Adult Stem Cells in Primary Culture From Human Dental Tissues

Stem cells are clonogenic cells capable of self-renewal and differentiation into multiple cell lineages. Recently stem cells have been found in human dental tissues. Objective: To establish primary cell cultures of human dental pulp and periodontal ligament (PDL) and to identify multipotential adult stem cells in these cultures during the course of multiple passages. Methods: Cell cultures were established using single cell suspensions derived from pulp tissue and PDL of human extracted third molars. PDL tissue was separated from root surfaces using sterile scalpels. The pulp was opened chamber using sterilized dental fissure burs. The pulp tissue was gently separated from the pulp chamber. Both tissues were digested in a collagenase type I and dispase containing solution for 1 h at 37°C. We used 70 mm strainers to obtain single-cell suspensions. Dental pulp and PDL cells were seeded into 6-well plates and 6 cm culture dishes, respectively. Cell cultures were established under standard circumstances using á-MEM supplemented with FCS and incubated at 37°C in 5% CO2. To assess colony-forming efficiency, day 14 cultures were fixed with 4% formalin, and then stained with 0.2% Giemsa. Mesenchymal stem cell marker STRO-1 immunohistochemistry was performed on both cell cultures with primary antibody anti-STRO-1 (mouse IgM), secondary antibody anti-mouse (rabbit IgG), combined with CLSM. Results: Cell cultures were successfully established from both pulp tissue and periodontal ligament, then cultivated for up to 20 passages. Both cell cultures showed typical fibroblast-like morphology, showing clonogenic activity. Colony-forming efficiency levels were 25-30 colonies per 105 cells. We detected STRO-1 immunoreactivity in both cell cultures in 6-8% of the cells thorough the passages. Conclusion: We were able to culture pulp tissue and PDL cells under standardized circumstances, identifying colony-forming cells. STRO-1 immunoreactivity indicates the steady level of stem cells in the primary cultures for up to many passages.