The choroid of the eye is a vascularized and pigmented connective tissue lying between the retina and the sclera. Increasing evidence demonstrates that, beyond supplying nutrients to the outer retina, the different choroidal cells contribute to the retina's homeostasis, especially by paracrine signaling. However, the precise role of each cell type is currently unclear. Here, we developed a choroidal substitute using the self-assembly approach of tissue engineering. Retinal pigment epithelial (RPE) cells, as well as choroidal stromal fibroblasts, vascular endothelial cells and melanocytes, were isolated from human eye bank donor eyes. Fibroblasts were cultured in a medium containing serum and ascorbic acid. After six weeks, cells formed sheets of extracellular matrix (ECM), which were stacked to produce a tissue-engineered choroidal stroma (TECS). These stromal substitutes were then characterized and compared to the native choroid. Their ECM composition (collagens and proteoglycans) and biomechanical properties (ultimate tensile strength, strain and elasticity) were similar. Furthermore, RPE cells, human umbilical vein endothelial cells and choroidal melanocytes successfully repopulated the stromas. Physiological structures were established, such as a confluent monolayer of RPE cells, vascular-like structures and a pigmentation of the stroma. Our TECS thus recaptured the biophysical environment of the native choroid, and can serve as study models to understand the normal interactions between the RPE and choroidal cells, as well as their reciprocal exchanges with the ECM. This will consequently pave the way to derive accurate insight in the pathophysiological mechanisms of diseases affecting the choroid. STATEMENT OF SIGNIFICANCE: The choroid is traditionally known for supplying blood to the avascular outer retina. There has been a renewed attention directed towards the choroid partly due to its implication in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Since AMD involves the dysfunction of the choroid/retinal pigment epithelium (RPE) complex, a three-dimensional (3D) model of RPE comprising the choroid layer is warranted. We used human choroidal cells to engineer a choroidal substitute. Our approach takes advantage of the ability of cells to recreate their own environment, without exogenous materials. Our model could help to better understand the role of each choroidal cell type as well as to advance the development of new therapeutics for AMD.

中文翻译：

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组织工程脉络膜的特征。

眼睛的脉络膜是位于视网膜和巩膜之间的带血管的色素结缔组织。越来越多的证据表明，除了向外部视网膜提供营养外，不同的脉络膜细胞特别是通过旁分泌信号传导促进了视网膜的稳态。但是，目前尚不清楚每种细胞类型的确切作用。在这里，我们使用组织工程的自组装方法开发了脉络膜替代品。从人眼银行供体的眼睛中分离出视网膜色素上皮（RPE）细胞，脉络膜基质成纤维细胞，血管内皮细胞和黑素细胞。在含有血清和抗坏血酸的培养基中培养成纤维细胞。六周后，细胞形成了细胞外基质（ECM）片，堆叠起来产生组织工程脉络膜基质（TECS）。然后表征这些基质替代物并将其与天然脉络膜进行比较。它们的ECM成分（胶原蛋白和蛋白聚糖）和生物力学特性（最终抗张强度，应变和弹性）相似。此外，RPE细胞，人脐静脉内皮细胞和脉络膜黑色素细胞成功地重新填充了基质。建立了生理结构，例如融合的RPE细胞单层，血管样结构和基质的色素沉着。因此，我们的TECS重新捕获了天然脉络膜的生物物理环境，并且可以用作研究模型，以了解RPE和脉络膜细胞之间的正常相互作用以及它们与ECM的相互交换。因此，这将为在影响脉络膜的疾病的病理生理机制中获得准确的见解铺平道路。意义声明：脉络膜传统上因向无血管的外部视网膜供应血液而闻名。脉络膜受到了新的关注，部分原因是脉络膜与年龄相关性黄斑变性（AMD）的发展有关，这是工业化国家失明的主要原因。由于AMD涉及脉络膜/视网膜色素上皮（RPE）复合体的功能障碍，因此需要一个包含脉络膜层的RPE的三维（3D）模型。我们使用了人类脉络膜细胞来设计脉络膜替代物。我们的方法利用了细胞在没有外来物质的情况下重建自身环境的能力。