Eye Institute and Affiliated Xiamen Eye Center, Xiamen University School of Medicine, Xiamen, Fujian, China.
Invest Ophthalmol Vis Sci. 2013 Jun 10;54(6):4041-7. doi: 10.1167/iovs.13-11648.
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.
我们研究了低温气升式保存人眼角膜缘组织用于体外扩增和同种异体角膜缘移植的效果。
人眼角膜缘组织要么浸没在 Optisol-GS 培养基中，要么气升式保存于 4°C 下，最长可达 8 天。进行苏木精和伊红染色和 E-钙黏蛋白染色，以研究上皮结构和细胞-细胞连接。使用标志物如 K10、K12、K14、Ki67 和 p63 研究上皮细胞分化和增殖。用 TUNEL 检测细胞凋亡。通过在 3T3 饲养层上克隆培养上皮细胞来评估上皮祖细胞库。为了临床应用，采用气升式保存的角膜缘组织，对 3 例部分角膜缘干细胞缺乏患者进行了角膜缘移植。通过裂隙灯显微镜和荧光素染色进行术前和术后评估。
8 天后，仅在气升式保存的组织中保留了具有强细胞-细胞连接的完整上皮。气升式保存维持了正常的角膜分化模式，同时增殖活性低、增殖潜力增加，但凋亡很少。从气升式保存中收获的上皮细胞培养至 8 天仍具有稳定的克隆形成能力。气升式保存的角膜缘组织成功重建了部分角膜缘干细胞缺乏患者的角膜和缘部表面。
在低温气升条件下保存的角膜缘组织保持了角膜缘上皮的完整结构、正常表型、高活力和干细胞库。这种方法可用于眼库组织处理和角膜上皮组织工程。
