Mechano-Stimulation of Fibroblasts by Adjusting Viscous Drag of Mobile Cell Linkers in Biomembrane-Mimicking Substrates

Abstract

Recent advancements in the design of polymeric substrates of tunable rigidity have shown that cells probe the viscoelasticity of their environment through an adaptive process of focal contact assembly/disassembly that critically affects cell adhesion and morphology. However, the specific mechanisms of this process of mechano-sensitivity have not yet been fully uncovered, in part due to the limitations of existing engineered cell substrates, which are characterized by immobilized cell linkers. To overcome this limitation, we here present a biomembrane-mimicking cell substrate based on polymer-tethered lipid muli-bilayers, in which laterally mobile cell linkers enable the free assembly and disassembly of focal adhesions. In this experimental platform, the mechano-stimulation of plated cells is accomplished by altering the viscous drag of cell linkers through the number of lipid bilayers in the solid-supported multi-bilayer stack. Results from microscopy experiments are discussed, which illustrate that the number of bilayers in the multi-bilayer stack has a profound impact on various cellular properties of plated 3T3 fibroblasts, including adhesion, morphology, shape fluctuations, migration, and cytoskeletal organization. Furthermore, this biomembrane-mimicking substrate is integrated into a force traction microscopy assay, which confirms that the presence of the fluid multi-bilayer system leads to a notable reduction in cellular traction forces. Our experiments illustrate that the described biomembrane-mimicking cell substrate is particularly well suited to monitor plated cells under conditions of weak force transduction conditions between cells and underlying substrate.