Temperature Sensitive Simian Virus 40 Large T Antigen Immortalization of Murine Odontoblast Cell Cultures: Establishment of Clonal Odontoblast Cell Line

During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchymal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 x 10(5)/well) were plated as monolayers and grown in alpha-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 micrograms/ml ascorbic acid. Cultures were maintained for 6 days at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2, with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 micrograms/ml of polybrene, the media was replaced with selective media containing 300 micrograms/ml of G418, and the cultures incubated at 33 degrees C for one month with media changes every 3-5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)