最佳角膜缘干细胞功能的隔离和无异种培养条件。
Optimal isolation and xeno-free culture conditions for limbal stem cell function.
机构信息
The Scheie Eye Institute, Department of Ophthalmology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
出版信息
Invest Ophthalmol Vis Sci. 2014 Jan 20;55(1):375-86. doi: 10.1167/iovs.13-12517.
PURPOSE
To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions.
METHODS
Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype.
RESULTS
Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2.
CONCLUSIONS
The LSC function can be maintained in vitro under appropriate Xeno-free conditions.
目的
在无外源物的培养条件下，保持角膜缘干细胞（LSC）的功能。
方法
从 139 位供体中使用三种分离溶液的 15 种变体分离角膜缘上皮细胞。所有培养条件均与基线条件（20%氧气下的鼠 3T3-J3 饲养细胞与外源物（Xeno）角质细胞生长培养基）进行比较。在 5%、14%和 20%氧气下，评估了五种具有不同钙和表皮生长因子（EGF）浓度的 Xeno 和无外源物培养基。比较了人 MRC-5、真皮（胎儿、新生儿或成人）和角膜缘基质成纤维细胞。对最大连续每周传代数、中止集落的百分比、集落形成效率（CFE）、p63α（明亮）细胞和 p63α/K12 的 RT-PCR 比值进行了统计分析。通过免疫细胞化学和 RT-PCR 评估了 p63α、ABCG2、Bmi1、C/EBPδ、K12 和 MUC1 的表型。
结果
Dispase/TrypLE 是一种分离方法，该方法显示出最好的产量、活力和 CFE。在 3T3-J2 饲养细胞上，20%氧气下无外源物培养基，钙 0.1 mM 和 EGF 10 ng/mL 支持更多传代，中止集落的百分比、p63α（明亮）细胞和 p63α/K12 RT-PCR 比值与基线 Xeno 培养基相当。使用这种无外源物培养基，MRC-5 饲养细胞表现出最佳性能，其次是胎儿、新生儿、成人 HDF 和角膜缘成纤维细胞。MRC-5 饲养细胞支持连续传代，祖细胞标志物的表达持续高水平，与无明显差异的基线条件一样稳健20%和 5%氧气下。
结论
在适当的无外源物条件下，LSC 的功能可以在体外维持。
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