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

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Abstract
During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchy-mal 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 × 105well) were plated as monolayers and grown in α-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 μg/ml ascorbic acid. Cultures were maintained for 6 days at 37°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 μg/ml of polybrene, the media was replaced with selection media containing 300 μg/ml of G418, and the cultures incubated at 33°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°C. One established cell line, M06-G3, showed high constitutive expression of dentin phosphoprotein, type I collagen and alkaline phosphatase, characteristic of an odontoblast phenotype. These results demonstrate the establishment of a stable murine odontoblast cell line which retains the tissue-specific expression for a number of DECM proteins.