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.787-790

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 reagentsHigh cost of operation

robotics multi-well plates

Inexpensive chip-platforms High-density arraysOnly expose cells to a single compound at once

P. J. Lee, 2006 K. 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 inputsseven possible outputsOne control channel

Overpass Allow one microfluidic channel to cross over other microfluidic channels

Cell culture-chambersCells culture

Combinatorial mixerDeliver 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 : H2SO4:H2O2 = 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 sealing2. Adapter to connect the tubes3. 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 loadand the flow rate

Appliance

Outline

Introduction Device design and fabrication Experimental and discussion Conclusion

Combinatorial Mixer Operatedflow rate : 10L min−1 flow rate : 0.1L min−1

D : diffusion coefficientU : fluid velocityw: channel widthZ : distance during time period

D

UwZ

2

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 Assay1.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, 2007.

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, 2006.

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. 77-85, 2007.

T. Chapman, Lab automation and robotics: automation on the move, Nature 421 (2003) 661–666.