Human corneal endothelial cells (HCECs) in-vitro cultivation from donor corneas remnants after penetrating keratoplasty (PKP) and deep anterior lamellar keratoplasty (DALK)

Abstract

Purpose : To compare the rate of successful endothelium isolation of HCECs cultivated from donor corneal remnants after PKP and DALK to maximize the use of donated corneas.

Methods : Endothelial remnants from donor corneas were obtained from PKP corneal peripheral rings (CPR) and DALK corneal central discs (CCD). Donor corneas were stored in Optisol® and were processed immediately after surgery. Informed consent was obtained from donor relatives. The study was approved by our institutional ethics committee. HCECs were isolated by Liberase digestion and cultured using a dual media system for cell adaptation and proliferation. All cultures were maintained in 24-well plates, subculture cycle lasted 21-25 days and plated at same cell density. A failed subculture was when cells lost their adherence or developed a fibroblast-like morphology. Evaluations were performed with light microscopy and immunofluorescence using calnexin, actin and ZO-1 markers.

Results : Three single CPR (sCPR), 15 double CPR (dCPR) and 10 single CCD (sCCD) were analyzed. Mean donors age was 47.6±13.5 year, mean endothelial cell density was 2808±345 cells/mm² and mean death-to-transplantation time was 4±2 days. No significant differences in donor characteristic were observed between groups (Kruskal Wallis, p>0.05). The rate of successful HCECs isolation was 67, 87 and 100% in sCPR, dCPR and sCCD respectively. Isolations had a low yield below 8,000 HCECs. Successful HCECs subcultivation at 1st passage was 0, 46 and 60% in sCPR, dCPR and sCCD respectively. At 2nd passage was 23 and 30 %; and at 3rd passage was 15% and 10% for dCPR and sCCD respectively. Morphologic analysis (ImageJ software) on 1st passage subcultures showed that sCPR had a lower incidence of polymorphism (polimegathism and pleomorphism) when compared to sCCD (t-test, p<0.05).

Conclusions : Viable HCECs can be isolated from PKP and DALK donor cornea remnants. The small initial number of cells available in donor corneal remnants hinders subsequent subculture, compromising cell adhesion and morphology. Therefore, it is proposed to explore the feasibility of combining tissue remnants from different surgeries or donors as to adjust culture media formulations for low-yield tissues.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.