Cultivation of Human Oral Keratinocytes and Fibroblasts Using an Explant Technique

Objective: To report a simple and reliable method for culturing normal human oral keratinocytes in a culture system free from feeder cells, with separation of the contaminating fibroblasts and their propagation.Materials and Methods: The primary culture was initiated by explantation. The contaminating fibroblasts were isolated through trypsinization for their propagation. Subcultivation of oral keratinocytes by cell drops was done when 70% to 80% confluence was achieved. Morphological characteristics of both cell types were assessed under phase-contrast microscopy and the origin of the cultured cells was determined by cytohistochemical staining. The growth potential of both types of cells was monitored by growth curves.Results: Oral keratinocytes could be serially cultured through 4 passages, and the contaminating fibroblasts were propagated after separation through trypsinisation. The morphology of the oral keratinocytes was typical pavement-like phenotype and was positive to cytokeratin staining. The growth curves of both cell types were S shaped.Conclusion: This explant culture method may be a useful and economical option for serial cultivation of oral keratinocytes and fibroblasts.