Three-Dimensional Closed-Loop Microfabricated BioreactorMichael Hwang, Jenny Lu, Alex Makowski,

Advisors: Lisa McCawley1, Dmitry Markov2, Phil Samson2, John WikswoVanderbilt University, Department of Biomedical Engineering; Vanderbilt Medical Center, Department of Cancer Biology1;

Vanderbilt Institute for Integrated Biosystems Research and Education2

Microfabricated bioreactors offer significant advantages over traditional cell culture techniques:o constrained μ-scale dimensionso small volumes and cell numberso physiologically relevant environment

Our VIIBRE developed bioreactors are:o fabricated from biologically inert PDMS materialo 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 cultureo taking near real time pH measurementso growing 3D multicellular morphological structureso maintaining constant nutrient supply and waste removal

BACKGROUND MATERIALS AND METHODSpH Sensors – Iridium Oxide (IrOx)

o inexpensive and manufacturable o low sensitivity degradationo fast response timeo ease of integration

Flow-Through IrOxpH Sensor

Microfluidic Bioreactor Instrumentation System o bioreactoro computer controlled Cavro XLP 6000 syringe pumpso PDMS mixing manifoldo IrOx sensors working in differential modeo instrumentation amplifier circuito LabVIEW analysis and control software

3D Culture of MCF10A Human Breast Epithelialso epithelial cells are the origin of 80% of all breast tumors (Knauss)o excellent model system for multicellular 3D structure in our

bioreactoro fully develop acinar (hollow sphere) mammospheres after 20 days o relatively inexpensive to purchase and maintaino Approved growth protocols and cells readily available from ViCBC

Illustration of Acinar Morphology Formation (from Debnath and Brugge 2005)

MATERIALS AND METHODS

Bioreactor Componentso inert PDMS cell growth chamber contains Matrigel matrix and cellso nanopore filter provides diffusive nutrient exchange and prevents

clogging o microfluidic supply network provides media/waste exchangeo fluidic ports in acrylic lid interfaces device with pumps o brass clamp provides structural integration

RESULTS AND DISCUSSIONS Original Device Design and Fabricationo generated assembly and fabrication protocols repeatable characteristics o device cross-sections show appropriate feature dimensions and shapeo gravity feed tests show viable sealed fluidics and quantifiable flowo 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 characterizedo average responsivity of >60mV/pHo sensor drift was within ± 2mVo response time ~7sec with 2.0pH changeo no significant hysteresis

Graph of bioreactor pH measurements during pumping indicates cell health

MCF10A Cells Formed Acini in Bioreactor Culture WellOptimized 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 measurementso 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 Directionso run fully sterilized integrated bioreactor for 20 Dayso optimize low volume pH measurements and fabricate tube IrOx sensorso implement feedback loop automation (<.4 pH window from 7.0)

100x100 μm channels molded in PDMS

1.875 in.

8mmcentered

47mmcentered

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IrOx pH sensors on 125 μm diameter Titanium Wires

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y = -70.647x + 680.15R2 = 0.999

300μm1mm

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Access Ports

Plexiglas

Channel Layer - PDMS

Glass Slide~1.9 in. square

Matrigel with Cells (8mm diam.)

Bioreactor

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DAQDevice

Iridium OxidepH-SensingElectrode

pH 8CalibrationSolution

pH 6CalibrationSolution

Cell MediaReservoir

FaradayCages

Bias CurrentReturn Path

Cavro XLP6000 SyringePumps

Iridium OxideQuasi-ReferenceElectrode

AcidifiedMedia

Conclusionso supported 20 day cell culture in newly designed bioreactoro measured acidification with near real time pH measurementso grew MCF10A acini 3D structures using microfluidicso maintained consistent nutrient exchange via LabView modules

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