OBJECTIVETo explore a method for isolation and culture Dispase II and Trypsin-EDTA. Cells were seeded onto mitomycin C-treated of rat buccal mucosa stem cells and to identify the stem cells.

METHODSEpithelial cell mass were obtained by digesting rat buccal mucosa with 3T3 Swiss albino layer and cultured in DMEM for 24 hours, followed by K-SFM culturing. Some cells were induced to osteocytes and adipocytes and underwent ALP testing after 72 hours. Five days later, the primary cells were digested with trypsin and inoculated onto collagen IV-coated flasks and cultured at room temperature for 20 minutes. The adherent cells continued to be cultured with epithelial stem cell medium, then examined for identifying the clones, osteocytes, adipocytes, cytokeratin and ALP staining.

RESULTS83.96 percent of the primary epithelial cell mass were in G0/G1 phase by flow cytometry test. The clones were seen after 72 hours on 3T3 Swiss albino layer, and the osteocytes and adipocytes were positive. Cells were adhered quickly to collagen IV, in a shape of round or orbicular-ovate with strong refraction. The induced-osteocyte and adipocyte, cytokeratin and ALP were all positive.

CONCLUSIONSThe stem cell-like epithelial cells could be obtain using the 3T3 Swiss albino layer method. Sieved by collagen IV and cultured in epithelial stem cell medium could make the epithelial stem cells depurate and proliferate quickly.