Serial Expansion of Primary Dental Pulp cell Cultures

Postnatal dental pulp contains multipotent mesenchymal progenitor cells. Understanding the behaviour of pulp derived cells during serial expansion is important to their exploitation in regeneration and tissue engineering for pulp and dentine. Objectives: Investigate the nature and behaviour of the cell populations obtained during expanded primary pulpal cell culture. Methods: Cells were isolated from extirpated pulps of young adult male Wistar rat incisors following dissociation in trypsin-EDTA for 30mins. Isolated primary cells were seeded at 1x10⁶ cells/ml and expanded as a monolayer culture in aMEM medium with 10% FCS, glutamine and antibiotics. Cultures were maintained at 37^oC, in 5% CO₂ in air with media changes every 48 hours until confluent. Once confluent, cells were isolated and further expanded to passage 20. Growth curves were determined at each passage and RNA was isolated for downstream sqRT-PCR analysis. Expression levels of markers of cell lineage, genes associated with mineralising tissues / dentinogenic potential and stem / progenitor cells were analysed. Results: The growth rate of cells demonstrated an increase from primary culture up to passage 3. At passage 4 onwards the growth rate decreased until passage 7 where further cell division was minimal. Once subcultivated and reseeded at passage 8, cells demonstrated a gradual increasing exponential growth rate, becoming constant at passages 15-20. Gene expression analysis through increasing passage number indicated a decrease in expression of mineralization / dentinogenic related gene transcripts with differential expression patterns observed for mesenchymal cell and progenitor / stem cell markers. Conclusions: These data indicate changes in the cell population during expansion of pulp cells and suggest a degree of variation in the mineralization and reparative potential during expansion. Selection of specific sub-populations of cells from these cultures may provide a more effective strategy for sourcing larger numbers of cells suitable for tissue engineering and regeneration applications.