Epithelial-mesenchymal transition (EMT) is a key phenotypic switch in cancer metastasis, leading to fatal consequences for patients. Under geometric constraints, the morphology of cancer cells changes in both cellular and subcellular levels, whose effects on EMT are, however, not fully understood. Herein, we designed and fabricated chimeric micropatterns of polystyrene (PS) with adhesion contrast to reveal the impacts of cell shapes and nuclear shapes on EMT in a decoupled way. Cell elongation was modulated via microwell aspect ratios (ARs), and nuclear deformation was generated through a micropillar array in the microwell. Human non-small cell lung cancer cells (A549) were cultured on the quasi-three dimensional micropatterned surfaces, and transforming growth factor-β1 (TGF-β1) was added to induce EMT. We found that chimeric micropatterns upregulated EMT with an increase of cellular AR and nuclear indentation under given TGF-β1. The subsequent assessment of the contractility and oriented assembly of microfilaments elucidated the key role of mechanotransduction in cell elongation and EMT, as proved by myosin inhibition, while it was obstructed by micropillars in the chimeric micropattern. Hence, the micropillar array possessed a nonmonotonic influence, enhancing the EMT of cells with AR of 1, but hindering the EMT with an impact more significant on microwells with large ARs due to the impeded cytoskeleton assembly. This fundamental research has illustrated the complex of cellular and subcellular geometries on cell behaviors including phenotype transition in cancer metastasis.

Most melanomas that occur on the skin surface originate from a newly formed nevus and grow outward in a circular pattern and metastasize from the nevus center. Herein, a circular microfabricated substrate is constructed to explore the growth behavior of melanoma cells. Modeling software is used to calculate appropriate parameters, including shape and size, and then the substrates are processed with microfabrication technologies. The results show that the melanoma cells on the circular adhesion substrate are oval and are significant changes in cell spread length, nuclei, area, aspect ratio, Young's modulus, and orientation angles, indicating inhibition of cell polarization. Moreover, three different layers from circular adhesion substrates are selected to construct new substrates, which indicates that the polarization degree of cells is closely related to the number of micropillar arrays on the circular geometric substrate. In addition, flow cytometry demonstrates that the circular substrate reduced the transition from resting/gap 1 phase (G0/G1) to synthesis phase (S phase), thereby decreasing DNA synthesis and proliferation, reminding a potential method for treatment strategy. More importantly, the circular adhesion substrate influences the integrin signaling pathway, which has a potential application and research prospect in the treatment of melanoma.

Human engineered tissues hold great promise for therapeutic tissue regeneration and repair. Yet, development of these technologies often stalls at the stage of in vivo studies due to the complexity of engineered tissue formulations, which are often composed of diverse cell populations and material elements, along with the tedious nature of in vivo experiments. We introduce a "plug and play" platform called parallelized host apposition for screening tissues in vivo (PHAST). PHAST enables parallelized in vivo testing of 43 three-dimensional microtissues in a single 3D-printed device. Using PHAST, we screen microtissue formations with varying cellular and material components and identify formulations that support vascular graft-host inosculation and engineered liver tissue function in vivo. Our studies reveal that the cellular population(s) that should be included in engineered tissues for optimal in vivo performance is material dependent. PHAST could thus accelerate development of human tissue therapies for clinical regeneration and repair.

Humans excel at categorizing objects by shape. This facility involves identifying shape features that objects have in common with other members of their class and relies-at least in part-on semantic/cognitive constructs. For example, plants sprout branches, fish grow fins, shoes are moulded to our feet. Can humans parse shapes according to the processes that give shapes their key characteristics, even when such processes are hidden? To answer this, we investigated how humans perceive the shape of cells from the olfactory system of Xenopus laevis tadpoles. These objects are novel to most humans yet occur in nature and cluster into classes following their underlying biological function. We reconstructed three-dimensional (3D) cell models through 3D microscopy and photogrammetry, then conducted psychophysical experiments. Human participants performed two tasks: they arranged 3D-printed cell models by similarity and rated them along eight visual dimensions. Participants were highly consistent in their arrangements and ratings and spontaneously grouped stimuli to reflect the cell classes, unwittingly revealing the underlying processes shaping these forms. Our findings thus demonstrate that human perceptual organization mechanisms spontaneously parse the biological systematicities of never-before-seen, natural shapes. Integrating such human perceptual strategies into automated systems may enhance morphology-based analysis in biology and medicine.

Electrochemiluminescence (ECL) is a chemiluminescence phenomenon triggered by electrochemical reactions and is widely used for (bio)chemical analyses and electrochemical bioimaging. Compared to fluorescence sensing, ECL sensing reduces background noise by eliminating autofluorescence associated with excitation light. In addition, compared with conventional electrochemical imaging with scanning electrochemical microscopes, ECL imaging is faster as it requires no scanning. Furthermore, unlike electrode arrays, ECL devices can function without complex wiring, simplifying their construction. These characteristics render ECL sensing a useful analytical tool. Recently, ECL sensing has been widely used for in vitro cell analysis due to high demand for biochips in regenerative medicine, drug screening, and microphysiological systems. This review focuses on recent advancements in ECL-based cell analysis with applications for the detection of H2O2, respiration activity, cell adhesion, lipid membranes, and bipolar electrochemistry-based devices.

Epithelial tissues serve as the first line of host against bacterial infections. The self-organization of epithelial tissues continuously adapts to the architecture and mechanics of microenvironments, thereby dynamically impacting the initial niche of infections. However, the mechanism by which tissue geometry regulates bacterial infection remains poorly understood. Here, we showed geometry-guided infection patterns of bacteria in epithelial tissues using bioengineering strategies. We discovered that cellular traction forces play a crucial role in the regulation of bacterial invasive sites and marginal infection patterns in epithelial monolayers through triggering co-localization of mechanosensitive ion channel protein Piezo1 with bacteria. Further, we developed precise mechanobiology-based strategies to potentiate the antibacterial efficacy in animal models of wound and intestinal infection. Our findings demonstrate that tissue geometry exerts a key impact on mediating spatiotemporal infections of bacteria, which has important implications for the discovery and development of alternative strategies against bacterial infections.

Tissue deformations are a central feature of development, from early embryogenesis, growth and building the body plan to the establishment of functional organs. These deformations often result from active contractile forces generated by cells and cell collectives, and are mediated by changes in their mechanical properties. Mechanical forces drive the formation of functional organ architectures, but they also coordinate cell behaviour and fate transitions, ensuring robustness of development. Advances in microscopy, genetics and chemistry have enabled increasingly powerful tools for measuring, generating and perturbing mechanical forces. Here we discuss approaches to measure and manipulate mechanical forces with a focus on developmental processes, ranging from quantification of molecular interactions to mapping the mechanical properties of tissues. We focus on contemporary methods, and discuss the biological discoveries that these approaches have enabled. We conclude with an outlook to methodologies at the interface of physics, chemistry and biology to build an integrated understanding of tissue morphodynamics.

Recent advancements in single-cell analysis have underscored the need for precise isolation and manipulation of individual cells. Traditional techniques for single-cell manipulation are often limited by the number of cells that can be parallel trapped and processed and usually require complex devices or instruments to operate. Here, we introduce an acoustic microfluidic platform that efficiently traps and selectively releases individual cells using spherical air cavities embedded in a polydimethylsiloxane (PDMS) substrate for large scale manipulation. Our device utilizes the principle of acoustic impedance mismatches to generate near-field acoustic potential gradients that create trapping sites for single cells. These single cell traps can be selectively disabled by illuminating a near-infrared laser pulse, allowing targeted release of trapped cells. This method ensures minimal impact on cell viability and proliferation, making it ideal for downstream single-cell analysis. Experimental results demonstrate our platform's capability to trap and release synthetic microparticles and biological cells with high efficiency and biocompatibility. Our device can handle a wide range of cell sizes (8-30 μm) across a large active manipulation area of 1 cm2 with 20 000 single-cell traps, providing a versatile and robust platform for single-cell applications. This acoustic microfluidic platform offers a cost-effective and practical method for large scale single-cell trapping and selective releasing with potential applications in genomics, proteomics, and other fields requiring precise single-cell manipulation.

Despite their genetic diversity, metastatic cells converge on similar physical constraints during tumor progression. At the nanoscale, these forces can induce substantial molecular deformations, altering the structure and behavior of cancer cells. To address the challenges of osteosarcoma (OS), a highly aggressive cancer, we explored the mechanobiology of OS cells, in vitro. Using uniaxial-stretching technology, we examined the biophysical modulation of metastatic traits in SAOS-2, U-2 OS, and non-tumorigenic hFOB cells. Changes in cell morphology were quantified using confocal and fluorescence microscopy. To elucidate the molecular mechanisms that translate biomechanical alterations into biochemical responses, we employed Western blotting, real-time quantitative RT-PCR, reactive oxygen species ROS assay, and the mechanosensitive channel blocker Grammostola MechanoToxin4 (GsMTx-4). Our study reveals that mechanical stimulation uniquely affects OS cells, increasing nuclear size and altering the N/C ratio. We found that mechanosensitive (MS) channels are activated, leading to ROS accumulation, Src protein modulation, and histone H3 acetylation. These changes influence OS cell motility and adhesion but not proliferation. Importantly, mechanical preconditioning differentially impacts doxorubicin resistance, correlating with the Src-H3 acetylation axis. This study underscores the critical role of MS channels in OS cells and highlights the importance of mechanobiology in identifying molecular pathways that traditional biochemical approaches may not reveal. Notably, the GsMTx-4 venom peptide effectively countered mechanically induced responses, particularly by inhibiting OS cell migration, without harming healthy cells. Thus, suggesting its potential as a promising therapeutic agent for targeting osteosarcoma metastasis.

Biological processes are inherently dynamic, necessitating biomaterial platforms capable of spatiotemporal control over cellular organization and matrix stiffness for accurate study of tissue development, wound healing, and disease. However, most in vitro platforms remain static. In this study, a dynamic biomaterial platform comprising a stiffening hydrogel is introduced and achieved through a stepwise approach of addition followed by light-mediated crosslinking, integrated with an elastomeric substrate featuring strain-responsive lamellar surface patterns. Employing this platform, the response of human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) is investigated to dynamic stiffening from healthy to fibrotic tissue stiffness. The results demonstrate that culturing hIPSC-CMs on physiologically relevant healthy stiffness significantly enhances their function, as evidenced by increased sarcomere fraction, wider sarcomere width, significantly higher connexin-43 content, and elevated cell beating frequency compared to cells cultured on fibrotic matrix. Conversely, dynamic matrix stiffening negatively impacts hIPSC-CM function, with earlier stiffening events exerting a more pronounced hindering effect. These findings provide valuable insights into material-based approaches for addressing existing challenges in hIPSC-CM maturation and have broader implications across various tissue models, including muscle, tendon, nerve, and cornea, where both cellular alignment and matrix stiffening play pivotal roles in tissue development and regeneration.

Stem cells adapt to their local mechanical environment by rearranging their cytoskeleton, which underpins the evolution of their shape and fate as well as the emergence of tissue structure and function. Here, in the second part of a two-part experimental series, we aimed to elucidate spatiotemporal cytoskeletal remodeling and resulting changes in morphology and mechanical properties of cells and their nuclei. Akin to mechanical testing of the most basic living and adapting unit of life, i.e., the cell, in situ in model tissue templates, we probed native and microtubule-stabilized (via Paclitaxel, PAX, exposure) stem cells' cytoskeletal adaptation capacity on substrates of increasing compliance (exerting local tension on cells) and with increased target seeding densities (exerting local compression on cells). On 10 and 100 kPa gels, cells seeded at 5000 cells/cm2 and cells proliferated to 15 000 cells/cm2 exhibited bulk moduli that nearly matched those of their respective substrates; hence, they exhibited a greater increase in Young's Modulus after microtubule stabilization than cells cultured on glass. Culture on compliant substrates also reduced microtubule-stabilized cells' F-actin, and microtubule concentration increases compared to cells seeded on glass. On gels, F-actin alignment decreased as more randomly oriented, short actin crosslinks were observed, representing emergent adaptation to the compliant substrate, mediated through myosin II contractility. We conclude that stem cell adaptation to compliant substrates facilitates the accommodation of larger loads from the PAX-stabilized polymerizing microtubule, which, in turn, exerts a larger effect in determining cells' capacity to stiffen and remodel the cytoskeleton. Taken as a whole, these studies establish correlations between cytoskeleton and physical and mechanical parameters of stem cells. Hence, the studies progress our understanding of the dynamic cytoskeleton as well as shape changes in cells and their nuclei, culminating in emergent tissue development and healing.

A variety of potential biological roles of mechanical forces have been proposed in the field of cell biology. In particular, mechanical forces alter the mechanical conditions within cells and their environment, exerting a strong effect on the reorganization of the actin cytoskeleton. Single-molecule imaging studies have provided evidence that an actin filament may act as a mechanosensor. An increase in the tension within actin filaments causes changes in their conformation and affinity to their regulatory proteins. However, our current understanding of the molecular mechanisms of the tension sensing and the affinity change of regulatory proteins is still incomplete. In this study, we employed fluorescence polarization microscopy and magnetic tweezers to directly quantify the torsional fluctuations of single actin filaments and cofilin binding to the filament under several distinct mechanical conditions. When an actin filament was severed by scratching the filament with a pipette tip, the amplitude of twisting/torsional fluctuations and the rate of cooperative cofilin binding increased. On the other hand, when a piconewton force was applied to single actin filaments by magnetic tweezers, the amplitude of twisting fluctuations and the rate of cooperative cofilin binding decreased. This may be involved in the molecular mechanism behind the mechanical force-dependent severing of actin filaments in cells.

DNA nanostructures (DNs) have gained popularity in various biomedical applications due to their unique properties, including structural programmability, ease of synthesis and functionalization, and low cytotoxicity. Effective utilization of DNs in biomedical applications requires a fundamental understanding of their interactions with living cells and the mechanics of cellular uptake. Current knowledge primarily focuses on how the physicochemical properties of DNs, such as mass, shape, size, and surface functionalization, affect uptake efficacy. However, the role of cellular mechanics and morphology in DN uptake remains largely unexplored. In this work, we show that cells subjected to geometric constraints remodel their actin cytoskeleton, resulting in differential mechanical force generation that facilitates DN uptake. The length, number, and orientation of F-actin fibers are influenced by these constraints, leading to distinct mechanophenotypes. Overall, DN uptake is governed by F-actin forces arising from filament reorganisation under geometric constraints. These results underscore the importance of actin dynamics in the cellular uptake of DNs and suggest that leveraging geometric constraints to induce specific cell morphology adaptations could enhance the uptake of therapeutically designed DNs.

3D scaffolds enable biological investigations with a more natural cell conformation. However, the porosity of synthetic hydrogels is often limited to the nanometer scale, which confines the movement of 3D encapsulated cells and restricts dynamic cell processes. Precise control of hydrogel porosity across length scales remains a challenge and the development of porous materials that allow cell infiltration, spreading, and migration in a manner more similar to natural ECM environments is desirable. Here, a straightforward and reliable method is presented for generating kinetically-controlled macroporous biomaterials using liquid-liquid phase separation between poly(ethylene glycol) (PEG) and dextran. Photopolymerization-induced phase separation resulted in macroporous hydrogels with tunable pore size. Varying light intensity and hydrogel composition controlled polymerization kinetics, time to percolation, and complete gelation, which defined the average pore diameter (Ø = 1-200 µm) and final gel stiffness of the formed hydrogels. Critically, for biological applications, macroporous hydrogels are prepared from aqueous polymer solutions at physiological pH and temperature using visible light, allowing for direct cell encapsulation. Human dermal fibroblasts in a range of macroporous gels are encapsulated with different pore sizes. Porosity improved cell spreading with respect to bulk gels and allowed migration in the porous biomaterials.

Cell micro-patterning controls cell fate and function and has potential for generating therapeutically usable mesenchymal stromal cell (MSC) populations with precise functions. However, to date, the micro-patterning of human cells in a translational context has been impossible because only ruminant media supplements, e.g. fetal bovine serum (FBS), are established for use with micro-patterns (MPs). Thus, there are currently no good manufacturing practice (GMP)-compliant media available for MPs. This study tested a xenogenic-free human plasma and platelet lysate (hP + PL) medium supplement to determine its compatibility with MPs. Unfiltered hP + PL medium resulted in significant protein deposition, creating a 'carpet-like' layer that rendered MPs ineffective. Filtration (3×/5×) eliminated this effect. Importantly, quantitative comparison using droplet digital PCR revealed that human MSCs in all media types exhibited similar profiles with strong myogenic Calponin 1/Transgelin 2 (TAGLN2) and weaker osteogenic alkaline phosphatase/Runt-related transcription factor 2 marker expression, and much weaker adipogenic (lipoprotein lipase/peroxisome proliferator-activated receptor gamma) and chondrogenic (collagen type II/aggrecan) expression, with profiles being dominated by myogenic markers. Within these similar profiles, an even stronger induction of the myogenic marker TAGLN2 by all hP + PL- compared to FBS-containing media. Overall, this suggested that FBS can be replaced with hP + PL without altering differentiation profiles. However, assessing individual MSC responses to various MP types with defined categories revealed that unfiltered hP + PL medium was unusable. Importantly, FBS- and 3× filtered hP + PL media were comparable in each differentiation category. Summarized, this study recommends 3× filtered hP + PL as a xenogenic-free and potentially GMP-compliant alternative to FBS as a culture medium supplement for micro-patterning cell populations in both basic and translational research that will ensure consistent and reliable MSC micro-patterning for therapeutic use.

In the era of a constantly growing number of reports on the therapeutic properties of dedifferentiated, ontogenetically rejuvenated cells and their use in the treatment of neurological diseases, the optimization of their derivation and long-term culture methods seem to be crucial. One of the solutions is seen in the use of dedifferentiated fat cells (DFATs) that are characterized by a greater homogeneity. Moreover, these cells seem to possess a higher expression of transcriptional factors necessary to maintain pluripotency (stemness-related transcriptional factors) as well as a greater ability to differentiate in vitro into 3 embryonic germ layers, and a high proliferative potential in comparison to adipose stem/stromal cells. However, the neurogenic and neuroprotective potential of DFATs is still insufficiently understood; hence, our research goal was to contribute to our current knowledge of the subject. To recreate the brain's physiological (biomimetic) conditions, the cells were cultured at 5% oxygen concentration. The neural differentiation capacity of DFATs was assessed in the presence of the N21 supplement containing the factors that are typically found in the natural environment of the neural cell niche or in the presence of cerebrospinal fluid and under various spatial conditions (microprinting). The neuroprotective properties of DFATs were assessed using the coculture method with the ischemically damaged nerve tissue.

The physical microenvironment plays a crucial role in tumor development, progression, metastasis and treatment. Recently, we proposed four physical hallmarks of cancer, with distinct origins and consequences, to characterize abnormalities in the physical tumor microenvironment: (1) elevated compressive-tensile solid stresses, (2) elevated interstitial fluid pressure and the resulting interstitial fluid flow, (3) altered material properties (for example, increased tissue stiffness) and (4) altered physical micro-architecture. As this emerging field of physical oncology is being advanced by tumor biologists, cell and developmental biologists, engineers, physicists and oncologists, there is a critical need for model systems and measurement tools to mechanistically probe these physical hallmarks. Here, after briefly defining these physical hallmarks, we discuss the tools and model systems available for probing each hallmark in vitro, ex vivo, in vivo and in clinical settings. We finally review the unmet needs for mechanistic probing of the physical hallmarks of tumors and discuss the challenges and unanswered questions associated with each hallmark.

The glass-like slow dynamics in confluent epithelial monolayers is crucial for wound healing, embryogenesis, cancer progression, etc. Experiments have indicated several unusual properties in these systems. Unlike ordinary glasses, the glassiness in cellular systems strongly correlates with their static properties and is sub-Arrhenius. These results imply that the slow dynamics in epithelial monolayers is either not glassy or the underlying mechanism is different from ordinary glasses. Combining the analytical mode-coupling theory (MCT), vertex model simulations, and cellular experiments, we show that the slow dynamics is glassy, though the mechanism differs from ordinary glasses. The structure-dynamics feedback mechanism of MCT, and not the barrier-crossing mechanism, dominates the glassy dynamics, where the relaxation time diverges as a power law with a universal exponent 3/2 and naturally explains the sub-Arrhenius relaxation. Our results suggest the possibility of describing various complex biological processes like cell division and apoptosis via the static properties of the systems, such as average cell shape or shape variability.

Single-cell technology (SCT), which enables the examination of the fundamental units comprising biological organs, tissues, and cells, has emerged as a powerful tool, particularly in the field of biology, with a profound impact on stem cell research. This innovative technology opens new pathways for acquiring cell-specific data and gaining insights into the molecular pathways governing organ function and biology. SCT is not only frequently used to explore rare and diverse cell types, including stem cells, but it also unveils the intricacies of cellular diversity and dynamics. This perspective, crucial for advancing stem cell research, facilitates non-invasive analyses of molecular dynamics and cellular functions over time. Despite numerous investigations into potential stem cell therapies for genetic disorders, degenerative conditions, and severe injuries, the number of approved stem cell-based treatments remains limited. This limitation is attributed to the various heterogeneities present among stem cell sources, hindering their widespread clinical utilization. Furthermore, stem cell research is intimately connected with cutting-edge technologies, such as microfluidic organoids, CRISPR technology, and cell/tissue engineering. Each strategy developed to overcome the constraints of stem cell research has the potential to significantly impact advanced stem cell therapies. Drawing on the advantages and progress achieved through SCT-based approaches, this study aims to provide an overview of the advancements and concepts associated with the utilization of SCT in stem cell research and its related fields.

Cells can sense geometrical cues with sizes of several tens of micrometers in their vicinity. Recent in vitro studies show that cells can adapt their shape, align along specific directions, or regulate other cellular functions when grown on surfaces with curvatures larger than their size. Although possible mechanisms for such responses like the alignment along axial cues have been suggested, a detailed understanding of the involved cellular processes remains open. This work addresses this gap by systematically investigating mesenchymal cell and nucleus orientation responses using a low-cost model surface platform, the CurvChip. Using an array of cylindrically curved topographies with radii of curvatures ranging from tens to hundreds of micrometers, the contact guidance response of cells and nuclei is quantified in dependence on substratum curvature and manipulation of cytoskeletal components. Results suggest a desired perceived curvature for the investigated cells, and a very sensitive and robust curvature perception mechanism, as the effect of pharmacological manipulation of cytoskeletal components is relatively small. Furthermore, a comparison with previously published work strengthens the hypothesis of an involvement of the nucleus in the cell response to three-dimensional (3D) curvatures.

Microbubble-facilitated sonoporation is a rapid, versatile, and non-viral intracellular delivery technique with potential for clinical and ex vivo cell engineering applications. We developed a micropatterning-based approach to investigate the impact of cell shape on sonoporation efficacy. Cationic microbubbles were employed to enhance sonoporation by binding to the cell membrane electrostatically. NIH/3T3 fibroblasts were micropatterned into circle, square, triangle, and rectangle. A two-plate system ensured high-throughput and efficient sonoporation by controlling cationic microbubble-cell attachment. High-speed video microscopy captured the acoustic dynamics of microbubbles under short ultrasound pulses. Our findings reveal that for NIH/3T3 fibroblasts, rectangular cells achieved the highest sonoporation and survival rate, while square-shaped cells demonstrated the greatest propidium iodide uptake. Triangle-shaped NIH/3T3 fibroblasts displayed an initial rise then a plateau in the sonoporation and survival rate as the ultrasound pulse duration increased from 10 cycles to 100 cycles, and then to 200 cycles. Conversely, rectangle-shaped cells showed a decrease followed by a stabilization. Circle-shaped and rectangle-shaped HeLa cells exhibited similar sonoporation outcomes, which were not as effective as NIH/3T3 fibroblasts. This study underscores the significance of cell shape in optimizing sonoporation efficiency and highlights the potential of combining micropatterning with controlled targeting sonoporation to advance intracellular delivery technologies.

Cells in the body reside in a dynamic microenvironment subjected to various physical stimuli, where mechanical stimulation plays a crucial role in regulating cellular physiological behaviors and functions.

Investigating the mechanisms and interactions of mechanical transmission is essential for understanding the physiological and functional interplay between cells and physical stimuli. Therefore, establishing an in vitro biomechanical stimulation cell culture system holds significant importance for research related to cellular biomechanics.

In this review, we primarily focused on various biomechanically relevant cell culture systems and highlighted the advancements and prospects in their preparation processes. Firstly, we discussed the types and characteristics of biomechanics present in the microenvironment within the human body. Subsequently, we introduced the research progress, working principles, preparation processes, potential advantages, applications, and challenges of various biomechanically relevant in vitro cell culture systems. Additionally, we summarized and categorized currently commercialized biomechanically relevant cell culture systems, offering a comprehensive reference for researchers in related fields.

Collective cell invasion underlies several biological processes such as wound healing, embryonic development, and cancerous invasion. Here, we investigate the impact of cell motility on invasion in epithelial monolayers and its coupling to cellular mechanical properties, such as cell-cell adhesion and cortex contractility. We develop a two-dimensional computational model for cells with active motility based on the cellular Potts model, which predicts that the cellular invasion speed is mainly determined by active cell motility and is independent of the biological and mechanical properties of the cells. We also find that, in general, motile cells out-compete and invade non-motile cells, however, this can be reversed by differential cell proliferation. Stable coexistence of motile and static cell types is also possible for certain parameter regimes.

Despite remarkable advances in Organ-on-a-chip (Organ Chip) microfluidic culture technology, recreating tissue-relevant physiological conditions, such as the region-specific oxygen concentrations, remains a formidable technical challenge, and analysis of tissue functions is commonly carried out using one analytical technique at a time. Here, we describe two-channel Organ Chip microfluidic devices fabricated from polydimethylsiloxane and gas impermeable polycarbonate materials that are integrated with multiple sensors, mounted on a printed circuit board and operated using a commercially available Organ Chip culture instrument. The novelty of this system is that it enables the recreation of physiologically relevant tissue-tissue interfaces and oxygen tension as well as non-invasive continuous measurement of transepithelial electrical resistance, oxygen concentration and pH, combined with simultaneous analysis of cellular metabolic activity (ATP/ADP ratio), cell morphology, and tissue phenotype. We demonstrate the reliable and reproducible functionality of this system in living human Gut and Liver Chip cultures. Changes in tissue barrier function and oxygen tension along with their functional and metabolic responses to chemical stimuli (e.g., calcium chelation, oligomycin) were continuously and noninvasively monitored on-chip for up to 23 days. A physiologically relevant microaerobic microenvironment that supports co-culture of human intestinal cells with living Lactococcus lactis bacteria also was demonstrated in the Gut Chip. The integration of multi-functional sensors into Organ Chips provides a robust and scalable platform for the simultaneous, continuous, and non-invasive monitoring of multiple physiological functions that can significantly enhance the comprehensive and reliable evaluation of engineered tissues in Organ Chip models in basic research, preclinical modeling, and drug development.

Cells and tissues are often under some level of confinement, imposed by the microenvironment and neighboring cells, meaning that there are limitations to cell size, volume changes, and fluid exchanges. 3D cell culture, increasingly used for both single cells and organoids, inherently impose levels of confinement absent in 2D systems. It is thus key to understand how different levels of confinement influences cell survival, cell function, and cell fate. It is well known that the mechanical properties of the microenvironment, such as stiffness and stress relaxation, are important in activating mechanosensitive pathways, and these are responsive to confinement conditions. In this review, we look at how low, intermediate, and high levels of confinement modulate the activation of known mechanobiology pathways, in single cells, organoids, and tumor spheroids, with a specific focus on 3D confinement in microwells, elastic, or viscoelastic scaffolds. In addition, a confining microenvironment can drastically limit cellular communication in both healthy and diseased tissues, due to extracellular crowding. We discuss potential implications of extracellular crowding on molecular transport, extracellular matrix deposition, and fluid transport. Understanding how cells sense and respond to various levels of confinement should inform the design of 3D engineered matrices that recapitulate the physical properties of tissues.

Cell shape and function are intimately linked, in a way that is mediated by the forces exerted between cells and their environment. The relationship between cell shape and forces has been extensively studied for cells seeded on flat 2D substrates, but not for cells in more physiological 3D settings. Here, a technique called 3D micropatterned traction force microscopy (3D-µTFM) to confine cells in 3D wells of defined shape, while simultaneously measuring the forces transmitted between cells and their microenvironment is demonstrated. This technique is based on the 3D micropatterning of polyacrylamide wells and on the calculation of 3D traction force from their deformation. With 3D-µTFM, it is shown that MCF10A breast epithelial cells exert defined, reproducible patterns of forces on their microenvironment, which can be both contractile and extensile. Cells switch from a global contractile to extensile behavior as their volume is reduced are further shown. The technique enables the quantitative study of cell mechanobiology with full access to 3D cellular forces while having accurate control over cell morphology and the mechanical conditions of the microenvironment.

The myotendinous junction (MTJ) facilitates force transmission between muscle and tendon to produce joint movement. The complex microarchitecture and regional mechanical heterogeneity of the myotendinous junction pose major challenges in creating this interface in vitro. Engineering this junction in vitro is challenging due to substantial fabrication difficulties in creating scaffolds with intricate microarchitecture and stiffness heterogeneity to mimic the native muscle-tendon interface. To address the current challenges in creating the MTJ in vitro, digital light processing (DLP)-based 3D printing was used to fabricate poly(glycerol sebacate)acrylate (PGSA)-based muscle-tendon scaffolds with physiologically informed microstructure and mechanical properties. Local mechanical properties in various regions of the scaffold were tuned by adjusting the exposure time and light intensity used during the continuous DLP-based 3D printing process to match the mechanical properties present in distinct regions of native muscle-tendon tissue using printing parameters defined by an artificial intelligence-trained algorithm. To evaluate how the presence of zonal stiffness regions can affect the phenotype of a 3D-printed MTJ in vitro model, three 3D-printed PGSA-based scaffold conditions were investigated: (1) a scaffold with muscle-informed mechanical properties in its entirety without zonal stiffness regions, (2) a scaffold with one end possessing native muscle stiffness and the other end possessing native tendon stiffness, and (3) a scaffold with three distinct regions whose stiffness values correspond to those of muscle on one end of the scaffold, MTJ in the middle junction of the scaffold, and tendon on the other end of the scaffold. The scaffold containing regional mechanical heterogeneity most similar to the native MTJ (condition 3) was found to enhance the expression of MTJ-related markers compared to those without the presence of zonal stiffness regions. Overall, the DLP-based 3D printing platform and biomaterial system developed in this study could serve as a useful tool for mimicking the complexity of the native MTJ, which possesses inherent geometric and mechanical heterogeneity.

Cell shape reflects the spatial configuration resulting from the equilibrium of cellular and environmental signals and is considered a highly relevant indicator of its function and biological properties. For cancer cells, various physiological and environmental challenges, including chemotherapy, cause a cell state transition, which is accompanied by a continuous morphological alteration that is often extremely difficult to recognize even by direct microscopic inspection. To determine whether deep learning-based image analysis enables the detection of cell shape reflecting a crucial cell state alteration, we used the oral cancer cell line resistant to chemotherapy but having cell morphology nearly indiscernible from its non-resistant parental cells. We then implemented the automatic approach via deep learning methods based on EfficienNet-B3 models, along with over- and down-sampling techniques to determine whether image analysis of the Convolutional Neural Network (CNN) can accomplish three-class classification of non-cancer cells vs. cancer cells with and without chemoresistance. We also examine the capability of CNN-based image analysis to approximate the composition of chemoresistant cancer cells within a population. We show that the classification model achieves at least 98.33% accuracy by the CNN model trained with over- and down-sampling techniques. For heterogeneous populations, the best model can approximate the true proportions of non-chemoresistant and chemoresistant cancer cells with Root Mean Square Error (RMSE) reduced to 0.16 by Ensemble Learning (EL). In conclusion, our study demonstrates the potential of CNN models to identify altered cell shapes that are visually challenging to recognize, thus supporting future applications with this automatic approach to image analysis.

Inspired by iron gall ink (IGI), the Fe2+ ion is utilized for the continuous formation of Fe3+-tannic acid (TA) films through its in situ oxidation to the Fe3+ ion. Although several IGI-based strategies have been developed to meet different application requirements, there remains a need for the precise kinetic control for Fe2+ oxidation, along with one-step spatial controllability. This work demonstrates a visible light-induced method for oxidizing Fe2+ to Fe3+ ions, achieving the kinetic control over Fe3+-TA film formation. Photoexcitation of eosin Y leads to significantly accelerated film formation in a continuous manner, as shown by an 11-fold increase in film thickness compared with the air oxidation-based IGI method. Mechanistic studies reveal that the process is O2-dependent, and the kinetics of the film-forming process could be easily tuned by changing the light intensity or eosin Y concentration. Furthermore, the spatiotemporal controllability of the photoreaction allows for the pattern generation of Fe3+-TA complexes, proteins, and cells.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer great potential for drug screening and disease modeling. However, hiPSC-CMs remain immature compared to the adult cardiac cells. Cardiomyocytes isolated from adult human hearts have a typical rod-shaped morphology. Here, we sought to develop a simple method to improve the architectural maturity of hiPSC-CMs by using a rod-shaped cell micropatterned substrate consisting of repeated rectangles (120 μm long × 30 μm wide) surrounded by a chemical cell repellent. The generated hiPSC-CMs exhibit numerous characteristics similar to adult human cardiomyocytes, including elongated cell shape, well-organized sarcomeres, and increased myofibril density. The improvement in structural properties correlates with the enrichment of late ventricular action potentials characterized by a more hyperpolarized resting membrane potential and an enhanced depolarization consistent with an increased sodium current density. The more mature hiPSC-CMs generated by this method may serve as a useful in vitro platform for characterizing cardiovascular disease.

The progression of many solid tumors is accompanied by temporal and spatial changes in the stiffness of the extracellular matrix (ECM). Cancer cells adapt to soft and stiff ECM through mechanisms that are not fully understood. It is well known that there is significant genetic heterogeneity from cell to cell in tumors, but how ECM stiffness as a parameter might interact with that genetic variation is not known. Here, we employed experimental evolution to study the response of genetically variable and clonal populations of tumor cells to variable ECM stiffness. Proliferation rates of genetically variable populations cultured on soft ECM increased over a period of several weeks, whereas clonal populations did not evolve. Tracking of DNA barcoded cell lineages revealed that soft ECM consistently selected for the same few variants. These data provide evidence that ECM stiffness exerts natural selection on genetically variable tumor populations. Soft-selected cells were highly migratory, with enriched oncogenic signatures and unusual behaviors such as spreading and traction force generation on ECMs with stiffness as low as 1 kPa. Rho-regulated cell spreading was found to be the directly selected trait, with yes-associated protein 1 translocation to the nucleus mediating fitness on soft ECM. Overall, these data show that genetic variation can drive cancer cell adaptation to ECM stiffness.

Untreated primary carcinomas often lead to progression, invasion and metastasis, a process that involves the epithelial-to-mesenchymal transition (EMT). Several transcription factors (TFs) mediate the development of EMT, including SNAIL1/SNAIL2, TWIST1/TWIST2 and ZEB1/ZEB2, which are overexpressed in various carcinomas along with the under expression of the metastasis suppressor Raf Kinase Inhibitor Protein (RKIP). Overexpression of RKIP inhibits EMT and the above associated TFs. We, therefore, hypothesized that there are inhibitory cross-talk signaling pathways between RKIP and these TFs. Accordingly, we analyzed the various properties and biomarkers associated with the epithelial and mesenchymal tissues and the various molecular signaling pathways that trigger the EMT phenotype such as the TGF-β, the RTK and the Wnt pathways. We also presented the various functions and the transcriptional, post-transcriptional and epigenetic regulations for the expression of each of the EMT TFs. Likewise, we describe the transcriptional, post-transcriptional and epigenetic regulations of RKIP expression. Various signaling pathways mediated by RKIP, including the Raf/MEK/ERK pathway, inhibit the TFs associated with EMT and the stabilization of epithelial E-Cadherin expression. The inverse relationship between RKIP and the TF expressions and the cross-talks were further analyzed by bioinformatic analysis. High mRNA levels of RKIP correlated negatively with those of SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2 in several but not all carcinomas. However, in these carcinomas, high levels of RKIP were associated with good prognosis, whereas high levels of the above transcription factors were associated with poor prognosis. Based on the inverse relationship between RKIP and EMT TFs, it is postulated that the expression level of RKIP in various carcinomas is clinically relevant as both a prognostic and diagnostic biomarker. In addition, targeting RKIP induction by agonists, gene therapy and immunotherapy will result not only in the inhibition of EMT and metastases in carcinomas, but also in the inhibition of tumor growth and reversal of resistance to various therapeutic strategies. However, such targeting strategies must be better investigated as a result of tumor heterogeneities and inherent resistance and should be better adapted as personalized medicine.

Molecular motor amphiphiles have already been widely attempted for dynamic nanosystems across multiple length-scale for developments of small functional materials, including controlling macroscopic foam properties, amplifying motion as artificial molecular muscles, and serving as extracellular matrix mimicking cell scaffolds. However, limiting examples of bola-type molecular motor amphiphiles are considered for constructing macroscopic biomaterials. Herein, this work presents the designed two second generation molecular motor amphiphiles, motor bola-amphiphiles (MBAs). Aside from the photoinduced motor rotation of MBAs achieved in both organic and aqueous media, the rate of recovering thermal helix inversion step can be controlled by the rotor part with different steric hindrances. Dynamic assembled structures of MBAs are observed under (cryo)-transmission electron microscopy (TEM). This dynamicity assists MBAs in further assembling as macroscopic soft scaffolds by applying a shear-flow method. Upon photoirradiation, the phototropic bending function of MBA scaffolds is observed, demonstrating the amplification of molecular motion into macroscopic phototropic bending functions at the macroscopic length-scale. Since MBAs are confirmed with low cytotoxicity, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can grow on the surface of MBA scaffolds. These results clearly demonstrate the concept of designing MBAs for developing photoresponsive dynamic functional materials to create new-generation soft robotic systems and cell-material interfaces.

Micropatterning is a sophisticated technique that precisely manipulates the spatial distribution of cell adhesion proteins on various substrates across multiple scales. This precise control over adhesive regions facilitates the manipulation of architectures and physical constraints for single or multiple cells. Furthermore, it allows for an in-depth analysis of how chemical and physical properties influence cellular functionality. In this comprehensive review, we explore the current understanding of the impact of geometrical confinement on cellular functions across various dimensions, emphasizing the benefits of micropatterning in addressing fundamental biological queries. We advocate that utilizing directed self-organization via physical confinement and morphogen gradients on micropatterned surfaces represents an innovative approach to generating functional tissue and controlling morphogenesis in vitro. Integrating this technique with cutting-edge technologies, micropatterning presents a significant potential to bridge a crucial knowledge gap in understanding core biological processes.

Mesenchymal stem cells (MSCs) hold promise for regenerative medicine, particularly for bone tissue engineering. However, directing MSC differentiation towards specific lineages, such as osteogenic, while minimizing undesired phenotypes remains a challenge. Here, we investigate the influence of micropatterns on the behavior and lineage commitment of rat bone marrow-derived MSCs (rBMSCs), focusing on osteogenic differentiation. Linearly aligned triangular micropatterns (TPs) and circular micropatterns (CPs) coated with fibronectin were fabricated to study their effects on rBMSC morphology and differentiation and the underlying mechanobiological mechanisms. TPs, especially TP15 (15 μm), induced the cell elongation and thinning, while CPs also promoted the cell stretching, as evidenced by the decreased circularity and increased aspect ratio. TP15 significantly promoted osteogenic differentiation, with increased expression of osteogenic genes (Runx2, Spp1, Alpl, Bglap, Col1a1) and decreased expression of adipogenic genes (Pparg, Cebpa, Fabp4). Conversely, CPs inhibited both osteogenic and adipogenic differentiation. Mechanistically, TP15 increased Piezo1 activity, cytoskeletal remodeling including the aggregates of F-actin and myosin filaments at the cell periphery, YAP1 nuclear translocation, and integrin upregulation. Piezo1 inhibition suppressed the osteogenic genes expression, myosin remodeling, and YAP1 nuclear translocation, indicating Piezo1-mediated the mechanotransduction in rBMSCs on TPs. TP15 also induced osteogenic differentiation of BMSCs from aging rats, with upregulated Piezo1 and nuclear translocation of YAP1. Therefore, triangular micropatterns, particularly TP15, promote osteogenesis and inhibit adipogenesis of rBMSCs through Piezo1-mediated myosin and YAP1 pathways. Our study provides novel insights into the mechanobiological mechanisms governing MSC behaviors on micropatterns, offering new strategies for tissue engineering and regenerative medicine.

Crack is found on the soil when severe drought comes, which inspires the idea to rationalize patterning applications using dried deoxyribonucleic acid (DNA) film. DNA is one of the massively produced biomaterials in nature, showing the lyotropic liquid crystal (LC) phase in highly concentrated conditions. DNA nanostructures in the hydrated condition can be orientation controlled, which can be extended to make dryinginduced cracks. The controlled crack generation in oriented DNA films by inducing mechanical fracture through organic solvent-induced dehydration (OSID) using tetrahydrofuran (THF) is explored. The corresponding simulations show a strong correlation between the long axis of DNA due to the shrinkage during the dehydration and in the direction of crack propagation. The cracks are controlled by simple brushing and a 3D printing method. This facile way of aligning cracks will be used in potential patterning applications.

Cell-based medicinal products (CBMPs) are a growing class of therapeutics that promise new treatments for complex and rare diseases. Given the inherent complexity of the whole human cells comprising CBMPs, there is a need for robust and fast analytical methods for characterization, process monitoring, and quality control (QC) testing during their manufacture. Existing techniques to evaluate and monitor cell quality typically constitute labor-intensive, expensive, and highly specific staining assays. In this work, we combine image-based deep learning with flow imaging microscopy (FIM) to predict cell health metrics using cellular morphology "fingerprints" extracted from images of unstained Jurkat cells (immortalized human T-lymphocyte cells). A supervised (i.e., algorithm trained with human-generated labels for images) fingerprinting algorithm, trained on images of unstained healthy and dead cells, provides a robust stain-free, non-invasive, and non-destructive method for determining cell viability. Results from the stain-free method are in good agreement with traditional stain-based cytometric viability measurements. Additionally, when trained with images of healthy cells, dead cells and cells undergoing chemically induced apoptosis, the supervised fingerprinting algorithm is able to distinguish between the three cell states, and the results are independent of specific treatments or signaling pathways. We then show that an unsupervised variational autoencoder (VAE) algorithm trained on the same images, but without human-generated labels, is able to distinguish between samples of healthy, dead and apoptotic cells along with cellular debris based on learned morphological features and without human input. With this, we demonstrate that VAEs are a powerful exploratory technique that can be used as a process monitoring analytical tool.

Cells exhibit various morphological characteristics due to their physiological activities, and changes in cell morphology are inherently accompanied by the assembly and disassembly of the actin cytoskeleton. Stress fibers are a prominent component of the actin-based intracellular structure and are highly involved in numerous physiological processes, e.g., mechanotransduction and maintenance of cell morphology. Although it is widely accepted that variations in cell morphology interact with the distribution and localization of stress fibers, it remains unclear if there are underlying geometric principles between the cell morphology and actin cytoskeleton. Here, we present a machine learning system that uses the diffusion model to convert the cell shape to the distribution and alignment of stress fibers. By training with corresponding cell shape and stress fibers datasets, our system learns the conversion to generate the stress fiber images from its corresponding cell shape. The predicted stress fiber distribution agrees well with the experimental data. With this conversion relation, our system allows for performing virtual experiments that provide a visual map showing the probability of stress fiber distribution from the virtual cell shape. Our system potentially provides a powerful approach to seek further hidden geometric principles regarding how the configuration of subcellular structures is determined by the boundary of the cell structure; for example, we found that the stress fibers of cells with small aspect ratios tend to localize at the cell edge while cells with large aspect ratios have homogenous distributions.

From embryonic development, postnatal growth and adult homeostasis to reparative and disease states, cells and tissues undergo constant changes in genome activity, cell fate, proliferation, movement, metabolism and growth. Importantly, these biological state transitions are coupled to changes in the mechanical and material properties of cells and tissues, termed mechanical state transitions. These mechanical states share features with physical states of matter, liquids and solids. Tissues can switch between mechanical states by changing behavioural dynamics or connectivity between cells. Conversely, these changes in tissue mechanical properties are known to control cell and tissue function, most importantly the ability of cells to move or tissues to deform. Thus, tissue mechanical state transitions are implicated in transmitting information across biological length and time scales, especially during processes of early development, wound healing and diseases such as cancer. This Review will focus on the biological basis of tissue-scale mechanical state transitions, how they emerge from molecular and cellular interactions, and their roles in organismal development, homeostasis, regeneration and disease.

Microfluidic devices with integrated membranes that enable control of mass transport in constrained environments have shown considerable growth over the last decade. Membranes are a key component in several industrial processes such as chemical, pharmaceutical, biotechnological, food, and metallurgy separation processes as well as waste management applications, allowing for modular and compact systems. Moreover, the miniaturization of a process through microfluidic devices leads to process intensification together with reagents, waste and cost reduction, and energy and space savings. The combination of membrane technology and microfluidic devices allows therefore magnification of their respective advantages, providing more valuable solutions not only for industrial processes but also for reproducing biological processes. This review focuses on membrane-based microfluidic devices for biomedical science with an emphasis on microfluidic artificial organs and organs-on-chip. We provide the basic concepts of membrane technology and the laws governing mass transport. The role of the membrane in biomedical microfluidic devices, along with the required properties, available materials, and current challenges are summarized. We believe that the present review may be a starting point and a resource for researchers who aim to replicate a biological phenomenon on-chip by applying membrane technology, for moving forward the biomedical applications.