Perreault N, Beaulieu J F
Département of d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.
Exp Cell Res. 1998 Nov 25;245(1):34-42. doi: 10.1006/excr.1998.4221.
The epithelium of the small intestinal mucosa is a highly dynamic system particularly well suited for analyzing key biological phenomena such as cell differentiation and migration, cell-matrix interactions, and apoptosis. However, in vitro models of fully differentiated normal human enterocytes are still lacking. The objective of the present study was to investigate the possibility of generating such differentiated intestinal cell cultures from the fetal small intestine. For this purpose, various dissociation methods were tested in order to obtain pure, viable, and functional enterocytes. One of these methods, based on the procedure to recover epithelial cells grown on Matrigel and involving the use of Matrisperse, a nonenzymatic dissociating solution, was found to allow the isolation of the integral epithelial lining from the mesenchyme. In culture, these epithelial fractions plated on collagen I spread rapidly and reached confluence after 3-4 days. When tested after 5-7 days, these primary cultures of differentiated enterocytes (PCDE) remained well preserved. Both goblet and absorptive cells exhibit all the main characteristics of intact villus intestinal cells as assessed by electron microscopy. Indirect immunofluorescence and Western blot analyses confirmed the purity of PCDE. The functional status of these cells was demonstrated by the presence of uniformly distributed tight junction, zonula adherens, and desmosomal components at the region of cell to cell contact as well as expression of various brush border enzymes, namely sucrase-isomaltase and lactase, and goblet cell mucins. As expected, cell proliferation was found to be negligible as assessed by DNA synthesis. Taken together, these data show that primary cultures of pure and viable differentiated enterocytes can be generated from the human fetal small intestine.
小肠黏膜上皮是一个高度动态的系统，特别适合分析细胞分化与迁移、细胞与基质相互作用以及细胞凋亡等关键生物学现象。然而，完全分化的正常人肠上皮细胞的体外模型仍然缺乏。本研究的目的是探讨从胎儿小肠生成这种分化的肠细胞培养物的可能性。为此，测试了各种解离方法，以获得纯净、有活力且功能正常的肠上皮细胞。其中一种方法基于回收生长在基质胶上的上皮细胞的程序，并使用了一种非酶解离溶液Matrisperse，结果发现该方法能够从间充质中分离出完整的上皮层。在培养过程中，接种在I型胶原上的这些上皮组分迅速铺展，并在3 - 4天后达到汇合。在培养5 - 7天后进行测试时，这些分化肠上皮细胞的原代培养物（PCDE）保存良好。通过电子显微镜评估，杯状细胞和吸收细胞均表现出完整绒毛肠细胞的所有主要特征。间接免疫荧光和蛋白质印迹分析证实了PCDE的纯度。这些细胞的功能状态通过细胞间接触区域均匀分布的紧密连接、黏着小带和桥粒成分以及各种刷状缘酶（即蔗糖酶 - 异麦芽糖酶和乳糖酶）和杯状细胞黏蛋白的表达得以证明。正如预期的那样，通过DNA合成评估发现细胞增殖可忽略不计。综上所述，这些数据表明可以从人胎儿小肠生成纯净且有活力的分化肠上皮细胞的原代培养物。
