Dental Pulp-Derived Mesenchymal Stem Cell Culture

Explant culture of human dental pulp derived MSC was performed according to our previous published protocol.8 (link) Briefly, pulps were extirpated, treated with antibiotic antimycotic solution (penicillin 100U/mL, streptomycin 100μg/mL, amphotericin B 0.25μg/mL), rinsed in 1X phosphate buffered saline (PBS) (Sigma-Aldrich, Inc, USA) and minced into approximately 1–2 mm³ pieces using surgical blade. Minced fragments were cultured in Dulbecco’s modified essential medium F12 (DMEM-F12) (Sigma-Aldrich, Inc, USA) supplemented with 20% fetal bovine serum (FBS), penicillin 100U/mL, streptomycin 100μg/mL, amphotericin B 0.25μg/mL, 1mM sodium pyruvate and 2mM L-glutamine (Sigma-Aldrich, Inc, USA). DPSC and SHED were cryopreserved (90% FBS and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Inc., USA) in liquid nitrogen after establishing primary culture and were subsequently thawed and expanded for later use.