Cultivation and characterisation of human peripheral cornea derived endothelial cells
Cultivation and characterisation of human peripheral cornea derived endothelial cells
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人外周角膜来源的内皮细胞的培养和表征
DOI：
10.1111/j.1755-3768.2013.4766.x
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发表时间：
2013
影响因子：
3.4
通讯作者：
ALBERT R
中科院分区：
文献类型：
--
作者：
ALBERT R
证实DALK/DSEK/PK手术遗留的人角膜缘可能是体外内皮细胞扩增的有用来源。使用DALK/DSEK/PK手术后剩余的人角膜缘（1:1性别比例，年龄63+20岁，内皮细胞密度bb0 2,500细胞/mm2）。从死亡到使用的时间从3天到1.5个月不等。内皮细胞的分离采用两步剥离和消化方法，即在解剖显微镜下剥离内皮细胞和内皮细胞，然后在胶原酶中消化。将分离的细胞悬浮在TrypLE中，然后将其镀到FNC包被的组织培养板上。然后将细胞培养于含抗坏血酸、转铁蛋白、亚硒酸钠和bFGF的Ham’s F12:M199（1:1）培养基中。通过RT - qPCR和免疫荧光染色对培养细胞进行表征。分离的内皮细胞数反复偏低（< 2万个）。然而，改进的技术允许减少基质细胞污染。结果表明，随着培养面积的减小，内皮细胞的增殖能力增强。当细胞密度较高时，可见典型的内皮鹅卵石形态。细胞形态和生长与供体年龄和保存时间有关。ZO‐1，Na/K‐atp酶和PITX2被用来确认内皮表型。保存的人角膜缘可以用于角膜内皮细胞的体外扩增，但需要进一步优化。
To confirm that human corneal rims left over from DALK/DSEK/PK surgeries could be useful sources for ex vivo endothelial cell expansion. Human corneal rims remaining from DALK/DSEK/PK surgeries were utilized (1:1 sex ratio, age 63+20 years, endothelial cell density >2,500 cells/mm2). The time from death to use varied between 3 days and 1.5 months. Endothelial cells isolated using a two‐step, peel‐and‐digest method, whereby the Descemet’s membrane and endothelial cells were peeled off under a dissecting microscope, followed by digestion in collagenase. The isolated cells were suspended in TrypLE prior to plating onto FNC‐coated tissue culture plates. The cells were then cultured in Ham’s F12:M199 (1:1) media supplemented with, ascorbic acid, transferrin, sodium selenite and bFGF. Characterisation of the cultured cells was performed by RT‐qPCR and immunofluorescence staining accordingly. The number of isolated endothelial cells was repeatedly low (< 20,000 cells). However, improved techniques allowed to reduce stromal cell contamination. It was observed that endothelial cell proliferation was improved when the culture surface area was reduced. Furthermore, typical endothelial cobble stone morphology was observed when the cell density was high. Cell morphology and growth showed notable difference related to donor age and preservation time. ZO‐1, Na/K‐ATPase and PITX2 were used to confirm the endothelial phenotype. Preserved human corneal rims can be utilized for ex vivo expansion of corneal endothelial cells but further optimization is needed.