Immortalization of bovine dental papilla cells with simian virus 40 large t antigen.

Primary cultures of dental papilla-derived cells have a limited lifespan in vitro and can be maintained only up to passage 7-9 before showing senescence, but in vitro investigations often require a large number of cells showing phenotypic characteristics of the original tissue. To overcome this shortcoming, second-passage cells established from calf molar tooth germs by enzymatic pretreatment of the dental papilla were transfected by electroporation with pSV3neo, coding for the oncogene simian virus 40 large t antigen and a neomycin-resistance gene. Under selection by G418 (neomycin), four cell clones were isolated by single cell dilution at passage 15. Integration of simian virus 40 large t antigen and expression of the gene products were determined in cell clones by polymerase chain reaction (PCR) and immunohistochemistry. Four transfected cell lines (clones B, C, D and no. 12) were maintained in culture for over 1.5 years. For cell characterization, gene expression of procollagen alpha1 (I) and osteocalcin was evaluated by reverse transcriptase (RT)-PCR with cDNA obtained from the established cell lines at passage 20. Expression of collagen type I, osteocalcin and dentine phosphoprotein was evaluated immunohistochemically at passage 20 and after 1.5 years of continuous cell culture. Gene expression and the expression of mineralized tissue-specific proteins was demonstrated with RT-PCR and immunohistochemistry within all four immortalized cell lines. Expression of dentine phosphoprotein was observed in three simian virus 40 large t antigen-transfected cell lines, suggesting the immortalization of odontoblast-like cells in vitro. Thus, transfection of bovine dental papilla-derived cells resulted in immortal cell lines exhibiting phenotypic characteristics of the original tissue.