小肠类器官培养技术的建立和优化

Establishment and Optimization of Culture Technique for Enteroids

背景:小肠类器官是体外研究肠道上皮最好的工具,应用前景广泛,然而目前国内尚无相关研究报道。目的:在国内建立并优化小肠类器官培养技术,为小肠上皮细胞的基础研究提供平台。方法:常规培养L-WRN细胞,收集含不同浓度胎牛血清(FBS)的条件培养基。处死6~8周龄C57BL/6小鼠,自末端回肠起取约15 cm肠段,纵向剖开,EDTA法分离、收集隐窝上皮,以基质胶包埋多聚化后加入不同浓度梯度的L-WRN条件培养基,显微镜下动态观察出芽情况,待出芽达一定长度后,重新包埋传代培养。结果:与含20%FBS的L-WRN条件培养基相比,含10%FBS的条件培养基更有利于小肠类器官的体外培养。条件培养基浓度为10%、15%、20%、25%、30%均可促使小肠类器官形成,15%条件培养基的出芽率最高。结论:本研究在国内首次成功建立了小肠类器官培养技术,并发现15%L-WRN条件培养基(含10%FBS)更有利于小肠类器官出芽。

Background: Enteroids are considered to be the best tool for studies on intestinal epithelium in vitro and have a widely application prospects,however,there are no associated reports in China. Aims: To establish and optimize the culture technique for enteroids and provide a fantastic platform for the basic research of small intestinal epithelial cells in China. Methods: L-WRN cells were cultured routinely and the conditioned medium with different concentrations of fetal bovine serum( FBS) was collected. Six to eight weeks old C57 BL /6 mice were sacrificed and 15 cm small intestine from the terminal ileum was removed and cut longitudinally. Crypts were digested with EDTA and then collected and embedded in MatrigelMatrix; after polymerization of MatrigelMatrix,L-WRN conditioned medium at different concentration gradient was added. The budding ratio and length of buds were measured dynamically under microscope. The enteroids were re-embedded for subculture when certain length of buds was reached. Results: Compared with L-WRN conditioned medium containing 20% FBS,the conditioned medium containing 10% FBS was more favorable for enteroids culture in vitro. When conditioned medium accounted for 10%,15%,20%,25% or 30% of the mixed medium,they all promoted the growth of enteroids and the 15% one seemed to yield better result. Conclusions: An enteroids culture technique was successfully established for the first time in China. When the L-WRN conditioned medium containing 10% FBS accounts for 15% of the mixed medium,it might promote budding better than the others.