Caco‐2 cells cultured in serum‐free medium as a model for the study of enterocytic differentiation in vitro

Abstract

Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.

A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability

“…We took advantage of the ordered system of enterocyte differentiation to assess MLCK isoform expression, both in the in vitro Caco-2 cell model of enterocyte differentiation (21,30) and in vivo, as assessed in human intestine. Analysis of mRNA from Caco-2 cells and human intestinal epithelia showed that long MLCK expression was limited to isoforms 1 and 2.…”

Epithelial: Lamina Propria Lymphocyte Interactions Promote Epithelial Cell Differentiation

“…The protein content of each sample was quantified using the Biorad DC kit (Biorad, Hercules, CA). Caco-2 cells exhibit a constitutively high level of IAP activity 23,24 , so, we used this cell line as a positive control in our experiments.…”

“…Intestinal Alkaline Phosphatase (IAP) is exclusively expressed in villus enterocytes in normal mucosa and hence serves as an excellent marker of crypt-villus differentiation 23,24 . The putative effect of normal LPL on epithelial differentiation was investigated using T84, a cryptlike intestinal epithelial cell line, co-cultured with freshly isolated normal LPL for 1 hour, 4 and 8 days (Fig 1A).…”

Expression of Calnexin Reflects Paneth Cell Differentiation and Function

“…Because primary cultures of differentiated human Paneth cells are not yet well established, the intestinal epithelial cell line CaCo2 was used (Cario et al, 1999;Jumarie and Malo, 1991). In mitotic CaCo2 cells, immunoreactivity to Cnx was found in numerous dispersed cisternae and vesicles, a common phenomenon observed for proteins associated with the ER (Jesch and Linstedt, 1998).…”