Long-term culture and growth kinetics of murine corneal epithelial cells expanded from single corneas.
Long-term culture and growth kinetics of murine corneal epithelial cells expanded from single corneas.
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DOI：
10.1167/iovs.08-2139
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发表时间：
2009-06
影响因子：
4.4
通讯作者：
Xiaoli Ma;S. Shimmura;H. Miyashita;S. Yoshida;M. Kubota;T. Kawakita;K. Tsubota
中科院分区：
文献类型：
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作者：
Xiaoli Ma;S. Shimmura;H. Miyashita;S. Yoshida;M. Kubota;T. Kawakita;K. Tsubota
目的：建立一种可重复的长期培养小鼠角膜上皮细胞的方法。方法C57BL6/J小鼠角膜缘外植体在添加EGF和霍乱毒素的无血清低ca（2+）培养基中培养。上皮细胞按1:3的比例传代至传代(P)4， P4传代后呈低密度传代。测定了菌落形成效率、种群倍增次数和种群倍增次数。采用RT-PCR、免疫细胞化学和Western blotting分析p63、角蛋白(K)19、K12和天花素的表达。通过将培养基切换到血清或高Ca（2+）含量的培养基来检测分化电位。采用气举培养法分析其分层能力。结果32个角膜外植体中有30个（93.8%）成功传代到P1。没有霍乱毒素的培养物没有增殖超过P2 (n = 12)，但55%的添加霍乱毒素的培养物达到了P4 （n = 20）。P4后，细胞稳定传代25代。集落形成效率由P5处理下的9.7% +/- 2.6%提高到P20处理下的29.0% +/- 3.3%。细胞呈鹅卵石状，表达p63、K19和involucrin，但K12不表达。血清和高Ca（2+）诱导细胞分化，在DMEM/F12中培养的细胞有K12 mRNA表达。气举形成层状上皮。结论：通过这种方法，单个角膜上皮细胞可以长期培养，并保留分化和分层的潜力。对于需要在体外比较正常小鼠和转基因小鼠角膜上皮细胞的研究来说，这一过程是一个强大的工具。
PURPOSE To develop a reproducible procedure for the long-term culture of corneal epithelial cells from a single mouse cornea. METHODS Corneal limbal explants of C57BL6/J mice were cultured in serum-free, low-Ca(2+) medium supplemented with EGF and cholera toxin. Epithelial cells were subcultured at a 1:3 split until passage (P)4 and at lower densities after P4. Colony-forming efficiency, population-doubling times, and population doublings were determined. The expression of p63, keratin (K)19, K12, and involucrin was analyzed by RT-PCR, immunocytochemistry, and Western blotting. Differentiation potential was examined by switching the medium to serum or high Ca(2+)-containing medium. Stratification ability was analyzed by air-lift culture. RESULTS Thirty of 32 (93.8%) corneal explants were successfully subcultured to P1. Cultures without cholera toxin did not proliferate past P2 (n = 12), but 55% of cultures supplemented with cholera toxin achieved P4 (n = 20). After P4, cells were stably subcultured over 25 passages. Colony-forming efficiency increased from 9.7% +/- 2.6% at P5 to 29.0% +/- 3.3% at P20. The cells showed cobblestone appearance and expressed p63, K19, and involucrin but were negative for K12. Serum and high Ca(2+) induced differentiation, and cells cultured in DMEM/F12 with serum showed K12 mRNA expression. Stratified epithelium was formed by air-lifting. CONCLUSIONS With this procedure, corneal epithelial cells from a single cornea can be cultured long term and can retain the potential to differentiate and stratify. This procedure can be a powerful tool for studies that require comparison of corneal epithelial cells from normal and transgenic mice in vitro.