Human corneal endothelial cell proliferation: potential for use in regenerative medicine.
Human corneal endothelial cell proliferation: potential for use in regenerative medicine.
复制标题
DOI：
10.1097/01.ico.0000136666.63870.18
复制
发表时间：
2004-11-01
期刊：
影响因子：
2.8
通讯作者：
Zhu, Cheng Chris
中科院分区：
文献类型：
--
作者：
Joyce, Nancy C;Zhu, Cheng Chris
目得：回顾和更新我们的实验室在培养人角膜内皮细胞（HCEC）的经验，从年轻人和老年人donors.METHODS：角膜获得国家疾病研究交流，费城，PA。回顾了过去3年的数据，以制定用于内皮细胞培养的供体角膜的选择标准。使用mAb 9.3.E的免疫细胞化学定位鉴定了内皮细胞，Ki 67染色显示了活跃的细胞周期。细胞计数证明了生长促进剂对年轻（50岁）供体细胞增殖的影响。相差显微镜记录原代培养细胞的形态特征和生长因子对细胞形态的影响。结果：排除标准的制定，以增加成功培养的机会HCEC。分离方法，以消除后弹力层与附着的内皮细胞避免污染与其他角膜细胞类型。EDTA处理结合机械破碎促进细胞分离。含有FBS、EGF、NGF和牛垂体提取物的培养基刺激最大生长并促进正常单层形成。在原代培养中汇合细胞的密度和对生长促进剂的增殖反应中，检测到了与HCEC相关的差异。结论：未转化的HCEC可以成功地从年轻人和老年人的角膜培养供体材料的选择中使用护理。在内皮细胞分离的早期阶段也必须小心，以获得最大数量的健康细胞用于培养。似乎有真正的年龄相关的整体增殖能力的差异，但是，特定的生长因子的相对反应是相似的细胞从年轻和老年供体。这些研究的结果为未转化的HCEC在再生医学中的成功生长提供了指导。
PURPOSE: To review and update the experience of our laboratory in culturing human corneal endothelial cells (HCEC) from young and older donors.METHODS: Corneas were obtained from National Disease Research Interchange, Philadelphia, PA. Data from the past 3 years were reviewed to develop criteria for selecting donor corneas to be used for endothelial cell culture. Immunocytochemical localization using mAb 9.3.E identified endothelial cells, and Ki67 staining demonstrated actively cycling cells. Cell counts demonstrated the effect of growth-promoting agents on proliferation of cells from young (50 years old) donors. Phase-contrast microscopy documented morphologic characteristics of cells in primary culture and the effect of growth factors on cell morphology.RESULTS: Exclusion criteria were developed to increase the chance of successful culture of HCEC. Isolation methods to remove Descemet membrane with attached endothelial cells avoided contamination with other corneal cell types. EDTA treatment combined with mechanical disruption facilitated isolation of cells. Culture medium containing FBS, EGF, NGF, and bovine pituitary extract stimulated maximal growth and facilitated normal monolayer formation. Age-related differences were detected in the density of confluent cells in primary culture and in the proliferative response to growth-promoting agents.CONCLUSIONS: Untransformed HCEC can be successfully cultured from the corneas of both young and older donors by using care in the selection of donor material. Care must also be taken in the early phases of endothelial cell isolation to obtain maximal numbers of healthy cells for culture. There appear to be true age-related differences in overall proliferative capacity; however, the relative response to specific growth factors was similar in cells from young and older donors. Results of these studies provide guidelines for successful growth of untransformed HCEC for use in regenerative medicine.