Study On New Cryoprotectant Agent Of Adipose-derived Stem Cells

Aims: Adipose-derived stem cells(ADSCs)not only have the function of promoting tissue repair and regeneration,but also have the potential of multidirectional differentiation,which plays a very wide role in clinical application.Due to the need of clinical application,ADSCs often need to be cryopreserved.Cryoprotectants agent(CPA)play a key role in it.However,most of CPA contain toxic concentrations of dimethyl s ulfoxide(DMSO)limiting the possibility of their clinical application.Therefore,the purpose of this study is to explore a non-toxic,easy to elute and suitable for clinical use CPA aiming at achieving high-efficiency cryopreservation of ADSCs.Methods : We explored the efficiency of different concentrations of trehalose(Tre)(0.3 M,0.6 M,1.0 M,1.25 M)and glycerol(Gly)(10%,20%,30% v/v)to cryopreserve ADSCs.The ADSCs were thawing after 30 days.We selected the optimal cryopreservation concentration of trehalose and glycerol by viability.The we compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycero l and the commonly used CPA DMSO(10%)+ fetal bovine serum(FBS)(90%).The effectiveness was evaluated by the proliferation,migration,expression of surface markers and multi-potential differentia tion of the ADSCs after thawing.The resuscated ADSCs of the experimental group were compared with fresh ADSCs to verify the biological functions of ADSCs by the gross observation and histological analysis of the wound healing.Results: Among different concentrations of trehalose and glycerol,the group of 1.0 M trehalose and 20% glycerol showed the highest viability of ADSCs after thawing.Compared with individual reagents,the 1.0 M trehalose(Tre)+ 20% glycerol(Gly)group as a new CPA to cryopreserve ADSCs showed significantly higher efficiency.Compared wi th the 10%DMSO + 90% FBS group,the ADSCs preserved in 1.0 M Tre +20% Gly showed similar cell viability,surface markers,and m ulti-potential differentiation but a significantly higher migration capability(p<0.001).The results of resuscitation ADSCs tran splantation experiment showed that compared with the fresh ADSCs group,ADSCs preserved in the 1.0 M Tre+20% Gly group had the same ability to promote wound healing(p=0.325)and angiogenesis(p=0.528).Conclusion: 1.0 M Tre + 20% Gly,as a new CPA,can achieve a higher efficiency in ADSCs cryopreservation.At the sametimes,the CPA can keep the cytological and biological functions of ADSCs after thawing.As a safe and non-toxic CPA,1.0 M Tre + 20% Gly may more suitable for clinical application.