Introduction

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.

Methods

We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). 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.

Results

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.

Conclusions

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.

中文翻译：

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用于控制单细胞 3D 几何和细胞内组织的 3D 微孔平台

介绍

细胞结构和迁移受细胞粘附环境的机械性能和几何形状的影响。迄今为止，大多数研究 3D 环境对细胞的影响的研究都没有控制单细胞水平的几何形状，因此很难理解 3D 环境线索对单细胞的影响。在这里，我们开发了微孔平台来研究 2D 与 3D 几何形状对单细胞 F-肌动蛋白和核组织的影响。

方法

我们使用微细加工技术制造了三个聚丙烯酰胺平台：具有 3D 粘合环境的 3D 微孔（3D / 3D）、仅在底部具有 2D 粘合区域的 3D 微孔（3D / 2D）和带有 2D 图案粘合区域的扁平 2D 凝胶（2D / 2D）。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，并评估工程微环境对 F-肌动蛋白结构和核形状的影响。

结果

我们通过操纵凝胶配方来调整微制造平台的机械特性。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。在比较这些平台中的细胞时，我们发现对 F-肌动蛋白和核结构有显着影响。我们的分析表明，增加肌动蛋白和核高度需要 3D/3D 环境。与 2D/2D 环境相比，3D/2D 环境足以增加肌动蛋白排列和核纵横比。

结论

使用我们的新型聚丙烯酰胺平台，我们能够解耦 3D 限制和粘附环境的影响，发现它们都会影响肌动蛋白和核结构。