Isolation, immortalization and preliminary characterization of porcine epithelial cells.

LAURA C BARTEL; PABLO STEINBERG

Viena

Simposio; 22. VETPHARM-SYMPOSIUM; 2012

Institut für Pharmakologie und Toxikologie. Veterinärmedizinische Universität Wien

In many zoonoses, the intestine plays a relevant role in the establishment of infections as well as in the elimination and spreading of the pathogens that cause these infections. For the study of these processes, up to now, only rat, mouse and human cell culture systems are available. In the area of bovine, porcine or avian applied gastrointestinal research can only be used primary cell cultures or whole animals, both of which have many disadvantages, like being used only for short term studies or having many ethical constraints. This model would be also of great importance not only for the study of infections but also for the study of the molecular mechanisms involved in chemically-induced colon carcinogenesis. The aim of the present work was to establish a new porcine intestinal cell line and to microscopically and biochemically characterize it. The isolation of cells was carried out with colonic tissue from pig fetuses, 10 weeks of gestation. After modifying a protocol from the literature (Weng XH et al, 2005), the dissection steps covered the incubation with EDTA/Tripsin, scraping the surface of the tissue and several centrifugation steps with media to wash the cells. At the end, cells were digested enzymatically with collagenase and further washed through different centrifugation steps in order to obtain only the crypts. Cells were grown on Optimem® media with low serum content (4%) and different growth factors and cultured in collagenated plates, under normal culture conditions. After the first passage, cells were immortalized with SV40 large T antigen. After a selection phase with puromicin, cells were maintained under the same culture conditions and subcultured for more than 15 times without showing senescence process. In order to discard the fibroblasts from the epithelial cultures, different clones were obtained through subclonation steps without complete success. Cell proliferation was evaluated by doubling time determination and the epithelial character of the cells was evaluated with different cell markers (cytokeratin 18 and 20), as well as the comparison with a mesenchymal marker (vimentin). The observed morphology appeared as compact cuboidal to spindle-shaped cells, exhibiting the expression of both epithelial markers in a high percentage but also vimentin. As these methods are not sufficient to characterize it, we aim to analyze other markers as e.g. the evaluation of cell growth until monolayer is built with the TEER measurement, or the use of other epithelial or mesenchymal markers (Zona Ocludens-1, α-smooth muscle actin) among others.