Objective To establish the human intestinal epithelial cells-derived organoid culture system ex-vivo.Methods The chelating buffer containing 2 mmol/L EDTA was used to isolate the crypts from human intestinal mucosa.The isolated crypts were embedded in Matrigel and then covered by the culture medium enriched with growth factors.The growth of organoids was observed under the microscope.The expression of the stem cell and mature epithelial cell markers was analyzed by immunohistochemistry or in situ hybridization.Cell proliferation was labeled with EdU staining.Cell apoptosis was detected by TUNEL assay.Results After 4-5 days,crypts embedded in Matrigel grew into organoids.On day 8,organoids were digested,re-embedded in Matrigel,and cell fractions grew into organoids again.Lgr5 + cells were detected by in situ hybridization.On day 5 after the removement of Wnt3a,R-Spodin,SB202190,Nicotinamide,organoids were differentiated to mature cells which were labeled as Fabp1+ cells,Muc2+ cells,Chga+ cells and Lyz+ cells by immunofluorescence.Proliferating cells were detected in organoids while few apoptotic cells were detected.Conclusions The human intestinal epithelial organoid culture system can be successfully established.The cuhure system is a reliable ex-vivo model to study the pathophysiology of digestive tract diseases.%目的 构建人肠上皮细胞类器官三维培养体系.方法 采用含2 mmol/L乙二胺四乙酸的分离液分离肠镜活检标本或手术标本肠黏膜的隐窝,并用基质胶包埋隐窝后覆盖富含生长因子的培养液.光镜下观察记录类器官生长情况,并采用免疫组织荧光及核酸原位杂交分析检测肠上皮及类器官成熟细胞及干细胞标记物的表达情况.分别采用5-乙炔基-2’脱氧尿嘧啶核苷染色法和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法标记增殖或凋亡的细胞.结果 隐窝细胞包埋于基质胶内培养4～5d可形成类器官结构,第8天消化重新包埋后可继续生长.核酸杂交法显示类器官富含Lgr5阳性细胞.撤去Wnt3a、R-Spodin、SB202190、Nicotinamide 5 d后类器官细胞可分化成Fabp1阳性细胞、Muc2阳性细胞、Chga阳性细胞、Lyz阳性细胞.类器官可见增殖细胞,细胞凋亡少见.结论 该培养体系可成功构建人肠上皮细胞类器官,可为研究肠道疾病肠上皮病理生理机制提供可靠的体外研究模型.
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