Isolation of Stem Cells from Exfoliated Deciduous Teeth

To obtain stem cells from human exfoliated deciduous teeth (SHED), human deciduous incisors were obtained from a 7-year-old child at the Nippon Dental University Hospital at Tokyo under approved guidelines set by the Committee of Ethics, the Nippon Dental University School of Life Dentistry at Tokyo (authorization number: NDU-T2012-35, August 13, 2015) [19 (link)]. Similar to our previous reports, the incisors were washed three times in ice cold PBS (−) and then cut into halves from the tooth cervix with a diamond disc. Dental pulp tissues were carefully moved out from the pulp cavity. After washing three times with growth medium MEM-α (Thermo Fisher Scientific, Tokyo, Japan) containing 20% FBS, 100 units/mL penicillin, 10 mg/mL streptomycin and 1% Gibco^® GlutaMAX™ Supplement (Thermo Fisher Scientific), the dental pulp tissues were minced into 1- to 3-mm² fragments, plated on 10-cm dishes with the growth medium, and cultured at 37 °C in a humidified tissue culture incubator with 5% CO₂ and 95% O₂. After 7–10 days of cultivation, the plastic-adherent confluent cells were treated with 0.05% trypsin containing 1 mM EDTA for 5 min to harvest pure mesenchymal cells. SHED were passaged and continuously subcultured and maintained in the complete growth medium. SHED from third to 14 passages were used in the experiments [19 (link)].