PriCells: Postnatal human dental pulp stem cells (DPSCs)

1. Normal human impacted third molars were collected from adults.
2. Tooth surfaces were cleaned and cut around the cementum-enamel junction by using sterilized dental fissure burs to reveal the pulp chamber.
3. The pulp tissue was gently separated from the crown and root and then digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 h at 37°C (PriCells Isolation Kit).
4. Single-cell suspensions were obtained by passing the cells through a 70-μm strainer. Bone marrow cells, processed from marrow aspirates of normal human adult volunteers were purchased and then washed in growth medium.
5. Single-cell suspensions (0.01 to 1 × 10⁵/well) of dental pulp and bone marrow were seeded into 6-well plates with alpha modification of Eagle's medium supplemented with 20% FCS/100 μM l-ascorbic acid 2-phosphate/2 mM l-glutamine/100 units/ml penicillin/100 μg/ml streptomycin, and then incubated at 37°C in 5% CO_² (PriCells Culture System).
6. To assess colony-forming efficiency, day 14 cultures were fixed with 4% formalin, and then stained with 0.1% toluidine blue.
7. Aggregates of ≥50 cells were scored as colonies.
8. Conditions for the induction of calcified bone matrix deposition in vitro were as reported.
9. The proliferation rate of subconfluent cultures (first passage) of DPSCs and BMSCs was assessed by bromodeoxyuridine (BrdUrd) incorporation for 24 h by using a BrdUrd staining kit.