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 × 10 5 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 × 10 3 cells) were cultured in 60 cm 2 dishes for 14 days for colony formation assays. Additional 62.5 μL samples of cell suspensions (1.25 × 10 5 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. Electronic supplementary material The online version of this article (10.1186/s12891-019-2700-3) contains supplementary material, which is available to authorized users.

Thawed Cryopreserved Synovial Mesenchymal Stem Cells Show Comparable Effects to Cultured Cells in the Inhibition of Osteoarthritis Progression in Rats

“…Similar results have been reported previously for human synovial MSCs [25]. DMSO in the cryopreservation uid substantially increased the viability of synovial MSCs.…”

“…The tubes were removed from the freezing vessel and the frozen cells were thawed using a cell-thawing device (ThawSTAR, Astero Bio, Menlo Park CA, USA). As a negative control for in vitro studies, the cells were also frozen in 100% FBS [25].…”

Thawed cryopreserved synovial mesenchymal stem cells show comparable effects to cultured cells in the inhibition of osteoarthritis progression in rats

“…The reason for testing 100% FBS was that we expected that it could serve as a negative control for the cryopreservation solution. We have previously reported that 95% FBS plus 5% DMSO was effective, whereas 100% FBS was not effective, for the cryopreservation of human MSCs 22 . The present study confirmed similar results for rat MSCs.…”

Cryopreserved Spontaneous Spheroids from Compact Bone-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

“…Since spheroids consist of dozens to hundreds of cells, the optimal freezing protocol for a single cell suspension cannot be applied. The optimal conditions should be determined based on the size of spheroids and the type/status of cells [36][37][38] . Use of lower concentrations of cryoprotectant cause insufficient permeation to the central part and may result in a lower survival rate.…”

“…Our results demonstrated that the 5% DMSO group had the highest cell survival, confirmed by the lowest percentage of dead and apoptotic cells. For the single-cell suspensions, the reported optimal concentrations of DMSO were between 5 to 10% for most of the cells under various freezing protocols 25,37,39,40 . Interestingly, we found that the cell viability of the 10% DMSO group was significantly lower than that of the 5% DMSO group.…”