Characterization of Partially Lifted Cell Sheets

Abstract

There is a need to characterize biomechanical cell–cell interactions, but due to a lack of suitable experimental methods, relevant in vitro experimental data are often masked by cell–substrate interactions. This study describes a novel method to generate partially lifted substrate-free cell sheets that engage primarily in cell–cell interactions, yet are amenable to biological and chemical perturbations and, importantly, mechanical conditioning and characterization. A polydimethylsiloxane (PDMS) mold is used to isolate a patch of cells, and the patch is then enzymatically lifted. The cells outside the mold remain attached, creating a partially lifted cell sheet. This simple yet powerful tool enables the simultaneous examination of lifted and adherent cells. This tool was then deployed to test the hypothesis that the lifted cells would exhibit substantial reinforcement of key cytoskeletal and junctional components at cell–cell contacts, and that such reinforcement would be enhanced by mechanical conditioning. Results demonstrate that the mechanical strength and cohesion of the substrate-free cell sheets strongly depend on the integrity of the actomyosin cytoskeleton and the cell–cell junctional protein plakoglobin. Both actin and plakoglobin are significantly reinforced at junctions with mechanical conditioning. However, total cellular actin is significantly diminished on dissociation from a substrate and does not recover with mechanical conditioning. These results represent a first systematic examination of mechanical conditioning on cells with primarily intercellular interactions.