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摘要
A fully integrated microchip device for performing a complete and automated sample-perfusion experiment on living cells is presented. Cells were trapped and immobilized in a defined grid pattern inside a small 0.5 mu l volume incubation chamber by pneumatic anchoring on 1000 5-mu m orifices. This new cell trapping technique assures a precise and repeatable cell quantity for each experiment and enables the formation of a homogeneous cell population in the incubation chamber. The microsystem includes a perforated silicon chip seamlessly integrated by a new embedding technique in a larger elastomer substrate, which features the microfluidic network. The latter forms the incubation chamber and allows for economic logarithmic dilution of the sample reagent over a range of three orders of magnitude with subsequent perfusion of the cell population. First, the logarithmic dilution stage was validated using quantitative fluorescent imaging of fluorescein solution. Then, the cell adhesion and culturing inside the incubation chamber was studied using primary normal human dermal fibroblasts (NHDFs). The cells adhered well on laminin-coated surfaces and proliferated to form a confluent cell layer after 6 days in vitro. Finally, the complete system was tested by a perfusion experiment with cultured NHDFs, which were exposed to a fluorescent cell tracker at dilutions of 100 mu M, 10 mu M, 1 mu M, 0.1 mu M and 0 mu M at a flow rate of 1.25 mu l min(-1) for 20 min. Fluorescence imaging of the cell array after incubation and image analysis showed a logarithmic relationship between sample concentration and the fluorescence signal. This paper describes the fabrication of the components and the assembly of the microsystem, the design approach and the validation of the sample diluter, cell-adhesion and cell-culturing experiments over several days.
- 出版日期2007-8
