Department of Biomedical Engineering, University of California Irvine, Irvine, CA, 92697-2730, United States.
Department of Biomedical Engineering, University of California Irvine, Irvine, CA, 92697-2730, United States; Center for Complex Biological Systems, University of California Irvine, Irvine, CA, 92697-2280, United States.
Acta Biomater. 2022 Mar 15;141:39-47. doi: 10.1016/j.actbio.2021.12.028. Epub 2021 Dec 28.
Cells are known to constantly interact with their local extracellular matrix (ECM) and respond to a variety of biochemical and mechanical cues received from the ECM. Nonetheless, comprehensive understanding of cell-ECM interactions has been elusive. Many studies rely on analysis of cell behavior on 2D substrates, which do not reflect a natural cell environment. Further, lack of dynamic control over local stiffness anisotropies and fiber alignment hinders progress in studies in naturally derived fibrous 3D cultures. Here, we present a cell-safe method of patterned photocrosslinking, which can aid in studying biological hypotheses related to mechanotransduction in 3D hydrogels. As previously described by our group, ruthenium-catalyzed photocrosslinking (RCP) of selected ECM regions promotes localized increase in stiffness mediated by focused blue laser light in a confocal microscope. In this study, we further demonstrate that RCP can induce localized strain stiffening and fiber alignment outside of the selected crosslinked region and induce stiffness anisotropy biased towards the direction of fiber alignment. MDA-MB-231 cells are shown to respond to RCP-induced changes in local ECM architecture and display directional bias towards the direction of fiber alignment, as compared to control cells. Further, the effect of patterned crosslinking on a stiffness landscape is measured using multi-axes optical tweezers active microrheology (AMR) with backscattered laser beam illumination. AMR validates RCP as a suitable tool for creating distinct stiffness anisotropies which promote directed migration of cells, further underscoring the usefulness of RCP in cell-ECM studies. STATEMENT OF SIGNIFICANCE: Studies on cell-ECM interactions in 3D cultures have often been hindered by the lack of available tools to dynamically alter local ECM stiffness and fiber alignment. Here, we present a non-invasive, cell-safe and easily applicable method of patterned photocrosslinking, which can aid in studying biological hypotheses in fibrous 3D hydrogels. Ruthenium-catalyzed crosslinking (RCP) of selected fibrin ECM regions promotes localized increase in stiffness and creates distinct stiffness anisotropies in the presence of the focused blue laser light. Outside of the crosslinked region, RCP causes fiber alignment and strain stiffening in the ECM, verified using multi-axes optical tweezers active microrheology (AMR). Following RCP, human breast cancer MDA-MB-231 exhibit directed cell migration, validating usefulness of this method in cell-ECM studies.
细胞不断与局部细胞外基质（ECM）相互作用，并对 ECM 传递的各种生化和机械线索做出反应。尽管如此，对细胞-ECM 相互作用的全面理解仍然难以捉摸。许多研究依赖于对 2D 基质上细胞行为的分析，而这并不能反映自然的细胞环境。此外，缺乏对局部刚度各向异性和纤维排列的动态控制，阻碍了对天然衍生纤维 3D 培养物中研究的进展。在这里，我们提出了一种细胞安全的图案光交联方法，可辅助研究与 3D 水凝胶中的机械转导相关的生物学假设。如我们小组之前所述，所选 ECM 区域的钌催化光交联（RCP）可通过共聚焦显微镜中的聚焦蓝光促进刚度的局部增加。在这项研究中，我们进一步证明 RCP 可以在选定的交联区域之外诱导局部应变硬化和纤维排列，并诱导偏向纤维排列方向的各向异性刚度。与对照细胞相比，MDA-MB-231 细胞对 RCP 诱导的局部 ECM 结构变化做出反应，并表现出朝向纤维排列方向的定向偏差。此外，使用具有背散射激光束照明的多轴光学镊子主动微流变（AMR）测量图案化交联对刚度景观的影响。AMR 验证了 RCP 作为创建独特各向异性刚度的合适工具，可促进细胞的定向迁移，进一步强调了 RCP 在细胞-ECM 研究中的有用性。
在 3D 培养物中进行细胞-ECM 相互作用的研究常常受到缺乏可用工具来动态改变局部 ECM 刚度和纤维排列的限制。在这里，我们提出了一种非侵入性、细胞安全且易于应用的图案光交联方法，可辅助研究纤维状 3D 水凝胶中的生物学假设。选定纤维蛋白 ECM 区域的钌催化交联（RCP）在聚焦蓝光的存在下促进刚度的局部增加，并在存在聚焦蓝光的情况下产生独特的各向异性刚度。在交联区域之外，RCP 导致 ECM 中的纤维排列和应变硬化，这通过多轴光学镊子主动微流变学（AMR）得到验证。在 RCP 之后，人乳腺癌 MDA-MB-231 细胞表现出定向细胞迁移，验证了该方法在细胞-ECM 研究中的有用性。
