Ali M Yakut, Chuang Chih-Yuan, Saif M Taher A
Department of Mechanical Science and Engineering, College of Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA61801.
Soft Matter. 2014 Nov 28;10(44):8829-37. doi: 10.1039/c4sm01602e.
A variety of cell types exhibit phenotype changes in response to the mechanical stiffness of the substrate. Many cells excluding neurons display an increase in the spread area, actin stress fiber formation and larger focal adhesion complexes as substrate stiffness increases in a sparsely populated culture. Cell proliferation is also known to directly correlate with these phenotype changes/changes in substrate stiffness. Augmented spreading and proliferation on stiffer substrates require nuclear transcriptional regulator YAP (Yes associated protein) localization in the cell nucleus and is tightly coupled to larger traction force generation. In this study, we show that different types of fibroblasts can exhibit spread morphology, well defined actin stress fibers, and larger focal adhesions even on very soft collagen gels (modulus in hundreds of Pascals) as if they are on hard glass substrates (modulus in GPa, several orders of magnitude higher). Strikingly, we show, for the first time, that augmented spreading and other hard substrate cytoskeleton architectures on soft collagen gels are not correlated with the cell proliferation pattern and do not require YAP localization in the cell nucleus. Finally, we examine the response of human colon carcinoma (HCT-8) cells on soft collagen gels. Recent studies show that human colon carcinoma (HCT-8) cells form multicellular clusters by 2-3 days when cultured on soft polyacrylamide (PA) gels with a wide range of stiffness (0.5-50 kPa) and coated with an extracellular matrix, ECM (collagen monomer/fibronectin). These clusters show limited spreading/wetting on PA gels, form 3D structures at the edges, and eventually display a remarkable, dissociative metastasis like phenotype (MLP), i.e., epithelial to rounded morphological transition after a week of culture on PA gels only, but not on collagen monomer coated stiff polystyrene/glass where they exhibit enhanced wetting and form confluent monolayers. Here, we show that HCT-8 cell clusters also show augmented spreading/wetting on soft collagen gels and eventually form confluent monolayers as on rigid glass substrates and MLP is completely inhibited on soft collagen gels. Overall, these results suggest that cell-material interactions (soft collagen gels in this case) can induce cellular phenotype and cytoskeleton organization in a remarkably distinct manner compared to a classical synthetic polyacrylamide (PA) hydrogel cell culture model and may contribute in designing new functional biomaterials.
多种细胞类型会因底物的机械硬度而表现出表型变化。在稀疏培养中，随着底物硬度增加，除神经元外的许多细胞会出现铺展面积增大、肌动蛋白应力纤维形成以及更大的粘着斑复合物。细胞增殖也与这些表型变化/底物硬度变化直接相关。在更硬的底物上增强铺展和增殖需要核转录调节因子YAP（Yes相关蛋白）定位于细胞核，并且与更大的牵引力产生紧密相关。在本研究中，我们表明，即使在非常柔软的胶原蛋白凝胶（模量为数百帕斯卡）上，不同类型的成纤维细胞也能呈现铺展形态、清晰的肌动蛋白应力纤维和更大的粘着斑，就好像它们在坚硬的玻璃底物（模量为吉帕斯卡，高出几个数量级）上一样。令人惊讶的是，我们首次表明，在柔软的胶原蛋白凝胶上增强的铺展和其他硬底物细胞骨架结构与细胞增殖模式无关，并且不需要YAP定位于细胞核。最后，我们研究了人结肠癌细胞（HCT - 8）在柔软胶原蛋白凝胶上的反应。最近的研究表明，人结肠癌细胞（HCT - 8）在涂有细胞外基质ECM（胶原单体/纤连蛋白）的具有广泛硬度（0.5 - 50千帕）的柔软聚丙烯酰胺（PA）凝胶上培养时，2 - 3天内会形成多细胞簇。这些簇在PA凝胶上显示出有限的铺展/浸润，在边缘形成三维结构，并最终呈现出显著的、解离性转移样表型（MLP），即仅在PA凝胶上培养一周后从上皮形态转变为圆形形态，但在涂有胶原单体的刚性聚苯乙烯/玻璃上不会出现这种情况，在那里它们表现出增强的浸润并形成汇合的单层。在这里，我们表明HCT - 8细胞簇在柔软的胶原蛋白凝胶上也显示出增强的铺展/浸润，并最终像在刚性玻璃底物上一样形成汇合的单层，并且在柔软的胶原蛋白凝胶上MLP被完全抑制。总体而言，这些结果表明，与经典的合成聚丙烯酰胺（PA）水凝胶细胞培养模型相比，细胞 - 材料相互作用（在这种情况下是柔软的胶原蛋白凝胶）可以以一种非常独特的方式诱导细胞表型和细胞骨架组织，并且可能有助于设计新的功能性生物材料。
