sulfo-Succinimidyl-6-[(4'-azido-nitrophenyl)amino]hexanoate

Purpose: The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. Methods: A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor β (TGF-β [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. Results: MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-β1 and TGF-β2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. Conclusions: Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments.

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.