MONOLITHIC 3-D MICROFLUIDIC DEVICE FOR CELL ASSAY WITH AN INTEGRATED COMBINATORIAL MIXER 陳睿鈞 Mike C. Liu, Dean Ho, Yu-Chong Tai Department of Bioengineering, California Institute of Technology, Pasadena, USA Department of Biomedical and Mechanical Engineering, Northwestern University, Evanston, USA Department of Electrical Engineering, California Institute of Technology, Pasadena, USA Transducers’07 pp

Outline Introduction Device design and fabrication Experimental and discussion Conclusion

Outline Introduction Device design and fabrication Experimental and discussion Conclusion

Biological Assays Devices Drug screening and biological assays often include multiple combinations of different compounds. Traditional screening tools Shane J. Stafslien, 2005T. Chapman, 2003 Microfluidic devices Poor small-volume liquid handling ability Large consumption of reagents High cost of operation robotics multi-well plates Inexpensive chip-platforms High-density arrays Only expose cells to a single compound at once P. J. Lee, 2006K. R. King,2007

3-D Microfluidic Combinatorial Mixer Combinatorial Mixer LOC device Individually chamber Streams control

Outline Introduction Device design and fabrication Experimental and discussion Conclusion

Design Three inputs seven possible outputs One control channel Overpass Allow one microfluidic channel to cross over other microfluidic channels Cell culture-chambers Cells culture Combinatorial mixer Deliver different solution combinations to the culture-chambers 1 cm×1 cm chip

Device Fabrication 2.Parylene-coated Si : 3μm Sacrificial photoresist AZ4620 : 15μm Parylene : 10μm 1.Si wafer clean : H 2 SO 4 :H 2 O 2 = 3:1 Promote adhesion : DI water:IPA:A-174 = 100:100:1 3.Pattern parylene : oxygen plasma 4.Sacrificial photoresist AZ4620 : 32μm Parylene : 10μm 5.SU-8 : 100μm Elute AZ4620 : IPA

Packaging PDMS layer 1. Gasket layer to provide proper sealing 2. Adapter to connect the tubes 3. Adjusted as open or blocked Transparent acrylic Milled with a computer-numerical controlled (CNC) machine Teflon tubes Plugged into the holes of the PDMS layer Programmable syringe pumps Controll the food coloring solutions load and the flow rate Appliance

Outline Introduction Device design and fabrication Experimental and discussion Conclusion

Combinatorial Mixer Operated flow rate : 10L min−1 flow rate : 0.1L min−1 D : diffusion coefficient U : fluid velocity w: channel width Z : distance during time period

Microfluidic Cell Culture The cells were grown with continuous perfusion of culture media and pictures were taken 4 h, 16 h and 42 h after cells were loaded. 1.UV irradiation 70% ethanol solution PBS solution 0.05% polyethyleneimine (PEI) : 24h 2.B35 cells adhered to the culture-chamber : 4 h 3.Continuous perfusion of culture media at flow rate of 33 nL/min, 37 ° C.

Simple Cell Assay 1.B35 cells injected 4 h. 2.Injecting 3 cell stains : crystal violet, methylene blue, neutral red. 3.The combinatorial mixer 4.The various combinatorial streams into the cell culture- chambers. 5.Cells were stained with different color patterns

Conclusion The ability to simultaneously treat arrays of cells with different combinations of compounds. The fruition of such system will enable LOC devices to perform highly parallel and combinatorial chemical or biochemical reactions with reduced labors, reagents and time. The fabrication technology can enhance the functionalities of current LOC devices by integrating the devices with complex 3-D microfluidic networks. Future work Monitoring cell growth, more complicated cellular response Real-time monitoring of gene expression

References Mike C. Liu, Dean Ho, Yu-Chong Tai, “Monolithic fabrication of three-dimensional microfluidic networks for constructing cell culture array with an integrated combinatorial mixer”, Sensors and Actuators B, P. J. Lee, P. J. Hung, V. M. Rao and L. P. Lee, “Nanoliter scale microbioreactor array for quantitative cell biology,” Biotechnology and Bioengineering, Vol. 94, No. 1, pp. 5-14, K. R. King, S. Wang, D. Irimia, A. Jayaraman, M. Toner and M. L. Yarmush, “A high-throughput microfluidic real-time gene expression living cell array,” Lab on a Chip, Vol. 7, pp , T. Chapman, Lab automation and robotics: automation on the move, Nature 421 (2003) 661–666.