Sarker Bapi, Walter Christopher, Pathak Amit
Department of Mechanical Engineering & Materials Science, Washington University, St. Louis, Missouri 63130, United States.
Department of Biomedical Engineering, Washington University, St. Louis, Missouri 63130, United States.
ACS Biomater Sci Eng. 2018 Jul 9;4(7):2340-2349. doi: 10.1021/acsbiomaterials.8b00331. Epub 2018 Jun 6.
Microcontact printing of extracellular matrix (ECM) proteins in defined regions of a substrate allows spatial control over cell attachment and enables the study of cellular response to irregular ECM geometries. Over the past decade, numerous micropatterning techniques have emerged that conjugate ECM proteins on hydrogel substrates of tunable stiffness, which have revealed a range of cellular responses to varying matrix stiffness and geometry. However, micropatterning of ECM proteins on polyacrylamide (PA) hydrogel remains inconsistent due to its unreliable conjugation with the commonly used protein cross-linkers, particularly at low stiffness. To address these problems, we developed a micropatterning technique in which the PA gel is functionalized by incorporating oxidized -hydroxyethylacrylamide, which allows direct protein binding through reactive aldehyde groups without any exogenous cross-linkers. As a result, a uniform and consistent protein transfer onto the hydrogel substrates of defined geometries is achieved, even for soft PA gels. We formed square, rectangular, and triangular patterns of two constant areas on soft and stiff PA gels that provide large and small adhesive areas for the MCF10A human mammary epithelial cell pairs. We measured intercellular E-cadherin (E-cad) expression and found that cell-cell junctions could be deteriorated independently by either the stiff ECM of any shape or the elongated cell morphology, accompanied by increased cell-generated tractions, on rectangular soft ECM patterns. Inhibition of nonmuscle myosin II reduced the E-cad junctional localization in cell pairs. When the cell spreading was restricted by reducing the adhesive area of the patterns, we observed an overall rise in E-cad expression at cell-cell junctions. Our findings present an improved micropatterning technique which reveals a geometric regulation of cell-cell junctions in epithelial cell pairs.
在基质的特定区域进行细胞外基质（ECM）蛋白的微接触印刷，能够对细胞黏附进行空间控制，并有助于研究细胞对不规则ECM几何形状的反应。在过去十年中，出现了许多微图案化技术，这些技术将ECM蛋白结合到具有可调刚度的水凝胶基质上，揭示了细胞对不同基质刚度和几何形状的一系列反应。然而，由于ECM蛋白在聚丙烯酰胺（PA）水凝胶上的微图案化与常用的蛋白质交联剂的结合不可靠，尤其是在低刚度时，其结果仍然不一致。为了解决这些问题，我们开发了一种微图案化技术，其中通过掺入氧化的羟乙基丙烯酰胺使PA凝胶功能化，这允许通过反应性醛基直接结合蛋白质而无需任何外源交联剂。结果，即使对于柔软的PA凝胶，也能实现将蛋白质均匀且一致地转移到具有特定几何形状的水凝胶基质上。我们在柔软和坚硬的PA凝胶上形成了两个恒定面积的正方形、长方形和三角形图案，为MCF10A人乳腺上皮细胞对提供了大小不同的黏附区域。我们测量了细胞间E-钙黏蛋白（E-cad）的表达，发现无论是任何形状的坚硬ECM还是长方形柔软ECM图案上拉长的细胞形态，都能独立地破坏细胞间连接，同时细胞产生的牵引力增加。抑制非肌肉肌球蛋白II可减少细胞对中E-cad连接的定位。当通过减小图案的黏附面积来限制细胞铺展时，我们观察到细胞间连接处E-cad表达总体上升。我们的研究结果提出了一种改进的微图案化技术，该技术揭示了上皮细胞对中细胞间连接的几何调控。
