Materials and methods in quantifying cell mechanobiology

时空机械底物和力显微镜在量化细胞收缩和粘弹性方面取得了进展。 在过去的几十年里，生物系统的力和机械特性已经被认为是生命的所有尺度上的重要组成部分。特别是，弹性细胞培养底物的出现催化了细胞生物学的复兴，揭开了生物科学中一个以前未被认识到的维度。理解驱动生物过程的物理机制是21世纪的一个关键挑战，将通过一个全新的力和力学角度来解决生物系统，然而，量化这些相互作用的材料和方法是必不可少的。 大多数真核细胞的活性特性的核心是它们是可收缩的，不断施加动态力，拉动邻近的细胞及其底物。通过在可变形的基质上培养细胞，研究人员使用牵引力显微镜(TFM)来表征细胞的刚性依赖的收缩能力，以及这种收缩能力对从分化、增殖到癌症转移的各种生物学过程的影响。然而，底物稳定性、孔隙率、产量和技术复杂性的瓶颈阻碍了这些基本指标在更广泛的生命科学应用中的采用。我的实验室已经生产出了机械可调的硅胶衬底，用于在不同的生理和病理环境中高通量测量收缩力，而NSERC DG的支持在这些进展中起到了关键作用。 这项DG建议的重点是通过开发(1)量化细胞力学的新材料和方法来扩展这一前沿，目标是将这些应用于(2)量化细胞粘弹性。为此，我们将创造基于硅胶的细胞基板，a)在空间上弹性地构图；b)在时间上弹性地切换；c)粘合图案化以直接指向单个和集体细胞结构维度；以及d)局部收缩以拉伸细胞并探测其机械响应的水凝胶基板。我的实验室将以我们在TFM方面的专业知识为基础，简化和增强实时细胞收缩和机械性能的量化。我们将选择性地修饰关键的细胞内和细胞间蛋白质，并量化它们对收缩力和细胞粘弹性的贡献。 这个DG项目建立在我的实验室在硅胶衬底力学和细胞力测量方面的专业知识的坚实基础上，并得到了广泛的显微镜和机械表征仪器的良好支持。我的DG计划将推动细胞收缩和粘弹性的基于材料的量化。由于这里提出的创新具有广泛和基本的效用，我预计将在生物物理、定量生物学、材料科学和实验医学方面产生深远影响。

Spatiotemporal mechanical substrates and force microscopy advances to quantify cell contractility and viscoelasticity. Over the past decades, the forces and mechanical properties of biological systems have been recognized as an essential component across all scales of life. In particular, advents in elastic cell culture substrates have catalyzed a renaissance of cell biology, unlocking a previously unrecognized dimension in biological sciences. Understanding the physical mechanisms driving biological processes is a key challenge of the 21st century, and will resolve biological systems through an entirely new perspective of forces and mechanics, however, materials and methods to quantify these interactions are essential. Central to the active properties of most eukaryotic cells is that they are contractile, continuously exerting dynamic forces that pull on neighboring cells and their substrate. By culturing cells on deformable substrates, researchers have employed Traction Force Microscopy (TFM) to characterize the stiffness-dependent contractility of cells and the influence this contractility has on diverse biological processes from differentiation and proliferation to cancer metastasis. Bottlenecks of substrate stability, porosity, throughput, and technical complexity nevertheless have hampered adoption of these essential metrics in broader life-science applications. My lab has produced mechanically tunable silicone substrates for high-throughput contractile force measurements in diverse physiological and pathological contexts, and NSERC DG support has been critical in these advances. This DG proposal is focused on extending this frontier by developing (1) new materials and methodologies to quantify cell mechanics, with the goal of applying these to (2) quantify cell viscoelasticity. To do so, we will create silicone-based cell substrates that are a) elastically patterned in space; b) elastically switchable in time; c) adhesively patterned to direct single and collective cell structure dimensionality; and d) hydrogel substrates that locally contract to stretch cells and probe their mechanical response. My lab will build on our expertise with TFM to both simplify and enhance quantification of real-time cell contractility and mechanical properties. We will selectively modify key intracellular and intercellular proteins and quantify their contributions to contractile forces and cellular viscoelasticity. This DG program is built on a firm foundation of my lab's expertise in silicone substrate mechanics and cell force measurements, and well-supported by extensive microscopy and mechanical-characterization instrumentation. My DG program will drive a materials-based quantification of cell contractility and viscoelasticity. Due to the broad and fundamental utility of the innovations proposed here, I anticipate far-reaching impact in biological physics, quantitative biology, materials science, and experimental medicine.