Dental Pulp Stem Cell Isolation and Culture

Cells from ten non-erupted open-apex teeth donated from patients aged 18 to 20 years, after signing the consent form, were used. Patients with third molars presenting carious lesions, fractures or periodontal disease were not invited to the study. After extraction, the teeth were placed in a Falcon tube containing 10 mL of Dulbecco’s Modified Eagle Medium—DMEM (Gibco®, Grand Island, NY, USA), with 200 µL of amphotericin B fungizone (Sigma Aldrich®, St. Louis, USA) and 10 µL of gentamicin (Sigma Aldrich®). The set was transported to the university laboratory and cultured within 2 h of extraction. Cells were cultured using the explant technique^{66 (link)}. Dental pulp was removed and washed in phosphate-buffered saline (PBS). The pulp was sectioned with a scalpel blade and transferred to a 6-well plate in DMEM high glucose (25 mM) medium (Sigma Aldrich, St. Louis, MO, USA), 50 U mL⁻¹ penicillin (Gibco, Grand Island, NY, USA), 50 µg mL⁻¹ streptomycin (Gibco) and 20% fetal bovine serum (Gibco). Confluent cells were subcultured in DMEM medium supplemented with antibiotics and 10% fetal bovine serum. Cell cultures between the 3^rd and 6^th passages were used for cell viability experiments, cell morphology, migration, and proliferation. Pulp cells between the 3^rd and 4^th passage were used for odontogenic differentiation assays^{67 (link)}.