Ludwig Jessica E, Verma Sudhir, Thibodeau Alexa, Evonuk Kirsten S, Nallasamy Nambi, Mian Shahzad I, Coulson-Thomas Vivien J, Sawant Onkar B
Center for Vision and Eye Banking Research, Eversight, Cleveland, OH.
College of Optometry, University of Houston, Houston, TX.
Cornea. 2025 May 9;44(9):1158-1163. doi: 10.1097/ICO.0000000000003888.
Limbal stem cell deficiency is a blinding corneal disease that requires transplantation to replenish the limbal epithelial stem cells (LESCs) and restore vision. Keratolimbal allografts from cadaveric donors are commonly used as a source for LESCs. The goal of this study was to verify the efficacy of Food and Drug Administration-approved hypothermic corneal storage solutions in preserving LESCs within human donor corneas over time.
Limbal segments from deceased donor corneas were stored in Food and Drug Administration-approved corneal storage solutions for 0, 4, or 7 days. Tissues were then processed for immunohistochemistry, and number of p63α+ cells within the limbus were calculated. LESC density was calculated as the number of p63α+ cells per total number of cells. LESCs were isolated from donor corneas after 7 days of hypothermic storage and subjected to a colony formation assay, and the numbers of holoclones were calculated.
The average density of p63α+ cells was 37.4% on day 0, 27.3% on day 4, and 10.9% on day 7 ( P < 0.05) in limbal segments. Holoclones (15.33 ± 3.5) were formed by cells isolated from the limbal region of donor corneas after 7 days in the hypothermic storage conditions, indicating that viable LESCs are still present in human donor corneas after 7 days in storage.
Although the density of p63α+ LESCs significantly decreases over time, viable LESCs are present in the donor corneas after 7 days in hypothermic storage conditions. Thus, failure of detection or absence of expression of standard marker(s) does not necessarily indicate the loss of functional LESCs.
角膜缘干细胞缺乏症是一种致盲性角膜疾病，需要通过移植来补充角膜缘上皮干细胞（LESCs）并恢复视力。来自尸体供体的角膜缘移植片通常用作LESCs的来源。本研究的目的是验证美国食品药品监督管理局（FDA）批准的低温角膜保存液随着时间推移在保存人供体角膜内LESCs方面的有效性。
将已故供体角膜的角膜缘节段保存在FDA批准的角膜保存液中0天、4天或7天。然后对组织进行免疫组织化学处理，并计算角膜缘内p63α+细胞的数量。LESC密度计算为p63α+细胞数量占细胞总数的比例。低温保存7天后从供体角膜中分离出LESCs并进行集落形成试验，计算全克隆的数量。
角膜缘节段中p63α+细胞的平均密度在第0天为37.4%，第4天为27.3%，第7天为10.9%（P<0.05）。在低温保存条件下7天后，从供体角膜的角膜缘区域分离出的细胞形成了全克隆（15.33±3.5），这表明在保存7天后人供体角膜中仍存在有活力的LESCs。
尽管p63α+LESCs的密度随时间显著降低，但在低温保存条件下7天后供体角膜中仍存在有活力的LESCs。因此，标准标志物检测失败或表达缺失不一定表明功能性LESCs丧失。
