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Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

机译：通过透析精制培养基以扩增悬浮培养物中人诱导的多能干细胞

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摘要

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 +/- 0.1) x 10(6) cells mL(-1) was obtained, with 99.5 +/- 0.2 % OCT 3/4 and 78.3 +/- 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.
机译：人类诱导的多能干细胞（hiPSC）分泌必需的自分泌因子，并且每天更换生长培养基时，这些自分泌因子会与有毒代谢物一起被清除。在这项研究中，在确定乳酸对hiPSCs的最低抑制水平后，构建了培养基精制系统，通过该系统从使用过的培养基中去除有毒代谢物，并回收自分泌因子以及其他生长因子。具体而言，透析后，精制培养基中保留了约87％的碱性成纤维细胞生长因子和80％的转化生长因子β1。精制培养基可有效增强贴壁培养中hiPS细胞的增殖。当使用精制系统刷新悬浮培养中的培养基时，最终细胞密度为（1.1 +/- 0.1）x 10（6）细胞mL（-1），OCT 3/4为99.5 +/- 0.2％在培养的第4天时，其表达为78.3 +/- 1.1％TRA-1-60。这些表达水平类似于常规悬浮培养中观察到的表达水平。通过这种方法，建立了通过透析提纯培养基的方法，以去除有毒代谢物，回收自分泌因子以及其他生长因子，并减少大分子在悬浮培养中扩增hiPSC的用途。

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