Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered‐cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio‐membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL‐25 and TSLP), inhibitory (IL‐10 and TGF‐β), or pro‐inflammatory mediators (IL‐1β and TNF) were induced. In conclusion, this novel protocol using cow's collagen‐ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell‐microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489–2496, 2017. © 2016 Wiley Periodicals, Inc. We have developed a method for culturing primary intestinal epithelial cells useful for the study of their interaction with microbes.