An effective method for culturing functional human corneal endothelial cells using a xenogeneic free culture medium

Abstract

Endothelial dysfunction is a leading cause of corneal blindness in developed countries and the only available treatment is the endothelial transplantation. However, the limited availability of suitable donors remains a significant challenge, driving the exploration of alternative regenerative therapies. Advanced Therapy Medicinal Products show promise but must adhere to strict regulations that prohibit the use of animal-derived substances. This study investigates a novel culture methodology using Plasma Rich in Growth Factors (PRGF) as the only source of growth factors for primary cultures of human corneal endothelial cells (CECs). CECs were obtained from discarded corneas or endothelial rings and cultured in two different media: one supplemented with xenogeneic factors and other xenogeneic-free, using PRGF. Comprehensive characterization through immunofluorescence, morphological analyses, trans-endothelial electrical resistance measurements, RNA-seq, and qPCR was conducted on the two groups. Results demonstrate that CECs cultured in the xenogeneic-free medium exhibit comparable gene expression, morphology, and functionality to those cultured in the xenogeneic medium. Notably, PRGF-expanded CECs share 46.9% of the gene expression profile with native endothelium and express all studied endothelial markers. In conclusion, PRGF provides an effective source of xenogeneic-free growth factors for the culture of CECs from discarded corneal tissue. Further studies will be necessary to demonstrate the applicability of these cultures to cell therapies that make clinical translation possible.