小肠类器官培养技术的建立和优化*

宋东娟#童锦禄&冉志华郑青&

上海交通大学医学院附属仁济医院消化内科上海市消化疾病研究所

上海市炎症性肠病研究中心(200001)

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。

关键词小肠类器官；原代细胞培养；L-WRN细胞；培养基，条件性；隐窝；干细胞

Establishment and Optimization of Culture Technique for Enteroids

SONGDongjuan,TONGJinlu,RANZhihua,ZHENGQing.DivisionofGastroenterologyandHepatology,RenJiHospital,SchoolofMedicine,ShanghaiJiaoTongUniversity;ShanghaiInstituteofDigestiveDisease;ShanghaiInflammatoryBowelDiseaseResearchCenter,Shanghai(200001)

Co-correspondence to: TONG Jinlu, Email: tongjinlu490@126.com; ZHENG Qing, Email: qingzheng101@163.com

Background: Enteroids are considered to be the best tool for studies on intestinal epitheliuminvitroand 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 cultureinvitro. 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.

Key wordsEnteroids;Primary Cell Culture;L-WRN Cells;Culture Media, Conditioned;Crypts;

Stem Cells

肠道上皮由隐窝和绒毛单元组成，具有快速自我更新能力，其中隐窝为增殖区域，包括小肠干细胞及其子代细胞，绒毛则是由各种终末分化细胞组成的分化区域。相对于转化细胞和肿瘤细胞株，小肠类器官(enteroids)是目前体外研究肠道上皮最好的工具，应用前景广泛。既往已有众多国内外学者探讨了肠上皮细胞的原代培养问题，然而均无法做到长期体外培养以及维持原代细胞的分化能力[1]。……