Design and Construction of an Equibiaxial Cell Stretching System That Is Improved for Biochemical Analysis

We describe the design and validation of an equibiaxial stretching device in which cells are confined to regions of homogeneous strain. Using this device, we seek to overcome a significant limitation of existing equibiaxial stretching devices, in which strains are not homogeneous over the entire region of cell culture. We cast PDMS in a mold to produce a membrane with a cylindrical wall incorporated in the center, which was used to confine cell monolayers to the central membrane region subjected to homogeneous equibiaxial strain. We demonstrated that the presence of the wall to hold the culture medium did not affect strain homogeneity over the majority of the culture surface and also showed that cells adhered well onto the PDMS membranes. We used our device in cyclic strain experiments and demonstrated strain-dependent changes in extracellular signal-regulated kinase (ERK) and tyrosine phosphorylation upon cell stretching. Furthermore, we examined cell responses to very small magnitudes of strain ranging from 1% to 6% and were able to observe a graduated increase in ERK phosphorylation in response to these strains. Collectively, we were able to study cellular biochemical response with a high degree of accuracy and sensitivity to fine changes in substrate strain. Because we have designed our device along the lines of existing equibiaxial stretching technologies, we believe that our innovations can be incorporated into existing systems. This device would provide a useful addition to the set of tools applied for in vitro studies of cell mechanobiology.

本研究详述了一款双轴拉伸装置的设计与验证流程，该装置可将细胞限制于均匀应变区域内。借助该装置，我们旨在克服现有双轴拉伸装置的显著缺陷：现有装置无法在细胞培养的全域实现均匀应变。我们通过模具浇铸聚二甲基硅氧烷（PDMS）制备得到一种膜结构，其中心集成有圆柱形侧壁，用于将单层细胞限制于承受均匀双轴应变的中央膜区域内。实验证实，用于盛放培养基的侧壁不会在绝大多数培养表面上影响应变均匀性，同时还证明细胞可在PDMS膜上良好黏附。我们将本装置应用于循环应变实验，证实细胞受拉伸后，细胞外调节蛋白激酶（ERK）及酪氨酸磷酸化水平会随应变发生依赖性变化。此外，我们还探究了细胞对1%至6%极低应变幅度的响应，成功观测到ERK磷酸化水平随应变提升呈现梯度递增的现象。综上，本装置可高精度、高灵敏度地研究细胞生化响应，并能精准捕捉基底应变的细微变化。由于本装置的设计遵循现有双轴拉伸技术的框架，我们认为其创新点可集成至现有系统中。该装置可为细胞力学生物学体外研究所用的工具库提供极具价值的补充。