A Novel Method for Preservation of Human Corneal Limbal Tissue

Abstract

We investigated the efficacy of low-temperature airlift preservation of human corneal limbal tissue for ex vivo expansion and allograft keratolimbal transplantation. Human limbal tissue either was submerged or airlifted in Optisol-GS medium and preserved at 4°C for up to eight days. Hematoxylin and eosin, and E-cadherin staining was performed to investigate epithelial structure and cell-cell junction. Epithelial cell differentiation and proliferation were studied using the biomarkers, such as K10, K12, K14, Ki67, and p63. Cell apoptosis was detected with the TUNEL assay. The epithelial progenitor cell pool was evaluated by clonal culture of epithelial cells on 3T3 feeder layers. For clinical application, keratolimbal transplantation was performed in three patients with partial limbal stem cell deficiency, using limbal tissues preserved under the airlift manner. Pre- and postoperative evaluations were conducted by slit-lamp microscopy and fluorescein staining. After eight days, intact epithelia with strong cell-cell junctions were retained only in airlifted tissues. Airlifting maintained a normal corneal differentiation pattern, along with low proliferation activity and increased proliferation potential, but little apoptosis. Epithelial cells harvested from the airlift preservation for up to eight days exhibited stable clonogenicity. Limbal tissues preserved under the airlift manner successfully reconstructed corneal and limbal surfaces in partial limbal stem cell-deficient patients. Limbal tissues preserved under hypothermic airlift conditions maintain the intact structure, normal phenotype, high viability, and stem cell pool of limbal epithelia. Such a method may be used in eye bank tissue processing and corneal epithelial tissue engineering.