人脐带来源间充质干细胞分离培养方法的优化

背景：关于人脐带间充质干细胞的分离培养方法不一,如何提高人脐带来源间充质干细胞获得效率的问题尚未解决。目的：优选人脐带来源间充质干细胞制备的消化酶组分。方法：无菌条件下取正常足月剖腹产的脐带近胎儿段,按不同混合酶浓度比值划分为3组,混合酶Ⅰ组成分为0.4%Ⅱ型胶原酶、0.1%胰酶、0.02%EDTA和0.1%透明质酸酶;混合酶Ⅱ组成分为0.1%Ⅱ型胶原酶、0.4%胰酶、0.02%EDTA和0.1%透明质酸酶;混合酶Ⅲ组成分为0.3%Ⅱ型胶原酶、0.1%胰酶、0.02%EDTA、0.1%透明质酸酶和0.1%DNA酶。每组再按消化时间分为1,2,3 h 3个亚组。最后重悬细胞终体积均定位4 mL并计数,采用含血清的DMEM培养液体外培养细胞。结果与结论：采用3种不同混合酶浓度进行消化,发现相同消化时间内,混合酶Ⅲ组消化细胞最多(P < 0.05)。在作用3 h时,混合酶Ⅲ组获取的细胞中活细胞比值显著低于混合酶Ⅰ组和混合酶Ⅱ组(P < 0.01)。提示0.3%Ⅱ型胶原酶、0.1%胰酶、0.02%EDTA、0.1%透明质酸酶和0.1%DNA酶混合液消化脐带组织块2 h为获取脐带间充质干细胞的最佳条件。

Optimized study on isolation and cultivation of human umbilical cord mesenchymal stem cells

BACKGROUND:There are different methods to isolate and culture human umbilical cord mesenchymal stem cells (hUC-MSCs), so how to fast and efficiently harvest hUC-MSCs have become a research hotspot.OBJECTIVE:To optimize digestive enzymes components for the preparation of hUC-MSCs, in order to lay the foundation of preparation for hUC-MSCs. METHODS:Human umbilical cords were collected from full term deliveries under aseptic conditions. According to different mixed enzyme concentration ratio, the samples were divided into three groups: mixed enzyme Ⅰ group, mixed enzyme Ⅱ group, mixed enzyme Ⅲ group. Then according to digest time each group into three subgroups: 1 hour, 2 hours, and 3 hours. Finally, suspended cell volume was decided as 4 mL to count cells. Dulbecco’s Modified Eagle Medium with fetal bovine serum was used for cell culture. RESULTS AND CONCLUSION:Based on the three digestion enzyme concentration, at the duration of 1 hour, 2 hours and 3 hours, mixed enzyme Ⅲ group had the highest total cell number and the total cell rate was statistically different from the other groups (P < 0.05). At the duration of 3 hours, live cell rate was the lowest in the mixed enzyme Ⅲ group, and there was statistically significant differences among these groups (P < 0.01). The optimal isolation for hUC-MSCs is 0.3% collagenase Ⅱ, 0.1% trypsin, 0.02% EDTA, 0.1% hyaluronidase and 0.1% DNA enzymeⅠ digestion for 2 hours.