Isolation of Corneal Endothelial Cells

HCEnCs were isolated from research grade donor corneas using a peel-and-digest method similar to previously published methods, [7 (link), 13 (link)–15 (link)] with limited modifications. Firstly, the corneas [n = 30] were washed in sterile PBS and Descemet's membrane with endothelium was dissected with a fine forceps, similar to the stripping technique used for Descemet's membrane endothelial keratoplasty (DMEK). Secondly, the excised pieces were incubated in 2 mg/mL collagenase type 1 (Thermo Fisher Scientific, Rochester, NY, USA) solution for 2-3 hours at 31°C, 5% CO₂. Once Descemet's membrane was digested, the solution was centrifuged for 5 minutes at 1000 rpm. The supernatant was removed, and the cells were resuspended in TrypLE Express (1x) for 10 minutes at 37°C, (Life Technologies, Monza, Italy) to obtain single cell suspension suitable for seeding. An overview of the performed experiments can be seen in Figure 1.