The post-mortem interval influences the properties of human corneal epithelial cells in vitro.

Abstract

Purpose : The cornea is crucial for visual acuity as it allow light refraction to the retina and constitutes the first ocular barrier. It ruptures frequently cause infection and in severe cases, lead to the complete loss of vision. In North America, corneal wounds accounts for 37% of visual deficiency. Normal homeostasis of the corneal epithelium is ensured by corneal epithelial stem cells. According to the severity of the trauma, stem cell integrity can be compromised and a corneal transplantation may be required. In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an tissue-engineered human cornea (hTECs) is of prime importance. For transplantation purposes or to conduct research studies, the human corneal epithelial cells (hCECs) used in the hTECs production must be of the highest possible quality. The present study was intended to evaluate the impact of the post-mortem interval (PMI) on the properties of hCECs in vitro.

Methods : hCECs were isolated from the limbal area of donor corneas at different PMI (from 0 to 19 days) and grown as monolayers or on hTECs. The gene expression pattern was determined in ten populations. Growth rate measurements and colony-forming efficiency assays were conducted to evaluate the hCECs proliferative potential whereas the histological integrity of the hTECs was analysed by immunofluorescence. The stem cell population from cultured hCECs with different PMI was evaluated by p63 staining and the PMI impact on the wound closure dynamic studied on both hCECs and hTECs.

Results : Short PMI hCECs have a gene expression pattern distinct from that of long PMI hCECs grown as monolayers, and also displayed an increased number of daily doublings and generated more colonies per seeded cell compared with long PMI hCECs. Immunofluorescence analyses revealed that components of the basement membrane and extracellular matrix were deposited similarly in all hTECs, irrespective of the hCECs PMI. Interestingly, short PMI hCECs have more stem cells than the long PMI cells, which significantly accelerated wound closure in the short PMI group (in both hCECs and hTECs).

Conclusions : These results suggest that hTECs reconstructed using short PMI hCECs have a higher number of limbal stem cells and exhibit a more efficient wound healing response in vitro, making them the best candidates to produce substitutes for clinical studies.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.