Clinically relevant preservation conditions for mesenchymal stem/stromal cells derived from perinatal and adult tissue sources

Abstract The interplay between mesenchymal stem/stromal cells (MSCs) and preservation conditions is critical to maintain the viability and functionality of these cells before administration. We observed that Ringer lactate (RL) maintained high viability of bone marrow–derived MSCs for up to 72 h at room temperature (18°C–22°C), whereas adipose‐derived and umbilical cord‐derived MSCs showed the highest viability for 72 h at a cold temperature (4°C–8°C). These cells maintained their adherence ability with an improved recovery rate and metabolic profiles (glycolysis and mitochondrial respiration) similar to those of freshly harvested cells. Growth factor and cytokine analyses revealed that the preserved cells released substantial amounts of leukaemia inhibitory factors (LIFs), hepatocyte growth factor (HGF) and vascular endothelial growth factor‐A (VEGF‐A), as well as multiple cytokines (eg IL‐4, IL‐6, IL‐8, MPC‐1 and TNF‐α). Our data provide the simplest clinically relevant preservation conditions that maintain the viability, stemness and functionality of MSCs from perinatal and adult tissue sources.


| INTRODUC TI ON
Mesenchymal stem/stromal cells (MSCs), first discovered in the 1960s, are a plastic-adherent cell population possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the International Society for Cell and Gene Therapy (ISCT). 1,2 In the last decade, MSC therapy has emerged as the most promising cell therapy due to its capabilities of in vitro expansion, direct interaction of MSCs with immunological cells to modulate the functions of the latter population, and secretion of growth factors and cytokines that promote cell survival, proliferation and function. MSCs for use in therapy including autologous and allogeneic administration originate from several sources, including adipose tissue (AD), bone marrow (BM), umbilical cord (UC), dental pulp, placenta and peripheral blood. In the last 25 years, more than 950 registered MSC-based clinical trials have supported the safety profiles of treating over 10,000 patients with MSCs under clinical trial conditions, 3 and 1,040 MSC-based clinical trials targeting approximately 48,000 patients globally (searching term 'mesenchymal stem cells' at clinicaltrials.gov) have been registered to date. 4 A recent study related to the production and application of MSCs in the United States revealed that AD-, BM-and UC-derived MSCs are the most widely used MSC sources for therapeutic applications and regenerative medicine. 5 The lack of major histocompatibility (MHC) class II in combination with the expression of early embryonic surface markers in the naive form of these MSCs provides solid evidence for their multipotency and immunological privilege. 6 Accessibility, easy and reproducible expansion, safety and potential effective treatment are considered major advantages of MSC therapy in various diseases, including orthopaedic injuries, cardiovascular disease, 7 pulmonary disease (bronchopulmonary dysplasia), 8,9 graft-vs.-host disease 10 and autoimmune disease, 11 among others.
The majority of MSC products or therapies describe the use of cryopreservation conditions to store and transport the final product, which is usually thawed within a few hours prior to infusion. Many groups have identified challenges regarding the potential functionality of MSC products after preservation and thawing processes, particularly when bioactivity measurements are commonly conducted on MSCs before or without cryopreservation or following culture post-thaw. 12 The effects of storage and transport conditions, such as preservation solutions, temperature and duration of storage, play significant roles in cell viability and functionality. MSCs derived from various sources (such as AD-, BM-and UC-MSCs) might behave differently under these conditions, adding another layer of complexity to the quality control of stem cell therapies, especially in clinical trial settings. Hence, it is important to find the optimal conditions to maintain the quality of MSCs prior to performing clinical therapy while requiring minimal processing steps. Few studies have identified the optimal conditions to maintain the viability of AD-, 13   and UC-MSCs. 14 To our best knowledge, no study has reported the optimal conditions to maintain all three sources of MSCs, including AD-, BM-and UC-MSCs, for therapeutic applications. Therefore, our study aimed to compare the effects of NaCl and Ringer Lactate (RL) and their combination with 0.4% human albumin (HA) on the viability, proliferation, marker expression, metabolic profile and paracrine functions of MSCs from three main sources: AD, BM and UC. The concentration of 0.4% HA was chosen because it has been used intensively as a supplement at our institute for preserving immune cells and is effective in maintaining the high quality of these cells before administration as a suspension solution. 15, 16 We sought to determine how MSCs from these three sources behave under preservation conditions for up to 72 h and explore the metabolic activities and growth factor and cytokine secretion profiles from these cells before and after preservation; addressing these issues will allow us to identify suitable, clinically relevant preservation conditions for MSCs and support their therapeutic application in clinical trials. To this end, the experiments were designed to evaluate how MSCs derived from AD, BM and UC behave under different storage conditions and durations based on (1) cell viability (measured using 7-ADD and Trypan blue), (2) cell proliferation and recovery,

| Study approval
Adipose tissue, BM and UC tissues were obtained from healthy donors and collected at Vinmec International Hospital in 2019 after patients signed an informed consent form as previously reported. 17 The collection of human samples was approved by the Ethics Board of Vinmec International Hospital (approval number: 122/2019/ QD-VMEC).

| MSC culture and characterization
The three MSC lines from each respective tissue were randomly selected from the MSC biobank, were carefully characterized according to the ISCT guidelines, and were cultured under xeno-free and serum-free conditions as previously described. 17 Culture reagents were purchased from Thermo Fisher Scientific (https://www.therm ofish er.com/) and MACS Miltenyi Biotec (StemMACS TM MSC expansion media kit XF, human, https://www.milte nyibi otec.com/) unless otherwise specified. To confirm the surface marker expression of the in vitro-expanded cells isolated from AD, BM and UC, the harvested cells were subjected to flow cytometry analysis at P4 using a human BD Mesenchymal Stem kit (BD Biosciences; 562245) according to the manufacturer's protocol. Flow cytometry was performed using a BD FACSCanto flow cytometer (BD Biosciences). Data were analysed using FlowJo software (BD Biosciences). The trilineage differentiation assay was performed as previously described 17

| Evaluation of the effects of storage medium composition and temperature on stem cell quality
The MSCs from each respective tissue were thawed at P3 and cultured under xeno-free and serum-free conditions until 80% confluence followed by harvested using CTS TM TrypLE TM Select Enzyme (Gibco; A12859-01). Next, the harvested MSCs (P4) were aliquoted into the 1.8 mL cryovials (Corning, New York, USA) to meet the requirement of a minimal cell infusion density of 2 × 10 6 cells/mL. The MSCs were then resuspended in preservation media (NaCl, RL, NaCl+0.4%HA or F I G U R E 1 Experimental design and testing conditions. (A) hMSCs from AD, BM and UC tissues were cultured and suspended in preparation for various experiments in this study. Four transport media compositions and two transport temperatures (eight conditions per hMSC type, 32 total conditions) were prepared as follows: RL, RL supplemented with 0.4% HA, NaCl (0.9%), and NaCl (0.9%) supplemented with 0.4% HA; each of the media were stored at 4-8°C and 18-24°C. (B) Testing conditions of hMSCs at 0 h, 24 h, 48 h and 72 h after harvest to identify the optimal preservation conditions for MSCs from all three tested sources (AD, BM and UC) RL+0.4%HA) and then stored at two different temperatures, 2°C-8°C and 18°C-22°C, without shaking and agitation. Three technical repeats per condition per tissue source were performed. The duration of storage was 24, 48 or 72 h. Cell viability was evaluated based on two independent methods, including an automated cell counter (Countess FL II, Thermo Fisher Scientific) using Trypan blue staining and flow cytometry measurement of 7-AAD staining detected by a BD FACSCanto flow cytometer (BD Biosciences).

| Cell recovery assessment
After subjecting the cells to the storage conditions in the four media and two temperature conditions, the cells were collected from the cryovials and plated into CTS CellStart-coated 24-well plates (Gibco; #A1014201) at a seeding density of 5,000 viable cells/cm 2 . Cell confluence was measured every 24 h using a Spark ® Multimode Microplate reader (Tecan) until the culture reached 80% confluence.
The environmental settings were 37°C and 5% CO 2 throughout the experimental process. The medium at 0, 24, 48 and 72 h was frozen for the analysis of growth factors and cytokines. Three technical repeats were performed for all the experiments.

| Analysis of glycolysis and mitochondrial respiration
Cell mitochondrial stress and glycolytic stress were evaluated using an Agilent Seahorse XFe96 Analyzer (Agilent Technologies) according to the manufacturer's recommended protocol. Cells were seeded at 1 × 10 4 cells per well onto Seahorse XF96 cell culture microplates (Agilent Technologies) coated with CTS TM CellStart TM and were incubated overnight at 37°C in 5% CO 2 . The XFe96 Extracellular Flux sensor cartridges (Agilent Technologies) were hydrated with distilled water and stored at 37°C without CO 2 for 24 h and incubated with Agilent Seahorse XF Calibrant (pH 7.4) (Agilent Technology) for 1 h at 37°C without CO 2 before the assay. On the day of the experiment, XF assay medium was supplemented with 10 mM glucose (Agilent Technologies, #103577-100), 2 mM glutamine (Agilent Technologies, #103579-100) and 1 mM pyruvate (Agilent Technologies, #103578-100) and warmed to 37°C. The growth medium was removed, and the wells were rinsed with assay medium before they were then filled with 180 μL of assay medium; the cell culture plates were incubated for 1 h at 37°C without CO 2 .
The Seahorse XF Cell Glycolysis Stress test (Agilent Technologies, After the assay was completed, the cells were fixed with 4% paraformaldehyde (PFA) and stained with DAPI staining solution (1:5000, Abcam; #ab228549). The DAPI signals were captured using the ImageXpress ® Micro Confocal High-content Imaging System (Molecular Devices). The total cell number in each well was then calculated and used for normalization. At least three technical repeats were performed per storage condition for all the experiments.

| Growth factor and cytokine quantification by multiplex immunoassay
Quantitative analysis of growth factors and cytokines was performed using the ProcartaPlex Human Immunoassay (Affymetrix, eBioscience). The supernatant from the MSCs used in the cell confluence experiment was collected on day five and stored at −80°C until further analysis. Samples were thawed on ice and subsequently centrifuged at 10,000 ×g for 10 min to remove particulates according to the manufacturer's protocol. We measured the levels of brain-derived neurotrophic factor (BDNF), LIF, stem cell factor (SCF), vascular endothelial growth factor-D (VEGF-D), nerve growth factor-beta (NGFβ), epidermal growth factor (EGF), fibroblast growth factor 2 (FGF-2), hepatocyte growth factor (HGF), platelet-

| Statistics
Statistical analyses were performed using two-sided Student's t test with GraphPad Prism 8, unless otherwise stated. The data were presented as means ±SEMs. All the experiments were performed in biological triplicates with at least three technical repeats per biological sample for each individual tissue source. To compare the means of multiple groups, the data were analysed using twoway ANOVA as indicated in the text. Significant differences in means are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

| Ringer lactate supports the survivability of MSCs derived from BM, AD and UC for up to 72 h
Mesenchymal stem/stromal cells from AD, BM and UC tissues were expanded in vitro to passage 4 (P4) and equally distributed into 4 preservation media followed by storage at either room temperature (RT, 18°C-22°C) or cold temperature (CT, 4°C-8°C) for 24 h, 48 h and 72 h as illustrated in Figure 1A. Cell viability, proliferation and the expression of MSC markers were measured as described in Figure 1B. To evaluate the impact of preservation conditions, cell viability was first analysed using Trypan blue staining (Table S1).
Live and dead cells were counted using an automated cell counter ( Figure 2A) and were confirmed by flow cytometry analysis of nucleic acid staining by 7-AAD (Table S2). All data are presented as the means ±SEM with three biological replicates.
Harvested AD-MSCs stored in NaCl-based conditions showed a significant reduction in viability after 24 h in RT and CT (77% ± 2.5% and 70% ± 5.5%, respectively; p = 0.001). After 72 h of preservation, while 66% ± 3% of AD-MSCs stored in NaCl at RT were viable, their CT counterparts showed a reduced viability to 62% ± 3%. RL+HA at RT showed 70% ± 1% viability, which is higher than that of their counterparts stored at CT (60% ± 1.5%; Figure 2B Table S1). Using RL-based media improved the viability of UC-MSCs, especially at CT ( Figure 2B). UC-MSCs maintained in RL+HA showed the highest survival rate after 72 h at CT (67% ± 4%) compared to other conditions. Taken together, the results from the viability assays illustrated that the viabilities of AD-, BM-and UC-MSCs were best maintained in RL-based media for up to 72 h (>70% viability) and that supplementation with 0.4% HA did not dramatically improve the viability of MSCs under the tested conditions. Interestingly, MSCs derived from BM exhibited the highest viability when stored at RT, whereas AD-and UC-MSCs showed improved viability when stored at colder temperatures. Similar results were obtained in the 7-AAD analysis, although the results showed a tendency of higher cell viability than that of the Trypan blue technique ( Figure 2B, Table S2).

| Distinct cell recovery profiles after 72 h of preservation
Cell attachment and proliferation are two important characteristics of MSCs that are directly affected by cell storage. To evaluate these two factors, dissociated MSCs in each condition were replated, and confluence was measured every 24 h until the cells reached 80% confluence. Table 1 presents the extracted data describing the preservation conditions allowing the stored cells to most rapidly reach 80%. Three distinct cell proliferation profiles were observed for MSCs derived from different origins, reflecting their recovery rate. to reach 80% confluence, whereas cells stored in NaCl at RT could not attach and proliferate (Table 1; Figure S1).  Figure S1).
The sensitivities of UC-MSCs under different preservation conditions were also observed (  The results showed that all tested MSCs were able to differentiate into osteogenic, adipogenic and chondrogenic lineages, with no difference between before and after storage, or between RL and RL supplemented with 0.4% HA. (Figure 2C and Figure S3).
Consequently, MSCs preserve their fundamental biological features after 72 h of storage in RL-based media at optimum temperature, as recommended by ISCT.

| Metabolic analysis of hMSCs under different storage conditions
To determine whether the survival and recovery rate in hMSCs was accompanied by changes in metabolism, we examined the two major metabolic pathways in the cells glycolysis and mitochondrial respira-     Our study successfully identified acceptable and clinically relevant preservation conditions for MSCs derived from the three most commonly used tissues (AD, BM and UC), as shown in Table 3 In our study, we noted a significant difference in viability measurement using Trypan blue and 7-AAD, which was in line with a previous study. 18  Recent studies indicate that inhibition of the Na + /K + -ATPase pump in low K + medium resulted in a reduction in intracellular K + concentration, which, in turn, slowed the proliferation rate of cell lines in vitro. 25,26 It was reported that a K + concentration of approximately 500 µmole/g protein is the threshold by which it would directly prevent the proliferation process. 27,28 Regarding the MSC proliferation potential, recent research suggests that the growthdependent cell K + concentrations dropped within the range between 600 and 1,100 µmol/g protein, which was associated with the accumulation of G1 cells in the population and resulted in reduced proliferation and delays in cell cycle progression. 29   (for AD-and UC-MSCs) in RL or RL+HA solution. We also analysed the phenotype, metabolism and paracrine profile of MSCs after they were stored under the aforementioned conditions, providing a basis for future studies to investigate the potential mechanism that enhances the effectiveness of preserved MSC therapy. As elucidated in this study, MSCs under the optimal preservation conditions described here harness diverse adaptations to maintain the functionality of these cells in response to the surrounding environment. Future studies are still required to explore the mechanism underlying our observations, especially with regard to metabolic alteration and paracrine secretion of these MSCs.

ACK N OWLED G EM ENTS
The authors would like to thank our collaborating clinicians and nurses at the Vinmec International Hospital (Times City) for collecting samples for this study and the Department of Cellular Therapy (High-tech centre) for supporting our study. The authors appreciate the support from Vinmec Tissue Bank for their high-class service of the cryopreservation of our samples. Finally, the authors would like to thank all the volunteers who donated primary materials for the research.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.