Hypothermic Storage of 3D Cultured Multipotent Mesenchymal Stromal Cells for Regenerative Medicine Applications

The regulatory requirements in cell processing, in the choice of a biomaterial scaffold and in quality control analysis, have to be followed in the clinical application of tissue-engineered grafts. Confirmation of sterility during quality control studies requires prolonged storage of the cell-based construct. After storage, preservation of the functional properties of the cells is an important prerequisite if the cells are to be used for cell-based tissue therapies. The study presented here shows the generation of 3D constructs based on Wharton’s jelly multipotent mesenchymal stromal cells (WJ-MSCs) and the clinically-acceptable HyaloFast® scaffold, and the effect of two- and six-day hypothermic storage of 3D cell-based constructs on the functional properties of populated cells. To study the viability, growth, gene expression, and paracrine secretion of WJ-MSCs within the scaffolds before and after storage, xeno-free culture conditions, metabolic, qPCR, and multiplex assays were applied. The WJ-MSCs adhered and proliferated within the 3D HyaloFast®. Our results show different viability of the cells after the 3D constructs have been stored under mild (25 °C) or strong (4 °C) hypothermia. At 4 °C, the significant decrease of metabolic activity of WJ-MSCs was detected after 2 days of storage, with almost complete cell loss after 6 days. In mild hypothermia (25 °C) the decrease in metabolic activity was less remarkable, confirming the suitability of these conditions for cell preservation in 3D environment. The significant changes were detected in gene expression and in the paracrine secretion profile after 2 and 6 days of storage at 25 °C. The results presented in this study are important for the rapid transfer of tissue engineering approaches into clinical applications.


Introduction
Multipotent mesenchymal stromal cells (MSCs) are currently the focus of many studies seeking novel approaches for the treatment of various diseases. It has already been confirmed that the main mechanism of the therapeutic action of MSCs is based on their paracrine activity. MSCs secrete a cocktail of growth factors, cytokines, extracellular vesicles, or mitochondria, which target the host cells and improve their functional activity [1][2][3]. Being isolated from various human tissues, umbilical cord Wharton's jelly (WJ) is a unique source of MSCs with high proliferation potential and paracrine activity [4,5]. We recently compared the growth kinetics, the immune properties, and the paracrine activity of MSCs derived from human adult tissues (bone marrow, BM, and adipose tissue, AT) and WJ (fetal adnexa tissue), and we demonstrated a significantly higher expansion rate of WJ-MSCs compared to BM-and AT-MSCs, while maintaining the neuroprotective and Such viability rates are insufficient for achieving the optimal therapeutic outcomes. The storage of 3D MSC-based grafts under hypothermic conditions is an alternative strategy for short-term graft preservation. It is crucial to choose an optimal storage temperature that is able to preserve the function of the cells at maximum levels.
The aim of our study was to generate 3D allogeneic grafts based on WJ-MSCs and HyaloFast ® non-woven scaffolds under xeno-free conditions, and to study the effect of short-term storage under mild hypothermia (25 • C) and under strong hypothermia (4 • C) on the viability, the paracrine secretion profile, and the gene expression of 3D cultured cells.

Tissue Collection and Source of Chemicals
Discarded human umbilical cords (UC) were obtained from healthy neonates after spontaneous delivery at the University Hospital in Pilsen (Czech Republic). All donors provided written informed consent before UC collection. The human UCs were donated anonymously. All studies involving human tissues or cells were approved by the Ethical Committee of the Institute of Experimental Medicine Academy of Sciences Czech Republic, Prague. All methods were performed in accordance with the relevant guidelines and regulations. Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).

Cell Culture
Umbilical cord collection, WJ-MSC isolation and characterization have already been described in our previous publications [4,26]. The cells were cultured in a xeno-free complete culture medium (CCM) consisting of α-minimal essential medium (αMEM; LONZA, Basel, Switzerland), 5% pooled human platelet lysate (PL; Bioinova, Ltd., Prague, Czech Republic), and 10 µg/mL gentamicin (Sandoz, Holzkirchen, Germany). The cells were cultured at 37 • C in a humidified atmosphere containing 5% CO 2 with regular media changes twice a week. After reaching 90% of confluence, the cells were trypsinized using 0.05% Trypsin/EDTA (Life Technologies, Carlsbad, CA, USA), were counted, and were used for seeding the scaffolds.

Scaffolds, Cell Seeding, and Culture
The commercially-available ready-to-use 3D product HyaloFast ® (Anika Therapeutics inc.) was used as a scaffold for this study. The scaffold was sterilely cut into 0.5 × 0.5 cm squares in a laminar box, placed in bacterial dishes (P-Lab, Prague, Czech Republic) with a non-adherent surface (five squares in each), and immersed in CCM for 20-30 min to hydrate the scaffold. Cell seeding was performed by immobilizing the cells with a blood plasma-based hydrogel. Briefly, 300,000 cells were resuspended in 29.7 µL human blood plasma (Thomayer University Hospital, Prague, Czech Republic), 0.8 µL 10% CaCl 2 (HBM Pharma, Martin, Slovak Republic), and 2.5 µL human serum (Biowest, Nuaillé, France), and were pipetted onto one moistened scaffold. The 3D constructs were left to polymerize for 5-10 min and were then cultured in 10 mL CCM in a CO 2 incubator at 5% CO 2 and 37 • C for 8 days. The culture medium was changed twice a week.

Hypothermic Storage of 3D Constructs
After 8 days of culture, the 3D constructs were transferred to sterile screw-cap cryotubes (Nunc, Roskilde, Denmark) with 1 mL fresh CCM, one construct per tube, and the cell-seeded constructs were incubated at 37 • C and 5% CO 2 with the lid remaining ajar to allow air exchange and saturation of the medium with CO 2 . After 1 h of incubation, the cryovials were tightly sealed and were stored at either 4 • C or 25 • C for 2 or 6 days.

Resazurin Assay
Cell metabolic activity, proliferation, and recovery of WJ-MSCs after storage in 3D constructs were assessed using the resazurin assay [27]. To evaluate the cell growth within the scaffold, the metabolic activity was measured repeatedly on days 1, 4, and 7 in three identical randomly selected samples and was measured on day 8 completely in all samples. The post-storage recovery was measured after 2 or 6 days of hypothermic storage at 4 • C and 25 • C. Each sample was labelled to allow pairing of pre-and post-storage measurements. Briefly, the samples were transferred to a new 24-well plate containing 0.5 mL of fresh CCM with 40 µM resazurin in each well. The samples were incubated for 2 h at 37 • C in a humidified atmosphere with 5% CO 2 . Then, 150 µL of the solution was transferred to a 96-well plate, and the fluorescence (Ex/Em = 530/590) was measured on a Synergy TM HT Multi-Mode Microplate reader (BioTek, Santa Clara, CA, USA). The measured values were corrected for the background control (CCM with resazurin). Each experiment was performed in triplicate.

Total RNA Isolation
Total mRNA was isolated from the 3D constructs using the Animal Tissue RNA Purification Kit (Norgen Biotek, Ontario, Canada). Due to the small amount of mRNA in one 3D construct, three HyaloFast ® -cell constructs were pooled into a single experimental sample. Samples were solubilized twice (repeatedly) in 200 µL of RL buffer. Isolation was then carried out according to the manufacturer's protocol. The amounts of RNA were quantified using a NanoDrop™ OneC Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using the Omniscript Reverse Transcription Kit (205113; Qiagen, Hilden, Germany) using random hexamers (New England Biolabs, Inc, Ipswich, MA, USA).

Real-Time PCR
Real-time PCR was performed to investigate the changes in the relative mRNA expression of 15 genes selected to perform the multi-sided screening of different cell functions (Table 1) in samples isolated before and after 2 and 6 days of storage.
The mRNA level was quantified using 5x HOT FIREPol ® Probe qPCR Mix Plus (ROX) (08-36-00001; Solis BioDyne, Tartu, Estonia) and by TaqMan Gene Expression Assays (4331182; Thermo Fisher Scientific), labelled with the FAM reporter dye specific to the following human genes: CXCL12 (Hs00171022_m1), CCL2 (Hs002341410_m1), ICAM (Hs00164932_m1), (Hs00174131_m1), VEGFA (Hs00900055_m1), HGF (Hs00300159_m1), FGF2 (Hs00266645_m1), P21 (Hs01040810_m1), HIF1A (Hs00153153_m1), and BAX (Hs00180269_m1), with GAPDH (Hs00187842_m1) as the reference gene. The experiments were performed using LightCycler ® 480 System Technology (Roche, Basel, Switzerland) in a 96-well optical reaction plate. The qPCR was carried out in a final volume of 20 µL. All amplifications were run under the same cycling conditions: 12 min at 95 • C, followed by 42 cycles of 15 s at 95 • C and 1 min at 60 • C. The data for each experimental point are the mean of 4 measurements of 2 independent samples. The relative gene expression was calculated as 2 −∆∆Ct , and the results are presented as the log2-fold change. The results are related to the reference gene and to the non-stored cell samples (d 0) as a control. The results are expressed as Mean ± SEM.

Gene Symbol Full Gene Name
Chemokines:

Conditioned Medium (CM) Production and Proteomic Analysis
After 2 and 6 days of storage at 25 • C, immediately after the resazurin assay, the cellseeded constructs were transferred to 1 mL of fresh CCM and were incubated at 37 • C/5% CO 2 . After 24 h, the conditioned medium was collected and was stored at −80 • C until use. The concentrations of the growth factors and cytokines (SDF-1α, VEGF-A, VCAM-1, FGF-2, HGF, MCP-1, IL-6, and bNGF) were determined using the Luminex ® -based multiplex ProcartaPlex ® Immunoassay (Thermo Fisher Scientific). Nine samples were measured for each time interval. CMs collected on day 8 from the same samples that were later measured after 2 and 6 days of storage at 25 • C served as controls. The measurement was performed on a Bio-Plex 200 Instrument (Bio-Rad, Prague, Czech Republic) according to the manufacturer's instructions. All samples were analyzed in duplicate. The cytokine and growth factor concentrations (pg/mL or ng/mL) in the samples were derived from measured MFIs using fitted standard curves.

Statistical Analysis
Statistical analyses were performed using SigmaStat (Systat Software, San Jose, CA, USA). Multiple-comparison procedures were conducted by analysis of variance, using the Student-Newman-Keuls method. p ≤ 0.05 was considered significant.

Growth and Paracrine Activity of WJ-MSCs within 3D HyaloFast ®
The proliferation of WJ-MSCs within HyaloFast ® was assessed by resazurin assay ( Figure 1A).

Growth and Paracrine Activity of WJ-MSCs within 3D HyaloFast ®
The proliferation of WJ-MSCs within HyaloFast ® was assessed by resazurin assay ( Figure 1A). After 4 days of 3D culture, a remarkable two-fold increase in resazurin activity was observed, confirming the growth of cells within the scaffold. During the subsequent culture, the rate of cell proliferation slowed down, likely due to density-dependent growth inhibition. The complete population of the scaffold by WJ-MSCs was confirmed by a microscopic evaluation ( Figure 1C). To confirm the paracrine secretion activity of the WJ-MSCs within 3D HyaloFast ® , the content of growth factors and cytokines was assessed in a conditioned medium collected after 24 h of culture ( Figure 1B). We detected a high content of HGF, VEGF-A, and MCP-1 (in the range of 3.5-4.6 ng/mL), a lower amount of IL-6 (2.4 ng/mL), and a low content of FGF-2, bNGF, and SDF-1α (less than 1 ng/mL). Thus, 7 days of culture of WJ-MSCs in HyaloFast ® allowed the generation of a viable 3D graft with remarkable paracrine activity. After 4 days of 3D culture, a remarkable two-fold increase in resazurin activity was observed, confirming the growth of cells within the scaffold. During the subsequent culture, the rate of cell proliferation slowed down, likely due to density-dependent growth inhibition. The complete population of the scaffold by WJ-MSCs was confirmed by a microscopic evaluation ( Figure 1C). To confirm the paracrine secretion activity of the WJ-MSCs within 3D HyaloFast ® , the content of growth factors and cytokines was assessed in a conditioned medium collected after 24 h of culture ( Figure 1B). We detected a high content of HGF, VEGF-A, and MCP-1 (in the range of 3.5-4.6 ng/mL), a lower amount of IL-6 (2.4 ng/mL), and a low content of FGF-2, bNGF, and SDF-1α (less than 1 ng/mL). Thus, 7 days of culture of WJ-MSCs in HyaloFast ® allowed the generation of a viable 3D graft with remarkable paracrine activity.

The Effect of Hypothermic Storage on the Recovery of WJ-MSCs in 3D Conditions
To select the optimal temperature for hypothermic storage of 3D constructs based on WJ-MSCs within HyaloFast ® , the samples were placed in sealed vials and were stored at 4 • C and 25 • C for 2 and 6 days. The metabolic activity was determined for the constructs by resazurin assay, and the data were presented as mean relative fluorescence units (RFU) per each sample (Figure 2).

The Effect of Hypothermic Storage on the Recovery of WJ-MSCs in 3D Conditions
To select the optimal temperature for hypothermic storage of 3D constructs based on WJ-MSCs within HyaloFast ® , the samples were placed in sealed vials and were stored at 4 °C and 25 °C for 2 and 6 days. The metabolic activity was determined for the constructs by resazurin assay, and the data were presented as mean relative fluorescence units (RFU) per each sample (Figure 2).     After 2 days of hypothermic storage, we detected significant upregulation of the PDL1, BAX, P21, HGF, VEGFA, TGFB1, and HIF1A genes. Simultaneously, the expression of COX2, SDF1A (CXCL12), MCP1 (CCL2), TWIST, VCAM, ICAM, and IL6 genes was downregulated or unchanged. The duration of storage also affected the expression of separate genes. Here, the expression of PDL1, HGF, MCP1 (CCL2), HIF1A, VCAM1, COX2, SDF1A, and TGFB1 was significantly reduced after 6 days compared to 2 days of storage. Conversely, the expression of P21 was increased after prolonged storage.
The effect of hypothermic storage on the paracrine activity of WJ-MSCs within Hy-aloFast ® is shown in Figure 4. . The data are presented as Mean ± SD. Solid line-non-stored cells, dashed line-background control (the medium without cells). *, **, ***-the data are significantly different compared to the non-stored cells (***-p < 0.001; **-p < 0.01; *-p < 0.05); °°, °°°-the data are significantly different between the 2d group and the 6d group (°°°-p < 0.001; °°-p < 0.01). No significant changes in the levels of MCP-1, HGF, and SDF-1α were detected in the conditioned medium of 3D cultured WJ-MSCs after 2 and 6 days of storage. A significant decrease was indicated for bNGF content. However, even in the control group, the values did not exceed 100 pg/mL. A significant decrease was detected for IL-6 for both storage periods ( Figure 4). However, a significant increase in the content of VEGF-A, FGF-2, and VCAM-1 was indicated after hypothermic storage. The levels of these growth factors became even higher after 6 d of storage. Based on the obtained data, we may conclude that short-term storage of 3D WJ-MSC grafts did not have any detrimental effects on the paracrine secretion capacity of the cells.

Discussion
In this study we have shown the generation of a 3D paracrine construct based on WJ-MSCs and HyaloFast ® using xeno-free clinically-acceptable conditions, and we propose a way to preserve the viability and the paracrine properties of the cells for a period of 6 days, as required to complete sterility testing. We chose HyaloFast ® as a 3D scaffold due to its high applicability in clinical applications, and also its confirmed biocompatibility and biodegradation [17,18,28,29]. HyaloFast ® is based on a semisynthetic insoluble polymer obtained by the esterification of all carboxyl groups of HA with benzyl alcohol (HYAFF-11 ® ). It was previously shown that HYAFF-11 ® provides an appropriate environment, supporting growth, migration, proliferation, and differentiation of MSCs into chondrogenic lineage [30][31][32]. Successful fabrication of tissue-engineered osteochondral grafts has been shown for MSCs loaded into a HYAFF-11 ® sponge [33]. The bone marrow concentrate grown within the HYAFF11 ® scaffolds was able to differentiate into chondrogenic [34] and osteogenic [35] lineages.
As a cellular component, WJ-MSCs were considered optimal for fast generation of the 3D graft due to the high proliferation rate and the strong paracrine secretion capacity, as shown in our previous study [4]. In the present study, we confirmed the proliferation of WJ-MSCs in a 3D environment and complete population of HyaloFast ® during 7-8 days of culture. The overall process of cell seeding, complete population of the scaffold, and subsequent storage for quality control analysis took a total of 14 days. We detected high levels of angiogenic factors HGF, VEGF-A, as well as MCP-1 and IL-6 responsible for the immunomodulatory activity of MSCs in the conditioned medium obtained from the 3D cultures [36].
In this study, we used blood plasma-based hydrogel to improve the seeding efficiency and immobilize the WJ-MSCs within the HyaloFast ® . It is widely accepted that the 3D culture of MSCs in fibrin-based hydrogels supports the proliferation, differentiation, and paracrine activity of cells (reviewed in [37,38]). In our previous study, we found remarkable increase of FGF-2 and VEGF secretion by adipose tissue MSCs, cultured in human blood plasma-based hydrogels [39]. Therefore, we believe that the high paracrine secretion of WJ-MSCs within the HyaloFast ® occurs via the combinatorial action of 3D environment, composed of HyaloFast ® and human plasma-based hydrogels. The fast manufacturing times and the high paracrine activity would be promising for wide application of the proposed 3D WJ-MSC/HyaloFast ® grafts in allogeneic applications.
As mentioned before, the clinical application of cell-based grafts requires the completion of sterility testing of the final packed product, and the storage conditions may significantly affect the therapeutic properties of the graft. It is known that a decrease in temperature slows down the metabolic processes in cells, and the hypothermic storage approach may allow the state of the cells in the pre-storage stage to be preserved. Therefore, in the second part of our study we compared two temperature regimes (4 • C and 25 • C) for the 2 and 6 days of hypothermic storage of the generated 3D constructs. We found that storage at ambient, mild hypothermic conditions (25 • C) was superior to storage at 4 • C. The metabolic activity of cells stored under strong hypothermic conditions for a period of 48 h decreased significantly, whereas at the ambient temperature (25 • C), the metabolic activity remained almost unchanged. Subsequent storage of cells at 4 • C until day 6 resulted in the complete loss of cell viability within the scaffolds, confirming that 25 • C is the temperature of choice during hypothermic storage of 3D grafts.
It is known that irreversible changes in cellular structures may occur at 2-8 • C and may lead to cell apoptosis. In cell suspensions, profound hypothermia causes perturbation of ion balances, leading to osmotic cell swelling, oxidative stress, and an extracellular and intracellular pH switch [40,41]. We have previously shown that the storage of bone marrow MSC suspensions in a conventional saline medium led to a dramatic drop in cell viability after 48 h at 4 • C. In this case, only the use of specially-designed hypothermic solutions could improve the cell viability [42]. However, little is known about the effects of hypothermic preservation on cells populated within 3D scaffolds. Although we have shown that alginate encapsulation improves the viability of MSCs after short-term storage [43], the cells were not attached and were distributed in alginate beads as an entrapped suspension. Tam et al. showed high viability of induced pluripotent stem cell-derived MSCs within a tissue-engineered bone graft [44]. Another report showed poor osteogenic capacity of tissue-engineered bone preserved in an osteogenic medium at 4 • C [45]. Lower survival of MSCs at 4 • C compared to 25 • C has been shown for encapsulated cell aggregates [46]. We showed around 90% recovery of WJ-MSCs after 6 d storage at 25 • C, which we believe to be clinically-acceptable values. Further study was directed towards a deeper evaluation of the effect of mild hypothermic storage on cell characteristics -gene expression and the paracrine secretion profile.
We have not observed a remarkable decrease in the content of most of the growth factors and cytokines analyzed in our study. Only significantly lower secretion of bNGF and IL-6 was detected after both storage periods. Interestingly, however, the secretion of FGF-2 and VEGF-A remarkably increased after 6 d of storage at 25 • C. The secretion of high FGF-2 amounts is not a typical feature of MSCs, and before the storage, the FGF-2 content in the conditioned medium comprised only around 250 pg/mL. Therefore, the remarkable increase in FGF-2 secretion was probably connected with processes that occurred during storage. It is known that the prolonged storage of cells and tissues below physiological temperatures results in the moderation of metabolic activity and cell cycle deceleration or the initiation of apoptotic processes [47]. In this study, the significant upregulation of P21 was detected after 2 and 6 days of storage, indicating the possible temperature-induced cell cycle arrest. Vice versa, it was shown previously that FGF-2 release might lead to the upregulation of P21 [48]. The upregulation of P21 expression may also correspond to the initiation of apoptotic processes in cells within the 3D construct [49]. It was recently shown that an increase in FGF-2 release could be associated with the initiation of apoptotic processes within the cells [50,51]. In our study, we have indicated the upregulation of pro-apoptotic BAX, which aligns with these data and can affect FGF-2 release. The apoptotic nature of the enhanced paracrine activity of MSCs has been recently proposed in several studies [52,53]. In parallel, the VEGF signaling is closely connected with hypoxia and the expression of HIF1A, and the significant upregulation of the HIF1A gene during storage may explain the increase in VEGF-A secretion [54]. Interestingly, the expression of PDL1 involved in the immunomodulatory activity of MSCs was significantly upregulated after storage, and the PDL1 expression can also be associated with the increased expression of HIF1A [55]. Conversely, the level of IL-6 in the conditioned medium decreased significantly after storage, which was accompanied by downregulation of the IL6 gene (Figures 3 and 4). Although many reports have been focused on evaluating the effect of profound hypothermia (at 4 • C) on different types of cells and tissues, little is currently known about the exact mechanisms of cell responses, which occur in MSCs during the storage at moderate hypothermia.
The data therefore suggests that hypothermic storage may affect the gene expression and the paracrine secretion, however more studies are needed to reveal the exact mechanisms of these changes.

Conclusions
We have shown a clinically-acceptable procedure for the fast generation of a paracrine 3D graft based on WJ-MSCs and HyaloFast ® for applications in regenerative medicine. We have further evaluated the effect of hypothermic storage of 3D cell-based constructs on the functional properties of populated cells, and we have shown the different viability of cells after storage under mild hypothermia (25 • C) and under strong hypothermia (4 • C). At 25 • C, the viability of the WJ-MSCs was significantly higher than at 4 • C. We then indicated the changes of gene expression of WJ-MSCs after storage, and we observed the changes in the paracrine profile of the cells. The results presented in this study are important for accelerating the transfer of tissue engineering approaches towards clinical applications.