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 C57BL/ 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.%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。
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