Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces
Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces
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DOI：
10.1096/fj.04-3035fje
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发表时间：
2006-02-01
期刊：
影响因子：
4.8
通讯作者：
Tano, Yasuo
中科院分区：
文献类型：
--
作者：
Sumide, Taizo;Nishida, Kohji;Tano, Yasuo
这项研究报告了一种制造适用于眼科手术和修复的生物工程人角膜内皮细胞片的新方法。我们最初在 IV 型胶原涂层培养皿上培养人角膜内皮细胞，经过几次传代后，将扩增的细胞接种到新型温度响应培养皿上。达到汇合后四个星期，通过简单的降温而不进行酶处理，将这些培养的内皮细胞收获为完整的单层细胞片。扫描电子显微镜表明这些细胞主要是六角形的，具有大量微绒毛和纤毛，类似于天然角膜内皮。 Na+、K+-ATP 酶泵位点与体内一样位于细胞边界。此外，在优化条件下，细胞密度和泵位点数量与体内人角膜内皮相同。 A3H-哇巴因结合分析表明，细胞泵密度在 575 个细胞/mm2 和 3070 个细胞/mm2 的汇合细胞密度之间呈线性比例关系。我们还在体外证实了片材中的 Na+、K+-ATP 酶活性。异种移植结果表明，制造的片材保留了在体内维持适当基质水合的功能。我们已经建立了一种培养和增殖人角膜内皮细胞的方案，并离体制造形态和功能与天然角膜内皮相似的内皮片。我们的结果支持了收获的细胞片在眼内皮失代偿患者眼部重建手术中的临床应用价值。
This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen‐coated dishes and, after several passages, expanded cells were then seeded onto novel temperature‐responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+‐ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A3H‐ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2and 3070 cells/mm2. We also confirmed Na+, K+‐ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.