Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Abstract

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

HUIEC, Human intestinal epithelial cell line with differentiated properties: process of isolation and characterisation

“…2). The characteristic shape was polygonal to round human physiology and metabolic processes that would otherwise not be possible in vivo [36,37]. The isolation of these cells is demanding because in vitro epithelial intestinal cells die rapidly, mainly due to the lack of attachment to the substrate, which is crucial for their survival [28].…”

Human Prenatal Small Intestine Cells as a Valuable Source of Stem Cells and Epithelial Cells: Phenotypic and Functional Characterization

“…15,19 Presently, there is no standard in vitro cell culture system for small intestine epithelial cells although several have been reported in the literature. [5][6][7]19 Sato et al described a small intestine cell culture system that is in a 3D matrix and requires organoid formation and maintenance. Although this system may have limitless potential, it does not appear to be a simplified in vitro model and to the best of our knowledge they have yet to test the ability of this model for specific in vitro studies such as GLP-1 secretion and multi-drug transporter assays in human cells.…”

“…2 Small intestine epithelial cells are potentially a suitable model for researching inflammatory bowel disease, colitis, intestinal cancer, intestinal infections and more importantly, toxicity and drug discovery. Although recent research has described a variety of culture methods, [3][4][5][6][7] no simplified long-term culture system has been established from human prenatal intestinal tissue that maintains basic crypt-villus physiology and metabolic functionality in the differentiated state. The majority of studies are not performed on primary intestinal epithelial cells that have been derived directly from human intestinal tissue.…”

“…12 Since the use of these cell lines as tools has limitations, 2,5,9,10 efforts have been directed at developing alternative sources of human intestinal epithelial cells that maintain their functions and are easy to work with. Although culture systems of human intestinal cells have been described, [2][3][4][5][6][7][8][9] an adequate and easily obtainable primary cell culture model that is characterized and demonstrates functionality does not exist. The ideal primary cell model would contain the complete flora of cells normally found in the small intestine, be reproducible, expandable, and functional in vitro.…”

Normal Mouse Intestinal Epithelial Cells as a Model for the in vitro Invasion of Trichinella spiralis Infective Larvae

“…Firstly, fetal tissues can be obtained under sterile conditions. Secondly, fetal tissues seem to be more effective for the generation of epithelial cells because of their rapid metabolism and self-renewing rate [17]. Thirdly, the structure of the villus at E15–E16 develops rapidly and the epithelium changes from a stratified to a monolayer shape [18], [19].…”