Development of primary keratinocyte cell culture method on fibrin matrix

Abstract

The use of cultured epithelial grafts in major burn cases is a promising alter native. Keratinocyte cells have the ability to proliferate and form an epithelial layer when suitable conditions are provided and simulate as a dermal substitute. However, this layer, which is produced by the cell culture method, creates difficulties in surgical application due to its fragile structure. In this thesis, a biopolymer fibrin matrix was used as a carrier layer due to the advantages it offers in terms of biocompatibility, non-toxicity, biodegradability, and benefit for cell culture. In order to produce fibrin, fresh frozen plasma (FFP) was obtained from volunteers and with only CaCl2 solution, coagulation of the FFP has been achieved. In the second stage, to create an autologous cell culture method, a primary keratinocyte source is acquired directly from the human skin. Instead of a cell line, skin samples were taken from volunteers who had undergone abdominal panniculectomy surgery, and keratinocyte isolation was performed. After the formation of fibrin and the isolation of keratinocyte cells, keratinocyte cells are seeded on the fibrin matrix surface. The formation of the fibrin matrix scaffold and the feasibility of primary cell culture on this scaffold was examined and a sustainable and accessible method was established. FT-IR analysis was made to observe chemical bonds of polymerized fibrin structure. The success of cell isolation, cell culture, and the effect of fibrin scaffold on cell proliferation was demonstrated in optical microscopy, scanning electron microscopy (SEM), and pan-cytokeratin staining. The interaction of the keratinocyte cells and the feeder layer cells were visualized by SEM. In this thesis, methods of fibrin matrix formation from FFP, keratinocyte cell isolation and culture from a primary source, and keratinocyte culture on fibrin matrix were developed.|Keywords : Keratinocyte Cell, Primary Cell Culture, Fibrin Matrix, Cultured Epithelial Graf.