Bovine dental papilla-derived cells immortalized with HPV 18 E6/E7

In vitro investigations of cell-specific metabolism and cell interactions a s well as biocompatibility studies are often hampered by the limited lifesp an of primary cells originating from target tissues like the oral mucosa, g ingiva or pulp. Pulp cells, as do other primary cells, undergo senescence a fter several passages in vitro. However, senescence can be overcome by tran sfection of primary cells with oncogenes like the HPV 18 (human papillomavi rus 18) E6/E7 oncogene, resulting in immortalized cell lines. The purpose o f our study was to establish and preliminary characterize an immortalized b ovine dental papilla-derived cell line by transfection with HPV 18 E6/E7 fo r future use in biocompatibility testing of dental materials. First passage dental papilla-derived cells from molar tooth germs of 6-month-old calves were transfected by electroporation with pUC18 LCR E6/E7 coding for the HPV Is E6/E7 oncogene. Cells underwent crisis after 5 wk in culture. Distinct cell colonies arose after about 9 wk. Cells were cloned by single cell dilu tion in passage 15 and 17. Out of three cell clones maintained in culture, two cell clones showed cell death after 28 and 30 wk, respectively. One cel l clone overcame a second crisis after 38 wk and was maintained in culture until passage 75. Stable gene expression of HPV 18 E6/E7 oncogenes was veri fied by polymerase chain reaction (PCR) and immunohistochemistry. Reverse t ranscription (RT)-PCR revealed that the established cell line (passage 50) expresses procollagen type I, alkaline phosphatase and osteocalcin. This su ggests that the immortalization with HPV Is E6/E7 results in a cell line, w hich maintained phenotypic characteristics of odontoblast-like cells.