Cryopreservation in 95% serum with 5% DMSO maintains colony formation and chondrogenic abilities in human synovial mesenchymal stem cells

Abstract

Background: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. The optimum cryopreservation medium has not been determined, but dimethylsulfoxide (DMSO) should be excluded, if possible, because of its toxicity. The purposes of our study were to examine the possible benefits of higher concentrations of serum and the effectiveness of 100% serum (without DMSO) for the cryopreservation of synovial MSCs.

Methods: Human synovium was harvested from the knees of four donors with osteoarthritis during total knee arthroplasty. Synovial MSCs (8 × 10⁵ cells) were suspended in 400 μL medium and used as a Time 0 control. The same number of synovial MSCs was also suspended in 400 μL α-MEM medium containing 10% fetal bovine serum (FBS) (5% DMSO, and 1% antibiotic), 95% FBS (and 5% DMSO), or 100% FBS (no DMSO) and cryopreserved at - 80 °C for 7 days. After thawing, the cell suspensions (1.5 μL; 3 × 10³ cells) were cultured in 60 cm² dishes for 14 days for colony formation assays. Additional 62.5 μL samples of cell suspensions (1.25 × 10⁵ cells) were added to tubes and cultured for 21 days for chondrogenesis assays.

Results: Colony numbers were significantly higher in the Time 0 and 95% FBS groups than in the 10% FBS group (n = 24). Colony numbers were much lower in the 100% FBS group than in the other three groups. The cell numbers per dish reflected the colony numbers. Cartilage pellet weights were significantly heavier in the 95% FBS group than in the 10% FBS group, whereas no difference was observed between the Time 0 and the 95% FBS groups (n = 24). No cartilage pellets formed at all in the 100% FBS group.

Conclusion: Synovial MSCs cryopreserved in 95% FBS with 5% DMSO maintained their colony formation and chondrogenic abilities to the same levels as observed in the cells before cryopreservation. Synovial MSCs cryopreserved in 100% FBS lost their colony formation and chondrogenic abilities.

Fig. 1

Scheme for the colony formation and chondrogenesis assays. a Colony formation assay. Synovial mesenchymal stem cells (MSCs; 8 × 10⁵ cells) were suspended in 400 μL medium containing 10% fetal bovine serum (FBS) as a time 0 control. Synovial MSCs (8 × 10⁵ cells) suspended in 400 μL medium containing 10% FBS, 95% FBS, or 100% FBS were cryopreserved at − 80 °C for 7 days and thawed. A 1.5 μL volume of cell suspension (containing 3 × 10³ cells, including living and dead cells) was added to 60 cm² dishes for colony formation and cell proliferation assays. b Chondrogenesis assay. A 62.5 μL volume of cell suspension (containing 1.25 × 10⁵ cells, including living cells and dead cells) was added to 15 mm tubes for chondrogenesis assays

Fig. 2

Characteristics of synovial mesenchymal stem cells (MSCs) as MSCs. a Cell morphology. b Colony morphology. c Representative histograms for surface markers (d) Multidifferentiation

Fig. 3

Analysis of colony formation. a Representative dishes stained with crystal violet. Synovial mesenchymal stem cells (MSCs) were derived from four donors. b Colony numbers per dish. Data are shown as means ± SD (n = 4 for each donor). *p < .05 by the Friedman test followed by Dunn’s multiple comparisons. c Cell numbers per dish. d Cell numbers per colony

Fig. 4

Analysis of chondrogenesis. a Representative macroscopic appearance of cartilage pellet. Synovial mesenchymal stem cells (MSCs) were derived from four donors. In the 100% fetal bovine serum (FBS) group, no cartilage pellets were formed. b Pellet weight. Data are shown as means ± SD (n = 4 for each donor). *p < .05 by the Friedman test followed by Dunn’s multiple comparisons. ND: not detected. c Representative histological sections stained with toluidine blue and immunostained for collagen type II