Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts.

Cells cultured on stiff 2D substrates exert high intracellular force, resulting in mechanical deformation of their nuclei. This nuclear deformation (ND) plays a crucial role in the transport of Yes Associated Protein (YAP) from the cytoplasm to the nucleus. However, cells in vivo are in soft 3D environment with potentially much lower intracellular forces. Whether and how cells may deform their nuclei in 3D for YAP localization remains unclear. Here, by culturing human colon cancer associated fibroblasts (CAFs) on 2D, 2.5D, and 3D substrates, we differentiated the effects of stiffness, force, and ND on YAP localization. We found that nuclear translocation of YAP depends on the degree of ND irrespective of dimensionality, stiffness and total force. ND induced by the perinuclear force, not the total force, and nuclear membrane curvature correlate strongly with YAP activation. Immunostained slices of human tumors further supported the association between ND and YAP nuclear localization, suggesting ND as a potential biomarker for YAP activation in tumors. Additionally, we conducted quantitative analysis of the force dynamics of CAFs on 2D substrates to construct a stochastic model of YAP kinetics. This model revealed that the probability of YAP nuclear translocation, as well as the residence time in the nucleus follow a power law. This study provides valuable insights into the regulatory mechanisms governing YAP dynamics and highlights the significance of threshold activation in YAP localization. STATEMENT OF SIGNIFICANCE: Yes Associated Protein (YAP), a transcription cofactor, has been identified as one of the drivers of cancer progression. High tumor stiffness is attributed to driving YAP to the nucleus, wherein it activates pro-metastatic genes. Here we show, using cancer associated fibroblasts, that YAP translocation to the nucleus depends on the degree of nuclear deformation, irrespective of stiffness. We also identified that perinuclear force induced membrane curvature correlates strongly with YAP nuclear transport. A novel stochastic model of YAP kinetics unveiled a power law relationship between the activation threshold and persistence time of YAP in the nucleus. Overall, this study provides novel insights into the regulatory mechanisms governing YAP dynamics and the probability of activation that is of immense clinical significance.

Mind In Vitro Platforms: Versatile, Scalable, Robust, and Open Solutions to Interfacing with Living Neurons.

Motivated by the unexplored potential of in vitro neural systems for computing and by the corresponding need of versatile, scalable interfaces for multimodal interaction, an accurate, modular, fully customizable, and portable recording/stimulation solution that can be easily fabricated, robustly operated, and broadly disseminated is presented. This approach entails a reconfigurable platform that works across multiple industry standards and that enables a complete signal chain, from neural substrates sampled through micro-electrode arrays (MEAs) to data acquisition, downstream analysis, and cloud storage. Built-in modularity supports the seamless integration of electrical/optical stimulation and fluidic interfaces. Custom MEA fabrication leverages maskless photolithography, favoring the rapid prototyping of a variety of configurations, spatial topologies, and constitutive materials. Through a dedicated analysis and management software suite, the utility and robustness of this system are demonstrated across neural cultures and applications, including embryonic stem cell-derived and primary neurons, organotypic brain slices, 3D engineered tissue mimics, concurrent calcium imaging, and long-term recording. Overall, this technology, termed "mind in vitro" to underscore the computing inspiration, provides an end-to-end solution that can be widely deployed due to its affordable (>10× cost reduction) and open-source nature, catering to the expanding needs of both conventional and unconventional electrophysiology.

Synapses without tension fail to fire in an in vitro network of hippocampal neurons.

Neurons in the brain communicate with each other at their synapses. It has long been understood that this communication occurs through biochemical processes. Here, we reveal that mechanical tension in neurons is essential for communication. Using in vitro rat hippocampal neurons, we find that 1) neurons become tout/tensed after forming synapses resulting in a contractile neural network, and 2) without this contractility, neurons fail to fire. To measure time evolution of network contractility in 3D (not 2D) extracellular matrix, we developed an ultrasensitive force sensor with 1 nN resolution. We employed Multi-Electrode Array and iGluSnFR, a glutamate sensor, to quantify neuronal firing at the network and at the single synapse scale, respectively. When neuron contractility is relaxed, both techniques show significantly reduced firing. Firing resumes when contractility is restored. This finding highlights the essential contribution of neural contractility in fundamental brain functions and has implications for our understanding of neural physiology.

Mechanosensitive changes in the expression of genes in colorectal cancer-associated fibroblasts.

Most solid tumors become stiff with progression of cancer. Cancer Associated Fibroblasts (CAFs), most abundant stromal cells in the tumor microenvironment (TME), are known to mediate such stiffening. While the biochemical crosstalk between CAFs and cancer cells have been widely investigated, it is not clear if and how CAFs in stiffer TME promote metastatic progression. To gather insights into the process, we controlled the mechanical stiffness of the substrates and collected gene expression data with human colorectal CAFs. We cultured human primary CAFs on 2D polyacrylamide hydrogels with increasing elastic modulus (E) of 1, 10 and 40 kPa, and performed genome-wide transcriptome analyses in these cells to identify expression levels of ~16000 genes. The high-quality RNAseq results can be an excellent data-source for bioinformatic analysis for identifying novel pathways and biomarkers in cancer development and metastatic progression. With thorough analysis and accurate interpretation, this data may help researchers understand the role of mechanical stiffness of the TME in CAF-cancer cell crosstalk.

Astrocyte-mediated Transduction of Muscle Fiber Contractions Synchronizes Hippocampal Neuronal Network Development.

Exercise supports brain health in part by enhancing hippocampal function. The leading hypothesis is that muscles release factors when they contract (e.g., lactate, myokines, growth factors) that enter circulation and reach the brain where they enhance plasticity (e.g., increase neurogenesis and synaptogenesis). However, it remains unknown how the muscle signals are transduced by the hippocampal cells to modulate network activity and synaptic development. Thus, we established an in vitro model in which the media from contracting primary muscle cells (CM) is applied to developing primary hippocampal cell cultures on a microelectrode array. We found that the hippocampal neuronal network matures more rapidly (as indicated by synapse development and synchronous neuronal activity) when exposed to CM than regular media (RM). This was accompanied by a 4.4- and 1.4-fold increase in the proliferation of astrocytes and neurons, respectively. Further, experiments established that factors released by astrocytes inhibit neuronal hyper-excitability induced by muscle media, and facilitate network development. Results provide new insight into how exercise may support hippocampal function by regulating astrocyte proliferation and subsequent taming of neuronal activity into an integrated network.

Disorder to order transition in cell-ECM systems mediated by cell-cell collective interactions.

Cells in functional tissues execute various collective activities to achieve diverse ordered processes including wound healing, organogenesis, and tumor formation. How a group of individually operating cells initiate such complex collective processes is still not clear. Here, we report that cells in 3D extracellular matrix (ECM) initiate collective behavior by forming cell-ECM network when the cells are within a critical distance from each other. We employed compaction of free-floating (FF) 3D collagen gels with embedded fibroblasts as a model system to study collective behavior and found a sharp transition in the amount of compaction as a function of cell-cell distance, reminiscent of phase transition in materials. Within the critical distance, cells remodel the ECM irreversibly, and form dense collagen bridges between each other resulting in the formation of a network. Beyond the critical distance, cells exhibit Brownian dynamics and only deform the matrix reversibly in a transient manner with no memory of history, thus maintaining the disorder. Network formation seems to be a necessary and sufficient condition to trigger collective behavior and a disorder-to order transition. STATEMENT OF SIGNIFICANCE: Macroscopic compaction of in vitro collagen gels is mediated by collective mechanical interaction of cells. Previous studies on cell-induced ECM compaction suggest the existence of a critical cell density and phase transition associated with this phenomenon. Cell-mediated mechanical remodeling and global compaction of ECM has mostly been studied at steady state. Our study reveals a link between a transition in cell dynamics and material microstructure as cells collectively compact collagen gels. It underscores the significance of temporal evolution of these cell-ECM systems in understanding the mechanism of such collective action and provides insights on the process from a mechanistic viewpoint. These insights can be valuable in understanding dynamic pathological processes such as, cancer progression and wound healing, as well as engineering biomaterials and regenerative tissue mimics.

Principles for the design of multicellular engineered living systems.

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell-cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the "black box" of living cells.

Water as a "glue": Elasticity-enhanced wet attachment of biomimetic microcup structures.

Octopus, clingfish, and larva use soft cups to attach to surfaces under water. Recently, various bioinspired cups have been engineered. However, the mechanisms of their attachment and detachment remain elusive. Using a novel microcup, fabricated by two-photon lithography, coupled with in situ pressure sensor and observation cameras, we reveal the detailed nature of its attachment/detachment under water. It involves elasticity-enhanced hydrodynamics generating "self-sealing" and high suction at the cup-substrate interface, converting water into "glue." Detachment is mediated by seal breaking. Three distinct mechanisms of breaking are identified, including elastic buckling of the cup rim. A mathematical model describes the interplay between the attachment/detachment process, geometry, elasto-hydrodynamics, and cup retraction speed. If the speed is too slow, then the octopus cannot attach; if the tide is too gentle for the larva, then water cannot serve as a glue. The concept of "water glue" can innovate underwater transport and manufacturing strategies.

Adaptive biohybrid pumping machine with flow loop feedback.

Tissue-engineered living machines is an emerging discipline that employs complex interactions between living cells and engineered scaffolds to self-assemble biohybrid systems for diverse scientific research and technological applications. Here, we report an adaptive, autonomous biohybrid pumping machine with flow loop feedback powered by engineered living muscles. The tissue is made from skeletal muscle cells (C2C12) and collagen I/Matrigel matrix, which self-assembles into a ring that compresses a soft hydrogel tube connected at both ends to a rigid fluidic platform. The muscle ring contracts in a repetitive fashion autonomously squeezing the tube, resulting in an impedance pump. The resulting flow is circulated back to the muscle ring forming a feedback loop, which allows the pump to respond to the cues received from the flow it generates and adaptively manage its pumping performances based on the feedback. The developed biohybrid pumping system may have broad utility and impact in health, medicine and bioengineering.

Dose-independent threshold illumination for non-invasive time-lapse fluorescence imaging of live cells.

Fluorescent microscopy employs monochromatic light for excitation, which can adversely affect the cells being observed. We reported earlier that fibroblasts relax their contractile force in response to green light of typical intensity. Here we show that such effects are independent of extracellular matrix and cell lines. In addition, we establish a threshold intensity that elicits minimal or no adverse effect on cell contractility even for long-time exposure. This threshold intensity is wavelength dependent. We cultured fibroblasts on soft 2D elastic hydrogels embedded with fluorescent beads to trace substrate deformation and cell forces. The beads move towards cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio (λ r), in addition to traction force, as measures of cell response to red (wavelength, λ=635-650 nm), green (λ=545-580 nm) and blue (λ=455-490 nm) lights with varying intensities. Our results suggest that intensities below 57, 31 and 3.5 W/m2 for red, green and blue lights, respectively, do not perturb force homeostasis. To our knowledge, these intensities are the lowest reported safe thresholds, implying that cell traction is a highly sensitive readout of the effect of light on cells. Most importantly, we find these threshold intensities to be dose-independent; i.e., safe regardless of the energy dosage or time of exposure. Conversely, higher intensities result in widespread force-relaxation in cells with λ r > 1. Furthermore, we present a photo-reaction based model that simulates photo-toxicity and predicts threshold intensity for different wavelengths within the visible spectra. In conclusion, we recommend employing illumination intensities below aforementioned wavelength-specific thresholds for time-lapse imaging of cells and tissues in order to avoid light-induced artifacts in experimental observations.

Compliant 3D frameworks instrumented with strain sensors for characterization of millimeter-scale engineered muscle tissues.

Tissue-on-chip systems represent promising platforms for monitoring and controlling tissue functions in vitro for various purposes in biomedical research. The two-dimensional (2D) layouts of these constructs constrain the types of interactions that can be studied and limit their relevance to three-dimensional (3D) tissues. The development of 3D electronic scaffolds and microphysiological devices with geometries and functions tailored to realistic 3D tissues has the potential to create important possibilities in advanced sensing and control. This study presents classes of compliant 3D frameworks that incorporate microscale strain sensors for high-sensitivity measurements of contractile forces of engineered optogenetic muscle tissue rings, supported by quantitative simulations. Compared with traditional approaches based on optical microscopy, these 3D mechanical frameworks and sensing systems can measure not only motions but also contractile forces with high accuracy and high temporal resolution. Results of active tension force measurements of engineered muscle rings under different stimulation conditions in long-term monitoring settings for over 5 wk and in response to various chemical and drug doses demonstrate the utility of such platforms in sensing and modulation of muscle and other tissues. Possibilities for applications range from drug screening and disease modeling to biohybrid robotic engineering.

Development of an objective index, neural activity score (NAS), reveals neural network ontogeny and treatment effects on microelectrode arrays.

Microelectrode arrays (MEAs) are valuable tools for electrophysiological analysis, providing assessment of neural network health and development. Analysis can be complex, however, requiring intensive processing of large data sets consisting of many activity parameters, leading to information loss as studies subjectively report relatively few metrics in the interest of simplicity. In screening assays, many groups report simple overall activity (i.e. firing rate) but omit network connectivity changes (e.g. burst characteristics and synchrony) that may not be evident from basic parameters. Our goal was to develop an objective process to capture most of the valuable information gained from MEAs in neural development and toxicity studies. We implemented principal component analysis (PCA) to reduce the high dimensionality of MEA data. Upon analysis, we found the first principal component was strongly correlated to time, representing neural culture development; therefore, factor loadings were used to create a single index score-named neural activity score (NAS)-reflecting neural maturation. For validation, we applied NAS to studies analyzing various treatments. In all cases, NAS accurately recapitulated expected results, suggesting viability of NAS to measure network health and development. This approach may be adopted by other researchers using MEAs to analyze complicated treatment effects and multicellular interactions.

A novel method for sensor-based quantification of single/multicellular force dynamics and stiffening in 3D matrices.

Cells in vivo generate mechanical traction on the surrounding 3D extracellular matrix (ECM) and neighboring cells. Such traction and biochemical cues may remodel the matrix, e.g., increase stiffness, which, in turn, influences cell functions and forces. This dynamic reciprocity mediates development and tumorigenesis. Currently, there is no method available to directly quantify single-cell forces and matrix remodeling in 3D. Here, we introduce a method to fulfill this long-standing need. We developed a high-resolution microfabricated sensor that hosts a 3D cell-ECM tissue formed by self-assembly. This sensor measures cell forces and tissue stiffness and can apply mechanical stimulation to the tissue. We measured single and multicellular force dynamics of fibroblasts (3T3), human colon (FET) and lung (A549) cancer cells, and cancer-associated fibroblasts (CAF05) with 1-nN resolution. Single cells show notable force fluctuations in 3D. FET/CAF coculture system, mimicking cancer tumor microenvironment, increased tissue stiffness by three times within 24 hours.

A portable pen-sized instrumentation to measure stiffness of soft tissues in vivo.

Quantitative assessment of soft tissue elasticity is crucial to a broad range of applications, such as biomechanical modeling, physiological monitoring, and tissue diseases diagnosing. However, the modulus measurement of soft tissues, particularly in vivo, has proved challenging since the instrument has to reach the site of soft tissue and be able to measure in a very short time. Here, we present a simple method to measure the elastic modulus of soft tissues on site by exploiting buckling of a long slender bar to quantify the applied force and a spherical indentation to extract the elastic modulus. The method is realized by developing a portable pen-sized instrument (EPen: Elastic modulus pen). The measurement accuracies are verified by independent modulus measures using commercial nanoindenter. Quantitative measurements of the elastic modulus of mouse pancreas, healthy and cancerous, surgically exposed but attached to the body further confirm the potential clinical utility of the EPen.

Phase imaging with computational specificity (PICS) for measuring dry mass changes in sub-cellular compartments.

Due to its specificity, fluorescence microscopy has become a quintessential imaging tool in cell biology. However, photobleaching, phototoxicity, and related artifacts continue to limit fluorescence microscopy's utility. Recently, it has been shown that artificial intelligence (AI) can transform one form of contrast into another. We present phase imaging with computational specificity (PICS), a combination of quantitative phase imaging and AI, which provides information about unlabeled live cells with high specificity. Our imaging system allows for automatic training, while inference is built into the acquisition software and runs in real-time. Applying the computed fluorescence maps back to the quantitative phase imaging (QPI) data, we measured the growth of both nuclei and cytoplasm independently, over many days, without loss of viability. Using a QPI method that suppresses multiple scattering, we measured the dry mass content of individual cell nuclei within spheroids. In its current implementation, PICS offers a versatile quantitative technique for continuous simultaneous monitoring of individual cellular components in biological applications where long-term label-free imaging is desirable.

Performance of fabrics for home-made masks against the spread of COVID-19 through droplets: A quantitative mechanistic study.

Coronavirus Disease 2019 (COVID-19) may spread through respiratory droplets released by infected individuals during coughing, sneezing, or speaking. Given the limited supply of professional respirators and face masks, the U.S. Centers for Disease Control and Prevention (CDC) has recommended home-made cloth face coverings for use by the general public. While there have been several studies on aerosol filtration performance of household fabrics, their effectiveness at blocking larger droplets has not been investigated. Here, we ascertained the performance of 11 common household fabrics at blocking large, high-velocity droplets, using a commercial medical mask as a benchmark. We also assessed the breathability (air permeability), texture, fiber composition, and water absorption properties of the fabrics. We found that most fabrics have substantial blocking efficiency (median values >70%). In particular, two layers of highly permeable fabric, such as T-shirt cloth, blocks droplets with an efficiency (>94%) similar to that of medical masks, while being approximately twice as breathable. The first layer allows about 17% of the droplet volume to transmit, but it significantly reduces their velocity. This allows the second layer to trap the transmitted droplets resulting in high blocking efficacy. Overall, our study suggests that cloth face coverings, especially with multiple layers, may help reduce droplet transmission of respiratory infections. Furthermore, face coverings made from materials such as cotton fabrics allow washing and reusing, and can help reduce the adverse environmental effects of widespread use of commercial disposable and non-biodegradable facemasks.

Development of 3D neuromuscular bioactuators.

Neuronal control of skeletal muscle bioactuators represents a critical milestone toward the realization of future biohybrid machines that may generate complex motor patterns and autonomously navigate through their environment. Animals achieve these feats using neural networks that generate robust firing patterns and coordinate muscle activity through neuromuscular units. Here, we designed a versatile 3D neuron-muscle co-culture platform to serve as a test-bed for neuromuscular bioactuators. We used our platform in conjunction with microelectrode array electrophysiology to study the roles of synergistic interactions in the co-development of neural networks and muscle tissues. Our platform design enables co-culture of a neuronal cluster with up to four target muscle actuators, as well as quantification of muscle contraction forces. Using engineered muscle tissue targets, we first demonstrated the formation of functional neuromuscular bioactuators. We then investigated possible roles of long-range interactions in neuronal outgrowth patterns and observed preferential outgrowth toward muscles compared to the acellular matrix or fibroblasts, indicating muscle-specific chemotactic cues acting on motor neurons. Next, we showed that co-cultured muscle strips exhibited significantly higher spontaneous contractility as well as improved sarcomere assembly compared to muscles cultured alone. Finally, we performed microelectrode array measurements on neuronal cultures, which revealed that muscle-conditioned medium enhances overall neural firing rates and the emergence of synchronous bursting patterns. Overall, our study illustrates the significance of neuron-muscle cross talk for the in vitro development of neuromuscular bioactuators.

Engineering geometrical 3-dimensional untethered in vitro neural tissue mimic.

Formation of tissue models in 3 dimensions is more effective in recapitulating structure and function compared to their 2-dimensional (2D) counterparts. Formation of 3D engineered tissue to control shape and size can have important implications in biomedical research and in engineering applications such as biological soft robotics. While neural spheroids routinely are created during differentiation processes, further geometric control of in vitro neural models has not been demonstrated. Here, we present an approach to form functional in vitro neural tissue mimic (NTM) of different shapes using stem cells, a fibrin matrix, and 3D printed molds. We used murine-derived embryonic stem cells for optimizing cell-seeding protocols, characterization of the resulting internal structure of the construct, and remodeling of the extracellular matrix, as well as validation of electrophysiological activity. Then, we used these findings to biofabricate these constructs using neurons derived from human embryonic stem cells. This method can provide a large degree of design flexibility for development of in vitro functional neural tissue models of varying forms for therapeutic biomedical research, drug discovery, and disease modeling, and engineering applications.

Neuromuscular actuation of biohybrid motile bots.

The integration of muscle cells with soft robotics in recent years has led to the development of biohybrid machines capable of untethered locomotion. A major frontier that currently remains unexplored is neuronal actuation and control of such muscle-powered biohybrid machines. As a step toward this goal, we present here a biohybrid swimmer driven by on-board neuromuscular units. The body of the swimmer consists of a free-standing soft scaffold, skeletal muscle tissue, and optogenetic stem cell-derived neural cluster containing motor neurons. Myoblasts embedded in extracellular matrix self-organize into a muscle tissue guided by the geometry of the scaffold, and the resulting muscle tissue is cocultured in situ with a neural cluster. Motor neurons then extend neurites selectively toward the muscle and innervate it, developing functional neuromuscular units. Based on this initial construct, we computationally designed, optimized, and implemented light-sensitive flagellar swimmers actuated by these neuromuscular units. Cyclic muscle contractions, induced by neural stimulation, drive time-irreversible flagellar dynamics, thereby providing thrust for untethered forward locomotion of the swimmer. Overall, this work demonstrates an example of a biohybrid robot implementing neuromuscular actuation and illustrates a path toward the forward design and control of neuron-enabled biohybrid machines.