Cell culture technique for human labial mucosal epithelium for use in patients with bilateral limbal stem cell deficiency
Aim: to obtain a stable population of the human labial mucosal epithelium without feeder cells using explant method and simplified formulation of the culture media. Materials and methods. Labial mucosa were obtained from 6 patients in the operating room after signed informed contest. Samples were trimmed of the substantia propria and cut into uniformed explants. Cell culture were done using DMEM/F12 (1:1) (1.05 мМСя) and EpiLife (0.06 мМСя) media supplemented with 5% fetal bovine serum, antibiotic-antimycotic, insulin (5 μg/ mL), hydrocortisone (5 μg/mL), and epidermal growth factor (10 ng/mL). Primary cells were stained against stemness and proliferative marker (anti-p63), intermediate filaments (anti-vimentin), and tight junction protein-1 (anti-ZO-1). Image analysis was performed in Fiji (ImageJ). Results. Primary cell culture were obtained from all the samples in both media. Cellular morphology was characterized as a classic «cobblestone» phenotype. It was found that p63 expression in medium with 1.05 mM Ca were detected in 34.7% cells (median, n = 3), and ZO-1 expression was estimated as 17.05 mkm per cell (median, n = 3). In cells cultured in 0.06 mM Ca medium positive expression of the p63 were found as 39,2% (median, n = 3), and the length of the ZO-1 expression were calculated as 5.18 mkm per cell (median, n = 3). Conclusion. This study presents a detailed protocol for obtaining the cell culture of human labial mucosal epithelium with high proliferative activity from a small biopsy without feeder cells condition. 1.05 mM Ca medium promoted generation of the tight junction and may be used in in vitro epithelial differentiation models. In opposite, medium with 0.06 mM Ca sustained lowered level of maturation in cell culture. Thus, media formulations, source and method of cell culture described in this study, may be used for the transplantation of the autologous labial mucosal epithelium in patients with bilateral limbal insufficiency.