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

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Abstract

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) × 106 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.

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Acknowledgments

The authors would like to thank Toyobo Co., Ltd., Japan for providing the membrane filter for medium dialysis and cell separation and Fujimori Kogyo Co. Ltd., Japan for technical support during cell expansion. This work was supported by the project of “Development of Cell Production and Processing Systems for Commercialization of Regenerative Medicine” from the Japan Agency for Medical Research and Development, AMED.

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Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan

    Suman Chandra Nath, Eiji Nagamori & Masahiro Kino-oka

  2. Division of Biochemical Engineering, Radioisotope Research Centre, Kyoto University, Kyoto, 606-8501, Japan

    Masanobu Horie

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  1. Suman Chandra Nath
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  2. Eiji Nagamori
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Correspondence to Masahiro Kino-oka.

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Nath, S.C., Nagamori, E., Horie, M. et al. Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture. Bioprocess Biosyst Eng 40, 123–131 (2017). https://doi.org/10.1007/s00449-016-1680-z

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  • DOI: https://doi.org/10.1007/s00449-016-1680-z

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