Phenotype and behaviour of dental pulp cells during expansion culture

OBJECTIVE: Primary pulp cell cultures are frequently used to study cellular responses, odontogenic potential and stem cell responses. Their isolation and expansion via a range of technical approaches are widely reported. The purpose of this study was to investigate the influence of isolation approach and extended expansion on cell phenotype and behaviour. DESIGN: To determine viable cell isolation, enzymatic dissociation was performed on rodent incisor pulps using collagenase, trypsin, hyaluronidase and ficin. Extended expansion culture of released cells was performed in DMEM and alpha-MEM media. Cultures were subsequently analysed for gene expression, cell proliferation, cell morphology and differentiation capacity up to passage 20. RESULTS: Data indicated that incubation of extirpated and mechanically minced rodent pulpal tissue with 0.25% Trypsin:EDTA and subsequent culture in alpha-MEM medium provided optimal conditions for maximal cell growth and expansion. Under these conditions, extended culture decreased cellular proliferative capacity up to passage 7, whilst higher passages demonstrated recovered growth rates. In general gene expression analysis of osteogenic and dentinogenic associated markers decreased with increasing passage number. Notably expression of TGFbetas-1, -2 and -3 increased up to passage 10 as did the stem cell and pericyte/myofibroblast markers, CD74, Neuroserpin and alpha-SMA. Analysis of molecular phenotypes indicated little difference in lineage differentiation capacity between earlier and later passages. CONCLUSIONS: The present study characterizes conditions for primary pulp cell isolation and expansion and indicates that both earlier and later passages maintain differentiation capacity. Continued passage however may result in selection for cells with a pericyte/myofibroblast phenotype.