Isolation, immortalization, and initial characterization of uterine cell lines: An in vitro model system for the porcine uterus

The aim of this study was to develop immortalized cell lines from porcine u terus. Endometrial cells including luminal epithelium (LE), glandular epith elium (GE), stroma (ST), and myometrium (MYO) were enzymatically isolated f rom the uterus of a day 12 pregnant gilt. Primary cultures were immortalize d by transduction with a retroviral vector containing the E6 and E7 open re ading frames of human papillomavirus type 16 (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomyein analog G418 (0.4-1.5 mg/ml). Surviv ing cells were maintained in complete culture medium containing G418 (0.1 m g/ml) anti subcultured for 1 yr. Expression of the E7 protein was confirmed in all cell lines by Western blotting. Phase contrast microscopy revealed that LE and GE cells exhibited cobblestone morphology, whereas ST and MYO c ells exhibited spindle-shaped morphology. The epithelial origin of LE and G E was confirmed by positive immunostaining for cytokeratin. Stromal and MYO cells were vimentin-positive, but cytokeratin-negative. The MYO cell lines were positive for smooth muscle alpha -actin staining, whereas LE, GE, and ST cell lines were negative for alpha -actin. Western blotting indicated t hat all cell lines expressed both estrogen and progesterone receptors, but only GE cells secreted uteroferrin (UF). Collectively, these porcine uterin e cell lines provide an in vitro model for studying cell type-specific acti ons of hormones and cytokines, signal transduction pathways, cell-cell inte ractions, and gene expression.