冻存人脐带间充质干细胞的分离培养

Cryopreserved human umbilical cord mesenchymal stem cells: isolation and culture

背景:目前大多数报道是从新鲜脐带中分离培养脐带间充质干细胞,而从冻存脐带组织中分离培养间充质干细胞鲜有报道。目的:从冻存人脐带中分离培养间充质干细胞,并比较多种培养方法获得的脐带间充质干细胞的生物学特性。方法:采用组织块贴壁法从冻存脐带组织中分离培养脐带间充质干细胞,分别用有血清和无血清培养基培养,观察其形态特征,绘制其生长曲线;流式细胞仪检测第3代脐带间充质干细胞(无血清培养基培养)和第14代脐带间充质干细胞(含血清培养基培养)表面抗原表达;第3代脐带间充质干细胞(无血清培养基培养)分别用成脂、成骨、成软骨诱导培养液诱导其分化,qPCR鉴定分化标记基因表达;检测第7代脐带间充质干细胞(无血清培养基培养)及其培养上清中的细胞因子水平。结果与结论:(1)从冻存人脐带组织原代培养的脐带间充质干细胞,呈梭形纤维样细胞形态,生长力旺盛,脐带间充质干细胞可用无血清培养基传代培养,但增殖速率还远低于有血清培养基;(2)流式细胞术分析结果显示为脐带间充质干细胞CD90、CD73、CD105呈强阳性表达,CD45、CD34、CD14/CD11b、CD79a/CD19、HLA-DR呈阴性表达;(3)经成脂、成骨和成软骨诱导培养14-28 d后,脂肪细胞标记基因PPARγ、骨细胞标记基因Osteonectin、软骨细胞标记基因CollagenⅡ的表达量显著升高;(4)第7代脐带间充质干细胞及其培养上清可检测出粒细胞集落刺激因子、粒细胞-巨噬细胞集落刺激因子、干扰素α2、趋化因子IP-10、白细胞介素4、白细胞介素6等细胞因子;(5)结果提示可从冻存人脐带中培养扩增脐带间充质干细胞。

BACKGROUND:Current studies have addressed the mesenchymal stem cells(MSCs)mostly from fresh human umbilical cord,but rarely from cryopreserved human umbilical cord.OBJECTIVE:To isolate MSCs from cryopreserved human umbilical cord,and to compare the biological characteristics of human umbilical cord MSCs(hUC-MSCs)harvested using several culture methods.METHODS:hUC-MSCs isolated from cryopreserved umbilical cord tissues by explants culture method were cultured in serum-containing and serum-free media.Cell morphology was observed to draw the growth curve of cells in different media.Passage 3 hUC-MSCs cultured in serum-free medium and passage 14 hUC-MSCs in serum-containing medium were selected to detect the expression of surface biomarkers by flow cytometry.Passage 3 cells in the serum-free medium were induced for adipogenic,osteogenic and chondrogenic differentiation,and differentiation marker genes were identified by qPCR.Expression of cytokines in the passage 7 cells cultured in the serum-free medium and in the culture supernatant was detected.RESULTS AND CONCLUSION:The hUC-MSCs isolated from cryopreserved human umbilical cord exhibited a spindle-shaped appearance and exuberant growth,and moreover,the cells could be expanded in serum-free medium but the cell proliferation was certainly slower than that in the serum-containing medium.Flow cytometry analysis revealed that the hUC-MSCs were positive for CD73,CD90,CD103,but negative for CD45,CD34,CD14/CD11b,CD79a/CD19,and HLA-DR.Adipogenic(PPARγ),osteogenic(Osteonectin)and chondrogenic(Collagen II)biomarker genes were highly expressed at 14-28 days of induction.Passage 7 hUC-MSC and its supernatant secreted several cytokines including granulocyte colony-stimulating factor,granulocyte-macrophage colony-stimulating factor,interferonα2,chemokine IP-10,interleukin 4,and interleukin-6.In conclusion,we could successfully isolate and expand hUC-MSCs from cryopreserved human umbilical cord tissues.