Establishment of primary cultures of pulp cells from bovine permanent incisors

A primary culture method was established by comparing the different effects of four methods of enzymatic separation--trypsin, collagenase with and without trypsin pretreatment, and a trypsin-collagenase mixture--and five media: DMEM, DMEM and Ham's F 12 mixture, F 12, RPMI 1640 and Medium 199. The trypsin pretreatment/collagenase method was most preferable considering the high number of isolated cells, satisfactory adhesion, good growth and a single population at subconfluence. DMEM and the DMEM/F-12 mixture resulted in the best adhesion, cell growth and cell number at confluence. Primary cells separated by the trypsin pretreatment/collagenase method and cultured in DMEM were responsive to parathyroid hormone at the proliferating stage and had higher alkaline phosphatase activity than cells cultured from gingiva and mucosa after reaching confluence. The long-term cultured cells formed nodules that were slightly mineralized. These results indicate that the cultured pulp cells had properties characteristic of pulp cells in vivo. This enzymatic separation method may be useful in studies of the regulation of pulp metabolism and odontoblast differentiation.