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

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.

freeze-thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products.

K E Y W O R D S
cell therapy, cryopreservation, dimethyl sulfoxide, discogenic cell, intervertebral disc degeneration, N-acetylcysteine, nucleus pulposus cells, oxidative stress, regenerative medicine 1 | INTRODUCTION Low back pain (LBP) is a major cause of disability worldwide and occurs in all age groups, from young to elderly populations. 1 The health care and work disability costs associated with LBP constitute a tremendous socioeconomic problem. 2 Although the causes of LBP are multifactorial and often unidentifiable, 3,4 intervertebral disc (IVD) degeneration is considered the primary etiology of LBP. 5,6 Aging, mechanical stress, genetics, and other external stimuli can precipitate an imbalance in anabolism and catabolism within the IVDs, provoking a degenerative cascade involving biochemical, biomechanical, and inflammatory changes that can accelerate further degeneration of the disc. [7][8][9][10][11] Moreover, likely due to the disc's largely avascular nature, 12 the IVD possesses limited self-repair capacity. 13 Progressive IVD degeneration can lead to disc herniation, spinal canal stenosis, and degenerative spondylolisthesis, potentially resulting in lower extremity radicular pain, numbness, muscle weakness, and LBP. 14,15 Conventional treatments are primarily palliative (e.g., analgesics or physiotherapy) and generally fail to target the underlying pathology. Patients who do not experience pain improvement following conservative interventions, can as a last resort proceed to rather invasive interventions, that is, spinal fusion. However, the efficacy of these surgical treatments remains uncertain. [16][17][18] The lack of pharmacological or biological therapies targeting IVD degeneration to resolve discogenic LBP and sequential degenerative spinal disease forms a considerable unmet medical need. The field of regenerative medicine has sought to develop new techniques, such as gene therapy, 19 tissue engineering, 20,21 and growth factor injection 22 to tackle the underlying pathogenesis of IVD degeneration in order to alleviate discogenic pain. In particular, cell therapy has shown rapid progression in recent decades 23 with multiple ongoing clinical trials using a variety of cell types and products, including autologous or allogeneic disc-derived cells and mesenchymal stem cells. [24][25][26][27][28] Although an optimal cell type for robust regenerative potential remains to be determined, IVD-derived nucleus pulposus cells (NPC) are a logical candidate due to their innate adaptability to the harsh IVD environment with the capacity to produce large quantities of IVD-specific extracellular matrix. 24,28 We have previously identified Tie2-positive human nucleus pulposus (NP) progenitor cells with clonal multipotency, optimized a whole-tissue culture method that enable the expansion and maintenance of Tie2-positive NPC, and demonstrated their therapeutic potential to treat degenerative disc disease. [29][30][31][32][33][34][35] Even so, cell transplantation still faces multiple translational and practical hurdles.
This includes the need for large quantities of highly potent, safe, and defined cells as a transplantation product to be ready at the time of intervention. This requires the need for reliable cell storage methods; therefore, it is important to develop effective cell cryopreservation techniques that have limited detrimental effects on cell potency.
Freeze-thawing cycles are well-known to affect cell viability and potential, which thereby could reduce therapeutic efficacy. 36,37 Maintaining cell viability after cryopreservation is a crucial step toward commercialization and off-the-shelf (OTS) availability of cell therapy products. The goal for commercialization should be mass production of cell therapy products, ideally producing cell transplantation products for ≥1000 patients from a single donor sample. 35 In addition, an OTS product that is directly transplanted after thawing without cell culture or removing cryoprotectants is desirable for clinical application, accessibility, and cost reduction. Indeed, some OTS cell products have now been approved for clinical trials. [38][39][40] Moreover, in mass production of cellular products, when a large volume of cells is cryopreserved at one time, it is inevitable that they are exposed to the cryopreservation medium at harmful temperatures for a certain period of time. This is a critical consideration because dimethyl sulfoxide (DMSO) is the most commonly used cryoprotective agent to prevent intracellular ice formation and has been reported to cause cytotoxicity via elevated oxidative stress and mitochondrial dysfunction. [41][42][43][44][45] Moreover, multiple studies of other cell types have highlighted the involvement of reactive oxygen species (ROS) as being involved in the detrimental effects of DMSO on cell viability. 45 While the direct effects of DMSO on human NPC have not been clarified, oxidative stress has been shown to impede NPC proliferation, promote cell senescence, and promote a catabolic phenotype. 46 Alternatively, DMSO-containing media have been shown to be detrimental to NPC viability 47 and differentiation potential. 48 As such, the purpose of this study was to investigate the effects of DMSOinduced oxidative stress on human NPC viability and whether the application of antioxidant N-acetylcysteine (NAC; a ROS scavenger) 49  NPC were isolated and cultured as described previously. 33,35 Briefly, the collected surgical NP tissue was washed with an excess of 0.9% saline and the tissue was cut into pieces of 3-5 mm in diameter with scissors and scalpels. Culture conditions were employed to mimic the culture conditions of our cell transplantation product in development, following the work of Sako et al. 35 As such, NP fragments were directly applied to a complete culture medium containing Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) and α-minimal essential medium (αMEM; Gibco), supplemented with 20% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/ streptomycin (Gibco) and cultured in polystyrene 6-well plates (IWAKI, Tokyo, Japan). About 0.3 g of NP tissue was seeded per 3 ml culture medium in a single well. Tissue fragments were cultured at 37 C in 5% CO 2 and 5% O 2 for 14 days without media replenishment. Subsequently, tissue fragments were collected, centrifuged, and the supernatant was removed. Next, the tissue was resuspended in 10 ml TrypLE Express (Thermo Fisher Scientific, Tokyo, Japan). The suspension was digested with gentle swirling at 37 C for 30 min. Next, the tissue was further digested in a mixture of 10 ml of αMEM supplemented with 10% (v/v) FBS and 0.25 mg/ml collagenase P (Roche, Basel, Switzerland), which was incubated for 2 h at 37 C. After digestion, the suspension was filtered through a 40 μm cell strainer (Corning, NY, USA), centrifuged, and the supernatant was removed. The resulting cells were seeded at a density of 3.0 Â 10 4 cells per well in 100-mm dishes (Corning) at 37 C in 5% CO 2 and 5% O 2 and cultured in medium as previously specified for 7 days without media change. IVD-derived cells were used following the third passage.

| Treatment with DMSO and/or NAC
To examine the dose-dependent effect of DMSO (Nacalai Tesque, Kyoto, Japan), NPC seeded at 1.5 Â 10 4 cells/well in 96-well plates or 5.0 Â 10 4 cells/well in 6-well plates were exposed to various concentrations of DMSO ranging from 0.01 to 10% (v/v) prepared in αMEM supplemented with 20% (v/v) FBS for 1 and 24 h. To evaluate the timedependent effect of DMSO, NPC were exposed to 5% or 10% DMSO for 3, 6, 12, and 24 h. Furthermore, NPC were treated with a combination of 5% or 10% DMSO and 10 mM NAC (Sigma-Aldrich) prepared in αMEM supplemented with 20% (v/v) FBS for 3, 6, 12, and 24 h. The concentration of NAC was based on previous work from Seol et al. 50 and confirmed most effective to retain cell viability ( Figure S1). Cells exposed to 0% DMSO were used as an experimental control.

| Apoptosis analysis
Apoptotic and necrotic cells were measured by flow cytometry using APC Annexin V Apoptosis Detection Kit with propidium iodide (PI; T A B L E 1 Summary of the patient characteristics of the donor IVD samples. Age, sex, pathology, total weight of IVD tissue collected at the time of surgery, and weight of NP tissue after selection are listed for each sample  Figure 1A, Sample values were normalized by the Ct value for the housekeeping gene 18S (4352930E; Applied Biosystems). The Ct value of control (0% DMSO) was used as a reference, and then relative mRNA expression was calculated using the 2 ÀΔΔCt method.

| Cryopreservation and thawing
The cryopreservation process was performed as described previously. 52

| DMSO decreases cell viability of NPC in a dose-and time-dependent manner
To examine the dose-dependent effects of DMSO, NPC were exposed to various concentrations of DMSO ( Figure 1A   Exposure to DMSO significantly increased oxidized DHE (red) intensity, indicating an increase in intracellular ROS levels (Figure 2A, B).

| DMSO induces intracellular and mitochondrial ROS of NPC
This increase occurred in a dose-dependent manner. Furthermore, MitoSOX staining highlighted that exposure to 5% DMSO increased and 10% DMSO significantly increased mitochondrial ROS levels ( Figure 2C, D).

| NAC attenuates DMSO-induced cell viability loss and cell death of NPC
To examine whether DMSO-induced cell viability loss and cell death could be prevented by inhibiting ROS generation, DMSO-treated NPC were subjected to 10 mM NAC. 50 The addition of NAC significantly decreased DMSO-induced intracellular ROS generation ( Figure 4A, B) and tended to decrease mitochondrial intracellular ROS generation

| NAC ameliorates DMSO-induced cell viability loss during the freeze-thawing cycle
Finally, the effects on cell viability of NAC addition to cryopreservation medium containing 10% DMSO during the freeze-thawing cycle were examined. Cell viability appeared not to be affected by prefreeze exposure time to cryopreservation medium. However, the addition of NAC significantly increased cell viability for each condition ( Figure 5A). Furthermore, cell viability evaluated after 24 and 72 h of incubation with an equal volume of culture medium, aiming to mimic cell transplantation products, revealed that exposure to cryopreservation medium for 3 h prior to freezing did not cause a significant decrease in cell viability, whereas exposure for more than 6 h significantly decreased cell viability at both 24 and 72 h after thawing ( Figure 5B-E). Notably, the addition of NAC significantly increased cell viability at all time points ( Figure 5B-E), and in some cases even doubled viability rates ( Figure 5E).

| DISCUSSION
This study highlights the association of intracellular and mitochondrial ROS with the cytotoxic effects observed in human NPC when apoptosis. [56][57][58] Moreover, oxidative stress caused by excessive ROS generation is one of the major causes leading to cell death. 59,60 Previous studies have shown that DMSO induces apoptosis via elevated ROS and mitochondrial dysfunction. In adipocytes, for example, DMSO at concentrations ≥1% for 1 h reduced cell viability and accelerated cellular apoptosis by increasing mitochondrial membrane potential and ROS. 42 Elsewhere, in cardiomyoblasts and in breast cancer cells, 3.7% DMSO exposure for 6 days induced apoptosis driven by mitochondrial dysfunction and oxidative stress. 44 Finally, exposure of astrocytes to 5% DMSO for 24 h caused cell viability loss and mitochondrial integrity disruption, membrane potential impairment, ROS production, and subsequent cytochrome C release and caspase-3 activation. 45  F I G U R E 5 Cytoprotective effects of N-acetylcysteine (NAC) against DMSO-induced cell viability loss of NPC following freeze-thawing processes. To examine the effects of NAC addition and pre-freeze exposure times to cryopreservation medium containing 10% DMSO on cell viability following a freeze-thawing processes, cell viability was determined (A) immediately after thawing, or after 24 h of incubation with an equal volume of (B) αMEM (FBS free) or (C) αMEM supplemented with 20% (v/v) FBS. Also, (D) cell viability after 72 h of incubation with an equal volume of αMEM (FBS free) or (E) with an equal volume of αMEM supplemented with 20% (v/v) FBS, was determined. All data are expressed as mean ± SD. (n = 4, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. 0 h without NAC control, *p < 0.05, **p < 0.01, ***p < 0.001, NAC-treated vs. corresponding untreated cells) NAC is a well-established drug that was originally employed as a medical agent against acetaminophen overdose or as a mucolytic agent in bronchitis and pneumatic diseases. Moreover, NAC is well established as an antioxidant (precursor), although the primary mechanism involved in its antioxidative activities remains contested. 61 NAC's application is being examined in a wide range of different diseases and for different applications. 62 In our study, the rationale and benefits of NAC addition to the cryopreservation medium appears to be twofold. First, the addition of NAC may have increased cell viability after the freeze-thawing cycle by inhibiting DMSO-induced ROS generation or reducing oxidative stress associated with the freezethawing processes. Previous studies have demonstrated that the changes in temperature and osmolarity during the freeze-thawing process lead to increased ROS, mitochondrial damage, and cell apoptosis. 36,37,63,64 Thus, our results suggest that NAC could protect cells from oxidative stress by acting as a ROS scavenger. Second, direct administration of NAC, for example as part of an OTS cell therapy product, into the IVDs is expected to have some beneficial effects on IVD homeostasis. For example, Suzuki et al. 65 showed that oxidative stress contributes to the progression of IVD degeneration and oral administration of NAC was able to moderate induced IVD degeneration in a rat model. Furthermore, in scaffold and organ cultures of rat NPC, NAC inhibited ROS generation, cell senescence, and decreased matrix synthesis promoted by high-magnitude compression. 66 Based on the above, the addition of NAC to cryopreservation medium may be useful in consideration of direct transplantation of OTS cell therapy products into the IVDs.
Cryopreserving cells provides many benefits for clinical use and commercialization, such as long-term storage, OTS usability, and the ability to complete safety and functional testing of the cells prior to human dosing. 25 We have previously demonstrated that cell transplantation of human discogenic cells directly from their cryopreserved state is an effective and safe strategy, and has the potential to provide an OTS cell therapy product for the treatment of degenerative disc disease. 34,67 As such, the findings of this study may be a promising discovery for future clinical applications of cell product development toward IVD regeneration.
Our study has several limitations. First, the biological and functional characteristics of NPC after the freeze-thawing cycle, such as matrix synthesis capacity have not been evaluated. Second, our experiments were only executed through in vitro experiments. As such, the effect of DMSO and NAC on NPC in vivo or as cell therapy products was not evaluated. Future studies will need to evaluate their effect in vivo through transplantation of NPC loaded into DMSO and/or NAC-containing media.