Wilson Robin E, Denisin Aleksandra K, Dunn Alexander R, Pruitt Beth L
Department of Mechanical Engineering, Stanford University, Stanford, CA USA.
Department of Bioengineering, Stanford University, Stanford, CA USA.
Cell Mol Bioeng. 2020 Aug 20;14(1):1-14. doi: 10.1007/s12195-020-00646-9. eCollection 2021 Feb.
Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization.
We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (/), 3D microwells with 2D adhesive areas at the bottom only (/), and flat 2D gels with 2D patterned adhesive areas (/). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape.
We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment.
Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.
细胞结构和迁移受细胞黏附环境的力学特性和几何形状影响。迄今为止，大多数研究三维环境对细胞影响的实验并未在单细胞水平上控制几何形状，因此难以理解三维环境线索对单个细胞的影响。在此，我们开发了微孔平台来研究二维与三维几何形状对单细胞F-肌动蛋白和细胞核组织的影响。
我们使用微制造技术制造了三种聚丙烯酰胺平台：具有三维黏附环境的三维微孔（/）、仅底部有二维黏附区域的三维微孔（/）以及具有二维图案化黏附区域的平面二维凝胶（/）。我们测量了平台的几何膨胀和杨氏模量。然后，我们在每个平台上培养C2C12成肌细胞，并评估工程化微环境对F-肌动蛋白结构和细胞核形状的影响。
我们通过控制凝胶配方来调整微制造平台的力学特性。交联剂比例强烈影响几何膨胀，而总聚合物含量主要影响杨氏模量。比较这些平台上的细胞时，我们发现对F-肌动蛋白和细胞核结构有显著影响。我们的分析表明，三维/三维环境对于增加肌动蛋白和细胞核高度是必要的。与二维/二维环境相比，三维/二维环境足以增加肌动蛋白排列和细胞核纵横比。
使用我们新颖的聚丙烯酰胺平台，我们能够分离三维限制和黏附环境的影响，发现两者均影响肌动蛋白和细胞核结构。
