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This study reports on the development of a novel perfusion microfluidic cell culture device integrated drug delivering, fluid controlling and cell culturing functions on a two- layer PDMS chip. The device consists of an array of 6 X 6 cell culture chambers, a drug gradient generator and two control valves. Cells are physically trapped and cultured in the center of the chamber by a C-shaped dam when cells loading. Drug gradient network is able to generate 6 gradient drug concentrations to treat the culture cells. And the hydraulic valves can not only make control of the fluid flow to mimic the microenvironment of cells, but keep different kind of cells separately cultured. We have investigated the effect of the hydraulic valves on fluid control, and the chip's ability to generate a range of chemical gradient concentrations. Three kinds of Human cells (L-02, SMMC-7721 and HUVEC-2C) are grown well after three-day continuous perfusion culturing in the chip. Therefore the microfluidic cell culture device shows the potential to make significant impacts in cell-based research of high throughput drug screening, tissue engineering and clinical medicine.