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.

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.

Three-dimensionally printed biological machines powered by skeletal muscle.

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼ 156 µm s(-1), which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design.

Necking and failure of constrained 3D microtissues induced by cellular tension.

In this paper we report a fundamental morphological instability of constrained 3D microtissues induced by positive chemomechanical feedback between actomyosin-driven contraction and the mechanical stresses arising from the constraints. Using a 3D model for mechanotransduction we find that perturbations in the shape of contractile tissues grow in an unstable manner leading to formation of "necks" that lead to the failure of the tissue by narrowing and subsequent elongation. The magnitude of the instability is shown to be determined by the level of active contractile strain, the stiffness of the extracellular matrix, and the components of the tissue that act in parallel with the active component and the stiffness of the boundaries that constrain the tissue. A phase diagram that demarcates stable and unstable behavior of 3D tissues as a function of these material parameters is derived. The predictions of our model are verified by analyzing the necking and failure of normal human fibroblast tissue constrained in a loop-ended dog-bone geometry and cardiac microtissues constrained between microcantilevers. By analyzing the time evolution of the morphology of the constrained tissues we have quantitatively determined the chemomechanical coupling parameters that characterize the generation of active stresses in these tissues. More generally, the analytical and numerical methods we have developed provide a quantitative framework to study how contractility can influence tissue morphology in complex 3D environments such as morphogenesis and organogenesis.

Modeling the mechanosensitive collective migration of cells on the surface and the interior of morphing soft tissues.

Cellular contractility, migration, and extracellular matrix (ECM) mechanics are critical for a wide range of biological processes including embryonic development, wound healing, tissue morphogenesis, and regeneration. Even though the distinct response of cells near the tissue periphery has been previously observed in cell-laden microtissues, including faster kinetics and more prominent cell-ECM interactions, there are currently no models that can fully combine coupled surface and bulk mechanics and kinetics to recapitulate the morphogenic response of these constructs. Mailand et al. (Biophys J 117(5):975-986, 2019) had shown the importance of active elastocapillarity in cell-laden microtissues, but modeling the distinct mechanosensitive migration of cells on the periphery and the interior of highly deforming tissues has not been possible thus far, especially in the presence of active elastocapillary effects. This paper presents a framework for understanding the interplay between cellular contractility, migration, and ECM mechanics in dynamically morphing soft tissues accounting for distinct cellular responses in the bulk and the surface of tissues. The major novelty of this approach is that it enables modeling the distinct migratory and contractile response of cells residing on the tissue surface and the bulk, where concurrently the morphing soft tissues undergo large deformations driven by cell contractility. Additionally, the simulation results capture the changes in shape and cell concentration for wounded and intact microtissues, enabling the interpretation of experimental data. The numerical procedure that accounts for mechanosensitive stress generation, large deformations, diffusive migration in the bulk and a distinct mechanism for diffusive migration on deforming surfaces is inspired from recent work on bulk and surface poroelasticity of hydrogels involving elastocapillary effects, but in this work, a two-field weak form is proposed and is able to alleviate numerical instabilities that were observed in the original method that utilized a three-field mixed finite element formulation.

Targeted worker removal reveals a lack of flexibility in brood transport specialisation with no compensatory gain in efficiency.

Division of labour is widely thought to increase the task efficiency of eusocial insects. Workers can switch their task to compensate for sudden changes in demand, providing flexible task allocation. In combination with automated tracking technology, we developed a robotic system to precisely control and spatiotemporally manipulate floor temperature over days, which allowed us to predictably drive brood transport behaviour in colonies of the ant Camponotus floridanus. Our results indicate that a small number of workers, usually minors belonging to the nurse social group, are highly specialised for brood transport. There was no difference in the speed at which workers transported brood, suggesting that specialisation does not correlate with efficiency. Workers often started to transport the brood only after having identified a better location. There was no evidence that workers shared information about the presence of a better location. Notably, once brood transporters had been removed, none of the remaining workers performed this task, and the brood transport completely stopped. When brood transporters were returned to their colony, brood transport was immediately restored. Taken together, our study reveals that brood transport is an inflexible task, achieved through the synchronous actions of a few privately informed specialist workers.

3D Printed Acoustically Programmable Soft Microactuators.

The concept of creating all-mechanical soft microrobotic systems has great potential to address outstanding challenges in biomedical applications, and introduce more sustainable and multifunctional products. To this end, magnetic fields and light have been extensively studied as potential energy sources. On the other hand, coupling the response of materials to pressure waves has been overlooked despite the abundant use of acoustics in nature and engineering solutions. In this study, we show that programmed commands can be contained on 3D nanoprinted polymer systems with the introduction of selectively excited air bubbles and rationally designed compliant mechanisms. A repertoire of micromechanical systems is engineered using experimentally validated computational models that consider the effects of primary and secondary pressure fields on entrapped air bubbles and the surrounding fluid. Coupling the dynamics of bubble oscillators reveals rich acoustofluidic interactions that can be programmed in space and time. We prescribe kinematics by harnessing the forces generated through these interactions to deform structural elements, which can be remotely reconfigured on demand with the incorporation of mechanical switches. These basic actuation and analog control modules will serve as the building blocks for the development of a novel class of micromechanical systems powered and programmed by acoustic signals.

Colloidal self-assembly of soft neural interfaces from injectable photovoltaic microdevices.

Biomimetic retinas with a wide field of view and high resolution are in demand for neuroprosthetics and robot vision. Conventional neural prostheses are manufactured outside the application area and implanted as a complete device using invasive surgery. Here, a minimally invasive strategy based on in situ self-assembly of photovoltaic microdevices (PVMs) is presented. The photoelectricity transduced by PVMs upon visible light illumination reaches the intensity levels that could effectively activate the retinal ganglion cell layers. The geometry and multilayered architecture of the PVMs along with the tunability of their physical properties such as size and stiffness allow several routes for initiating a self-assembly process. The spatial distribution and packing density of the PVMs within the assembled device are modulated through concentration, liquid discharge speed, and coordinated self-assembly steps. Subsequent injection of a photocurable and transparent polymer facilitates tissue integration and reinforces the cohesion of the device. Taken together, the presented methodology introduces three unique features: minimally invasive implantation, personalized visual field and acuity, and a device geometry adaptable to retina topography.

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.

Actuated 3D microgels for single cell mechanobiology.

We present a new cell culture technology for large-scale mechanobiology studies capable of generating and applying optically controlled uniform compression on single cells in 3D. Mesenchymal stem cells (MSCs) are individually encapsulated inside an optically triggered nanoactuator-alginate hybrid biomaterial using microfluidics, and the encapsulating network isotropically compresses the cell upon activation by light. The favorable biomolecular properties of alginate allow cell culture in vitro up to a week. The mechanically active microgels are capable of generating up to 15% compressive strain and forces reaching 400 nN. As a proof of concept, we demonstrate the use of the mechanically active cell culture system in mechanobiology by subjecting singly encapsulated MSCs to optically generated isotropic compression and monitoring changes in intracellular calcium intensity.

Microengineered devices enable long-term imaging of the ventral nerve cord in behaving adult Drosophila.

The dynamics and connectivity of neural circuits continuously change on timescales ranging from milliseconds to an animal's lifetime. Therefore, to understand biological networks, minimally invasive methods are required to repeatedly record them in behaving animals. Here we describe a suite of devices that enable long-term optical recordings of the adult Drosophila melanogaster ventral nerve cord (VNC). These consist of transparent, numbered windows to replace thoracic exoskeleton, compliant implants to displace internal organs, a precision arm to assist implantation, and a hinged stage to repeatedly tether flies. To validate and illustrate our toolkit we (i) show minimal impact on animal behavior and survival, (ii) follow the degradation of chordotonal organ mechanosensory nerve terminals over weeks after leg amputation, and (iii) uncover waves of neural activity caffeine ingestion. Thus, our long-term imaging toolkit opens up the investigation of premotor and motor circuit adaptations in response to injury, drug ingestion, aging, learning, and disease.

Correction: Actuated 3D microgels for single cell mechanobiology.

Correction for 'Actuated 3D microgels for single cell mechanobiology' by Berna Özkale et al., Lab Chip, 2022, 22, 1962-1970, https://doi.org/10.1039/D2LC00203E.

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.

Active biomaterials for mechanobiology.

Active biomaterials offer novel approaches to study mechanotransduction in mammalian cells. These material systems probe cellular responses by dynamically modulating their resistance to endogenous forces or applying exogenous forces on cells in a temporally controlled manner. Stimuli-responsive molecules, polymers, and nanoparticles embedded inside cytocompatible biopolymer networks transduce external signals such as light, heat, chemicals, and magnetic fields into changes in matrix elasticity (few kPa to tens of kPa) or forces (few pN to several µN) at the cell-material interface. The implementation of active biomaterials in mechanobiology has generated scientific knowledge and therapeutic potential relevant to a variety of conditions including but not limited to cancer metastasis, fibrosis, and tissue regeneration. We discuss the repertoire of cellular responses that can be studied using these platforms including receptor signaling as well as downstream events namely, cytoskeletal organization, nuclear shuttling of mechanosensitive transcriptional regulators, cell migration, and differentiation. We highlight recent advances in active biomaterials and comment on their future impact.

Investigating Tissue Mechanics in vitro Using Untethered Soft Robotic Microdevices.

This paper presents the design, fabrication, and operation of a soft robotic compression device that is remotely powered by laser illumination. We combined the rapid and wireless response of hybrid nanomaterials with state-of-the-art microengineering techniques to develop machinery that can apply physiologically relevant mechanical loading. The passive hydrogel structures that constitute the compliant skeleton of the machines were fabricated using single-step in situ polymerization process and directly incorporated around the actuators without further assembly steps. Experimentally validated computational models guided the design of the compression mechanism. We incorporated a cantilever beam to the prototype for life-time monitoring of mechanical properties of cell clusters on optical microscopes. The mechanical and biochemical compatibility of the chosen materials with living cells together with the on-site manufacturing process enable seamless interfacing of soft robotic devices with biological specimen.

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.

Cancer-cell stiffening via cholesterol depletion enhances adoptive T-cell immunotherapy.

Malignant transformation and tumour progression are associated with cancer-cell softening. Yet how the biomechanics of cancer cells affects T-cell-mediated cytotoxicity and thus the outcomes of adoptive T-cell immunotherapies is unknown. Here we show that T-cell-mediated cancer-cell killing is hampered for cortically soft cancer cells, which have plasma membranes enriched in cholesterol, and that cancer-cell stiffening via cholesterol depletion augments T-cell cytotoxicity and enhances the efficacy of adoptive T-cell therapy against solid tumours in mice. We also show that the enhanced cytotoxicity against stiffened cancer cells is mediated by augmented T-cell forces arising from an increased accumulation of filamentous actin at the immunological synapse, and that cancer-cell stiffening has negligible influence on: T-cell-receptor signalling, production of cytolytic proteins such as granzyme B, secretion of interferon gamma and tumour necrosis factor alpha, and Fas-receptor-Fas-ligand interactions. Our findings reveal a mechanical immune checkpoint that could be targeted therapeutically to improve the effectiveness of cancer immunotherapies.

Immune evasion by designer microrobots.

Recent work is unveiling the interactions between magnetic microswimmers and cells of the immune system.

Addressable Acoustic Actuation of 3D Printed Soft Robotic Microsystems.

A design, manufacturing, and control methodology is presented for the transduction of ultrasound into frequency-selective actuation of multibody hydrogel mechanical systems. The modular design of compliant mechanisms is compatible with direct laser writing and the multiple degrees of freedom actuation scheme does not require incorporation of any specific material such as air bubbles. These features pave the way for the development of active scaffolds and soft robotic microsystems from biomaterials with tailored performance and functionality. Finite element analysis and computational fluid dynamics are used to quantitatively predict the performance of acoustically powered hydrogels immersed in fluid and guide the design process. The outcome is the remotely controlled operation of a repertoire of untethered biomanipulation tools including monolithic compound micromachinery with multiple pumps connected to various functional devices. The potential of the presented technology for minimally invasive diagnosis and targeted therapy is demonstrated by a soft microrobot that can on-demand collect, encapsulate, and process microscopic samples.