CCK-8检测新鲜脐带组织中细胞活性的初步研究

Preliminary study of CCK-8 assays on fresh umbilical cord tissue cell activity

目的探索采用Cell Counting Kit-8(CCK-8)试剂检测新鲜脐带不同大小与数量组织中细胞活性,选用合适的组织块大小与数量诱导培养间充质干细胞,以获得最佳的细胞数量与细胞活性。方法临床获取新鲜脐带加工处理剪切成1/8组织块大小,重量为(0.045±0.001)g,采用CCK-8检测试剂盒检测新鲜脐带组织块活性,对CCK-8试剂的剂量、加入试剂后孵育时间、脐带组织块剪切大小与数量等做了比较与分析,并在6孔板中加入3块不同脐带组织块大小(1/2组、1/4组、1/8组)诱导培养间充质干细胞,对3组生成的细胞数量和细胞活性做了实验研究。结果新鲜脐带组织块剪切成厚度为0.5 cm左右环形组织块的1/8组织块大小,重量为(0.045±0.001)g,24孔板内脐带组织放置3块,CCK-8试剂浓度为15%,于37℃下孵育7-8 h为检测新鲜脐带组织块活性的最佳检测条件;6孔板中加入3块不同脐带组织块大小(1/2组、1/4组、1/8组)诱导培养间充质干细胞,3组中培养生成的细胞活性无明显差异(P>0.05)。但3组中培养生成的细胞数量,其中1/8组与1/2组或1/4组相比,差异有统计学意义(P<0.01),且每克组织块生成的细胞数量最多。结论 CCK-8试剂盒可用于准确地评估新鲜脐带组织中细胞活性。

Objective To investigate the Cell Counting kit-8（ CCK-8） kit to test the cell activity of the umbilical cord tissue with appropriate tissue size and number,and to select the appropriate tissue size and number to induce mesenchymal stem cells to obtain the best optimization of cell quantity and activity. Methods Fresh umbilical cord obtained from Clinical was processed into 1 /8 of the tissue size,weight（ 0. 045 ±0. 001） g. The umbilical cord tissue activity was detected by the CCK-8 kit,and compared on the doses of CCK-8 reagent,incubation time,umbilical cord tissue size and number,Three pieces of umbilical cord of different tissue sizes were selected to induce mesenchymal stem cell differentiation（ MSCs）（ 1 /2,1 /4,1 /8 group） in 6-well plates. The generated cell quantity and viability were evaluated. Results The fresh umbilical cord tissue was sheared into 0. 5cm thick circular tissue and then cut into 1 /8 of the tissue size with the weight of（ 0. 045 ± 0. 001） g. They were then cultured in a 24-well plate placed within 3 pieces,15% concentration of CCK-8 reagent,at 37℃ and hatched for 7- 8 hours,which is also known as the optimal condition. Three pieces of different umbilical cord tissue size（ 1 /2 group,1 /4 group,1 /8 group） were cultured to induce mesenchymal stem cell differentiation in 6-well plates. The cell activity in the three groups had no significant difference（ P〈0. 05）. However,when comparing the cell number generated from the 1 /8 group with the 1 /2 group or 1 /4 group,the difference was statistically significant（ P〈0. 01）. The highest cell number per gram tissue was generated from the 1 /8 group.Conclusion The CCK-8 reagent kit can be used to accurately assess cell viability of fresh umbilical cord tissue.