Isolation of Human Corneal Endothelial Cells and Potential for Immediate Transplantation

Abstract

Purpose : Pioneering studies have shown that cultivated human corneal endothelial cells (HCEC) can be used to restore a functional human corneal endothelium in disease states. Inspired by these studies, we conducted this project to determine the feasibility of isolating HCECs for potential immediate transplantation (without ex-vivo propagation).

Methods : Donor corneas prepared for Descemet Membrane Endothelial Keratoplasty (DMEK) were digested in different concentrations of collagenase, in different media for different durations. Following digestion, the centrifugation process was optimized with different buffers, speeds, and time points. Isolated cells were labeled with a live cell tracker. These cells were then placed on the endothelial surface of donor corneas after scraping the endothelial cells ex vivo. The isolated cells were also cultured in vitro to monitor their morphology and characteristics.

Results : The optimized digestion condition was found to be 2 hours at 37°C in a medium consisting of OptiMEM reduced serum media, 8% human serum, 1mg/mL collagenase, 200µg/mL calcium chloride, and 50µg/mL gentamycin. Optimized cellular recovery (80±2%) and viability (89±1%) was obtained at 4000g in 10 minutes after adding 1:1 human serum to the digestion media containing the isolated cells. The isolated cells were viable and successfully attached to the endothelial surface of donor corneas ex vivo after 3 hours and remained viable and attached after 9 days. In vitro culture of the isolated cells resulted in characteristic morphology of hexagonal endothelial cells after 14 days.

Conclusions : These findings show that human corneal endothelial cells can be isolated with high viability which could potentially be used for endothelial cell transplantation immediately. This approach limits the in vitro manipulation of the cells which may be advantageous from a safety and regulatory standpoint.

This is a 2020 ARVO Annual Meeting abstract.