Yeh K Y, Chopra D P
In Vitro. 1980 Nov;16(11):976-86. doi: 10.1007/BF02619336.
Epithelial cells from the colon of suckling rats have been propagated in vitro. The colons were excised and cut longitudinally. The epithelial sheets were peeled off and dissociated in 0.1% trypsin solution at 25 degrees C for 10 min. The first cell suspension was discarded and the remaining fragments trypsinized again for an additional 20 min. The dissociated cells were washed and cultured. Forty-eight hours later, several epithelial colonies consisting of closely packed polygonal cells were formed. Transmission and scanning electron microscope examination of the colonies showed numerous regularly spaced microvilli on the surface and tight junctions and desmosomes between adjacent cells. Immunocytochemical studies with antiserum prepared against the brush-border membrane of the colonic epithelium showed specific staining of the epithelial colonies. Epithelial colonies were subcultured by the penicylinder method. Although the subcultured cells retained their epithelial characteristics, the proliferative activity of the cells gradually decreased. Currently, efforts are being made to determine the optimum nutritional requirements of the primary and low-passage cultures.
乳鼠结肠上皮细胞已在体外进行培养。切除结肠并纵向切开。将上皮片剥离，在25℃下于0.1%胰蛋白酶溶液中解离10分钟。弃去第一次细胞悬液，其余碎片再用胰蛋白酶处理20分钟。将解离的细胞洗涤后进行培养。48小时后，形成了几个由紧密排列的多边形细胞组成的上皮菌落。对菌落进行透射和扫描电子显微镜检查，结果显示其表面有许多规则排列的微绒毛，相邻细胞之间有紧密连接和桥粒。用针对结肠上皮刷状缘膜制备的抗血清进行免疫细胞化学研究，结果显示上皮菌落有特异性染色。上皮菌落采用笔管法进行传代培养。尽管传代培养的细胞保留了其上皮特征，但细胞的增殖活性逐渐降低。目前，正在努力确定原代和低代培养的最佳营养需求。
