: o automated sensor calibrations o pH measurements o Cavro pump control Debnath, J., Brugge, J. S., Modeling glandular epithelial cancers in three-dimensional cultures, Nature Reviews Cancer 5, 675-688 (2005). Knauss, U. “Cell Growth Control of Breast Epithelial Cells”. Abstract. California Breast Cancer Research Program. Differential expression and subcellular and localization of the GTPase Rac3. FASEB Meeting: Small G-Proteins & Cell Dynamics, 2002. Special Thanks to: Ron Reiserer, Eduardo Lima, Igor Ges, Franz Baudenbacher, Don Berry, S. Marzouk, David Schaffer, David Shifrin, Bryan Gorman, Steven Manuel for device machining, and Dr. King and NCIIA for additional funding. WORKS CITED / ACKNOWLEDGEMENTS Future Directions o run fully sterilized integrated bioreactor for 20 Days o optimize low volume pH measurements and fabricate tube IrOx sensors o implement feedback loop automation (<.4 pH window from 7.0) 100x100 μm channels molded in PDMS 1.875 in. 8mm centered 47mm centered Potential (mV) IrOx pH sensors on 125 μm diameter Titanium Wires pH y = -70.647x + 680.15 R 2 = 0.999 300μm 1mm 2mm 3mm Access Ports Plexiglas Channel Layer - PDMS Glass Slide ~1.9 in. square Matrigel with Cells (8mm diam.) Bioreactor IA + - DAQ Device Iridium Oxide pH-Sensing Electrode pH 8 Calibration Solution pH 6 Calibration Solution Cell Media Reservoir Faraday Cages Bias Current Return Path Cavro XLP 6000 Syringe Pumps Iridium Oxide Quasi-Reference Electrode Acidified Media Conclusions o supported 20 day cell culture in newly designed bioreactor o measured acidification with near real time pH measurements o grew MCF10A acini 3D structures using microfluidics o maintained consistent nutrient exchange via LabView modules Time (sec) Potential (mV)