Dimethyl sulfoxide-free cryopreservation solution containing trehalose, dextran 40, and propylene glycol for therapy with human adipose tissue-derived mesenchymal stromal cells

Study design

The present study was approved by the ethics committee of Otsuka Pharmaceutical Factory, Inc.

We performed three experiments. First, the effects as a cryoprotective vehicle with 10% PG were compared among LR, LR-3 T, and LR-3 T-5D. Second, the cell characteristics (viability, annexin V-positive ratio, colony-forming capacity, differentiation ability, cell surface markers, and mRNA expression level) after cryopreservation in liquid nitrogen were compared between LR-3 T-5D with Me2SO and LT-3 T-5D with PG. Third, the concentration of PG was optimized.

Components of the solutions

Lactated Ringer’s solution (LR; Lactec® Injection), lactated Ringer’s solution with 3% trehalose (LR-3 T; Cellstor-W), and lactated Ringer’s solution with 3% trehalose and 5% dextran 40 (LR-3 T-5D; Cellstor-S) were supplied by Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). Me2SO (CultureSure®DMSO) was purchased from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). PG was purchased from Maruishi Pharmaceutical. Co., Ltd. (Osaka, Japan) and Fujifilm Wako Pure Chemical Co. (Osaka, Japan). The base solution (LR, LR-3 T, or LR-3 T-5D) with 4 or 10% cryoprotectant was prepared by mixing base solution and cryoprotectant at ratios of 24:1 or 9:1 (vol/vol), respectively. The compositions of LR-3 T, LR-3 T-5D, LR-3 T-5D with 4 or 10% Me2SO, and LR-3 T-5D with 4 or 10% PG are described in Table 1.

Table 1 Compositions of preservation solutions (LR-3 T and LR-3 T-5D) and the cryopreservation solution (LR-3 T-5D with 10% Me2SO and LR-3 T-5D with 10% PG)Preparation of hADSCs

Human ADSCs (Female, 44Y or 32Y, PT5006, Lot No. 0000692059 or 19TL200176; Lonza Walkersville, Inc., Walkersville, MD, USA) were used in this study; 44Y, and 32Y indicate the ages of the donors. Human ADSCs were seeded in a 75 cm2 flask with 15 mL of medium prepared from a medium kit (PT-4505 ADSC BulletKit™, Lonza Walkersville, Inc.) and maintained at 37 °C in a humidified atmosphere of 5% CO2. All of the medium was changed every 3 or 4 days. Cells were passaged at approximately 90% confluency, and passages 2, 3, or 5 (3, 4, or 6 after cell preparation) were used for the experiments. Cells were collected as described in previous report (Fujita et al. 2021).

Cryopreservation of hADSCs

The hADSCs suspended in LR-3 T were transferred to a 15- or 50 mL conical tube, centrifuged (210×g, 5 min, at room temperature), and the supernatant was aspirated. hADSCs were resuspended with LR, LR-3 T, or LR-3 T-5D containing 10% Me2SO or 10% PG so that the final cell concentration was 1.0 to 3.0 × 106 cells/mL. One milliliter of suspension was dispensed into each cryovial (Nunc™ CryoTube™ Vials, size 1.8 mL, Thermo Fisher Scientific Inc., Waltham, MA, USA). Immediately after dispensing, the vial was placed in a BICELL (Nihon Freezer Co., Ltd., Tokyo, Japan) and stored at − 80 °C for approximately 24 h. Then the vial was transferred from the BICELL or the freeze box to a freeze box preincubated in liquid nitrogen and stored in liquid nitrogen for 46 days at the longest. The length of the storage period in each experiment was different among experiments and described in figure legends.

Thawing of cryopreservation solution

The frozen vials in which the hADSCs were stored were quickly thawed in a thermostat bath heated to 37 °C. After thawing, they were used for the following studies.

Cell viability and viable cell recovery ratio

The total numbers of cells and dead cells were counted manually with a plastic cell counting plate (OneCell Counter, Bio Medical Science, Ltd., Tokyo, Japan) after trypan blue staining. Cell viability and viable cell recovery ratio were calculated according to the formulae below.

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$$}\left[ \% \right] = }/} \times 100$$

Annexin V staining

Suspended cells were stained using an Annexin V-FITC Kit (Beckman Coulter, Brea, CA, USA). Measurements were performed using a Gallios flow cytometer (Beckman Coulter, Indianapolis, IN, USA).

Colony-forming capacity

Colony-forming capacity was evaluated as described in previous report (Fujita et al. 2021). Briefly, Cells were plated at a density of 15 cells/cm2 (315 cells in a 21 cm2 culture dish). After 8 days, the cells were stained with Giemsa. Colonies of more than 50 cells were counted. The colony-forming efficiency of cells was calculated by dividing the number of colonies per dish by the number of cells (315) seeded per dish.

Cell proliferation curve

Colony-forming capacity was evaluated as described in previous report (Fujita et al. 2021). Briefly, hADSCs were seeded at a density of 5.6 × 103 cells/cm2 (5.0 × 104 cells/well). The total number of cells was counted with a OneCell Counter on 1, 3, 5, and 7 days after seeding.

Adipogenic and osteogenic differentiation ability

Adipogenic differentiation was induced according to the Poietics™ human ADSCs adipogenesis protocol (Lonza Walkersville, Inc.) and evaluated by Oil Red O staining. Osteogenic differentiation was induced according to the Poietics™ human ADSCs osteogenesis protocol (Lonza Walkersville, Inc.) and evaluated with an alkaline phosphatase staining kit (AK20, Cosmo Bio Co., Ltd., Tokyo, Japan) and a calcified nodule staining kit (AK21, Cosmo Bio Co., Ltd.).

Cell surface markers

To examine the surface immunophenotypes of the cells, 2 × 105 cells in 20 µL of staining buffer with fetal bovine serum (BD Biosciences, San Jose, CA, USA) were incubated for 60–120 min on ice with phycoerythrin-labeled antibodies against human CD14, CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR (BD Biosciences) or the respective isotype controls (BD Biosciences). After washing, the labeled cells were analyzed using a Gallios flow cytometer (Beckman Coulter).

mRNA expression level after cytokine stimulation

The changes in mRNA expression of the following human genes (Table 2) were examined in hADSCs under the following conditions with subcultures both before and after frozen storage. Either freshly prepared or thawed hADSC suspensions (1 × 106 cells) were diluted with culture medium (1:9) and were centrifuged at 800×g for 5 min. Each cell pellet was diluted in medium and divided in half (5 × 105 cells each). hADSCs were cultured in medium with or without both 5 ng/mL recombinant human interferon gamma (IFN-γ; R&D Systems, Inc., Minneapolis, MN, USA) and 5 ng/mL recombinant human tumor necrosis factor alpha (TNF-α; R&D Systems, Inc.) using 25 cm2 culture flasks for 24 h. Then, hADSCs were collected with trypsinization and were lysed in RLT buffer (Qiagen Inc., Germantown, MD, USA) with 2-mercaptoethanol. Total RNA was isolated using RNeasy columns (Qiagen Inc.) following the manufacturer’s instructions, and the RNA amount was measured by a NanoDrop 200c (Thermo Fisher Scientific Inc.). cDNA was synthesized from 1 µg of RNA using a High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific Inc.) with the following conditions: 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min, and was stored at − 30 °C. 25 ng of total RNA equivalent cDNA per reaction was mixed with TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific Inc.) (1:1) into a TaqMan Array Plate 32 Plus (Fast, 0.1 mL) (Thermo Fisher Scientific Inc.). PCR assay was performed using the Applied Biosystems 7500 Fast Real-Time PCR System with the following profile: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 20 s, and 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The threshold cycle (Ct) was calculated by the instrument's software (7500 Fast System ver. 2.3). The relative expression of each mRNA was calculated using the ΔCt method (where ΔCt is the value obtained by subtracting the Ct value of hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA from the Ct value of the target mRNA). Specifically, 2−(ΔCt) is expressed as the amount of target mRNA relative to HPRT1 mRNA. Target genes were selected from immunomodulatory genes, indoleamine 2,3-dioxygenase 1 (IDO1), hepatocyte growth factor (HGF), prostaglandin E synthase (PTGES), prostaglandin-endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase 2, COX2), programmed death 1 ligand-1 (PD-L1), and chemokine (C–C motif) ligand 5 (CCL5, also known as regulated on activation, normal T cell expressed and secreted, RANTES).

Table 2 Real-time PCR primersAnalysis and statistics

Results are presented as the mean ± standard deviation (SD). Statistical analysis was performed using Dunnett’s multiple comparison test, Tukey’s multiple comparison test, and Student’s t test with a significance level of p < 0.05. Data were analyzed with SAS 9.4 (SAS Institute Inc., Cary, NC, USA).

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