不同消化方式对脐带华通胶间充质干细胞提取的影响

目的探讨使用不同酶浓度、消化不同时间及不同浓度胎牛血清的培养基稀释对消化脐带华通胶后提取培养间充质干细胞的影响。方法无菌条件下取出正常剖腹产胎儿脐带,剔除动脉、静脉及外膜,取其之间的胶状物,剪成1 mm3及更小的块状,分别加入0.1%和0.2%的Ⅱ型胶原酶,分为A、B两组,37℃水浴消化。每一组消化时间分别为4、81、2、16、202、4、28、323、6、40 h共10个消化时间点,每一个消化时间点又分为两组,分别为A1、A2组和B1、B2组。消化后的黏稠细胞悬液用完全DMEM稀释,并加入特级胎牛血清至浓度为10%(A1、B1)和20%(A2、B2),充分混匀。悬液移至75 cm2培养瓶,15 ml/瓶,37℃、5%CO2、饱和湿度培养,3 d换液。通过细胞计数观察原代细胞贴壁生长的最早时间和80%细胞铺满瓶底的时间和生长活力。结果 0.2%的Ⅱ型胶原酶消化脐带华通胶在消化时间为20h,且稀释血清浓度为20%时,获得的细胞悬液在培养36 h即有细胞贴壁,培养6 d细胞贴壁达到80%,其余各组细胞生长情况各不相等,均弱于此组。其中消化时间在8 h以内和32 h以上时不论使用多少的酶浓度和稀释时使用多少的血清浓度,细胞均不生长。结论在使用胶原酶消化脐带华通胶提取间充质干细胞的过程中,使用0.2%Ⅱ型胶原酶、消化16～24 h、稀释培养时加入至20%的血清对干细胞的提取效果最佳。

Effect in mesenchymal stem cells extraction from human umbilical cord Wharton's jelly by different digestion mode

Objective To explore effect of extraction and cultivation in mesenchymal stem cells from human umbilical cord Wharton's jelly by different enzyme concentration、different digestion time in Wharton's jelly and different fetal bovine serum concentration in culture media.Methods To take out of umbilical cord by abdominal delivery under asepsis,divest arteries and veins and tunica adventitia to obtain jelly,cut it into shiver smaller than 1mm3,add 0.1% and 0.2% type Ⅱ collagen respectively,divide into A and B group,then digestion in 37℃ water bath,digestion time of every group were 4、8、12、16、20、24、28、32、36、40h respectively,every time point were divided into two groups,A1 A2 groups and B1,B2 groups,post-digestive ropiness cell suspension were diluted by complete DMEM,admoveatur fetal bovine serum and percentage 10%(A1 B1 groups)and 20%(A2 B2 groups),then admixture thoroughly,put cell suspensions into 75cm2 culture bottle,15ml every bottle put it into incubator,hold temperature37℃ and 5%CO2 and saturated humidity,instead culture media every 3 days.To observe earliest-growth-time of primary cell adherence and cells growth vigor of 80% cell completely covers the bottom of culture bottle.Results Digestion time of 0.2% type Ⅱ collagen was 20h and fetal bovine serum percentage was 20%,cells adherence will be found after about 36h of cells suspension breeding and reach 80% after 6 days.There were different growth status among the rest groups for cells,but cells were not growth when digestion time less than 8h and exceed 32h,no matter how many enzyme concentration and serum levels we use. Conclusion At the process of type Ⅱ collagen digestion umbilical cord Wharton's jelly and obtain in mesenchymal stem cells,we can use 0.2% type Ⅱ collagen with digestion time 16~24h and fetal bovine serum percentage 20% to get the best effect.