-Reviving ageing corneas, a ray of hope for fading eyes.

Background:

Corneal endothelium, a vital layer responsible for maintaining clear vision. The researches on undifferentiated mesenchymal cells, such as Mesenchymal Stem Cells (MSCs), light up the limited regenerative capacity of this layer, which is also where it originates from. As the endothelial cells mature and form intercellular bridges, their ability to proliferate decreases. The presence of TGF-ß in the aqueous humour further hinders cell cycle progression. Notably, older donor corneas, typically from donors over 50 years of age, exhibit slower cell cycle entry and doubling time, which complicates cell therapy efforts. Simultaneously, this discovery also arouses the research enthusiasm to tackle these puzzles.

Introduction:

Corneal endothelial dysfunction (CED) is a significant cause of vision impairment, with endothelial keratoplasty (EK) being the primary treatment choice. However, the limited availability of donor corneas poses a major challenge, as only a small fraction of patients in need can benefit from EK due to the lack of corneas donated by the young group. To address this issue, researchers have explored the expansion of human corneal endothelial cells (hCEC) in vitro as an alternative approach. Growing evidence suggests that expanding hCEC for transplantation is feasible, even from elderly donor corneas that do not meet keratoplasty criteria, and most corneas available for transplantation come from elderly donors. Therefore, the following study discussed in this essay, in terms of its methods, results and conclusion, is aimed to assess the viability and functionality of hCEC cultures obtained from elderly donors (> 60 years) whose corneas were preserved using an organotypic culture method and did not meet the clinical criteria for keratoplasty.

Methods:

The research involved obtaining hCEC cultures from donor corneas that had undergone descemetorhexis and a controlled incubation with collagenase type I, followed by recombinant trypsin. These cells were then seeded on coated plates and cultured using a dual-supplemented medium approach comprising a maintenance medium and a growth medium. A 10 μM ROCK inhibitor (Y-27632) was included in the culture medium. Cell passages occurred at culture confluency, typically after two weeks. The study conducted a quantitative colorimetric WST-1 cell growth assay at various time points to monitor cell proliferation. Additionally, morphometric analysis, immunocytochemistry, and transendothelial electrical resistance (TEER) measurements were performed on confluent cell monolayers.

Results:

The study found that there were no significant differences in the cell growth profiles of hCEC cultures obtained from corneas of donors older than 60 years, regardless of whether the corneas were cold-preserved or cultivated organic typically. These primary cultures retained a cell circularity index close to 0.8, maintaining a hexagonal morphology resembling the corneal endothelial mosaic. Confocal staining revealed high expression of ZO-1 and Na+/K+ -ATPase pump markers in confluent cell monolayers at 21 days after isolation (P0). However, these markers significantly decreased in confluent monolayers after the first passage (P1). Ki67 expression, a marker of cell proliferation, remained weak in both P0 and P1 monolayers. TEER measurements indicated that the P0 monolayers showed a progressive increase in TEER values between days 6 and 11, which remained stable until day 18, signifying a state of controlled permeability. While the P1 monolayers exhibited some functional capacity, they had lower TEER values compared to those at P0.

Conclusions:

This study demonstrates that functional hCEC cultures can be successfully established in eye banks using simplified and standardised protocols. These cultures are obtained from older donor corneas (> 60 years of age), previously preserved under organotypic culture conditions. This approach addresses the pressing issue of cornea availability, particularly in elderly donor populations or corneas with low endothelial cell counts, offering a potential alternative to traditional keratoplasty techniques. By providing access to these innovative cell-based therapies, it is possible to alleviate the challenges of corneal blindness and advance the field of regenerative medicine. This approach opens up new possibilities for collaboration between tissue banks, research centres, and regulatory authorities, making advanced cell therapy for corneal disorders a viable and promising option.

Reference:

Aloy-Reverté, C., Bandeira, F., Otero, N., Rebollo-Morell, A., Nieto-Nicolau, N., Gomes, J. Á. P., Güell, J. L., & Casaroli-Marano, R. P. (2023). Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy. Ophthalmic Research, 66(1), 1254–1265. https://doi.org/10.1159/000533701.