Deconstructing body axis morphogenesis in zebrafish embryos using robot-assisted tissue micromanipulation.

Classic microsurgical techniques, such as those used in the early 1900s by Mangold and Spemann, have been instrumental in advancing our understanding of embryonic development. However, these techniques are highly specialized, leading to issues of inter-operator variability. Here we introduce a user-friendly robotic microsurgery platform that allows precise mechanical manipulation of soft tissues in zebrafish embryos. Using our platform, we reproducibly targeted precise regions of tail explants, and quantified the response in real-time by following notochord and presomitic mesoderm (PSM) morphogenesis and segmentation clock dynamics during vertebrate anteroposterior axis elongation. We find an extension force generated through the posterior notochord that is strong enough to buckle the structure. Our data suggest that this force generates a unidirectional notochord extension towards the tailbud because PSM tissue around the posterior notochord does not let it slide anteriorly. These results complement existing biomechanical models of axis elongation, revealing a critical coupling between the posterior notochord, the tailbud, and the PSM, and show that somite patterning is robust against structural perturbations.

Tissue Engineering with Mechanically Induced Solid-Fluid Transitions.

Epithelia are contiguous sheets of cells that stabilize the shape of internal organs and support their structure by covering their surfaces. They acquire diverse morphological forms appropriate for their specific functions during embryonic development, such as the kidney tubules and the complex branching structures found in the lung. The maintenance of epithelial morphogenesis and homeostasis is controlled by their remarkable mechanics-epithelia can become elastic, plastic, and viscous by actively remodeling cell-cell junctions and modulating the distribution of local stresses. Microfabrication, finite element modelling, light-sheet microscopy, and robotic micromanipulation are used to show that collagen gels covered with an epithelial skin serve as shape-programmable soft matter. The process involves solid to fluid transitions induced by mechanical perturbations, generates spatially distributed surface stresses at tissue interfaces, and is amenable to both additive and subtractive manufacturing techniques. The robustness and versatility of this strategy for engineering designer tissues is demonstrated by directing the morphogenesis of a variety of molded, carved, and assembled forms from the base material. The results provide insight into the active mechanical properties of the epithelia and establish methods for engineering tissues with sustainable architectures.

Engineered Extracellular Matrices with Integrated Wireless Microactuators to Study Mechanobiology.

Mechanobiology explores how forces regulate cell behaviors and what molecular machinery are responsible for the sensing, transduction, and modulation of mechanical cues. To this end, probing of cells cultured on planar substrates has served as a primary experimental setting for many decades. However, native extracellular matrices (ECMs) consist of fibrous protein assemblies where the physical properties spanning from the individual fiber to the network architecture can influence the transmission of forces to and from the cells. Here, a robotic manipulation platform that allows wireless, localized, and programmable deformation of an engineered fibrous ECM is introduced. A finite-element-based digital twin of the fiber network calibrated against measured local and global parameters enables the calculation of deformations and stresses generated by different magnetic actuation schemes across a range of network properties. Physiologically relevant mechanical forces are applied to cells cultured on the fiber network, statically or dynamically, revealing insights into the effects of matrix-borne forces and deformations as well as force-mediated matrix remodeling on cell migration and intracellular signaling. These capabilities are not matched by any existing approach, and this versatile platform has the potential to uncover fundamental mechanisms of mechanobiology in settings with greater relevance to living tissues.

A model for 3D deformation and reconstruction of contractile microtissues.

Tissue morphogenesis and regeneration are essentially mechanical processes that involve coordination of cellular forces, production and structural remodeling of extracellular matrix (ECM), and cell migration. Discovering the principles of cell-ECM interactions and tissue-scale deformation in mechanically-loaded tissues is instrumental to the development of novel regenerative therapies. The combination of high-throughput three-dimensional (3D) culture systems and experimentally-validated computational models accelerate the study of these principles. In our previous work [E. Mailand, et al., Biophys. J., 2019, 117, 975-986], we showed that prominent surface stresses emerge in constrained fibroblast-populated collagen gels, driving the morphogenesis of fibrous microtissues. Here, we introduce an active material model that allows the embodiment of surface and bulk contractile stresses while maintaining the passive elasticity of the ECM in a 3D setting. Unlike existing models, the stresses are driven by mechanosensing and not by an externally applied signal. The mechanosensing component is incorporated in the model through a direct coupling of the local deformation state with the associated contractile force generation. Further, we propose a finite element implementation to account for large deformations, nonlinear active material response, and surface effects. Simulation results quantitatively capture complex shape changes during tissue formation and as a response to surgical disruption of tissue boundaries, allowing precise calibration of the parameters of the 3D model. The results of this study imply that the organization of the extracellular matrix in the bulk of the tissue may not be a major factor behind the morphogenesis of fibrous tissues at sub-millimeter length scales.

Complete inclusion of bioactive molecules and particles in polydimethylsiloxane: a straightforward process under mild conditions.

By applying a slow curing process, we show that biomolecules can be incorporated via a simple process as liquid stable phases inside a polydimethylsiloxane (PDMS) matrix. The process is carried out under mild conditions with regards to temperature, pH and relative humidity, and is thus suitable for application to biological entities. Fluorescence and enzymatic activity measurements show that the biochemical properties of the proteins and enzyme tested are preserved, without loss due to adsorption at the liquid-polymer interface. Protected from external stimuli by the PDMS matrix, these soft liquid composite materials are new tools of interest for robotics, microfluidics, diagnostics and chemical microreactors.

Surface and Bulk Stresses Drive Morphological Changes in Fibrous Microtissues.

Engineered fibrous tissues consisting of cells encapsulated within collagen gels are widely used three-dimensional in vitro models of morphogenesis and wound healing. Although cell-mediated matrix remodeling that occurs within these scaffolds has been extensively studied, less is known about the mesoscale physical principles governing the dynamics of tissue shape. Here, we show both experimentally and by using computer simulations how surface contraction through the development of surface stresses (analogous to surface tension in fluids) coordinates with bulk contraction to drive shape evolution in constrained three-dimensional microtissues. We used microelectromechanical systems technology to generate arrays of fibrous microtissues and robot-assisted microsurgery to perform local incisions and implantation. We introduce a technique based on phototoxic activation of a small molecule to selectively kill cells in a spatially controlled manner. The model simulations, which reproduced the experimentally observed shape changes after surgical and photochemical operations, indicate that fitting of only bulk and surface contractile moduli is sufficient for the prediction of the equilibrium shape of the microtissues. The computational and experimental methods we have developed provide a general framework to study and predict the morphogenic states of contractile fibrous tissues under external loading at multiple length scales.

Universal Soft Robotic Microgripper.

Here, a soft robotic microgripper is presented that consists of a smart actuated microgel connected to a spatially photopatterned multifunctional base. When pressed onto a target object, the microgel component conforms to its shape, thus providing a simple and adaptive solution for versatile micromanipulation. Without the need for active visual or force feedback, objects of widely varying mechanical and surface properties are reliably gripped through a combination of geometrical interlocking mechanisms instantiated by reversible shape-memory and thermal responsive swelling of the microgel. The gripper applies holding forces exceeding 400 µN, which is high enough to lift loads 1000 times heavier than the microgel. An untethered version of the gripper is developed by remotely controlling the position using magnetic actuation and the contractile state of the microgel using plasmonic absorption. Gentle yet stable robotic manipulation of biological samples under physiological conditions opens up possibilities for high-throughput interrogation and minimally invasive interventions.

Synthetic Biology Platform for Sensing and Integrating Endogenous Transcriptional Inputs in Mammalian Cells.

One of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators.

Transplantation of prokaryotic two-component signaling pathways into mammalian cells.

Signaling pathway engineering is a promising route toward synthetic biological circuits. Histidine-aspartate phosphorelays are thought to have evolved in prokaryotes where they form the basis for two-component signaling. Tyrosine-serine-threonine phosphorelays, exemplified by MAP kinase cascades, are predominant in eukaryotes. Recently, a prokaryotic two-component pathway was implemented in a plant species to sense environmental trinitrotoluene. We reasoned that "transplantation" of two-component pathways into mammalian host could provide an orthogonal and diverse toolkit for a variety of signal processing tasks. Here we report that two-component pathways could be partially reconstituted in mammalian cell culture and used for programmable control of gene expression. To enable this reconstitution, coding sequences of histidine kinase (HK) and response regulator (RR) components were codon-optimized for human cells, whereas the RRs were fused with a transactivation domain. Responsive promoters were furnished by fusing DNA binding sites in front of a minimal promoter. We found that coexpression of HKs and their cognate RRs in cultured mammalian cells is necessary and sufficient to strongly induce gene expression even in the absence of pathways' chemical triggers in the medium. Both loss-of-function and constitutive mutants behaved as expected. We further used the two-component signaling pathways to implement two-input logical AND, NOR, and OR gene regulation. Thus, two-component systems can be applied in different capacities in mammalian cells and their components can be used for large-scale synthetic gene circuits.