摘要
背景:研究表明小鼠胚胎成纤维细胞的制作受多种因素的影响。目前对这些因素的研究均建立于半经验的基础上,缺乏客观定量的数据支持。目的:对小鼠胚胎成纤维细胞制作胚胎干细胞饲养层细胞条件进行优化。方法:用两种不同方法从孕13.5d昆明小鼠胚胎中分离胚胎成纤维细胞,采用锥虫蓝染色计数,比较两种分离方法在细胞活性和数量方面的差异。对分离得到的胚胎成纤维细胞用0.05%胰酶在室温下消化不同时间,分析胰酶消化时间解离贴壁细胞能力和其对细胞活性影响;采用梯度浓度国产丝裂霉素灭活饲养层细胞2h和2.5h并制作细胞生长曲线,探索丝裂霉素最佳浓度和处理时间;通过不同冻存方法比较复苏后胚胎成纤维细胞活性,筛选出适合小鼠胚胎成纤维细胞最佳冻存方法。结果与结论:胚胎成纤维细胞对胰酶极其敏感,短时间多次消化法方法显著提高细胞活性和数量;室温下4～6min胰酶消化时间有利于保存细胞活性;10mg/L2.5h和20mg/L2h丝裂霉素灭活适合制作饲养层细胞;程序性降温使细胞更能适应温度变化,显著提高解冻后细胞活性和数量。
BACKGROUND：Studies have demonstrated that many factors may influence mouse embryonic fibroblasts （MEF） separation.Current researches on exploring these factors are based on semi-empirical results, which are lack of objective and quantitative data support.OBJECTIVE：To investigate the optimal conditions in establishing MEF as mouse embryonic stem cells＇ feeder layer cells.METHODS：Two different methods were conducted to isolate MEF from 13.5 days post coitum （dpc） Kunming mouse embryos.Trypan blue staining and cell counting of MEF were performed to study the relationship between cell livability and quantity.The MEF was digested by 0.05% trypsin at room temperature at various time points to analyze effects of dissociation adherent cell ability and cell viability at various digestion time points.Cells in feeder layer were inactivated using mitomycin for 2 and 2.5 hours.The cell growth curve was drawn.The optimal concentration and disposal time of mitomycin were explored.By various cryopreservation methods, the livability of MEF was compared after thawing.The optimal cryopreservation methods of MEF were screened.RESULTS AND CONCLUSION：MEF was sensitive to trypsin.The shorter trypsinase digestion time and multiple times method obviously elevated cell viability and quantity.At room temperature, 4-6 minutes digestion time contributed to cell viability preservation.10 mg/L 2.5 hours and 20 mg/L 2 hours mitomycin inactivation was suitable for establishing cells in feeder layer.Progressive temperature reduction made MEF adaptive to temperature changes, which significantly increased MEF livability and quantity after thawing.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第36期6768-6771,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30872473)~~
