人脐带间充质干细胞低温冻存后生物学鉴定

目的观察人脐带间充质干细胞(HUC-MSCs)经低温冻存复苏后的生物学特性,验证其是否仍具有间充质干细胞的基本特征。方法用胶原酶和胰蛋白酶消化脐带,分离诱导获得HUC-MSCs,传代培养,取第3代细胞加入冷冻保护剂(10%DMSO+40%FBS+50%DMEM/F12)后将其冻存于-80℃冰箱过夜,再转入液氮气相中保存1个月后复苏。采用倒置显微镜观察复苏后贴壁生长细胞的形态;台盼蓝拒染法比较冻存前后细胞活力;MTT法测定HUC-MSCs的增殖活性,比较冻存前后HUC-MSCs增殖活性的差异;流式细胞仪检测HUC-MSCs经冻存复苏后表面抗原的表达情况;复苏后HUC-MSCs进行成骨诱导分化培养,检验其是否仍具有多向分化潜能。结果 HUC-MSCs经低温保存复苏后,细胞仍保持贴壁生长等外形特征,复苏后HUC-MSCs存活率为91.17%;增殖活性与未冻存细胞比较,差异无统计学意义;细胞表面高表达CD105、CD73、CD71、CD90、CD29、CD44、CD13抗原,低表达或不表达CD34、CD133、CD45、CD14,HLA-DR、CD86、CD83、CD80、CD1α;复苏后HUC-MSCs具有向成骨细胞诱导分化能力,细胞经茜素红、ALP和Von-Kossa′s染色呈阳性反应。结论经冻存复苏后的HUC-MSCs保持了间充质干细胞的基本形态,其表型满足间充质干细胞的基本要求,仍具有向成骨细胞分化的潜能。

Biological Characteristics of Human Umbilical Cord Mesenchymal Stem Cells Following Cryopreservation

Objective To investigate biological characteristics of HUC-MSCs after cryopreservation,and explore the possibility of differentiation in vitro.Methods Mesenchymal stem cells were separated from umbilical cord with collagense type Ⅳ and trypsin digestion to obtain the adherent cells in vitro.Early-passage 3 HUC-MSCs were digested with 0.25% trypsin and harvested.Fresh HUC-MSCs containing cryoprotective agents(10%DMSO+40%FBS+50%DMEM/F12) were cooled in a-80℃ refrigerator overnight and then the cryovials were directly transferred and stored into the vapour-phase of liquid nitrogen.The cells were stored for at least 1 month before they were thawed and assessed.The post-thawed cells were evaluated by(1) observation of morphology of HUC-MSCs under an inverted microscope,(2) determination of viability of HUC-MSCs by trypan blue exclusion assay,(3) capability of proliferation by MTT assay.The phenotypes on the cells were identified by flow cytometry,and the multiple potential of differentiation were induced by culture.Results Compared with fresh HUC-MSCs,post-thawed HUC-MSCs showed no distinct change in morphological characteristics.Viability of HUC-MSCs after cryopreservation was 91.17%.There was no significant difference between cryopreserved-thawed and fresh human HUC-MSCs on the proliferation rate;cryopreserved-thawed HUC-MSCs were all positive for MSC-related antigens,such as CD105,CD73,CD71,CD90,CD29,CD44,CD13,but negative for CD34,CD133,CD45,CD14,HLA-DR,CD86,CD83,CD80,CD1α.Alizarin red,Von-Kossa′s and ALP staining were positive,which showed that these cells could be induced to osteoblasts.Conclusion After cryopreservation,HUC-MSCs keep the biological characteristics,and preserve the phenotype of mesenchymal stem cells and the potential differentiation into osteoblasts in vitro.