Department of Functional Polymer Research Laboratory, Tosoh Corporation, 1-8 Kasumi, Mie 510-8540, Japan; Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Department of Functional Polymer Research Laboratory, Tosoh Corporation, 1-8 Kasumi, Mie 510-8540, Japan.
J Biosci Bioeng. 2022 May;133(5):502-508. doi: 10.1016/j.jbiosc.2022.02.005. Epub 2022 Mar 1.
Induced pluripotent stem (iPS) cells have been widely studied in regenerative medicine, pathology modeling, and drug screening. Stable mass culture of iPS cells is essential for these applications. iPS cells can spontaneously differentiate into other cells during culture, and removal of these differentiated cells is necessary. Herein, a cost-effective culture method suitable for mass culture and a detailed analysis of the selective detachment of iPS cells are presented. A simple method for coating the water-insoluble thermoresponsive polymer poly (butyl methacrylate-block-N-isopropylacrylamide) on commercially available polystyrene dishes was employed. Analysis of the effects of the polymer composition, coating thickness, and surface structure on iPS cell culture/detachment showed that a coating thickness of approximately 10-40 nm using a polymer with a high poly (N-isopropylacrylamide) content was suitable for iPS cell detachment. Moreover, an interesting change in surface morphology was observed following temperature variation, thereby affecting laminin adsorption. Second, selective detachment in cocultures of iPS cells and differentiated cells enabled collection of iPS cells with more than 98% purity. Finally, long-term iPS cell culture was conducted using temperature-responsive cell detachment. Overall, long-term maintenance-free culture of iPS cells was possible without manual removal of differentiated cells.
诱导多能干细胞（iPS 细胞）在再生医学、病理学建模和药物筛选方面得到了广泛的研究。iPS 细胞的稳定大量培养对于这些应用至关重要。在培养过程中，iPS 细胞会自发分化为其他细胞，因此需要去除这些分化细胞。本文提出了一种适用于大量培养的具有成本效益的培养方法，并对 iPS 细胞的选择性分离进行了详细分析。采用简单的方法将水溶性差的温敏性聚合物聚（正丁基甲基丙烯酸酯-嵌段-N-异丙基丙烯酰胺）涂覆在市售的聚苯乙烯培养皿上。分析了聚合物组成、涂层厚度和表面结构对 iPS 细胞培养/分离的影响，结果表明，使用高聚（N-异丙基丙烯酰胺）含量的聚合物，涂层厚度约为 10-40nm 时，有利于 iPS 细胞的分离。此外，在 iPS 细胞和分化细胞的共培养中，观察到表面形态随温度变化而发生有趣的变化，从而影响层粘连蛋白的吸附。其次，在分化细胞和 iPS 细胞的共培养物中进行选择性分离，能够获得纯度超过 98%的 iPS 细胞。最后，使用温度响应细胞分离进行了 iPS 细胞的长期培养。总之，无需手动去除分化细胞即可实现 iPS 细胞的长期无维护培养。
