© 2019 by SCITEPRESS - Science and Technology Publications, Lda. This paper presents a cell culture well-plate for three dimensional perfusion cell cultures. A concentration gradient generator, a microchannel system, is embedded in the plate bottom for not only the perfusion culture but transfer of reagents with linear concentration gradient to 4 wells of the plate. The concentration uniformity of gradient generated is guaranteed by adding microchannel mixers at the end of generator. Sudden expansion reservoirs, air bubble traps, make perfusion cell cultures plate long-term culture without interruption of perfusion flow caused by injection of air bubbles in the microchannels. The performance of the developed 3D microfluidic perfusion cell culture system is examined experimentally and compared with analytical results. Then, it is applied to test the cytotoxicity of cells infected with Ewing's sarcoma. Cell death is observed for different concentrations of H2O2. Finally, the 3D perfusion cell culture well-plate is presented with not only similar structure to conventional 3×4 well-plate but expansion of concentration range from a 4 fold of dilution in 4 wells to a 100 fold of dilution in 7 wells.
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