Corneas discarded by low endothelial count: a source of human corneal endothelial cells (hCECs).

Abstract

Purpose : Surgical treatment of patients with severe endothelial damage has evolved in just over 100 years from complete corneal transplantation or penetrating keratoplasty to selective transplantation of the damaged layer. Descemet Membrane Endothelial Keratoplasty, a less invasive technique offers better visual results, requires of corneas with better endothelial count. This fact has caused that in the last years our local tissue bank discards 30-50 corneas per year due to low endothelial count.
The purpose of the present study is to use these corneas as a source to obtain hCEC cultures which could be used for the treatment of corneal endothelial dysfunctions.

Methods : hCECs culture: hCECs were obtained from corneas discarded by our local tissue bank due to low endothelial count. Descemet membrane along with endothelial cells was dissected following the Schwalbe line and was maintained for 48-96h at 37^oC in a culture plate (4cm²) with 4mL of culture medium.
Descemet membrane along with endothelial cells was digested with trypsin/EDTA for 2 hours at 37°C. After that, trypsin was neutralized with culture medium. The detached cells were centrifuged at 0.4g for 10 minutes, the supernatant was removed and the cells were seeded on a culture plate previously treated with FNC coating mix®.
Cellular analysis: cellular growth was assessed by phase contrast microscopy. Confluent cultures of hCECs were fixed using ice-cold methanol for 10 minutes and immunocytochemistry with Na⁺/K⁺ ATPase and zonula occludens-1 (ZO-1) antibodies were performed in order to check their specific markers. Moreover, expression of major corneal endothelial markers (Na⁺/K⁺ ATPase, ZO-1, Aquaporin-1, Vimentin, Connexin-43) were checked by qPCR and compared with native endothelium.

Results : hCECs obtained from corneas discarded due to low endothelial count were able to attach and proliferate until confluence showing positive staining for ZO-1 and Na⁺/K⁺ ATPase, typical markers of hCECs and responsible for endothelial barrier and pump functions. In addition, similar expression of protein mRNA was observed in cultured cells compared with a native endothelium for each of the genes tested.

Conclusions : Corneas discarded by low endothelial count could provide a source of hCECs and be potentially used in cell therapy or tissue engineering therapy for the treatment of corneal dysfunctions.

This is a 2020 ARVO Annual Meeting abstract.