Efficient derivation and banking of clinical-grade human embryonic stem cell lines in accordance with Japanese regulations

Kei Takada; Ryoko Nakatani; Emiko Moribe; Shizuka Yamazaki-Fujigaki; Mai Fujii; Masayo Furuta; Hirofumi Suemori; Eihachiro Kawase

doi:10.1016/j.reth.2022.10.006

Efficient derivation and banking of clinical-grade human embryonic stem cell lines in accordance with Japanese regulations

Regen Ther. 2022 Nov 6:21:553-559. doi: 10.1016/j.reth.2022.10.006. eCollection 2022 Dec.

Authors

Kei Takada¹, Ryoko Nakatani¹, Emiko Moribe¹, Shizuka Yamazaki-Fujigaki¹, Mai Fujii¹, Masayo Furuta¹, Hirofumi Suemori¹, Eihachiro Kawase¹

Affiliation

¹ Division of Clinical Basis for ES Cell Research, Center for Human ES Cell Research, Institute for Life and Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

Abstract

Introduction: We recently established clinical-grade human embryonic stem cell (hESC) line KthES11 in accordance with current good manufacturing practice standards in Japan. Despite this success, the establishment efficiency was very low at 7.1% in the first period.

Methods: To establish clinical-grade hESC lines, we used xeno-free chemically defined medium StemFit AK03N with the LM-E8 fragments instead of feeder cells. The protocol was then optimized, especially in the early culture phase.

Results: We established five hESC lines (KthES12, KthES13, KthES14, KthES15, and KthES16) with 45.5% efficiency. All five hESC lines showed typical hESC-like morphology, a normal karyotype, pluripotent state, and differentiation potential for all three germ layers. Furthermore, we developed efficient procedures to prepare master cell stocks for clinical-grade hESC lines and an efficient strategy for quality control testing.

Conclusions: Our master cell stocks of hESC lines may contribute to therapeutic applications using human pluripotent stem cells in Japan and other countries.

Keywords: CPF, cell processing facility; Cell banking; Clinical grade; GMP, good manufacturing practice; Human embryonic stem cells; ICM, inner cell mass; LM, laminin; MHLW, Ministry of Health, Labour, and Welfare; QC, quality control; hESCs, human embryonic stem cells.