Optimized Study On Isolation And Cultivation Of Human Umbilical Cord Mesenchymal Stem Cells

Objective:To explore establish a stable reliable method to isolate and culture hUC-MSCs by optimizing digestive enzymes components, medium choice, incubation density, first time of medium change, in order to lay the foundation of preparation for human umbilical cord mesenchymal stem cells.Methods:1 Isolation and cultivation of hUC-MSCs by kinds of digestive enzymes components.1.1 Human umbilical cords were collected from full term deliveries under aseptic conditions.1.2 Human umbilical cords were divided equally into 9 divisions,0.5cm in length each portion, then removed ateries and vein carefully. Every portion was cut into pieces the size of lmm×1mm×1mm.1.3 According to different mixed enzyme concentration ratio was divided into three groups: mixed enzyme I group, mixed enzymeⅡgroup, mixed enzymelllgroup. Then according to digest time each group into three subgroups:1 hour.2 hours, and 3 hours.1.4 Suspended cell volume was decided as 4ml, and to count cells, each subunit counted 6 times. Dulbecco's Modified Eagle Medium with fetal bovine serum was used for cell culture.1.5 Observe, count and compare the total cell number and live cell rate at the same digestive time.2 Cultivation of hUC-MSCs by kinds of medium choice, incubation density, first time of medium change.2.1 Mononuclear cells were inoculated in the six-well plate by densities of 5×105,1×106,5×106,1×107. Observe the cell adhesion time and cell primary culture time.2.2 The mononuclear cells were cultured with kinds medium of MesencultTM,LDMEM,DMEM/F12, which all contains 10% FBS. Observe and record cell growth time.3 Identification and verification differentiation of hUC-MSCs3.1 The surface markers:CD34, CD44, CD45, CD29, CD 105 were analysed by flow cytometry.3.2 Vertification differentiation of hUC-MSCs into osteoblast by von kossa staining.Results:1 Based on the three digestion enzyme concentration,at the duration of 1 hour,2 hours and 3 hours, mixed enzymelllgroup 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 enzymelllgroup, and there was statistically significant differences among these groups(P<0.01).2 Compared with the 4 different densiy groups, we found that the 5×103 groups cells grew very slowly, and barely passaged. In the 5×106 group cell extension time(92.0±6.3h) and cell primary time(21.2±4.1d) were shorter significantly than other groups.3 Different mediums on human umbilical cord mesenchymal stem cells in vitro culture:54.5% samples cultured with MesencultTM had homogenous mesenchymal stem cells,27.2% samples cultured with DMEM/F12 and 18.2% samples cultured with L-DMEM.4 Flow cytometry examination showed that MSCs, cultured in vitro, were expressed CD44, CD29 and CD105, but not CD34, CD31, CD45, which was consistent with the umbilical cord mesenchymal stem cell growth characteristics.5 MSCs can be induced into adipoblast and osteoblast.Conclusions:The hUC-MSCs have strong self-renewal capacity. When the other conditions were the same, The optimal isolation for human UC-MSCs are 0.3% collagenaseⅡ,0.1% trypsin.0.02% EDTA,0.1% hyaluronidase and 0.1% DNA enzymeⅠ. MesencultTM was the appropriate medium, and the optimal density of cell culture was 5×106/cm2. Under optimized conditions, the cells were consistent with the umbilical cord mesenchymal stem cell growth characteristics. We have optimized an efficient isolation and expansion system of hUC-MSCs, which verified umbilical cord a new reliable source of MSCs for clinical application of adult stem cell in future...