Abstract
Purpose :
Deceased donor limbal stem cells (LSCs) are an under-utilized source of transplant tissue to surgically treat limbal stem cell deficiency (LSCD). This study sought to determine the time-dependent viability of LSCs in corneal storage solution. This will serve as a basis for improving the practicability of using eye-bank procured tissue as a suitable source of LSCs for allogenic simple limbal epithelial transplant and keratolimbal allograft.
Methods :
All work was completed in compliance with the Declaration of Helsinki and Eye Bank Association of America regulations. Research-consented donor (N=3, ages 52-58) eyes were collected and dissected to retain the limbus and a section of sclera in clock-hour sized pieces. Segments were stored in FDA-approved corneal storage solution for 0, 4, or 7 days. After storage, tissue was processed for immunohistochemistry. Cryosections of the limbus were incubated with p63-α primary antibody (4892S, Cell Signaling) and goat anti-rabbit Alexa 594 (A32740, Invitrogen) to mark LSCs. The total number of LSCs was quantified by counting all p63-α positive (+) cells in four 20X fields under a Leica DMi8 epifluorescence microscope. This was compared to all bisbenzimide+ nuclei in the same field of view to calculate the % of cells that are p63-α+. One-way ANOVA was performed among the three time points. Pairwise analysis between the groups were performed using Tukey’s multiple comparisons test.
Results :
One-way ANOVA indicated statistically significant differences in p63-α+ cells among the 3 groups (P=0.0006). At day 0 we observed that 35% of cells are p63-α+. The density of p63-α+ cells significantly decreased to 13% at day 4 (P=0.0057). By day 7 there was a complete loss of p63-α+ cells and averaged 0.6% (P=0.0005).
Conclusions :
Our preliminary analysis indicates that expression of the LSC marker p63-α decreases during corneal storage conditions, and that cadaveric donor corneal tissues as a source of LSCs should be utilized within 4 days of procurement. The composition of storage media used for eye banking was created with balancing the needs of the corneal endothelium, stroma, and epithelium in mind and may not be particularly conducive to maintaining stemness in the LSC population. An approach to increase LSC viability may include modifying storage media or storage conditions to better suit the needs of this niche cell type.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.