Comparison of adhesion of thawed and cultured synovial mesenchymal stem cells to the porcine meniscus and the relevance of cell surface microspikes

Abstract

Background: Placement of a cultured synovial mesenchymal stem cell (MSC) suspension on a repaired meniscus for 10 min accelerated meniscus repair. Upon placement of the MSC suspension on the meniscus, microspikes projecting from the MSC surface trap meniscus fibers and promote MSC adhesion. Thawed cryopreserved MSCs are preferred materials for meniscus repair, as they can be transplanted without additional culture. However, the adhesion ability of thawed cryopreserved MSCs is unknown. Here, we compared the proportion of cultured versus thawed MSCs adhering to a porcine meniscus immediately and 10 min after placement. We also investigated the relationship between adhesion and the number of microspikes on the synovial MSCs.

Methods: Synovial MSCs were prepared from the knees of four donors with osteoarthritis. The "cultured MSCs" were thawed MSCs that were re-cultured and suspended in PBS for transplantation. A similarly prepared suspension was cryopreserved, thawed again, suspended in PBS, and used without further culture as the "thawed MSCs." MSCs with at least three microspikes in SEM images were defined as microspike-positive MSCs. Porcine meniscus surfaces were abraded, cut into a cylindrical shape, and treated with MSC suspension. Non-adherent cells were counted immediately and again 10 min after placement to calculate the adhesion proportion.

Results: The proportion of microspike-positive MSCs was significantly higher in thawed (53 ± 3%) than in cultured (28 ± 5%) MSC suspensions. MSC adhesion to the meniscus was significantly better for the thawed than for the cultured MSC suspensions immediately after placement on the meniscus, but no differences were detected after 10 min. The proportion of MSCs with microspikes in the cell suspension was significantly correlated with the proportion of adhered MSCs immediately after the placement, but not 10 min later. Addition of FBS to the cryopreservation medium promoted a concentration-dependent increase in the proportion of microspike-positive cells.

Conclusions: Thawed MSCs adhered better than cultured MSCs immediately after placement, but adhesion was similar for both MSC preparations after 10 min. Immediately after placement, the presence of microspikes was associated with better adhesion of synovial MSCs to the meniscus.

Keywords: Adhesion; Cryopreservation; Meniscus; Mesenchymal stem cell; Microspike; Scanning electron microscopy; Synovium; Thawed cell.

Fig. 1

Schematic of the study design. For cultured MSCs, the thawed cells were cultured for 14 days, and then suspended in PBS. For thawed MSCs, the thawed cells were cultured for 14 days, cryopreserved in 95% FBS and 5% DMSO for 3 days, and then suspended in PBS without further culturing. The cultured and thawed MSCs were observed by SEM and TEM. The MSCs were placed on porcine menisci, and the proportion of adhering cells and the cell SEM morphology were analyzed immediately and 10 min after MSC placement

Fig. 2

Properties of human synovial MSCs. a Cell morphology. b Colony morphology. c Multidifferentiation potential. d Surface markers of cultured and thawed MSCs. The dotted line indicates the isotype control. MFI, mean fluorescent intensity

Fig. 3

Morphology of cultured and thawed synovial MSCs in suspension. a SEM images. b The proportion of cells with microspikes. Fifty cells were examined once per donor, and the results from five donors are shown independently. The proportion of MSCs with microspikes was 28 ± 5% in the cultured MSC suspension and 53 ± 3% in the thawed MSC suspension.*; p < 0.05 by paired t-test. c Representative TEM images

Fig. 4

Morphology and adhesion proportion of human synovial MSCs immediately after placement on the porcine meniscus. a SEM images. b Proportion of cells adhered to the meniscus. Six cylindrical menisci were examined once per donor, and the results from five donors are shown independently. The proportion of adhering cells was 20 ± 4% for the cultured MSCs and 30 ± 3% for the thawed MSCs. *; p < 0.05 by paired t-test. c Correlation between the proportion of adhered cells and the proportion of cells with microspikes in suspension

Fig. 5

Morphology and adhesion proportion of human synovial MSCs 10 min after placement on the porcine meniscus. a SEM images. b Proportion of cells adhered to the meniscus. Six cylindrical menisci were examined once per donor, and the results from five donors are shown independently. The proportion of adhering cells was 33 ± 3% for the cultured MSCs and 36 ± 3% for the thawed MSCs. *;p < 0.05 by paired t-test. c Association between the proportion of adhered cells and the proportion of cells with microspikes in suspension

Fig. 6

Morphology of MSCs cryopreserved in solutions containing with different concentrations of fetal bovine serum (FBS). a SEM images. Cells with microspikes are marked with arrowheads. b The proportion of cells with microspikes. Fifty cells were examined once per donor, and the results from four donors are shown independently. The proportion of MSCs with microspikes was 27 ± 6% in the 10% FBS, 52 ± 5% in the 50% FBS, and 52 ± 5% in the 95% FBS. *; p < 0.05 by Tukey’s test