Identification of senescence markers on cultured human corneal endothelial cells

Aims/Purpose: Corneal transplantation is currently the most frequent type of transplantation globally. However, a chronic shortage of donor corneas makes traditional methods unsustainable. Alternatives such as bio‐engineered grafts or direct injection of corneal endothelial cells (CECs) into the patient's eye require the primary culture of CECs. Current CEC culture techniques face a significant challenge: cell senescence. Cells often become senescent during in vitro culture, rendering them unsuitable for clinical use and limiting the yield. Identifying specific markers of senescent CECs is crucial for studying this process. This study aims at identify robust markers of senescence in CEC cultures. Methods: Primary CEC cultures were obtained from human corneas following published methods. Various senescence markers for general cells and/or CECs were selected from the literature and applied using immunofluorescence (IF) in CEC cultures exhibiting heterogeneous morphology (presence of both very small and large, senescence‐associated cells) at different cell passages. The selected senescence markers were validated using induced senescent CECs by either removing anti‐senescent agents from the culture medium or adding TGF‐β1 or carteolol. β‐Galactosidase activity staining (SPiDER‐β Gal) was also tested. Results: Two candidate markers appeared suitable for detecting CEC senescence, each with specific attributes. Transgelin marked TGF‐β‐induced senescence, indicating a senescence‐associated secretory phenotype (SASP)‐related senescence, while SPiDER‐β Gal seemed linked to replicative senescence. Conclusions: Transgelin and SPiDER‐β Galactosidase are promising markers for detecting CEC senescence in vitro. Using both markers may help identify the causes of senescence.