Isolation and characterization of human primary enterocytes from small intestine using a novel method

Abstract

Cell culture studies of enterocytes are important in many fields. However, there are difficulties in obtaining cell lines from adult human intestine, such as microbial contamination of cultures from the tissue samples, short life span of enterocytes, overgrowth of mesenchymal cells, etc. Various model used to obtain adult intestinal cell lines are very complex requiring use of feeder layer or gel matrices. The aim of this study was to establish a novel method for the simple and reproducible isolation of human enterocytes. Enterocytes were isolated from SI samples (n = 5) obtained from cadaveric donors using a mechanical procedure, and separation with immunomagnetic beads coated with anti-EpCAM antibodies. Light and electron microscopy, flow cytometry and immunocytochemistry techniques were used to characterize the isolated cells. Immunohistochemical staining of normal SB biopsies confirmed that the cell cultures maintained an in vivo phenotype as reflected in cytokeratin expression CK18, CK20 and expression of intestine-specific markers such as sucrase isomaltase and maltase glucoamylase. Furthermore, the cells strongly expressed TLR-5, 6, 7, 8 and 10 and several molecules such as CD40, CD86, CD44, ICAM-1 and HLA-DR which are important in triggering cell-mediated immune responses. This novel technique provides a unique in vitro system to study the biology of enterocytes in normal conditions as well as to study inflammatory processes in various small bowel disorders.

Generation of Enterocyte-Like Cells with Pharmacokinetic Functions from Human Induced Pluripotent Stem Cells Using Small-Molecule Compounds

“…4. Undifferentiated human iPS cells had little cytoplasm and were small; however, differentiated enterocyte-like cells exhibited a cobbled shape similar to that of human intestinal epithelial cells (Chougule et al, 2012;Takenaka et al, 2014).…”

“…The human small intestinal epithelial cell monolayer expressed various drug transporters and could be used to evaluate the permeability of paracellularly absorbed compounds; however, further exploration of its pharmacokinetic functional characteristics is desirable considering that accurate prediction of intestinal absorption in this cell line is difficult. Some reports on the isolation and cultivation of human primary enterocytes have been published (Perreault and Beaulieu, 1996;Grossmann et al, 2003;Chougule et al, 2012). These applications remain limited, however, because of difficulties such as poor viability, short life span, limitation of passage number, and difficulty of obtaining human tissue samples.…”

Human Small Intestinal Epithelial Cells Differentiated from Adult Intestinal Stem Cells as a Novel System for Predicting Oral Drug Absorption in Humans

“…The utilization of human primary normal enterocytes was expected to be a promising solution for the aforementioned discrepancy between in vivo and in vitro models. Although several methods have been used to isolate primary enterocytes from the human small intestine (Perreault and Beaulieu, 1998;Aldhous et al, 2001;Grossmann et al, 2003;Chougule et al, 2012), their applications remain limited, which has been attributed to their poor viability and short life span (Aldhous et al, 2001;Grossmann et al, 2003).…”

Derivation and basic characterization of colorectal carcinoma primary cell lines

“…EpCAM) or antibodies that allow selective separation of required cell populations. Magnetic beads are also supplied in ready-to-use kits 9 . Nevertheless, the isolation of cells by nano-beads selects a narrow cell population and suppresses other heterogenic population of cells inside the tumour.…”

“…Several successful isolations of enterocytes have been described, using various procedures [9][10][11] . Most of these methods, such as separation using magnetic nano-beads, or separation on the specific density gradient 11 allows only for the isolation of specific cells types.…”