海藻糖-甘油复合型冷冻保护剂冻存脂肪来源干细胞在促创面愈合中的研究

Study on application of cryopreserved adipose-derived stem cells by trehalose-glycerol compound cryoprotective agent in wound healing

目的探究脂肪来源干细胞(adipose-derived stem cells,ADSCs)经复合型冷冻保护剂(cryoprotective agents,CPA)冻存后促进再生的生物学作用。方法自2019年6—9月,上海交通大学医学院附属第九人民医院整复外科对脂肪组织采用酶消化法分离,并体外培养ADSCs至第三代后进行冻存。复合型CPA组为海藻糖(trehalose,Tre)-甘油(glycerin,Gly)复合型CPA(1.0 mol/L Tre+20%Gly)冻存,传统CPA对照组为10%DMSO+90%胎牛血清(fetal bovine serum,FBS)冻存,新鲜ADSCs组为新鲜ADSCs,空白对照组不加任何CPA。冻存30 d后,检测复苏的ADSCs活性以及形态。在小鼠创面愈合模型中,通过愈合速度以及组织学染色比较ADSCs在创面愈合中的作用。结果复合型CPA组ADSCs的活性与传统CPA组比较,差异无统计学意义(P>0.05);而复合型CPA组具有与新鲜ADSCs组相同的促创面愈合以及血管化的能力。结论体内研究结果表明,复合型CPA可有效保存ADSCs的生物学作用,其成分安全无毒、无异种蛋白,适合临床转化应用。

Objective To explore the biological effects of adipose-derived stem cells(ADSCs)after cryopreservation by compound cryoprotective agent(CPA)on the promotion of regeneration.Methods From June to September 2019,the ADSCs were isolated from adipose tissue by enzymatic digestion and were cultured to the third passage for cryopreservation.The compound CPA group was treated by 1.0 mol/L trehalose(Tre)combined with 20%glycerin(Gly).The traditional CPA control group was treated by 10%DMSO combined with 90%FBS.The fresh ADSCs group was treated by fresh ADSCs.The blank control group was not treated by any CPA.After 30 days of cryopreservation,the cell viability and cell morphology of resuscitated ADSCs were assessed.In the mice model of wound healing,the biological effects of ADSCs were compared by speed of wound healing and histological staining.Results There was no significant difference of cell activity between compound CPA group and traditional CPA control group.The compound CPA group has the same biological effects as the fresh ADSCs group to promote wound healing and vascularization.Conclusion The results of in vivo study suggested that the compound CPA group can effectively preserve the biological effects of ADSCs.The ingredients are nontoxic and without heterologous protein.It is suitable for clinical trans formation applications.