| Summary: | 碩士 === 國立中央大學 === 化學工程與材料工程學系 === 107 === Human pluripotent stem cells (hPSCs), having the self-renewal ability and differentiation ability, have become promising candidates for regenerative medicine in these days. However, the number of the cells used for clinic treatment was large and the cell culture process was laborious. To solve these problems, the continuous culture system was developed in this study. hPSCs were cultured on the thermoresponsive surfaces and partially detached the cells by reducing the temperature below the lower critical solution temperature (LCST) of the thermoresponsive polymer. The remaining cells would be cultured in the same dishes by replacing the fresh medium.
Poly(N-isopropylacrylamide-co-butylacrylate) (polyNIPAM-BA) was selected as thermoresponsive polymer for cell cultivation. The various concentration of polyNIPAM-BA was investigated for determining the optimal concentration of polyNIPAM-BA for thermally-modulated cell adhesion/detachment. The different substrate surfaces prepared by coating the various concentration of polyNIPAM-BA were analyzed the surface properties by water contact angle goniometer, X-ray photoelectron spectra (XPS) and atomic force microscope (AFM). According to the thermally-induced cell detachment process, the thermoresponsive surface coated with 8 mg/ml of polyNIPAM-BA showed the highest detachment ratio. Therefore, 8 mg/ml of polyNIPAM-BA was chosen for coated concentration of long-term cultivation substrate.
In this study, hPSCs, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), were successfully cultured on the thermoresponsive substrates (polyNIPAM-BA (8 mg/ml)-rVN (5 μg/ml)) and partially detached from the thermoresponsive surfaces for 5-7 cycles. For each cycle, the partial detachment ratio could reach around 60 %. In addition, the cells indeed retained their pluripotency and differentiation ability after long-term cultivation. This continuous culture system might downsize the equipment requirement. Moreover, shifting two-dimensional culture to three-dimensional culture might be a great benefit for regenerative medicine in clinical treatment in the future.
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