Isolation and identification of human dental pulp stem cells

Abstract

Dental pulp tissue is a potential source of Mesenchymal stem cells (MSCs). The aim of this study was to isolate and culture dental cells from human dental pulp by various methods. We compared the effectiveness of human pulp cells isolated by either enzyme digestion or the outgrowth method. The primary culture of pulp cells in DMEM/F12 10% FBS (fetal bovine serum) with antibiotics was carried out. Cells in culture medium were strongly expanded after 7 days. When the cells reached 75- 80% confluence, dental pulp cells are subcultured by using trypsin/EDTA 0,05% to provide nutrients and surface for development. The culture medium was changed every two days. After 1 times of subculture, MSC candidates were identified by using RT-PCR in the presence of two kind of surface markers Oct4 and CD146. The result shown that DMEM solution supplemented with 300 IU/ml penicillin, 300 µg/ml streptomycin, 0.75 µg/ml amphotericin B (fungizone®) was the best medium for tooth preservation. The project accomplished succeeded in isolating stem cells from human dental pulp by using outgrowth method. Cells reached confluence after 30 days of primary culture and after 6 days of subculture.

Key words: dental pulp tissue, mesenchymal stem cell, pulp cell