In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

中文翻译：

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一种高通量微流体方法，用于制造排列整齐的胶原纤维以研究角质细胞行为。

在体内，角膜细胞被排列成薄片的 I 型胶原原纤维包围。越来越多的文献表明，角膜基质的独特地形是角膜细胞行为的重要调节因子。在这项研究中，我们描述了一种将排列整齐的 I 型胶原原纤维沉积到玻璃盖玻片上的微流体方法。这种高通量方法允许同时涂覆多达八个具有排列的胶原原纤维的基底。当这些基质集成到 PDMS 微孔培养系统中时，它们为角膜细胞行为的高分辨率成像提供了一个平台。通过使用宽视场荧光和微分干涉显微镜，我们观察到沉积的胶原原纤维的密度取决于胶原蛋白的灌注剪切速率和灌注时间。相反，在一定的剪切速率范围内观察到类似程度的原纤维排列。当原代正常兔角膜细胞 (NRK) 接种在具有高密度排列胶原纤维的基质上并在血小板衍生生长因子 (PDGF) 存在的情况下培养时，角膜细胞显示出与下面的胶原纤维共同排列的细长细胞体。相比之下，当 NRK 在具有低密度排列的胶原原纤维的基质上培养时，细胞没有显示出优先方向。这些结果表明，这种简单且廉价的方法可以提供一个通用平台来研究同时暴露于地形和可溶性线索如何影响细胞行为。