Reimer Michael, Petrova Zustiak Silviya, Sheth Saahil, Martin Schober Joseph
Department of Pharmaceutical Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, USA.
Department of Biomedical Engineering, Saint Louis University, St Louis, MO, USA.
Cell Biochem Biophys. 2018 Jun;76(1-2):197-208. doi: 10.1007/s12013-017-0834-1. Epub 2017 Oct 24.
In the continuous search for better tissue engineering scaffolds it has become increasingly clear that the substrate properties dramatically affect cell responses. Here we compared cells from a physiologically stiff tissue, melanoma, to cells isolated from a physiologically soft tissue, brain. We measured the cell line responses to laminin immobilized onto glass or polyacrylamide hydrogels tuned to have a Young's modulus ranging from 1 to 390 kPa. Single cells were analyzed for spreading area, shape, total actin content, actin-based morphological features and modification of immobilized laminin. Both cell types exhibited stiffness- and laminin concentration-dependent responses on polyacrylamide and glass. Melanoma cells exhibited very little spreading and were rounded on soft (1, 5, and 15 kPa) hydrogels while cells on stiff (40, 100, and 390 kPa) hydrogels were spread and had a polarized cell shape with large lamellipodia. On rigid glass surfaces, spreading and actin-based morphological features were not observed until laminin concentration was much higher. Similarly, increased microglia cell spreading and presence of actin-based structures were observed on stiff hydrogels. However, responses on rigid glass surfaces were much different. Microglia cells had large spreading areas and elongated shapes on glass compared to hydrogels even when immobilized laminin density was consistent on all gels. While cell spreading and shape varied with Young's modulus of the hydrogel, the concentration of f-actin was constant. A decrease in laminin immunofluorescence was associated with melanoma and microglia cell spreading on glass with high coating concentration of laminin, indicating modification of immobilized laminin triggered by supraphysiologic stiffness and high ligand density. These results suggest that some cell lines are more sensitive to mechanical properties matching their native tissue environment while other cell lines may require stiffness and extracellular ligand density well above physiologic tissue before saturation in cell spreading, elongation and cytoskeletal re-organization are reached.
在持续寻找更好的组织工程支架的过程中，越来越清楚的是，基质特性会显著影响细胞反应。在这里，我们将来自生理硬度组织（黑色素瘤）的细胞与从生理柔软组织（脑）分离的细胞进行了比较。我们测量了细胞系对固定在玻璃或聚丙烯酰胺水凝胶上的层粘连蛋白的反应，这些水凝胶的杨氏模量范围为1至390 kPa。对单个细胞的铺展面积、形状、总肌动蛋白含量、基于肌动蛋白的形态特征以及固定层粘连蛋白的修饰进行了分析。两种细胞类型在聚丙烯酰胺和玻璃上均表现出硬度和层粘连蛋白浓度依赖性反应。黑色素瘤细胞在柔软（1、5和15 kPa）水凝胶上铺展极少且呈圆形，而在坚硬（40、100和390 kPa）水凝胶上的细胞则铺展且具有极化的细胞形状，带有大的片状伪足。在刚性玻璃表面，直到层粘连蛋白浓度高得多时才观察到铺展和基于肌动蛋白的形态特征。同样，在坚硬水凝胶上观察到小胶质细胞铺展增加以及基于肌动蛋白结构的存在。然而，在刚性玻璃表面上的反应却大不相同。与水凝胶相比，即使所有凝胶上固定层粘连蛋白的密度一致，小胶质细胞在玻璃上也有大的铺展面积和细长的形状。虽然细胞铺展和形状随水凝胶的杨氏模量而变化，但f - 肌动蛋白的浓度是恒定的。在高浓度层粘连蛋白包被的玻璃上，黑色素瘤和小胶质细胞铺展时层粘连蛋白免疫荧光降低，表明超生理硬度和高配体密度引发了固定层粘连蛋白的修饰。这些结果表明，一些细胞系对与其天然组织环境匹配的机械特性更敏感，而其他细胞系在达到细胞铺展、伸长和细胞骨架重组饱和之前，可能需要远高于生理组织的硬度和细胞外配体密度。
