人脐带间充质干细胞生物特性比较:胰酶冷消化和组织块法体外培养

Biological characteristics of human umbilical cord mesenchymal stem cells: cold trypsin digestion versus tissue explant method in vitro

背景:以往采用胰酶冷消化法培养脐带间充质干细胞的研究较少。目的:比较两种方法体外培养人脐带间充质干细胞的生物学特性。方法:采用胰酶冷消化法和组织块法从人脐带中分离、纯化和传代培养人脐带间充质干细胞,记录首次出现贴壁细胞时间及原代培养周期,倒置相差显微镜观察脐带间充质干细胞的形态及生长情况,制作第3代脐带间充质干细胞生长曲线,流式细胞仪分析检测第3代脐带间充质干细胞表面标志的表达,第3代脐带间充质干细胞加入成神经诱导培养基诱导分化第3天行荧光免疫化学染色检测Nestin的表达。结果与结论:使用上述两种方法均可获得脐带间充质干细胞,胰酶冷消化法首次出现贴壁细胞时间早于组织块法(P<0.05);原代培养周期差异无显著性意义(P>0.05)。倒置相差显微镜下细胞均为贴壁生长,形态为成纤维细胞样,但胰酶冷消化法培养的少数原代细胞呈多角形,不规则,细胞的体积较组织块法大。组织块法获得第3代细胞的增殖速率显著快于胰酶冷消化法,在进入对数生长期后各个时间点细胞数量差异有显著性意义(P<0.05)。两种细胞具有均一的细胞表型,均表达CD29,CD105,不表达CD34,CD45。两种方法培养出来的第3代脐带间充质干细胞经诱导后,免疫荧光均显示神经干细胞特征性标志物nestin阳性表达。结果表明胰酶冷消化法与组织块法均能培养出脐带间充质干细胞,但组织块法更适合于此类细胞的培养,对细胞的伤害更低。

BACKGROUND：Cold trypsin digestion is rarely reported to culture umbilical cord mesenchymal stem cells. OBJECTIVE：To compare the biological characteristics of umbilical cord mesenchymal stem cells cultured by cold trypsin digestion and tissue explant method. METHODS：Human umbilical cord mesenchymal stem cells were isolated, purified and passaged using cold trypsin digestion and tissue explant method, and then the first adhesion time and cellcycle were recorded. Morphology of umbilical cord mesenchymal stem cells was observed under inverted phase contrast microscope to draw growth curve of cells at passage 3. Flow cytometry was used to detect the surface markers of passage 3 umbilical cord mesenchymal stem cells, and Nestin expression was detected in passage 3 cells after 3 days culture in neural induction medium by fluorescence immunochemistry staining. RESULTS AND CONCLUSION：These two methods were both successful to harvest umbilical cord mesenchymal stem cells, but the first adhesion time was earlier in cells cultured by cold trypsin digestion than tissue explant method （P〈0.05）, and there was no difference in primary cellcycle （P〉0.05）. Under the inverted microscope, cells grew adherently and presented fibroblast-like shape. However, the minority of primary cells under induction of cold trypsin digestion was polygonal, irregular, and had larger cellvolume than those cultured by tissue explant method. Passage 3 cells cultured by tissue explant method showed faster proliferation rate than those cultured by cold trypsin digestion, and at logarithmic growth phase, cells cultured by these two methods were significant different in cellnumber （P〈0.05）. Two types of cells had a uniform cellphenotype, both of which expressed CD29, CD105, but did not express CD34, CD45. Under induction, passage 3 cells cultured by these two methods were both positive for nestin. These findings indicate that these two methods can both be used to culture umbilical cord mesenchymal stem cells, but the tissue explant method is more suitable for culture of umbilical cord mesenchymal stem cells and exhibits less damage to cells.