Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal Epithelial Cells

Abstract

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.

Short-chain fatty acids oppositely altered expressions and functions of intestinal cytochrome P4503A and P-glycoprotein and affected pharmacokinetics of verapamil following oral administration to rats

“…Primary intestinal epithelial cells of mice were isolated according to method described previously with slight modification. Briefly, the mice were sacrificed by cervical dislocation.…”

“…[21] Contribution of intestinal P-gp to intestinal transport of verapamil in normal rats was also investigated in presence of P-gp inhibitor quinidine (200 lM). [22] Isolation and culture of primary intestinal epithelial cells from mice Primary intestinal epithelial cells of mice were isolated according to method described previously [23] with slight modification. Briefly, the mice were sacrificed by cervical dislocation.…”

Efficient Generation of Small Intestinal Epithelial-like Cells from Human iPSCs for Drug Absorption and Metabolism Studies

“…Thus, a model that could evaluate drug absorption and metabolism in the small intestine would be useful for drug discovery. Currently, experimental animals and cancer cell lines are widely used as intestinal models for small intestinal absorption and metabolism studies because primary human small intestinal epithelial cells are difficult to obtain and culture (Di Claudio et al., 2017, Hansen et al., 2000, Shirasaka et al., 2006, Taylor et al., 2010, Theodoropoulos et al., 2003, Yamashita et al., 2002). These existing models, however, do not accurately reflect the human small intestine functions (Gupta et al., 2008, Hartley et al., 2006, Sun et al., 2008).…”

Mucosal Immunoregulatory Properties of Tsukamurella inchonensis to Reverse Experimental Food Allergy

“…Intestinal epithelial cells (IEC) were isolated from adult Balb/c mice and cultured in a collagen membrane as described in Di Claudio et al (12). Briefly, animals were sacrificed, and the first 5 cm of the jejunum were removed under sterile conditions and washed with PBS supplemented with antibiotics (penicillin 100 U/mL and streptomycin 100 mg/mL) and 20% FBS for 10 min at room temperature.…”