Endodermal differentiation of human induced pluripotent stem cells using simple dialysis culture system in suspension culture

Marie Shinohara; Hyunjin Choi; Masato Ibuki; Shigeharu G Yabe; Hitoshi Okochi; Atsushi Miyajima; Yasuyuki Sakai

doi:10.1016/j.reth.2019.05.004

Endodermal differentiation of human induced pluripotent stem cells using simple dialysis culture system in suspension culture

Regen Ther. 2019 May 30:12:14-19. doi: 10.1016/j.reth.2019.05.004. eCollection 2019 Dec 15.

Authors

Marie Shinohara¹, Hyunjin Choi¹, Masato Ibuki^{1

2}, Shigeharu G Yabe³, Hitoshi Okochi³, Atsushi Miyajima⁴, Yasuyuki Sakai^{1

5}

Affiliations

¹ Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.
² Regenerative Medicine and Cell Therapy Laboratories, Kaneka Corporation, Kobe MI R&D Center 3F, 6-7-3, Minatojima Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
³ Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjyuku-ku, Tokyo 162-8655, Japan.
⁴ Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
⁵ Department of Chemical System Engineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

Abstract

A differentiation of human induced pluripotent stem cells (hiPSCs) into definitive endoderm linage is required for a preparation of metabolic organ derived cells. The differentiation consumed high-priced cytokines and small molecules, which have hampered the manufacturability of differentiated cells. Although the cytokines and small molecules are remained or cells produce the autocrine factors, daily culture medium change should be proceeded to remove toxic metabolites generated from cells. In this study, we developed a simple dialysis culture system to refine the medium during definitive endodermal differentiation. We demonstrated that dialysis culture prevented cell damage to remove lactate. The hiPSCs cultured with dialysis also differentiated similarly as usual differentiation without dialysis even if they were not supplied Activin A for latter culture days in the differentiation. With this dialysis culture system, hiPSCs were differentiated into endodermal lineage with medium refinement and recycling and autocrine factors as well as cytokines, which may lead to reduce differentiation cost.

Keywords: DE, definitive endoderm; Definitive endoderm; Dialysis culture; E8, essential 8 medium; Human induced pluripotent stem cell; KSR, knockout serum replacement; NEAA, non-essential amino acids; PSA, penicillin–streptomycin–amphotericin B suspension; Suspension culture; VTN-N, vitronectin human protein; hiPSCs, human induced pluripotent stem cells.