Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal Epithelial Cells.
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.
Instituto de Estudios Inmunológicos y Fisiopatológicos (IIFP), CONICET y Universidad Nacional de La Plata, La Plata, Argentina.
Fiorella
F
Di Claudio
Instituto de Estudios Inmunológicos y Fisiopatológicos (IIFP), CONICET y Universidad Nacional de La Plata, La Plata, Argentina.
Cecilia I
CI
Muglia
Instituto de Estudios Inmunológicos y Fisiopatológicos (IIFP), CONICET y Universidad Nacional de La Plata, La Plata, Argentina.
Paola L
PL
Smaldini
Instituto de Estudios Inmunológicos y Fisiopatológicos (IIFP), CONICET y Universidad Nacional de La Plata, La Plata, Argentina.
María Lucía
ML
Orsini Delgado
Centro de Investigación y Desarrollo en Tecnología de Alimentos (CIDCA), CONICET y Universidad Nacional de La Plata, La Plata, Argentina.
Fernando M
FM
Trejo
Centro de Química Inorgánica (CEQUINOR), CONICET y Universidad Nacional de La Plata, La Plata, Argentina.
J Raúl
JR
Grigera
Instituto de Estudios Inmunológicos y Fisiopatológicos (IIFP), CONICET y Universidad Nacional de La Plata, La Plata, Argentina.
Guillermo H
GH
Docena
D000818:Animals; D017209:Apoptosis; D000069985:Bifidobacterium bifidum / Q000502:physiology; D015415:Biomarkers / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D016360:Clostridioides difficile / Q000502:physiology; D003094:Collagen / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D020895:Enterocytes / Q000201:enzymology / Q000382:microbiology / Q000502:physiology*; D004798:Enzymes / Q000378:metabolism; D005260:Female; D054884:Host-Pathogen Interactions; D006801:Humans; D018836:Inflammation Mediators; D007633:Keratins / Q000378:metabolism; D008297:Male; D008567:Membranes, Artificial; D057507:Mice, 129 Strain; D008875:Middle Aged; D010641:Phenotype; D061251:Primary Cell Culture / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013997:Time Factors; D051193:Toll-Like Receptors / Q000378:metabolism
D015415:Biomarkers; D016207:Cytokines; D004798:Enzymes; D018836:Inflammation Mediators; C116884:LGR5 protein, human; C493597:Lgr5 protein, mouse; D008567:Membranes, Artificial; D043562:Receptors, G-Protein-Coupled; D051193:Toll-Like Receptors; D007633:Keratins; D003094:Collagen