Abstract
Purpose :
We have been developing cellular therapy for the treatment of corneal endothelial decompensation. In this study, our aim was to assess the feasibility of cryopreservation of cultured human corneal endothelial cells (HCECs) as an intermediate product for efficient manufacturing of cellular products.
Methods :
Three lots of human corneal endothelial cells (HCECs) were cultured from 3 pairs of donor corneas (donor ages: 27, 38, and 40). Descemet membranes, including the corneal endothelium, were peeled from corneas and digested enzymatically. The resulting HCECs were seeded onto a culture plate, cultured, and passaged 3 times. On the 10th day of passage 3 culture, the HCECs were harvested, washed with basal medium (Opti-MEM I) without serum, suspended in cryopreservation medium, and stored in liquid nitrogen. The cells were thawed after 3, 6, 9, 12, and 24 months of cryopreservation, and cell viability was assessed by evaluating cell growth after seeding the cells in a culture plate. Additionally, HCECs cryopreserved for 24 months were cultured for 14 days, suspended in a frozen medium supplemented with 100 μM Y-27632 (a ROCK inhibitor), and cryopreserved again at -80°C. After thawing those passage 4 cells, expression of CD166 (human corneal endothelial marker), ZO-1 (barrier function marker), and Na+/K+-ATPase (pump function marker) was assessed using a fluorescence-activated cell sorter (FACS).
Results :
The cell viability before cryopreservation was 99.0±0.15%. After cryopreservation, cell viability was maintained for 24 months (94.2±2.03% at 3 months, 96.6±0.69% at 6 months, 97.1±0.56% at 9 months, 98.4±0.23% at 12 months, and 98.0±1.01% at 24 months). After thawing and seeding, all 3 HCEC lots showed polygonal and monolayer morphology similar to that of non-cryopreserved control HCECs at every point in the 24-month assessment. FASC demonstrated that the expression rates of CD166, ZO-1, and Na+/K+-ATPase in HCECs cryopreserved for 24 months were 99.0 ± 0.08%, 98.0 ± 0.98%, and 98.6 ± 0.33%, respectively.
Conclusions :
Our findings suggest that HCECs can be cryopreserved for at least 2 years. This methodology can be utilized for manufacturing HCECs as a cellular product for endothelial decompensation and will facilitate the international transportation of the intermediate product and the stable supply of the final product.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.