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A monolayer of highly motile cells can establish long-range orientational order, which can be explained by hydrodynamic theory of active gels and fluids. However, it is less clear how cell shape changes and rearrangement are governed when the monolayer is in mechanical equilibrium states when cell motility diminishes. In this work, we report that rat embryonic fibroblasts (REF), when confined in circular mesoscale patterns on rigid substrates, can transition from the spindle shapes to more compact morphologies. Cells align radially only at the pattern boundary when they are in the mechanical equilibrium. This radial alignment disappears when cell contractility or cell-cell adhesion is reduced. Unlike monolayers of spindle-like cells such as NIH-3T3 fibroblasts with minimal intercellular interactions or epithelial cells like Madin-Darby canine kidney (MDCK) with strong cortical actin network, confined REF monolayers present an actin gradient with isotropic meshwork, suggesting the existence of a stiffness gradient. In addition, the REF cells tend to condense on soft substrates, a collective cell behavior we refer to as the ‘condensation tendency’. This condensation tendency, together with geometrical confinement, induces tensile prestretch (i.e. an isotropic stretch that causes tissue to contract when released) to the confined monolayer. By developing a Voronoi-cell model, we demonstrate that the combined global tissue prestretch and cell stiffness differential between the inner and boundary cells can sufficiently define the cell radial alignment at the pattern boundary. 

Cells are physically contacting with each other. Direct and precise quantification of forces at cell–cell junctions is still challenging. Herein, we have developed a DNA-based ratiometric fluorescent probe, termed DNAMeter, to quantify intercellular tensile forces. These lipid-modified DNAMeters can spontaneously anchor onto live cell membranes. The DNAMeter consists of two self-assembled DNA hairpins of different force tolerance. Once the intercellular tension exceeds the force tolerance to unfold a DNA hairpin, a specific fluorescence signal will be activated, which enables the real-time imaging and quantification of tensile forces. Using E-cadherin-modified DNAMeter as an example, we have demonstrated an approach to quantify, at the molecular level, the magnitude and distribution of E-cadherin tension among epithelial cells. Compatible with readily accessible fluorescence microscopes, these easy-to-use DNA tension probes can be broadly used to quantify mechanotransduction in collective cell behaviors. 

Recently, researchers have been attempting to control pluripotent stem cell fate or generate self-organized tissues from stem cells. Advances in bioengineering enable generation of organotypic structures, which capture the cellular components, spatial cell organization and even some functions of tissues or organs in development. However, only a few engineering tools have been utilized to regulate the formation and organization of spatially complex tissues derived from stem cells. Here, we provide a review of recent progress in the culture of organotypic structures in vitro , focusing on how microengineering approaches including geometric confinement, extracellular matrix (ECM) property modulation, spatially controlled biochemical factors, and external forces, can be utilized to generate organotypic structures. Moreover, we will discuss potential technologies that can be applied to further control both soluble and insoluble factors spatiotemporally in vitro . In summary, advanced engineered approaches have a great promise in generating miniaturized tissues and organs in a reproducible fashion, facilitating the cellular and molecular understanding of embryogenesis and morphogenesis processes.