小鼠肠道类器官三种不同条件培养基的比较研究

摘要 目的：比较三种不同条件培养基对小鼠类器官形态和增殖速度的影响。方法：取C57BL/6小鼠的小肠和结肠，EDTA法分离隐窝，以基质胶包埋，加入不同小鼠肠道类器官培养基培养7 天，使用光学显微镜记录和比较类器官形成率和出芽情况。随后进行二代类器官培养，使用TrypLE将类器官消化为单细胞，重新包埋和培养，使用光学显微镜记录和比较不同类器官培养基对二代类器官的培养效率。采用荧光定量PCR比较不同条件培养类器官中干细胞标志物Lgr5和分化标志物MUC2的表达。使用免疫荧光法检测类器官中ki-67的表达。结果：对于小肠类器官的培养，使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（18.2±4.5） %、（63.8±4.0） %和（82.1±8.4） %。其中使用IntestiCult条件培养基培养类器官的出芽率更高。对于结肠类器官的培养，使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（17.3±7.3） %、（58.0±6.1） %和（46.3±7.4） %。对于二代类器官的培养，IntestiCult条件培养基和L-WRN培养基都能够支持消化为单细胞后的二代类器官培养。干细胞标志物Lgr5和分化细胞（杯状细胞）标志物MUC2的表达无明显差异。使用L-WRN培养基的类器官ki-67阳性比例更高，增殖速度更快。结论：本研究比较了三种不同条件培养基对小鼠类器官形态和增殖速度的影响。经过对比，L-WRN培养基更有利于小鼠肠道类器官的形成和增殖速度。

Comparison of Mouse Intestinal Organoids Cultured Under Different Conditions

ABSTRACT Objective: To compare the effects of three different media on the morphology and proliferation of mouse organoids. Organoid culture is a new ex vivo research system for studying the differentiation and proliferation of adult intestinal stem cells. Methods: The crypts were separated from intestinal and colon of C57BL/6 mice using EDTA digestion buffer, and then embedded with Matrigel. Three different organoid culture medium were added and culture for 7 days. The organoid forming rate and budding number were compared under light microscope. After digesting with TrypLE, single cells were re-entrapped for the second generation culture using different organoid culture medium. The forming rate and budding number of second-generation organoid were compared under light microscope. Realtime-PCR was used to compare the expression of stem cell marker Lgr5 and Differentiation Marker Muc2 in organoids cultured with different conditional medium. Immunofluorescence assay was used to detect the ki-67 positive cells in colon organoids. Results: For the cultivation of small intestinal organoid, the formation rate were (18.2±4.5) %, (63.8±4.0) % and (82.1±8.4) %, respectively, using the conditioned medium 1, IntestiCult medium and L-WRN medium. Moreover, the budding rate and morphology were the best by IntestiCult medium. For colon organoid culture, the formation rate were (17.3±7.3) %, (58.0±6.1) % and (46.3±7.4) % in the conditioned medium 1, IntestiCult medium and L-WRN medium respectively. Under L-WRN medium, the colon organoids could form shorter buds, and the morphology was better than other culture conditions. Both IntestiCult conditional medium and L-WRN conditional medium can support the second-generation organoid culture after digestion into single cells. The expression of stem cell marker LGR5 and goblet cell marker Muc2 showed no significant difference under different conditions. The positive rate of ki-67 was higher and the proliferation rate was faster in L-WRN medium. Conclusion: This study compared the effects of three different media on the morphology and proliferation of mouse organoids. L-WRN medium was more favorable for the formation and proliferation of intestinal organoids in mice.