Adipose tissue samples were collected in accordance with a protocol approved by the Polish Ministry of Health and proceeded within 24 h after the collection. The samples were obtained anonymously from healthy volunteers (n = 5) by liposuction. Every sample of adipose tissue (lipoaspirate) was 100–150 ml of capacity. The lipoaspirate was washed several times until a clear yellow tissue was obtained. The tissue was then digested with 0.1% collagenase NB 6 (SERVA) for about 30–60 min at 37°C with a rotation (depending on the quality of the lipoaspirate and content of adipose tissue in it, the incubation time varies. The criterion for interrupting the digestion process is the achievement of the appropriate consistency and homogeneity by the processed lipoaspirate). The obtained suspension was centrifuged (300 g, 7 min) to remove the collagenase and to isolate the stromal vascular fraction (SVF), the heterogeneous cell population consisting with preadipocytes, macrophages, lymphocytes, endothelial cells, AT-MSC and many others. SVF was then suspended in StemMACS™ human MSC Expansion Media Kit XF (Miltenyi Biotec) supplemented with antibiotics/antimicotics (Thermo Fisher) and adhered AT-MSCs were expanded in the 75 cm² flasks (NUNC, Thermo Scientific) at 37°C, 5% CO₂ and 90% humidity. AT-MSCs were cultured in these standard conditions until passage 3, which was used in further experiments.

The freezing procedure was performed using the IceCube device (Sylab). Four DMSO-based cryoprotectants were selected for our purposes: Cryo1—commercially available from Biological Industries; Cryo2—commercially available from ZENOAQ/AMSBIO; Cry3—composed of 10% DMSO, dextran40 in NaCl and 5% human albumin Alburex 5 (CSL Bechring) (Gao and Critser, 2000; Fuller, 2004); Cryo4—composed of 10% DMSO in Alburex 5.

For the experiments AT-MSCs from the third passage (P3) were used. The cells were frozen at the density of 5 × 10⁶ cell/ml using our own freezing program formulated for the 2–4.5 ml tubes (Figure 1).

After an arbitrarily selected weekly cryopreservation period, all samples were placed in the 37°C water bath for rapid thawing until no ice was detectable. The thawed samples were slowly dissolved in the culture medium (1:20) and after centrifugation the supernatant containing DMSO was removed. The obtained cells were suspended in the medium using the same seeding density and cultured at 37°C, 5% CO2 and 90% humidity in the 75 cm² flasks (NUNC, Thermo Scientific) until the 5th passage. Non-frozen AT-MSCs from passages 4 and 5 were used as control.

The phenotypic profile of AT-MSCs was determined using the PE-conjugated antibodies against human antigens CD73 (catalog no. 550257, BD Pharmingen™, BD Bioscence United States), CD90 (catalog no. 561970, BD Pharmingen™, BD Bioscence United States) and CD105 (catalog no. 560839, BD Pharmingen™, BD Bioscence United States) and using the FITC-conjugated antibodies against human antigens (later referred as Lin cocktail) HLA-DR (catalog no. 555811, BD Pharmingen™, BD Bioscence United States), CD34 (catalog no. 345801, BD Pharmingen™, BD Bioscence United States), CD45 (catalog no. 345808, BD Pharmingen™, BD Bioscence United States), CD 19 (catalog no. 345788, BD Pharmingen™, BD Bioscence United States) and CD14 (catalog no. 345784, BD Pharmingen™, BD Bioscence United States) (BD Bioscience) according to protocol. At least 10 × 10³ cells per sample were analyzed in a FACSCalibur cytometer (BD Bioscience). The results were analysed with use of the CellQuest Pro v.6.0 software (BD Bioscence).

The proliferative potential of the cells was evaluated using the population doubling time (PDT) test. The results were calculated using the following formula: PDT = (CT*Ln2)/ln (Nf/Ni), where CT is the cell-culture time, Nf—the final number of cells, and Ni—the initial number of cells.

The colony-forming unit-fibroblast (CFU-F) assay was used for estimation of the cells’ proliferation capacity. The cells from each sample suspended in MACS medium were placed in the 12-well culture plates (NUNC, Thermo Scientific) at 500 cells/well. On day 5 of the culture the cells were fixed with 4% PFA (CHEMLAND) and stained with crystal violet (Gibco). Colonies containing at least 50 cells were counted.

The frozen cells were thawed in the 37°C water bath, centrifuged (300 g, 7 min) to remove the remaining cryoprotectant and suspended in the MACS medium. After 30 min of incubation at 37°C to restore their membranes the cells’ viability was estimated using the Trypan Blue test and the Nikon Eclipse E 200 (Nikon) light microscope at ×20 magnification. Using hemocytometer chamber all living and dead cells are counted and the alive are presented as a percentage of all cells.

Cell suspensions obtained from both fresh and frozen/thawed P4 cells were seeded in the 6-well culture plates (NUNC, Thermo Scientific) at 1 × 10⁴ cells per well and cultured until about 60% confluency. The coverslips were removed and the attached cells were fixed with 4% PFA. The cells were then permeabilized with Triton X-100 (Merck) and blocked with 1% BSA in the phosphate-buffered saline (PBS) for 30 min. The obtained cell suspensions were incubated with the primary antibody tagged with the TRITC-conjugated Phalloidin (Merck) for 60 min at room temperature, washed with a washing buffer (1xPBS containing 0.05% Tween-20) and incubated with the FITC-conjugated secondary antibody for 60 min. To stain the nuclei, the cells were additionally incubated with DAPI for 5 min at room temperature. The cell preparations were then examined under the Leica DMi8 (Leica) microscope.

The P4 cells were seeded at 5 × 10³ cells/cm² onto the wells of the 24-well plate (BD Falcon), grown to the 50% confluence and then cultured in the following media (Thermo Fisher): 1) the StemPro Adipogenesis Differentiation Medium for the evaluation of the cells’ adipogenic potential, 2) the StemPro Chondrogenesis Differentiation Medium for the evaluation of the chondrogenic potential, and 3) the StemPro Osteogenesis Differentiation Medium for the evaluation of the osteogenic potential. After 3 weeks of incubation in standard conditions the cultured cells were fixed with 4% Paraformaldehyde, stained with suitable dye and analyzed under the Leica DM IL LED light microscope (Leica) for the presence of adipocytes (after staining with Oil Red O, Sigma), chondrocytes (after staining with Alcain Blue, Sigma) and osteocytes (after staining with Alizarin Red, Sigma).

The expression of genes was analyzed with qPCR performed in triplicates from three independent experiments. Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA) from the control and frozen/thawed AT-MSCs (P4 and P5 cells). cDNA was synthesized using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, United States) according to the manufacturer’s instruction. After the reverse transcription, all the cDNA samples were diluted to a final concentration of 8.333 ng/μl and stored at −80°C until further use. The specified primers are presented in Table 1. PowerUp™ SYBR™ Green Master Mix (Thermo Fischer) was used under the following conditions: activation—95°C for 2 min, denaturation—95°C for 5 s, annealing—60°C for 30 s for a total of 40 cycles. Results were analyzed using the 2^−∆Ct method relating gene expression to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT 1).

Supernatants from the P4 and P5 cells were collected, centrifuged (10,000 g, 10 min) and stored at −80°C. According to the producer’s guideline, the MILLIPLEX MAP HCYTOMAG-60K and the TGF-β1,2,3 magnetic bead kits (Merck KGaA) were used to evaluate the following cytokines, chemokines, and growth factors: the pro-inflammatory IL-8, IFN-γ, IL-2, IL-1β, MCP-1, IL-3, IL-12, IL-17, IP-10, MIP-1β, and MIP-1α, the anti-inflammatory IL-4, IL-5, IL-6, IL-10, IL-13, IFN-α, TGF-β1, and TGF-β2, and other growth factors, such as PDGF AA, and G-CSF. All the reagents and samples were prepared according to the manufacturer’s protocol. The Belysa 1.0.19 program was used for the analysis of the results.

Results are presented as means ± standard error of the mean (SEM). The GraphPad Prism V6.0 software (GraphPad Software, La Jolla, CA) was used to perform t-tests and estimate statistical differences between the results. p-values less than 0.05 were considered statistically significant.

Cells from all the groups exhibited a typical fibroblast-like, spindle-shaped morphology (Figure 2) and adhered to plastic surfaces. No marked differences between the non-frozen (control) and frozen/thawed cells were noted with respect to these features.

Cells from the four experimental groups as well as from the control group demonstrated classical phenotype of cultured AT-MSCs, i.e., expressed on the surface the CD73, CD90, CD105 antigens and did not express the CD14, CD19, CD34, CD45, HLA-DR antigens (Lin cocktail) (Table 2). No significant differences in the expression of these antigens were detected between the groups.

Proliferative capacities of the control and frozen/thawed AT-MSCs are presented in Figure 3. In the cryopreserved cells obtained from passage 4 the population doubling times were slightly (but not significantly) shorter than that of the control cells (Figure 3, left side). Likewise, PDTs of the cells from passage 5, which were treated with cryoprotectants 1 through 3 were insignificantly less than in the control cells and comparable to those of the P4 cells. In contrast, in the passage 5 cells pre-treated with cryoprotectant 4 PDT was longer than in cells treated with cryoprotectans 1 through 3, but the differences were not statistically significant (Figure 3, right side).

As indicated in Figure 4 clonogenic potential of the non-frozen (Control) cells was slightly higher than those of the frozen/thawed cells. In the passage 4 cells clonogenic potential of cells treated with cryoprotectant 2 was significantly suppressed compared to the control cells; it was also lower than the potentials of the cells from other experimental groups, but the differences were not significant (Figure 4, left side). Clonogenic potential of the passage 5 cells was similar in all the experimental groups (including cryo 2) and did not differ from that of the non-frozen cells (Figure 4, right side).

As shown in Table 3, average survival of the frozen-thawed cells from the four experimental groups ranged from 77 to 92% and the differences in cell viability between the groups were not statistically significant.

Analysis of the cytoskeleton was done because this structure helps cells maintain their shape and internal organization. By analyzing the cytoskeleton, we wanted to check whether the composition of the cryoprotective fluid may have any impact on its structure. As indicated by the actin staining test there is no such relationship. No evident differences in the microfilament structure were detected between the non-frozen (control) and frozen/thawed AT-MSCs (Figure 5).

As shown in Figure 6, none of the four cryoprotectants used affected differentiation of the AT-MSCs along the chondrogenic, adipogenic and osteogenic lineages which was similarly expressed in all the three cases (Figure 6).

The impact of the freezing/thawing on the expression of the selected genes in the AT-MSCs is presented in Figure 7. The level of expression of C-MYC in the cryoprocessed cells was in most cases similar to that demonstrated by the non-processed control cells except for the cryo2 P4 and cryo4 P5 cells which exhibited the significantly increased expression of this gene (Figure 7).

Compared to the non-frozen control cells expression of COL10A was markedly albeit insignificantly suppressed in the cryopreserved cells from passage 5, especially in those treated with Cryo 1. In the latter cells, expression of COL10A was also significantly lower than in the cryopreserved cells P4 cells in which the gene was markedly but insignificantly better expressed than in the non-frozen control cells (Figure 7).

The pattern of expression of RUNX2 was similar to that of COL10A1 as demonstrated by the significantly suppressed expression of this gene in the passage 5 cells compared to their counterparts from passage 4 from all the four experimental groups. However, compared to the control level, expression of RUNX2 was lower in the passage 4 cells, esp. in those treated with cryo 1 and cryo2, but the differences were not statistically significant (Figure 7).

In cells treated with cryo1, cryo 3 and cryo4 expression of FABP4 was insignificantly lower than in the non-frozen control cells. In contrast, in cells treated with cryo2 and obtained from passages 4 and 5 this expression was slightly but insignificantly higher than in the control cells (Figure 7, panel AD).

With respect to BGLAP no statistically significant differences in the expression of this gene were noted between the experimental groups of the cells as well as between the experimental and the control cells (data non shown).

With respect to the pluripotency-associated gene SOX-2, the adipogenesis-associated gene ADIPOQ, and the chondrogenesis-associated gene COL2A no expression thereof was detected in the investigated cells from both the control and the experimental groups (data not shown).

As shown in Table 1, there was a noticeable variation in the secretion of IL-6, MCP-1, TGF-β1, fractalkine, G-CSF, IP-10, MIP-1α, MIP-1β, IL-10 and IFNα by the cryopreserved AT-MDCs obtained from passage 4, but generally the levels produced by these cells and that secreted by the non-frozen, control cells were not statistically significant. For example, average concentrations of IL-6 in the culture media of the cryopreserved cells ranged from 247 to 1781 pg/ml depending on the cryoprotectant used as compared to 625 pg/ml detected in the medium of the control cells. In contrast, compared to the control values, significantly lower levels of TGF-β2 and FLT-3L were detected in the media of the passage 4 AT-MDCs treated with either of the four cryoprotectants.

In the case of passage 5 cells no statistically significant differences were detected between the amounts of the tested cytokines and growth factors produced by the cryopreserved and the non-frozen, control AT-MDCs. For example, secretion of IL-6 by the cryoprocessed cells averaged from 306 to 356 pg/ml whereas in the medium of the control cells the average concentration of this cytokine equaled to 345 pg/ml. Likewise, no statistical differences could be detected between the levels of other cytokines/factors secreted by the cryoprocessed and the control cells obtained from passage 5, including TGF-β2 and Flt-3L the production of which was significantly suppressed in the frozen cells from passage 4 (Table 4).