Generation of a temperature-sensitive SV40-transduced human odontoblast cell line

Objectives: Odontoblasts, which are lifelong responsible for dentin matrix formation and mineralization, represent an integral part of the dentin-pulp complex.  However, a profound knowledge of the mineralization process and the biological interaction of odontoblasts to both bacteria and biomaterials used in dental therapy are still scarce. Since odontoblasts have a limited life-span in vitro, the aim of the present study was to establish a stable human cell line useful to study odontoblast reactions, using a temperature-sensitive mutant of SV40 for transduction controlling the expression of large T antigen by changes in temperature and therefore cells senescence.

Methods: Primary cultures of odontoblasts isolated from third human molars were transduced in the presence of polybrene (8µg/ml) with lentiviral particles containing sequences for the temperature-sensitive expression of the SV40 T oncogene. Transduced cells were selected in G418-containing culture medium (0.5mg/ml) at 33°C, and after 12 days selective pressure the expression of the large T transcript was determined by qRT-PCR.

Results:  Primary cells did not survive exposure to G418-containing medium at 33°C after a five-day incubation period. Expression of SV40 T was detected in transduced and viable odontoblasts (33°C), but no expression was seen in primary cell cultures by qRT-PCR. Microscopically, the spindle-like morphology and long extensions of primary odontoblasts and  SV40 T-transduced cell cultures are similar after a temperature shift to 37°C.

Conclusion: This new cell line may provide a useful tool for analysing both the biological mechanism of mineralization and cellular responses to dental biomaterials. Further analysis of the morphological and biochemical properties will be used to verify the odontoblastic phenotype.