Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

Microfluidic devices allow for the production of physiologically relevant cellular microenvironments by including biomimetic hydrogels and generating controlled chemical gradients. During transport, the biomolecules interact in distinct ways with the fibrillar networks: as purely diffusive factors in the soluble fluid or bound to the matrix proteins. These two main mechanisms may regulate distinct cell responses in order to guide their directional migration: caused by the substrate-bound chemoattractant gradient (haptotaxis) or by the gradient established within the soluble fluid (chemotaxis). In this work 3D diffusion experiments, in combination with ELISA assays, are performed using microfluidic platforms in order to quantify the distribution of PDGF-BB and TGF-β1 across collagen and fibrin gels. Furthermore, to gain a deeper understanding of the fundamental processes, the experiments are reproduced by computer simulations based on a reaction-diffusion transport model. This model yields an accurate prediction of the experimental results, confirming that diffusion and binding phenomena are established within the microdevice.

Cell migration through a three-dimensional (3-D) matrix depends strongly on the ability of cells to generate traction forces. To overcome the steric hindrance of the matrix, cells need to generate sufficiently high traction forces but also need to distribute these forces spatially in a migration-promoting way. This unit describes a protocol to measure spatial maps of cell traction forces in 3-D biopolymer networks such as collagen, fibrin, or Matrigel. Traction forces are computed from the relationship between measured force-induced matrix deformations surrounding the cell and the known mechanical properties of the matrix. The method does not rely on knowledge of the cell surface coordinates and takes nonlinear mechanical properties of the matrix into account. © 2017 by John Wiley & Sons, Inc.

Cell migration through the extracellular matrix is governed by the interplay between cell-generated propulsion forces, adhesion forces, and resisting forces arising from the steric hindrance of the matrix. Steric hindrance in turn depends on matrix porosity, matrix deformability, cell size, and cell deformability. In this study, we investigate how cells respond to changes in steric hindrance that arise from altered cell mechanical properties. Specifically, we measure traction forces, cell morphology, and invasiveness of MDA-MB 231 breast cancer cells in three-dimensional collagen gels. To modulate cell mechanical properties, we either decrease nuclear deformability by twofold overexpression of the nuclear protein lamin A or we introduce into the cells stiff polystyrene beads with a diameter larger than the average matrix pore size. Despite this increase of steric hindrance, we find that cell invasion is only marginally inhibited, as measured by the fraction of motile cells and the mean invasion depth. To compensate for increased steric hindrance, cells employ two alternative strategies. Cells with higher nuclear stiffness increase their force polarity, whereas cells with large beads increase their net contractility. Under both conditions, the collagen matrix surrounding the cells stiffens dramatically and carries increased strain energy, suggesting that increased force polarity and increased net contractility are functionally equivalent strategies for overcoming an increased steric hindrance.

Osteoblast migration is a crucial process in bone regeneration, which is strongly regulated by interstitial fluid flow. However, the exact role that such flow exerts on osteoblast migration is still unclear. To deepen the understanding of this phenomenon, we cultured human osteoblasts on 3D microfluidic devices under different fluid flow regimes. Our results show that a slow fluid flow rate by itself is not able to alter the 3D migratory patterns of osteoblasts in collagen-based gels but that at higher fluid flow rates (increased flow velocity) may indirectly influence cell movement by altering the collagen microstructure. In fact, we observed that high fluid flow rates (1 µl/min) are able to alter the collagen matrix architecture and to indirectly modulate the migration pattern. However, when these collagen scaffolds were crosslinked with a chemical crosslinker, specifically, transglutaminase II, we did not find significant alterations in the scaffold architecture or in osteoblast movement. Therefore, our data suggest that high interstitial fluid flow rates can regulate osteoblast migration by means of modifying the orientation of collagen fibers. Together, these results highlight the crucial role of the matrix architecture in 3D osteoblast migration. In addition, we show that interstitial fluid flow in conjunction with the matrix architecture regulates the osteoblast morphology in 3D.