CHARACTERIZATION OF PRIMARY MURINE ENTEROCYTES

DI CLAUDIO FIORELLA; GRIGERA RAúL; MUGLIA CECILIA; GUILLERMO H DOCENA

Los Cocos

Congreso; Food allergy is a worldwide immune-mediated adverse reaction to food and is a growing clinical concern. Sublingual allergen immunotherapy has been proposed as an alternative therapy and there is so far no standardized and approved procedure. In this work,; 2013

            The intestinal epithelium is a highly dynamic compartment, being the enterocytes the target for different analysis. Culturing intestinal epithelial cells is difficult, and efforts are being done to improve the survival of cells. In this work we have optimized culture conditions for primary culture of murine enterocytes. Cells obtained from different regions of the gut were cultured on collagen membranes with the presence of growth factors and oligoelements in the culture medium.Cell viability, functional phenotype and proliferation were evaluated. Results showed that cells adhere to the collagen membranes and growth with an organized distribution. Acridin orange/ethidium bromide staining gave 3 ± 2 % of orange nuclei at day 7, increasing to 7 ± 2 % of orange nuclei at day 14 by fluorescence microscopy (10 fields-200X) (P<0.05, N=10). Annexin V/PI staining, showed by flow cytometry a low apoptosis rate (less than 3%) during the culture. The percentage of live cells remained constant (70 ± 6) at day 7, with a sharp decrease at day 14 (20 ± 3 % of live cells) (P<0.01, N=4). We observed by optical microscopy that cells proliferated to cover the empty areas of the membrane. Confocal microscopy revealed that zonuline, ZO-1, is expressed on culture cells after 5 days, and phaloidine(F-actin probe) gave a homogeneous staining throughout the cells (day 5). A low number of cultured cells (less than 0.5 %) were positive for the stem cell marker Lgr5, as assessed by qPCR and confocal microscopy. Alkaline phosphatase expression was analyzed by qPCR and we found that Akp3 was increased at day 7, as compared to gene expression in the whole gut (2.5 ± 0.3 vs 1.5 ± 0.5 of relative expression to â-actin, respectively). In conclusion, our results indicate that the developed method to keep murine enterocytes alive promoted the selection of epithelial cells with the capacity to proliferate, form tight junctions, and express enzymes that are relevant for the tissue function.