This simple yet powerful tool enables us to investigate the effects of mechanical conditioning on cell sheet properties and permits a direct comparison of physiological parameters between lifted cells and their adherent controls, side by side. 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 cellCcell 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 U 73122 actomyosin cytoskeleton and the cellCcell 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. Introduction The recent development of cell-sheet tissue engineering has generated a need for a systematic characterization of cellCcell interactions in cell sheets to better mimic and condition them for applications. Rather than using conventional three-dimensional scaffolds for tissue reconstruction, an approach using thermo-responsive polymeric surfaces that facilitate the noninvasive harvest of cultured cells as intact tissue sheets was developed.1 Such cell sheets have been generated for a wide variety of laminar tissues, such as skin, heart, corneal, and renal components.2C5 In addition, cell sheet tissue engineering bears a striking resemblance to the embryonic cell sheet building machinery. In early development, embryonic morphogenesis results largely from deformation of analogs of cell sheets, via internally generated forces. 6 As a naturally existing cell sheet, the blastoderm consists of a layer of cells that are enclosed in a fluid-filled blastocoel cavity, lacking extracellular matrix (ECM) support.7 The rearrangement and deformation of the cell layer in blastoderms and later in blastopores involves a series of precisely orchestrated morphogenetic episodes.8,9 The parallels between tissue engineering and tissue morphogenesis suggest that force homeostasis across cellCcell junctions not only govern blastoderm and blastopore formation, but also may play crucial roles in regulating mechanical strength of the cell sheet constructs for tissue engineering purposes. Currently, cell sheets are fragile and are U 73122 typically handled by external supports.10 Direct experimental methods for understanding and improving the sheets’ biomechanical properties, such as cellCcell adhesion, mechanotransduction, and other baseline cellular properties, are essential for further development of these sheet constructs. However, comprehensive experimental data are still lacking due to lack of suitable experimental methods. First, research in cell sheet engineering primarily focuses on biological or chemical cues; Rabbit polyclonal to PAI-3 comparatively little is known about mechanical cues. In particular, how mechanical U 73122 cues may regulate, or be controlled by, the cytoskeleton remains incompletely resolved. Since components such as actin are responsible for certain mechanoresponses as well as for cell processes such as migration, contraction, and adhesion, it is imperative that their part be examined in more detail.11C17 Second, most studies are done in adherent cells that may primarily U 73122 maintain cellCsubstrate relationships and, as a result, they likely introduce mixed reactions into the readouts. Thus, the functions of important junctional proteins in desmosomes, adherens junctions, and so on are not well characterized. However, recent studies possess shown that such junctional proteins regulate a variety of processes such as viability and migration.18C20 Third, most cell sheets are generated for immediate use and not conditioningwithout supporting scaffolds, these sheets are too fragile to endure handling or significant manipulation. A recent study on characterizing the mechanics of cultured cell monolayers offers begun dropping light on this topic.21 By culturing cells on a sacrificial collagen scaffold between test rods, and subsequently dissolving away the scaffold, the investigators provided a novel method to measure monolayer elasticity and greatest strength. Despite this superb work, our knowledge of underlying mechanisms.