小肠类器官的构建及传代培养

Establishment and passage of small intestine organoids

目的探索小肠隐窝分离,小肠类器官培养、传代、冻存、复苏和蛋白提取的方法,研究R-Spondin1的浓度对小肠类器官成熟的影响。方法分离小鼠小肠隐窝,在模拟体内肠道干细胞生长、增殖的体外3D培养体系中培养,倒置显微镜观察小肠类器官的形成过程。用1 mL注射器切割小肠类器官进行传代。设置R-Spondin1浓度梯度,在不同时间点比较小肠类器官形态。结果成功的完成了小肠类器官的分离培养、传代、冻存、复苏及蛋白提取工作。确定了隐窝与单细胞可以进行培养的比例标准且发现在R-Spondin1浓度低于0.5μg/mL的培养基中,小肠隐窝不能成长为成熟的小肠类器官。结论小肠类器官体外培养体系的建立,尤其传代培养的成功将为在体外长期研究小肠上皮提供新的、更符合生理的实验模型。隐窝与单细胞比例标准的确定将大幅提高小肠类器官培养的成功率,蛋白质的提取将有利于蛋白组学的研究。

Objective To explore the method of small intestinal crypts isolation,organoids culture,passage,cryopreservation and recovery,and protein extraction,then study the effect of R-Spondin1 concentration on intestinal organoids maturation.Methods Isolate the small intestinal crypts,cultivate them in an in vitro 3 D culture system that simulates the growth and proliferation of small intestinal stem cells in vivo,and observe the formation of intestinal organoids with an inverted microscope.The samll organoids were cut with a 1 m L syringe for passage.Set R-Spondin1 concentration gradient and compare organoids morphology at different time points.Results Successfully completed the separation culture,passage,cryopreservation,resuscitation and protein extraction of intestinal organoids and found that the intestinal crypts in the medium with a bottom concentration of less than 0.5μg/m L cannot grow into mature intestinal organoids.Conclusions The establishment of an in vitro culture system for small intestine organoids,especially the success of subculture,will provide a new and more physiological experimental model for long-term study of intestinal epithelium in vitro.The determination of the ratio of crypts to single cells will greatly improve the success rate of intestinal organoids culture,and the extraction of proteins will be beneficial to the study of proteomics.