Limbal stem cells (LSCs) are the stem cell reservoir for corneal epithelium. The protocol to isolate LSCs from human cornea has been examined and optimized. However, the isolation protocol has not been optimized for mouse cornea, which is crucial for the downstream cell analysis. Here we compared four different isolation methods evolved from the previous reports to obtain mouse limbal epithelial cells which are heterogeneous and contain LSCs in a single-cell suspension: (1) the dissected limbal rim was cut into pieces and digested by 10-cycle incubation in trypsin; (2) after the removal of corneal epithelium by a rotating bur, the remaining eyeball was incubated in dispase at 4 °C for overnight to obtain limbal epithelial sheet, followed by trypsin digestion into a single-cell suspension; (3) same as method 2 except that the incubation was in dispase at 37 °C for 2h and an additional collagenase incubation at 37 °C for 20 min; (4) same as method 3 except that the corneal epithelium was punctured by a 1.5 mm trephine instead of being removed by a rotating bur. Method 1 showed the lowest cell yield, the lowest percentage of single cells, and the lowest number of limbal epithelial stem/progenitor cells in the harvested cells among the four methods, thus not a recommended protocol. Method 2, 3, and 4 isolated a comparable number of K14⁺ and p63α-bright stem/progenitor cells per eye. The remaining eye globe after cell collection in the three methods showed a complete removal of limbal epithelium albeit different extent of corneal and limbal stromal digestion. Among the three methods, method 2 showed a higher cell viability than method 4; method 3 yielded the lowest cell number; method 4 led to the highest percentage of single cells in cell suspension. Results suggest that method 2, 3, and 4 are preferred methods to isolate heterogeneous-LSCs from mouse corneas.

角膜缘干细胞 (LSC) 是角膜上皮的干细胞库。从人角膜中分离 LSCs 的协议已经过检查和优化。然而，隔离协议尚未针对小鼠角膜进行优化，这对下游细胞分析至关重要。在这里，我们比较了从以前的报告演变而来的四种不同的分离方法，以获得异质的小鼠角膜缘上皮细胞，并在单细胞悬浮液中含有 LSC：（1）将解剖的角膜缘边缘切成碎片并通过 10 个周期的孵育消化胰蛋白酶；(2)旋转钻头去除角膜上皮后，剩余眼球在分散酶中4℃孵育过夜，得到角膜缘上皮片，胰蛋白酶消化成单细胞悬液；(3)与方法2相同，不同之处在于37℃分散酶孵育2h，另外37℃孵育胶原酶20min；(4) 与方法 3 相同，只是用 1.5 mm 环钻刺穿角膜上皮，而不是用旋转车针去除。方法 1 显示四种方法中收获的细胞中细胞产量最低、单细胞百分比最低、角膜缘上皮干/祖细胞数量最低，因此不是推荐的方案。方法 2、3 和 4 分离了相当数量的 K14 并且在四种方法中收获的细胞中角膜缘上皮干/祖细胞的数量最少，因此不是推荐的方案。方法 2、3 和 4 分离了相当数量的 K14 并且在四种方法中收获的细胞中角膜缘上皮干/祖细胞的数量最少，因此不是推荐的方案。方法 2、3 和 4 分离了相当数量的 K14每只眼睛的⁺和 p63α-亮干/祖细胞。三种方法收集细胞后剩余的眼球显示完全去除角膜缘上皮，尽管角膜和角膜缘基质消化程度不同。三种方法中，方法2的细胞活力高于方法4；方法 3 产生的细胞数最少；方法 4 导致细胞悬液中单细胞的百分比最高。结果表明，方法 2、3 和 4 是从小鼠角膜中分离异质 LSCs 的首选方法。