Currently, endothelial keratoplasty is the gold standard for the surgical treatment of Fuchs endothelial corneal dystrophy (FECD). Despite the remarkable success in terms of surgical outcomes, a shortage of corneal donor tissue poses a limitation to performing endothelial keratoplasty in many parts of the world. Cell therapy is a potential alternative strategy to keratoplasty and is currently under investigation. Considering that corneas with FECD may contain relatively healthy endothelial cells, samples obtained by descemetorhexis of eyes undergoing EK for FECD can be used for ex vivo expansion of endothelial cells as an autologous cell culture.In this study, we established corneal endothelial cell cultures derived from 40 patients that underwent endothelial keratoplasty for advanced FECD. Several parameters were evaluated including patient characteristics such as age, gender, and endothelial cell density as well as various processing and cell culture protocols based on different combinations of shipping temperatures, stabilization periods and treatment methods for corneal endothelial cell dissociation. FECD cultures were classified into three groups as: (i) no cells, (ii) cell cultures with endothelial-like morphology or (iii) cell cultures with fibroblast-like features.Our data seem to suggest that some factors can influence FECD cell culture characteristics including young age, high paracentral endothelial cell density, low shipping temperature and short stabilization period prior to cell isolation. Treatment with type 1 collagenase for cell isolation can delay endothelial-to-mesenchymal transition, but does not increase proliferative capacity.Although heterologous corneal endothelial cultures from healthy donors have shown encouraging outcomes, the feasibility of autologous cell therapy as a potential treatment for FECD remains challenging. Low initial cell concentration as well as endothelial to mesenchymal transition are the main obstacles to the application of FECD cultures in the clinical setting.