Cryopreservation of undifferentiated and differentiated human neuronal cells

Kenji Yamatoya; Yuya Nagai; Naozumi Teramoto; Woojin Kang; Kenji Miyado; Kazuya Nakata; Tohru Yagi; Yoshitaka Miyamoto

doi:10.1016/j.reth.2021.12.007

Cryopreservation of undifferentiated and differentiated human neuronal cells

Regen Ther. 2022 Jan 7:19:58-68. doi: 10.1016/j.reth.2021.12.007. eCollection 2022 Mar.

Authors

Kenji Yamatoya^{1

2}, Yuya Nagai¹, Naozumi Teramoto³, Woojin Kang⁴, Kenji Miyado⁴, Kazuya Nakata^{1

5}, Tohru Yagi⁶, Yoshitaka Miyamoto^{3

4

6}

Affiliations

¹ Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
² Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba, 279-0021, Japan.
³ Department of Applied Chemistry, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba, 275-0016, Japan.
⁴ Department of Reproductive Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan.
⁵ Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo, 184-0012, Japan.
⁶ Department of Mechanical Engineering, Tokyo Institute of Technology, 12-2-1 Ookayama, Meguro-ku, Tokyo, 152-8552, Japan.

Abstract

The effective use of human-derived cells that are difficult to freeze, such as parenchymal cells and differentiated cells from stem cells, is crucial. A stable supply of damage-sensitive cells, such as differentiated neuronal cells, neurons, and glial cells can contribute considerably to cell therapy. We developed a serum-free freezing solution that is effective for the cryopreservation of differentiated neuronal cells. The quality of the differentiated and undifferentiated SK-N-SH cells was determined based on cell viability, live-cell recovery rate, and morphology of cultured cells, to assess the efficacy of the freezing solutions. The viability and recovery rate of the differentiated SK-N-SH neuronal cells were reduced by approximately 1.5-folds compared to that of the undifferentiated SK-N-SH cells. The viability and recovery rate of the differentiated SK-N-SH cells were remarkably different between the freezing solutions containing 10% DMSO and that containing 10% glycerol. Cryoprotectants such as fetal bovine serum (FBS), antifreeze proteins (sericin), and sugars (maltose), are essential for protecting against freeze damage in differentiated neuronal cells and parenchymal cells. Serum-free alternatives (sericin and maltose) could increase safety during cell transplantation and regenerative medicine. Considering these, we propose an effective freezing solution for the cryopreservation of neuronal cells.

Keywords: 5-CFDA-AM, 5-carboxyfluorescein diacetate; BSA, bovine serum albumin; Cryopreservation; D-MEM, Dulbecco's Modified Eagle's Medium with high glucose, l-glutamine, phenol red, and sodium pyruvate; D-PBS-free, Dulbecco's phosphate-buffered saline without Ca2+ and Mg2+ supplementation; DAPI, 4′,6-Diamidino-2-phenylindole, dihydrochloride; DMSO, dimethyl sulfoxide; Differentiation; EDTA, ethylene-diamine-tetraacetic acid; ESC, embryonic stem cells; FBS, fetal bovine serum; NeuN, a neuronal marker; Neuronal cells; Regenerative therapy; SDS, sodium dodecyl sulfate; Sericin; Serum-free; iPSC, induced pluripotent stem cells.