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ORIGINAL RESEARCH article

Front. Bioeng. Biotechnol.
Sec. Tissue Engineering and Regenerative Medicine
Volume 12 - 2024 | doi: 10.3389/fbioe.2024.1443795

The impact of repeated temperature cycling on cryopreserved human iPSC viability stems from cytochrome redox state changes

Provisionally accepted
Jun Okuda Jun Okuda 1,2,3Namiko Watanabe Namiko Watanabe 2,3Tetsuji Nakamura Tetsuji Nakamura 2,3Kenta Mizushima Kenta Mizushima 4Heqi Xi Heqi Xi 4Yasuaki Kumamoto Yasuaki Kumamoto 4Katsumasa Fujita Katsumasa Fujita 4Masahiro KINO-OKA Masahiro KINO-OKA 1,2*
  • 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan
  • 2 Research Base for Cell Manufacturability, Osaka University, Suita, Japan
  • 3 R&D Center, Iwatani Corporation, Amagasaki, Japan
  • 4 Department of Applied Physics, Osaka University, Suita, Japan

The final, formatted version of the article will be published soon.

    Human induced pluripotent stem cells (hiPSCs) are an attractive cell source for regenerative medicine. For its widespread use as a starting material, a robust storage and distribution system in the frozen state is necessary. For this system, managing transient warming during storage and transport is essential, but how transient warming affects cells and the mechanisms involved are not yet fully understood. This study examined the influence of temperature cyclings (from -80 °C to -150 °C) on cryopreserved hiPSCs using a custom-made cryo Raman microscope, flow cytometry, and performance indices to assess viability. Raman spectroscopy indicated the disappearance of mitochondrial cytochrome signals after thawing. A reduction in the mitochondrial membrane potential was detected using flow cytometry. The performance indices indicated a decrease in attachment efficiency with an increase in the number of temperature cycles. This decrease was observed in the temperature cycle range above the glass transition temperature of the cryoprotectant. Raman observations captured an increase in the signal intensity of intracellular dimethyl sulfoxide (DMSO) during temperature cycles. Based on these results, we proposed a schematic illustration for cellular responses to temperature fluctuations, suggesting that temperature fluctuations above the glass-transition temperature trigger the movement of DMSO, leading to cytochrome c oxidation, mitochondrial damage, and caspase-mediated cell death. This enhances our understanding of the key events during cryopreservation and informs the development of quality control strategies for hiPSC storage and transport.

    Keywords: Transient warming event, Human Induced Pluripotent Stem Cells, Raman spectroscopy, Cold chain, Cryopreservation, Dimethyl Sulfoxide

    Received: 05 Jun 2024; Accepted: 17 Jul 2024.

    Copyright: © 2024 Okuda, Watanabe, Nakamura, Mizushima, Xi, Kumamoto, Fujita and KINO-OKA. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Masahiro KINO-OKA, Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.