Epithelial Cell Culture From Reduced Enamel Epithelium

Objectives:
Our goal is to isolate and characterize epithelial cellular culture from reduced enamel epithelium in dental follicle of retained third molars
Methods:
Dental follicle of retained third molars were collected after routine extractions in Dental faculty (Medical university – Sofia) after informed consent was obtained from the patients. Follicle tissue was dissected with scalpel and minced, after which it was digested with 3 mg/ml Collagenase I / 4 mg/ml Dispase solution in PBS for 30 min. Heterogenous cell culture was then trypsinised and mesenchymal cells were separated from epithelial cells. Further epithelial cells were grown in KBM-Gold medium (Lonza,Basel, Switzerland ) supplemented according to the manufacturer’s instructions.
Epithelial cell cultures were characterised for expression of variety of dental specific and epithelial markers with immunofluorescence.
Results:
After enzymatic digestion the follicle tissues were allowed to attach to the bottom of 6 cm. dishes and after 3-7 days we observed cells migrating and proliferating. We recognised two types of cells: spindle like mesenchymal cells and oval, tightly lined next to each other epithelial cells. Trypsin disattached quickly the mesenchymal cells and the epithelial colonies remained attached longer. Morphological and immunofluorescent observations confirmed we managed to isolate epithelial cell culture, expressing CK10, CK14 , P63, Amelogenin, Ameloblastin, Tuftelin.
Conclusions:
Dental follicles of retained third molars have viable epithelial cells, relative to the enamel organ epithelium and rests of Malassaez, which can be isolated and studied further.