N-acetylcysteine attenuates oxidative stress-mediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production

Shota Tamagawa; Daisuke Sakai; Jordy Schol; Kosuke Sako; Yoshihiko Nakamura; Erika Matsushita; Takayuki Warita; Soma Hazuki; Hidetoshi Nojiri; Masato Sato; Muneaki Ishijima; Masahiko Watanabe

doi:10.1002/jsp2.1223

N-acetylcysteine attenuates oxidative stress-mediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production

JOR Spine. 2022 Oct 1;5(4):e1223. doi: 10.1002/jsp2.1223. eCollection 2022 Dec.

Authors

Shota Tamagawa^{1

2}, Daisuke Sakai², Jordy Schol², Kosuke Sako², Yoshihiko Nakamura³, Erika Matsushita², Takayuki Warita^{2

4}, Soma Hazuki^{2

4}, Hidetoshi Nojiri¹, Masato Sato², Muneaki Ishijima¹, Masahiko Watanabe²

Affiliations

¹ Department of Medicine for Orthopaedics and Motor Organ Juntendo University Graduate School of Medicine Tokyo Japan.
² Department of Orthopaedic Surgery, Surgical Science Tokai University School of Medicine Isehara Japan.
³ Research Center for Regenerative Medicine Tokai University School of Medicine Isehara Japan.
⁴ TUNZ Pharma Co., Ltd. Osaka Japan.

Abstract

Background: Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require long-term cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and off-the-shelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSO-induced cytotoxicity. Furthermore, we examined the potential of antioxidant N-acetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO.

Methods: NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed.

Results: DMSO concentrations ≤1% for 24 h did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24 h) and cell death in a dose- and time-dependent manner. DMSO increased intracellular and mitochondrial ROS (1.9-fold and 3.6-fold respectively after 12 h exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a dose-dependent manner. Tempering ROS through NAC treatment significantly attenuated DMSO-induced oxidative stress and supported maintenance of cell viability.

Conclusions: This study demonstrated dose- and time-dependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSO-induced oxidative stress in the freeze-thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products.

Keywords: N‐acetylcysteine; cell therapy; cryopreservation; dimethyl sulfoxide; discogenic cell; intervertebral disc degeneration; nucleus pulposus cells; oxidative stress; regenerative medicine.