Rotavirus, the leading cause of infectious gastroenteritis in children, infects mature villous epithelial cells (EC) of the small intestine. However, because primary villous epithelial cells are difficult to maintain in culture, studies of rotavirus pathogenesis and immunogenesis have used either epithelial cell lines from human colonic carcinomas (such as Caco2 or HT-29 cells), or monkey or canine kidney epithelial cells. The aim of this study was to establish primary cultures of murine small intestinal epithelial cells and determine their susceptibility to rotavirus infection. EC were isolated from the small intestines of both adult and suckling (7-day-old) Balb/c mice using a modification of the method of Evans et al. (J Cell Sci 1992, 101:219-231). Following digestion of the small intestine by collagenase and dispase, cells were cultured on plastic or Matrigel (a basement membrane matrix). EC detached as either single cells or small organoids. Greater than 90% of isolated cells were viable as determined by trypan-blue exclusion and conversion of intracellular calcein AM in culture. Consistent with their epithelial origin, cells stained positively for cytokeratin, and expressed sucrase, maltase, lactase and palatinase. Cultures of live EC were maintained for up to 5 days with minimal proliferation, but little contamination from mesenchymal cells. To determine the susceptibility of cultured EC to rotavirus infection, suckling and adult mice were inoculated with murine rotavirus strain EDIM, at a dose of 6 × 104 SD50, and sacrificed 6 or 18 hrs post-infection (pi). Cultures of EC from EDIM-infected suckling mice harvested 18hrs pi, showed progressive CPE over 12-72 hours in culture. CPE was not observed in suckling mice whose EC were harvested 6 hrs pi or in adult mice 6 or 18 hrs pi. CPE was observed as migration of cells into syncytial-like clumps which increased in size over 3 days. EC from animals inoculated with lower doses of EDIM showed evidence of decreased CPE that correlated with the virus concentration. Studies are ongoing to determine if the observed CPE is due to cell-to-cell spread of virus, or to cytokines elaborated by EC. This system may provide a useful in-vitro model for study of the interactions between rotavirus and the intestinal epithelium.
