| Objective:To explore the effects of cryopreservation on the cell survival rate,cell viability,early cell apoptosis,migration ability and tendon-related marker expression of tendon-derived stem cells(TDSCs)in rat patellar tendons.Methods:In the experimental group,the patellar tendon tissues of six 4-month-old Sprague-Dawley rats were taken out and placed in cryopreservation tubes containing cryoprotectant agent.They were placed in the refrigerator at 4? for 2 hours and at-80? overnight.They were stored in liquid nitrogen for one week and then removed for cell culture.In the control group,patellar tendon tissues of the same condition were taken out for cell culture without any intervention.The two groups of patellar tendons were minced,digested with type I collagenase(3mg/ml,Sigma)to obtain nucleated cells,the survival rate of nucleated cells was detected by trypan blue exclusion assay;nucleated cell colony-forming ability was tested by crystal violet staining;cells from passages 3(P3)cultured under the same conditions were used for the other experiments,cell viability of TDSCs was detected by Alamar Blue method;early cell apoptosis was determined with Annexin V-FITC/PI assay;transwell method was used to find the cell migration ability;the mRNA expressions of tendon-related markers Collal,Scx and Tnmd were detected by qRT-PCR.Results:Compared with the control group,the survival rate of nucleated cells in the cryopreserved patellar tendons was decreased(P<0.05);it is confirmed that the TDSCs could be successfully isolated and cultured because of the formation of the primary nucleated cell clones in the experimental group and the control group,however,the number of TDSCs in the cryopreserved patellar tendon tissues was decreased(P<0.05);there was no significant difference in cell viability between the experimental group and the control group(P>0.05)in the progress of Passage 3(P3)TDSCs cultured for 14 days;similarly,there was no significant difference in the early cell apoptosis rate,cell migration ability and expressions of tendon-related markers of TDSCs between the experimental group and the control group(P>0.05).Conclusion:The survival rate of nucleated cells and TDSCs in cryopreserved rat tendon tissues is lower,but the tendon tissues still retain a large number of active stem cells.Although the number of TDSCs in the experimental group decreased,its cell viability(P3),early cell apoptosis rate,migration ability in vitro and cell tenogenic differentiation ability were not different from those of TDSCs in the control group.The study can provide the relevant theoretical basis for the clinical application of cryopreserved allograft tendon transplantation to repair tendon defects. |
PDF Full Text Request