Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
Department of Surgery, Medical University of South Carolina, Charleston, SC, USA.
Methods Mol Biol. 2021;2367:235-247. doi: 10.1007/7651_2020_314.
With the realization that mechanical forces mediate many biological processes and contribute to disease progression, researchers are focusing on developing new methods to understand the role of mechanotransduction in biological systems. Despite recent advances in stretching devices that analyze the effects of mechanical strain in vitro, there are still possibilities to develop new equipment. For example, many of these devices tend be expensive, whereas few have been designed to assess the effects of mechanical strain driven by the extracellular matrix (ECM) to epithelial cell monolayers and to cell-cell adhesion. In this chapter, we introduce a cost-efficient, user-friendly, 3D-printed stretching device that can be used to test the effects of mechanical strain on cultured epithelial cells. Evaluation of the device using speckle-tracking shows homogeneous strain distribution along the horizontal plane of membranes at 2.5% and 5% strains, supporting the reliability of the device. Since cell-cell junctions are mechanosensitive protein complexes, we hereby used this device to examine effects on cell-cell adhesion. For this, we used colon epithelial Caco2 cell monolayers that well-differentiate in culture and form mature adherens junctions. Subjecting Caco2 cells to 2.5% and 5% strain using our device resulted in significant reduction in the localization of the core adherens junction component E-cadherin at areas of cell-cell contact and its increased translocation to the cytoplasm, which in agreement with other methodologies showing that increased ECM-driven strain negatively affects cell-cell adhesion. In summary, we here present a new, cost-effective, homemade device that can be reliably used to examine effects of mechanical strain on epithelial cell monolayers and cell-cell adhesion, in vitro.
随着人们意识到机械力介导许多生物过程并促进疾病进展，研究人员正专注于开发新方法来理解机械转导在生物系统中的作用。尽管在体外分析机械应变影响的拉伸设备方面最近取得了进展，但仍有可能开发新设备。例如，这些设备中的许多都很昂贵，而很少有设备被设计用来评估由细胞外基质（ECM）驱动的机械应变对上皮细胞单层和细胞-细胞黏附的影响。在本章中，我们介绍了一种成本效益高、用户友好的 3D 打印拉伸设备，可用于测试机械应变对培养上皮细胞的影响。使用散斑跟踪对该设备进行评估表明，在 2.5%和 5%的应变下，膜的水平平面上的应变分布均匀，这支持了该设备的可靠性。由于细胞-细胞连接是机械敏感的蛋白质复合物，我们在此使用该设备来研究对细胞-细胞黏附的影响。为此，我们使用结肠上皮 Caco2 细胞单层，这些细胞在培养中很好地分化并形成成熟的黏附连接。使用我们的设备使 Caco2 细胞承受 2.5%和 5%的应变会导致细胞-细胞接触区域核心黏附连接成分 E-钙黏蛋白的定位显著减少，并且其向细胞质的易位增加，这与其他方法学一致，表明 ECM 驱动的应变增加会负性影响细胞-细胞黏附。总之，我们在此提出了一种新的、具有成本效益的自制设备，可可靠地用于研究机械应变对上皮细胞单层和细胞-细胞黏附的影响，在体外。
