Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy

Abstract

Introduction: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty.

Methods: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 μ<sc>m</sc> Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers.

Results: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0.

Conclusions: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (&gt;60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (&lt;2,000 cells/mm2) of the donated cornea.

Keywords: Cell culture; Cell therapy; Corneal transplant; Endothelial barrier; Endothelial dysfunction; Eye bank.

Fig. 1.

Isolation and in vitro attachment and growth of hCEC. a Schematic diagram showing the different phases of the in vitro culture of hCEC after enzymatic isolation from the Descemet’s Membrane (DM)-endothelial layer (EC) layer (DM-EC). b Phase contrast images showing the hCEC still attached to the DM (top) after descemetorhexis, released clusters from the DM (middle) after controlled collagenase digestion, and into monolayer in vitro cultures within the first 48 h (bottom left), after trypsin separation, and after 3–4 weeks of culture confluence (bottom right). c Viability and cell growth of in vitro hCEC cultures assessed by WST-1 assay for cultures obtained from preserved (4°C) and cultivated (31°C) corneas older than 60 years (upper) and cultivated corneas from donors younger than 60 years of age (lower). Data are expressed as MD ± SD from 3 independent experiments (***p < 0.001 compared to day 1). Scale bar, 100 µm. ON, overnight; S medium, maintenance cell culture medium (M5); P medium, growth cell culture medium (M4).

Fig. 2.

Cellular morphometric and circularity analysis of cultured hCEC along the passages. a Scatter plots showing cellular area according to the donor age (40 to 50, >50 to 60, and >60 years of age). The variability of the cell area is shown throughout the isolation (P0), first (P1), second (P2), and third (P3) passages (each point of the graph corresponds to the area of a cell). Five fields of each cell monolayer at confluency (day 21 in culture) obtained from 3 donors per age group were analyzed to obtain the median. Data are expressed as median and interquartile ranges. b Phase contrast images of cultures over the passages from a 43-year-old donor (upper) and a 65-year-old donor (lower). c Cellular circularity analysis of expanded hCEC at P0 and P1 according to the donors age (40 to 50, 50 to 60, and >60 years of age). Data are expressed as MD ± SD. **p < 0.01; ***p < 0.001. Scale bar, 100 µm.

Fig. 3.

Characterization and functionality of hCEC monolayers. a Immunostaining for corneal endothelial markers such as zonula occludens (ZO-1) and sodium-potassium ATPase (Na⁺/K⁺-ATPase). b Immunostaining of cultured hCECs for nuclear proliferation marker Ki67. c Transendothelial electrical resistance (TEER) measurements in hCEC polarized confluent monolayers at isolation (P0) and the first passage (P1). Cultures obtained from corneas older than 60-year-old donors. *p < 0.05; **p < 0.01; ***p < 0.001 compared to day 1. Scale bar, 100 µm.