Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Germany.
Department of Ophthalmology, University of Freiburg, Freiburg im Breisgau, Germany.
Klin Monbl Augenheilkd. 2024 Aug;241(8):964-971. doi: 10.1055/a-2084-7168. Epub 2023 May 2.
To assess various potential factors on human limbal epithelial cell (LEC) outgrowth using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm.
Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm.
The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007).
The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures .
使用经过长期储存（器官培养）的角膜供体组织，通过逐步线性回归算法评估人角膜缘上皮细胞（LEC）生长的各种潜在因素。
在 215 名供体中，我们使用了 304 个角膜巩膜环进行实验。为了消化角膜缘组织并分离角膜缘上皮细胞，将组织片在 37°C 下用 4.0mg/mL 胶原酶 A 在 95%相对湿度和 5%CO 气氛中孵育过夜。此后，使用 20-μm CellTricks 过滤器将角膜缘上皮细胞与角膜缘成纤维细胞分离。分离的人 LEC 培养在无血清角质形成细胞培养基中，含有 1%青霉素/链霉素（P/S）、0.02%表皮生长因子（EGF）和 0.3%牛垂体提取物（BPE）。使用逐步线性回归算法分析供体年龄（协变量）、死后时间（协变量）、培养基时间（协变量）、使用的角膜巩膜环大小（360°、270°180°、120°、90°、小于 90°）（协变量）、内皮细胞密度（ECD）（协变量）、性别（因子）、器官培养过程中培养基更换次数（因子）以及供体来源（供体机构和储存机构，因子）对角膜缘上皮细胞生长的潜在影响。
人 LEC 成功生长的比例为 37.5%。从逐步线性回归算法中，我们发现对 LEC 生长有影响的相关参数是截距（p<0.001）、供体年龄（p=0.002）、器官培养过程中培养基更换次数（p<0.001）、总培养基时间（p=0.181）和使用的角膜巩膜环大小（p=0.007），以及培养基时间×角膜巩膜环大小（p=0.007）。
LEC 生长的成功率随着供体年龄降低、储存过程中器官培养培养基更换次数减少、器官培养培养基时间缩短以及角膜巩膜环尺寸减小而增加。我们的逐步线性回归算法可以帮助我们优化 LEC 培养。
