Investigation of the Mitigation of DMSO-Induced Cytotoxicity by Hyaluronic Acid following Cryopreservation of Human Nucleus Pulposus Cells

To develop an off-the-shelf therapeutic product for intervertebral disc (IVD) repair using nucleus pulposus cells (NPCs), it is beneficial to mitigate dimethyl sulfoxide (DMSO)-induced cytotoxicity caused by intracellular reactive oxygen species (ROS). Hyaluronic acid (HA) has been shown to protect chondrocytes against ROS. Therefore, we examined the potential of HA on mitigating DMSO-induced cytotoxicity for the enhancement of NPC therapy. Human NPC cryopreserved in DMSO solutions were thawed, mixed with equal amounts of EDTA-PBS (Group E) or HA (Group H), and incubated for 3–5 h. After incubation, DMSO was removed, and the cells were cultured for 5 days. Thereafter, we examined cell viability, cell proliferation rates, Tie2 positivity (a marker of NP progenitor cells), and the estimated numbers of Tie2 positive cells. Fluorescence intensity of DHE and MitoSOX staining, as indicators for oxidative stress, were evaluated by flow cytometry. Group H showed higher rates of cell proliferation and Tie2 expressing cells with a trend toward suppression of oxidative stress compared to Group E. Thus, HA treatment appears to suppress ROS induced by DMSO. These results highlight the ability of HA to maintain NPC functionalities, suggesting that mixing HA at the time of transplantation may be useful in the development of off-the-shelf NPC products.


Introduction
Low back pain (LBP) is a common and serious issue that affects people of all ages and can result in significant disability, leading to a considerable socioeconomic burden [1,2]. Although the origin of LBP is complex and often multifactorial [3,4], intervertebral disc (IVD) degeneration is considered one of the primary contributing factors [5,6]. This degenerative process can be precipitated by a variety of aspects, including age, mechanical stress, genetics, and other external stimuli, which may promote an imbalance between anabolic and catabolic processes within the disc. This disparity between anabolic and catabolic processes can lead to a cascade of alterations within the IVD, including biochemical, biomechanical, and inflammatory changes, which further accelerate degeneration. Although the exact mechanism of degeneration is not yet clearly understood, several contributing associated with knee osteoarthritis by, in part, reducing inflammation [53,54]. HA has therefore been proposed as a promising carrier for cell transplantation in the IVD, as highlighted by several cell transplantation trials [38,55,56]. Furthermore, studies have reported that HA protects mitochondrial DNA from oxidative stress in particular chondrocytes, and contributes to cell survival [57,58]. Based on these findings, we set out to examine the potential of HA to alleviate DMSO-induced oxidative stress for its future application to support and enhance NPC cell therapy products. Specifically, we aim to test whether HA addition can alleviate oxidative stress induced by exposure to DMSO, therefore, enhancing NPC viability and potency.

Cell Viability and Cell Proliferation Rates
Human NPC suspensions derived from a cryopreserved condition were thawed and mixed with either 1 mL of albumin-containing EDTA-PBS (A-EDTA) (Group E) or 1 mL of 1% HA (Group H) and were incubated for 3, 4, or 5 h at room temperature. We found no significant differences in cell viability following 3-5 h of DMSO exposure between the two groups ( Figure 1A). However, while there was no significant difference in cell viability after 5 days of culture ( Figure 1B), there was a significant difference in cell proliferation rate. Specifically, the cell proliferation rate was approximately 11.1 ± 1.3-fold in Group E after 3-h DMSO exposure, compared to a 22.3 ± 5.8-fold increase for Group H with the same exposure time, a two-fold increase (p < 0.001). Although the proliferation rate decreased with an increase in DMSO exposure time, a similar 2-fold difference was consistently observed between Group E and H at each incubation length ( Figure 1C). Inverted phase contrast microscopy confirmed these trends ( Figure 2). Figure 1. Cell viability after 3-5 h exposure and 5 days of culture, and cell proliferation rates after 5 days of culture. Group E and Group H represent thawed Human NPC suspensions mixed with either 1 mL of A-EDTA or 1 mL of 1% HA, respectively. Indicated time is the number of hours of exposure after mixing. Bars and error bars represent mean and standard deviation, respectively. (A) Cell viability after 3-5 h exposure was not significantly different at each exposure time. (n = 7, * p < 0.05) (B) Cell viability after 5 days of culture was not significantly different at each exposure time. (n = 7, n.s. not significant) (C) Cell proliferation rates after 5 days of culture were significantly higher in Group H than in Group E. (n = 7, ** p < 0.01, *** p < 0.001).

Figure 1.
Cell viability after 3-5 h exposure and 5 days of culture, and cell proliferation rates after 5 days of culture. Group E and Group H represent thawed Human NPC suspensions mixed with either 1 mL of A-EDTA or 1 mL of 1% HA, respectively. Indicated time is the number of hours of exposure after mixing. Bars and error bars represent mean and standard deviation, respectively. (A) Cell viability after 3-5 h exposure was not significantly different at each exposure time. (n = 7, * p < 0.05) (B) Cell viability after 5 days of culture was not significantly different at each exposure time. (n = 7, n.s. not significant) (C) Cell proliferation rates after 5 days of culture were significantly higher in Group H than in Group E. (n = 7, ** p < 0.01, *** p < 0.001).

Tie2 Positivity Rates and Tie2 Positive Cells Numbers
Harvested NPC following 5 days of culture were analyzed through flow cytometry for the number and rate of Tie2 positive cells. Here we found no significant difference in the rate of Tie2 positivity between the two groups, regardless of DMSO exposure times ( Figure 3A). However, a significant difference was observed in the overall number of Tie2 positive cells after 5 days of culture, favoring Group H compared to Group E. Moreover, both groups showed a decline in Tie2-positive NPC numbers with an increase in DMSO exposure times ( Figure 3B).

Tie2 Positivity Rates and Tie2 Positive Cells Numbers
Harvested NPC following 5 days of culture were analyzed through flow cytometry for the number and rate of Tie2 positive cells. Here we found no significant difference in the rate of Tie2 positivity between the two groups, regardless of DMSO exposure times ( Figure 3A). However, a significant difference was observed in the overall number of Tie2 positive cells after 5 days of culture, favoring Group H compared to Group E. Moreover, both groups showed a decline in Tie2-positive NPC numbers with an increase in DMSO exposure times ( Figure 3B).

Tie2 Positivity Rates and Tie2 Positive Cells Numbers
Harvested NPC following 5 days of culture were analyzed through flow cytom for the number and rate of Tie2 positive cells. Here we found no significant differenc the rate of Tie2 positivity between the two groups, regardless of DMSO exposure tim ( Figure 3A). However, a significant difference was observed in the overall number of T positive cells after 5 days of culture, favoring Group H compared to Group E. Moreo both groups showed a decline in Tie2-positive NPC numbers with an increase in DM exposure times ( Figure 3B).

The Intracellular and Mitochondrial ROS of NPC
Next, we examined mitochondrial superoxide levels via dihydroethidium (DHE) and MitoSOX labeling following exposure to DMSO. As a control, NPC samples directly after thawing were spun down and DMSO-containing media was removed before seeding and culturing. These cells were thus minimally subjected to DMSO. The intracellular-and mitochondrial superoxide tended to increase with longer exposure to DMSO, a trend more evident within Group E ( Figure 4A,B). Subsequent culture of the DMSO-exposed NPC showed in particular a significant increase in mitochondrial superoxide levels after 5 days of culture for Group E samples, which were significantly higher than Group H ( Figure 4D). DHE levels showed less evident differences ( Figure 4C).

The Intracellular and Mitochondrial ROS of NPC
Next, we examined mitochondrial superoxide levels via dihydroethidium (DHE) and MitoSOX labeling following exposure to DMSO. As a control, NPC samples directly after thawing were spun down and DMSO-containing media was removed before seeding and culturing. These cells were thus minimally subjected to DMSO. The intracellular-and mitochondrial superoxide tended to increase with longer exposure to DMSO, a trend more evident within Group E ( Figure 4A,B). Subsequent culture of the DMSO-exposed NPC showed in particular a significant increase in mitochondrial superoxide levels after 5 days of culture for Group E samples, which were significantly higher than Group H ( Figure  4D). DHE levels showed less evident differences ( Figure 4C). Group H represent thawed Human NPC suspensions mixed with either 1 mL of A-EDTA or 1 mL of 1% HA, respectively. Indicated time is the number of hours of exposure after mixing. Bars and error bars represent mean and standard deviation, respectively. (A) DHE fluorescence intensity after DMSO exposure showed no significant differences between the two groups but tended to increase with longer exposure to DMSO. (n = 7, n.s. not significant) (B) MitoSOX fluorescence intensity after 3 h exposure showed significant differences between the two groups and tended to increase with longer exposure to DMSO. (n = 7, * p < 0.05, ** p < 0.01) (C) DHE fluorescence intensity after 5 days of culture showed no significant differences between the two groups. (n = 7, * p < 0.05) (D) MitoSOX fluorescence intensity after 5 days of culture showed significantly lower in Group H than in Group E. (n = 7, * p < 0.05, ** p < 0.01). Group H represent thawed Human NPC suspensions mixed with either 1 mL of A-EDTA or 1 mL of 1% HA, respectively. Indicated time is the number of hours of exposure after mixing. Bars and error bars represent mean and standard deviation, respectively. (A) DHE fluorescence intensity after DMSO exposure showed no significant differences between the two groups but tended to increase with longer exposure to DMSO. (n = 7, n.s. not significant) (B) MitoSOX fluorescence intensity after 3 h exposure showed significant differences between the two groups and tended to increase with longer exposure to DMSO. (n = 7, * p < 0.05, ** p < 0.01) (C) DHE fluorescence intensity after 5 days of culture showed no significant differences between the two groups. (n = 7, * p < 0.05) (D) MitoSOX fluorescence intensity after 5 days of culture showed significantly lower in Group H than in Group E. (n = 7, * p < 0.05, ** p < 0.01).

Discussion
To summarize the results of this study, mixing HA after freeze-thawing maintained the proliferation of NPC, which in turn could increase the yield of Tie2-positive NPC. Moreover, HA was able to suppress ROS production following DMSO exposure.
DMSO remains the cryopreservation agent of choice according to a most recent consensus paper from the spine research field [46]. Nevertheless, high concentrations of DMSO (≥10% v/v) are known to induce cytotoxicity by increasing intracellular DMSO concentrations through the formation of cell membrane pores and increasing ROS levels in a concentration-dependent manner [48]. Even at 5-10% DMSO concentrations, which are commonly found in most cryopreservation media, DMSO has been found to promote ROS production and apoptosis, as well as decrease overall cell viability and potency [40,45,47]. Although we have previously shown that the transplantation of human NPC directly from a cryopreserved state can be an effective and safe strategy to restore induced disc degeneration [38], concerns remain on the impact of DMSO on the transplanted cells as well as the endemic cells within the IVD. Reporting from our previous clinical trial has indicated that processing off-the-shelf NPC from thawing in the cell processing center to the final injection took an average of 122.9 min, subjecting the cell products to DMSO-containing media during these procedures [35,45]. Furthermore, due to the limited diffusion of the disc in part owing to the disc's avascular nature [12,13,59], DMSO may induce oxidative stress locally for several hours after transplantation. This further emphasizes the need to limit the impact of DMSO to optimize the regenerative potential of cell therapeutic agents.
Therefore, it is likely beneficial to shorten the exposure time or limit the impact of DMSO to optimize the regenerative agent. One method to solve this issue is by removing the cryopreservation solution before transplantation. This can easily be done by simple centrifugation and subsequent washing of the samples; however, this may increase the cost, time, and medical staff burden of the procedure. Therefore, the removal of the cryopreservation solution could be a hurdle for the successful commercialization of cell transplantation products. However, a previous canine study has shown the potential of direct off-the-shelf cell transplantation, without any clear adverse events [38]. Whether or not removing cryopreservation solution remains a matter of further study.
HA and its derivatives have important medical and industrial applications [51]. It has been employed in multiple areas for its anti-inflammatory characteristic [51,60], painrelieving potential [61][62][63], and as a direct lubricant [54]. Moreover, it has been shown that HA exhibits antioxidant properties for articular chondrocytes [57,58,64]. Reports have shown that levels of H 2 O 2 and O 2 − , known ROSs, are lower in the synovial fluid following intra-articular injection of HA compared to the before-treatment levels. Moreover, cell death can be rescued by adding HA to chondrocytes exposed to various concentrations of H 2 O 2 [64]. HA antioxidant properties have similarly been shown in other cell types [65][66][67][68]. Therefore, we set out to determine if HA could limit the oxidative stress in NPC, resulting from DMSO, to increase the potency of a potential off-the-shelf transplantation product. Here, we mimicked a clinical setting in vitro by subjecting the cells to direct encapsulation in HA immediately after thawing without removing DMSO followed by incubation for several hours before culture. The results of this study showed that ROS levels after incubation were higher than those after 5 days of culture, and this was especially true regarding mitochondrial ROS. This suggests that the effects of the DMSO-based freezing/thawing procedure were still affecting the NPC after the 5-day culture, as measured ROS intensity patterns showed similar trends to those observed before cell seeding. This underlines the potential impact of this cell processing aspect on the long-term potency of cell transplantation products. Moreover, we showed that the addition of HA alleviated oxidative stress, and proliferative capacity was maintained, even in cells exposed to DMSO, without reducing Tie2 expression levels. As a result, the yield of Tie2-positive NPCs increased. Tie2, a tyrosine kinase receptor with angiopoietin-1 as a ligand, is a marker for NP progenitor cells [33]. NPCs with high Tie2 expression have been shown to possess a strong capacity to produce IVD-specific ECM components, such as type II collagen and aggrecan [37,39]. Moreover, these cells present an overall high regenerative and differentiation capacity [69]. They are thus expected to be a potent regenerative cell population for IVD repair [32], and as such multiple attempts have been made to enhance the yield or expansion of these NP progenitor cells [39,[70][71][72][73]. Here, we could show that the recovery from DMSO-induced cytotoxicity through HA exposure, can enhance Tie2 expressing NPC yields, therefore offering a promising and easy adoption to enhance the development of NP progenitor cell-based transplantation products.
Despite our promising results, some limitations should be considered. In this study, we did not evaluate the ECM production capacity of the resulting cells. In addition, our experiments were conducted only in vitro, and it is unclear how DMSO and HA will impact cells in the harsher IVD upon transplantation. Furthermore, since the mechanism by which the addition of HA reduces the oxidative stress of DMSO has not been yet elucidated, we are now considering examining the effects of neutralizing CD44, a known receptor for HA, and confirming the impact on our observed beneficial effects [58,64].
Moreover, additional care will be required for the method of transplantation. It has been reported that misplacement or leakage of transplanted cells from the IVD to the surrounding tissues may result in the formation of undesirable tissue formation e.g., osteophytes [74]. Therefore, it could be useful to prevent osteophyte formation that cellular MR imaging for tracking transplanted cells [75], and a technique for non-invasive monitoring of minimally invasive stem delivery into the vertebral disc [76]. Here HA is also likely to support cell transplantation, by forming an adhesive carrier supporting the retention of the de novo cells within the disc space.

Human NP Cell Isolation and Culture
The study received approval from the Institutional Review Board for Clinical Research at Tokai University (application number: 17R-173), indicating that all research procedures described in the study met ethical and safety standards established by our institution. The study involved the collection of human IVD tissue samples from 7 patients (mean age ± standard deviation, 17.6 ± 2.7 years) who underwent surgery for lumbar disc herniation at Tokai University Hospital and related facilities (Table 1). Before tissue collection, all patients provided informed written consent, indicating their consent for the use of surgical waste for research. Alternatively, for patients under the age of 18, informed consent was obtained from their parent (s) or legal guardian (s). NP cells were isolated and cultured using previously described methods [37,39]. The collected surgical NP tissue was first washed with saline and then cut into 3-5 mm diameter pieces using scissors and scalpels [37]. The culture conditions were designed to mimic the culture conditions of a cell transplantation product under development, as outlined in the work of Sako et al. [39]. NP fragments were added directly to a complete culture medium consisting of Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) and α-minimal essential medium (αMEM; Gibco, Grand Island, NY, USA) supplemented with 20% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). The tissue fragments were then cultured in polystyrene 6-well plates (IWAKI, Tokyo, Japan) with approximately 0.3 g of NP tissue seeded per 3 mL culture medium in a single well. The fragments were cultured for 14 days at 37 • C in 5% CO 2 and 5% O 2 without media replenishment.
After two weeks, tissue fragment suspensions were collected, centrifuged, and the supernatant was discarded. The tissue was then resuspended in 10 mL of TrypLE Express (Thermo Fisher Scientific, Tokyo, Japan) and digested at 37 • C for 30 min under gentle swirling. The resulting suspension was again collected, centrifuged, and subsequently digested for 2 h at 37 • C using 10 mL of αMEM supplemented with 10% (v/v) FBS and 0.25 mg/mL collagenase P (Roche, Basel, Switzerland). After digestion, the suspension was filtered using a 40 µm cell strainer (Corning, NY, USA), centrifuged, and the supernatant was removed. The resulting cells were then seeded at a density of 3.0 × 10 4 cells per well in 100-mm dishes (Corning) and cultured in a medium as previously specified for 7 days without any media change. IVD-derived cells were used after the third passage.
NPCs were treated with 5 mL of TrypLE Express for 3 min and then collected into a 15 mL conical tube. Samples were centrifuged at 1200 rpm for 5 min at 4 • C, resuspended in 5 mL of buffered saline, and centrifuged again at 1200 rpm for 5 min at 4 • C. After collecting the cultured cells, they were aliquoted into a cryotube at 3.0 × 10 5 cells in 1 mL of CryoStor ® CS10 (CS10) (STEMCELL Technologies, Vancouver, BC, Canada), which contains 10% DMSO, and the samples were cryopreserved in stages to −80 • C using a controlled-rate cryopreservation device (Bicell, Nihon Freezer, Tokyo, Japan). The next day, the samples were stored in a liquid nitrogen container at −196 • C. The cryopreservation period was set to 2 weeks.

Incubation and Culture for Transplantation Simulation
Conditions were chosen to mimic the environment transplanted cells would be subjected to in the IVD i.e., low nutrition and low oxygen levels. Although DMSO concentration is expected to thin out after transplantation due to diffusion, this experiment was set up in a more severe environment where DMSO concentrations did not thin out, until DMSO was removed. Frozen NPC solutions were thawed slowly in a water bath set to 37 • C for about one minute. When fully defrosted, the 1 mL samples were transferred 15 mL conical tube and either mixed with 1 mL of A-EDTA forming Group E or mixed with 1 mL of ARTZ Dispo ® (Seikagaku Corporation, Tokyo, Japan) forming Group H. ARTZ Dispo ® contains 1% sodium hyaluronate solution with an average molecular weight range of 5.0 × 10 5 to 1.2 × 10 6 Daltons, at a concentration of 25 mg/2.5 mL and is being applied as an effective intra-articular agent for knee OA [77,78]. The solutions were carefully mixed by pipetting and kept with the lids slightly open (for gas exchange) and incubated for 3, 4, or 5 h at 37 • C in a 5% CO 2 and 5% O 2 incubator. After incubation, NPC samples were spun down and DMSO-containing media was removed. The NPCs were resuspended and seeded at 3.0 × 10 4 cells per 100-mm dishes in 6 mL αMEM supplemented with 30% FBS, 10 ng/mL basic fibroblast growth factor (FGF2; PeproTech, Cranbury, NJ, USA), and 1% penicillin/streptomycin, and cultured for 5 days at 37 • C in 5% CO 2 and 5% O 2 ( Figure 5). The cultured NPCs were observed, and images were captured using an inverted phase contrast microscope at 1-and 5-days of culture.

Cell Viability and Cell Proliferation Rates
The cells were exposed for 3, 4, and 5 h and were collected, spun down (to remove medium), and suspended in 1 mL of A-EDTA. On the other hand, the cells cultured for 5 days were treated with 5 mL of TrypLE Express for 3 min and collected, and the medium was removed and mixed using the same method. The number of viable and non-viable cells was determined using the trypan blue exclusion method. The cell proliferation rates were calculated by dividing the total number of cells after culture by the number of cells seeded before culture (3.0 × 10 4 cells).

Cell Viability and Cell Proliferation Rates
The cells were exposed for 3, 4, and 5 h and were collected, spun down (to remo medium), and suspended in 1 mL of A-EDTA. On the other hand, the cells cultured for days were treated with 5 mL of TrypLE Express for 3 min and collected, and the mediu was removed and mixed using the same method. The number of viable and non-viab cells was determined using the trypan blue exclusion method. The cell proliferation rat were calculated by dividing the total number of cells after culture by the number of ce seeded before culture (3.0 × 10 4 cells).

Flow Cytometry Analysis
NPCs were examined using a FACS Calibur flow cytometer (BD Biosciences, Frankl Lakes, NJ, USA), following procedures described in a previous study [37]. Living ce were selectively analyzed by implementing a propidium iodide-negative gate. NPC w examined using flow cytometry to determine the proportion of cells expressing NP pr genitor cell marker Tie2 [33,37]. NPCs were comparatively analyzed to isotype contr antibodies, as previously stated in Sakai et al. [37]. The number of Tie2-positive NPC w estimated by multiplying the number of living cells counted at 5 days of culture by t Tie2-positive rates measured by flow cytometry.

Measurement of Intracellular and Mitochondrial ROS
NPC samples following either 3-5 h DMSO exposure or following 5 days of cultu were collected, washed twice in phosphate-buffered saline (PBS), and counted. A total 3.0 × 10 4 NPC per condition were incubated with 10 µM dihydroethidium (DHE; Invitr gen) or 5 µM MitoSOX Red (Invitrogen) for 30 min in the dark at 37 °C. NPC samples th were directly spun after thawing to quickly remove DMSO-containing media were r moved and were seeded and cultured, and functioned as a control. The levels of intrac lular and mitochondrial superoxide were analyzed using FACS. The mean fluorescen intensity of DHE and MitoSOX was determined through an excitation wavelength of 4 nm and the measurement of the emission at 578 nm wavelength [47].

Flow Cytometry Analysis
NPCs were examined using a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), following procedures described in a previous study [37]. Living cells were selectively analyzed by implementing a propidium iodide-negative gate. NPC was examined using flow cytometry to determine the proportion of cells expressing NP progenitor cell marker Tie2 [33,37]. NPCs were comparatively analyzed to isotype control antibodies, as previously stated in Sakai et al. [37]. The number of Tie2-positive NPC was estimated by multiplying the number of living cells counted at 5 days of culture by the Tie2-positive rates measured by flow cytometry.

Measurement of Intracellular and Mitochondrial ROS
NPC samples following either 3-5 h DMSO exposure or following 5 days of culture, were collected, washed twice in phosphate-buffered saline (PBS), and counted. A total of 3.0 × 10 4 NPC per condition were incubated with 10 µM dihydroethidium (DHE; Invitrogen) or 5 µM MitoSOX Red (Invitrogen) for 30 min in the dark at 37 • C. NPC samples that were directly spun after thawing to quickly remove DMSO-containing media were removed and were seeded and cultured, and functioned as a control. The levels of intracellular and mitochondrial superoxide were analyzed using FACS. The mean fluorescence intensity of DHE and MitoSOX was determined through an excitation wavelength of 488 nm and the measurement of the emission at 578 nm wavelength [47].

Statistical Analysis
All statistical analyses were performed with Easy R (EZR; Saitama Medical Center, Jichi Medical University, Saitama, Japan, version 1.61), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria, version 4.2.2). More precisely, it is a modified version of R commander (version 2.8-0) designed to add statistical functions frequently used in biostatistics [79]. All values are presented as mean (±standard deviation) unless specifically stated otherwise. Statistical differences were determined via one-way ANOVA, followed by Dunnett's multiple comparison or Tukey's comparison test. A p-value of <0.05 was considered statistically significant.

Conclusions
The addition of HA to thawed cryopreservation medium helped to mitigate the cytotoxicity of DMSO, and thus enhanced NPC proliferation. Moreover, HA was able to