Objectives: Fibroblasts sometimes occur after enzymatic isolation of epidermis. They proliferate quickly, overgrowing the culture. A pure epithelial culture is essential for therapy using a keratinocyte graft. The aim of this study was to determine the possibility of fibroblast elimination from culture to prevent fibroblast overgrowth and obtain a pure monolayer of keratinocytes.
Material and methods: We analyzed three epidermal-derived cultures. Cells were cultured in medium contained Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 at a 3:1 ratio with 5% autologous serum and additives. The epithelial culture was confirmed using pancytokeratin MMF. If fibroblast like cells were present, they were removed using 0.01% edetate disodium dihydrate (Na2EDDA). This procedure was repeated until we obtained pure primary keratinocyte cultures.
Results: Fibroblast detachment was observed after Na2EDDA treatment. The procedure was performed twice and pure primary cultures of keratinocyte were achieved in two cases. These two cultures maintained their epithelial-like morphology and cytokeratin expression. One culture was treated four times with Na2EDDA with no effect; the morphology of the cultures became fibroblast-like with no observed cytokeratin expression.
Conclusions: Unwanted dermal fibroblasts can be separated from primary keratinocyte cultures during the first few days after the isolation. Cocultures of unwanted dermal fibroblasts and epidermal keratinocytes can be reverted to pure keratinocyte monolayers suitable as grafts for transplantation.