Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures
Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures
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DOI：
10.1016/j.exer.2004.02.015
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发表时间：
2004-07-01
影响因子：
3.4
通讯作者：
Li, DQ
中科院分区：
文献类型：
--
作者：
Kim, HS;Song, XJ;Li, DQ
培养的人角膜上皮细胞已成功用于角膜重建。外植体和单细胞系统目前用于人角膜上皮培养。本研究的目的是表征这两种培养系统体外扩增的人角膜上皮细胞的表型，包括其生长潜力、形态和抗原表达模式。通过在丝裂霉素C处理的3 T3成纤维细胞饲养层上进行角膜缘外植体培养或角膜缘单细胞悬浮培养来扩增人角膜上皮细胞。原代培养细胞的表型通过形态学和免疫组织化学染色进行评价，免疫组织化学染色用的抗体提出的角质形成细胞干细胞标志物（p63，EGFR，K19和整合素PI）和分化标志物（K3，外皮蛋白和间隙连接蛋白连接蛋白43）。进行BrdU标记以鉴定标记保留细胞。人角膜上皮细胞生长从角膜缘组织保存长达16天的两种培养系统。生长速度取决于组织新鲜度、从死亡到保存的时间以及从死亡到培养的时间，而与供体年龄无关。在96（.）2%（n = 43）的单细胞悬浮培养物和90（.）8%（n = 213）的外植体培养物。在单细胞悬浮培养中，细胞扩增在10-14天内汇合，在外植体培养中，在14-21天内汇合。单细胞悬浮培养的细胞形态比外植体培养的细胞更小、更致密、更均匀。免疫组化结果显示，在两种培养体系中，表达p63、EGFR、K19和整合素0 - 1的小细胞数量较多，而表达K3、外皮蛋白和连接蛋白43的大细胞数量较多。在2（.）3 +/- 0（.）7%的外植体培养物和3（.）73 +/- 1（.）5%的单细胞培养物追踪21天。结论：角膜缘是体外扩增人角膜上皮细胞的宝贵材料。角膜上皮细胞的表型，从基底细胞到表面分化的细胞，在两种培养系统中都得到了很好的保持。在培养物中鉴定了慢循环的BrdU-标记保留细胞，其是干细胞的特征。(C)2004 Elsevier Ltd.保留所有权利。
Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin PI) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96(.)2% (n = 43) of single cell suspension cultures and in 90(.)8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell Suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin 0 1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-Iabel retaining cells were identified in 2(.)3 +/- 0(.)7% of explant cultures and 3(.)73 +/- 1(.)5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-Iabel retaining cells, that are characteristic of stem cells, were identified in the cultures. (C) 2004 Elsevier Ltd. All rights reserved.