Biology subjects

Chen, C. S.

Publications and source records attributed to Chen, C. S..

At least 19 recordsLinked to original sources

bioRxiv2026

A Biophysical Platform for Electromechanical Stimulation of Engineered Cardiac Tissues

Human engineered cardiac tissues (ECTs) provide an in vitro model for studying human cardiac physiology and drug responses, but their performance remains limited by culture systems that do not fully reproduce the heart's electrical and mechanical environment. Electrical stimulation (ES) and mechanical stimulation (MS) have each been used to improve ECT function. Their combination, referred to as electromechanical stimulation (ES+MS), can provide further benefits. However, ES+MS depends not only on the presence of both cues but also on how they are coordinated in time. Here, we developed an incubator-compatible biophysical platform that delivers ES and MS independently or in combination, with programmable control over timing, amplitude, frequency, duration, and waveform. Calibration and dynamic characterization demonstrated tissue-relevant strain delivery, rapid and repeatable motion, and minimal attenuation and timing lag at the designated frequency of 1.5 Hz. We then compared four 6 h conditioning regimens: unstimulated control, ES alone, unsynchronized ES+MS, and synchronized ES+MS. We hypothesized that the synchronized ES+MS group, in which electrical excitation was aligned with peak externally applied strain, would produce the greatest increase in contractile force. Consistent with this hypothesis, synchronized ES+MS increased normalized twitch force by approximately 44% on average, whereas the other groups showed no comparable improvement. Twitch-timing metrics did not exhibit coordinated enhancement after 6 h, suggesting that the force increase reflects an adaptive biomechanical response rather than broad tissue maturation. These findings identify ES-MS timing as an important design parameter for ECT conditioning.

bioengineering↗

bioRxiv2026

PROPEL: a high-throughput shear-stress platform reveals organotypic thresholds in endothelial mechano-adaptation

Endothelial cells exhibit organotypic specialization, yet the tools to decode whether hemodynamic shear shapes this diversity across vascular beds have remained limited. Here we present PROPEL, a tubing-free, magnetic stirrer-driven platform that delivers programmable laminar shear stress (2 to 60 dyn/cm{superscript 2}) to multiple cell types in parallel within the confines of a standard Petri dish, with the modular flexibility to easily introduce different substrate geometries including 3D vessel formats. Using this platform, we profiled six human endothelial subtypes across static, low, intermediate, and high shear. Phenotypic profiling revealed subtype-specific thresholds for alignment, elongation, and Golgi-nuclear polarization, including a dissociation between elongation and polarization in dermal microvascular and saphenous venous endothelial cells at low shear. Bulk RNA sequencing showed conserved transcriptional programs that shift progressively with shear magnitude, including induction of mechanotransduction pathways alongside suppression of proliferative programs. Phenotype-informed transcriptional comparisons further linked alignment transitions to engagement of cytoskeletal and metabolic signatures. In addition to conserved responses, organotypic-specific responses were also observed. Together, these findings establish PROPEL as a scalable platform for multi-endothelial, multi-shear transcriptomic and phenotypic profiling, and reveal that endothelial cells engage shear adaptation along two organotypic axes--a threshold axis governing when a subtype responds and a signature axis governing which the shear stress response--with implications for vascular bed-specific disease susceptibility.

cell biology↗

bioRxiv2026

Sphingosine-1-phosphate cross-talks to Notch via a S1PR1-Dll4-MPDZ complex to regulate endothelial barrier function

Sphingosine-1-phosphate (S1P) - a key bioactive component of high-density lipoproteins (HDL) - is instrumental in mediating HDLs cardiovascular benefits, largely by enhancing endothelial barrier integrity1, 2. Here, we discovered that S1P induces Notch1 activation, and this Notch activation is required to enhance Rac1 activity and adherens junction assembly, which in turn stimulates endothelial barrier integrity. S1P rapidly activates Notch1 by stimulating the G-coupled protein receptor, S1P Receptor 1 (S1PR1) to drive internalization of the Notch ligand Delta-like protein 4 (Dll4). Notably, this internalization of Dll4 and subsequent activation of Notch does not involve traditional G-protein signaling; instead, S1P-bound S1PR1 forms a complex with Dll4 via the scaffolding protein MPDZ, and the undergoes co-endocytosis. Importantly, the loss or inhibition of Notch, Dll4, S1PR1, or MPDZ results in barrier defects. These findings elucidate a novel S1PR1-Dll4-MPDZ-Notch1 signaling axis that coordinates S1P and Notch signaling to regulate of endothelial cell signaling and barrier function.

cell biology↗

bioRxiv2026

Image-Guided Injectable Niche for Hepatocyte Transplantation

Liver transplantation remains the standard of care for end-stage liver failure, yet it is constrained by donor scarcity, surgical complexity, and restricted access for many patients. Cell-based therapies offer a potential alternative, yet their translation has been hindered by low engraftment, poor localization, and a lack of delivery strategies that are both effective and minimally invasive. To address these challenges, we developed a new approach termed INSITE (Injected Self-assembled Image-guided Tissue Ensembles), an injectable platform composed of primary human hepatocytes and hydrogel microspheres that can be delivered by image-guided injection and assembled in situ into supportive, vascularizable scaffolds. In vivo, ultrasound-guided delivery into an ectopic site enabled precise graft localization, persistent visibility under noninvasive imaging, and vascular integration. Hepatocytes within these niches remained confined to the scaffold and maintained long-term functional activity. Furthermore, tuning material properties allowed control over scaffold remodeling and vascular recruitment, providing a means to enhance graft function. By integrating image-guided delivery with a modular and supportive scaffold, INSITE establishes a clinically compatible strategy for advancing minimally invasive cell therapies.

bioengineering↗

bioRxiv2025

Synthetic Control of Implanted Engineered Liver Tissue Growth

Despite the promise of engineered tissue implants for the treatment of organ failure, scaling of these constructs to sizes of therapeutic relevance remains a barrier to clinical translation. Here, we propose a strategy to circumvent this limitation: to instead implant a small-scale construct and then induce it to grow in situ after its engraftment into a host. Using engineered liver tissue as a proof-of-concept application, we integrated synthetic biology and tissue engineering tools to build liver tissues that can be expanded on-demand after implantation in vivo. To achieve this goal, we first identified the combination of YAP and growth factor signaling as sufficient to drive human hepatocyte proliferation in dense, 3D engineered tissues. We then engineered control of these signaling axes using synthetic biology tools to drive human liver tissue expansion both in vitro and in vivo. As such, this work establishes a genetic strategy for generating large organ implants through bioengineered, on-demand outgrowth using synthetic triggers (BOOST). TeaserPresenting bioengineered on-demand outgrowth via synthetic biology triggering (BOOST) for in situ solid cell therapy scale up.

bioengineering↗

bioRxiv2025

NMDA receptor ablation in medial prefrontal cortex disrupts value updating and reward history integration

Schizophrenia, a serious mental illness, is associated with evidence of NMDA receptor (NMDAR) dysfunction and characterized by cognitive impairments that reflect impaired value updating and feedback-driven control; however the cellular and circuit-level mechanisms underlying these disruptions remain unclear. Here we test how NMDA receptor (NMDAR) signaling in the medial prefrontal cortex (mPFC) contributes to adaptive decision-making by combining targeted genetic ablation in mice and systemic pharmacology. Using a CRISPR-Cas9 approach to eliminate the obligate GluN1 subunit, we induced spatially confined NMDAR hypofunction in mPFC and compared its effects to systemic pharmacological blockade with the NMDAR antagonist MK-801 during performance of a touchscreen-based restless bandit task. Prefrontal NMDAR ablation impaired value discrimination, weakened the use of negative feedback, and reduced mutual information between recent outcomes and current choices, indicating disrupted reward-history integration. Reinforcement-learning models incorporating a choice-kernel term best captured behavior and revealed that NMDAR ablation selectively dampened learning and choice-history parameters governing flexible updating. Systemic MK-801 produced broad impairments in control animals, reducing accuracy, mutual information, and outcome sensitivity, yet exerted only modest additional effects after NMDAR ablation, suggesting that prefrontal NMDAR loss occluded much of the pharmacological disruption. Simulations using fitted RLCK parameters reproduced these patterns, showing convergent flattening of choice dynamics under MK-801 and persistent deficits in GluN1 ablated animals. Together, these findings demonstrate that prefrontal NMDAR signaling is necessary for effective value updating and feedback-driven learning, and that its loss recapitulates core features of systemic NMDAR hypofunction. This work establishes a mechanistic bridge between localized cortical glutamatergic dysfunction and the reinforcement-learning disturbances characteristic of schizophrenia.

neuroscience↗

bioRxiv2025

Next-generation enhancer AAVs for selective interspecies targeting of midbrain dopaminergic neurons

Using single-nucleus multiomic profiling of the marmoset midbrain, we develop and benchmark enhancer-AAVs to selectively access dopamine (DA) neurons in wild-type animals without combinatorial systems. In vivo candidate screenings identified one highly specific DA enhancer, cjDAE8, in both mice and marmosets. To overcome low-level off-target (leaky) expression, a common limitation for enhancer AAVs, we engineered next-generation AAV backbones that strengthen expression while minimizing leakiness. Quantitative histology comparing natural versus antibody-amplified fluorescence defined AAV doses to achieve high labeling efficiency with greater than 90-95% DA-neuron specificity across species and injection routes. We further demonstrate applications of DA-enhancer-AAVs for (i) retrograde targeting of projection-defined DA populations in marmoset, (ii) fiber-photometric recording of divergent DA-axonal dynamics in striatal subregions, and (iii) optogenetic VTA-DA self-stimulation in mice. Our results establish a resource for cross-species DA targeting and two practical guidelines: backbone context critically shapes enhancer performance, and antibody-amplified readouts rigorously assess specificity.

neuroscience↗

bioRxiv2025

Electrical stimulation directs formation of perfused vasculature in engineered tissues

Effective, rapid and functionally perfusable vascularization remains a major challenge in tissue engineering. Current approaches to generate vasculature in vitro require multipart fabrication methods or complex and costly media supplements, limiting their scalability. Here, we demonstrate that exogenous electrical stimulation (estim) offers a promising alternative by enhancing 3D vasculogenesis in engineered human tissues. Exposing 3D endothelial-fibroblast cocultures to pulsed estim promoted the formation of dense and branched vascular networks. In a microfluidic device model, we show that estim induces the formation of an interconnected vascular network that can be perfused, whereas unstimulated control networks remained less mature. Importantly, we demonstrate that upon implantation, estim-pretreated vascular grafts exhibit elevated anastomosis with host and perfusion with blood relative to the untreated grafts. In addition, we use estim to promote engraftment of a vascularized 3D liver construct. Mechanistically, we find that estim induces membrane hyperpolarization in endothelial cells via voltage-gated potassium (KV) channels. Inhibiting KV channels abrogated estims pro-vasculogenic effects in endothelial cells. Conversely, pharmacologically activating hyperpolarization induced endothelial responses even without estim, directly linking KV channel-mediated hyperpolarization as a key mechanism by which estim drives vascular assembly and function. Ultimately, our work establishes estim as a new orthogonal approach to promote formation of perfusable vasculature in engineered tissues.

bioengineering↗

bioRxiv2025

A 3D in vitro model of the human hepatobiliary junction

Cholestasis, or disruption in bile flow, is a common yet poorly understood feature of many liver diseases and injuries. Despite this, many engineered human tissue models of liver disease fail to recapitulate physiological bile flow. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction, the interface between hepatocytes and cholangiocytes often disrupted in liver disease that is required for directing bile excreted by hepatocytes into the biliary ductal system. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes into adult hepatobiliary organoids (aHBOs) that structurally connect and functionally transport bile. aHBOs directionally transport bile from hepatocyte bile canaliculi to cholangiocyte-lined ductules, which we visualize through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using fluorescent bile acid analogs and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs recapitulate features of biliary dysfunction that mimics the cholestasis and ischemia-reperfusion injury that complicates liver transplant. Our findings suggest that 1) a reversible reduction in hepatocyte canalicular function under hypoxia, followed by 2) selective cholangiocyte death upon reoxygenation, are processes that potentially contribute to biliary dysfunction upon ischemic injury. This human-derived, scalable platform provides a phenotypically-relevant in vitro model for dissecting biliary pathophysiology and lays the groundwork for a therapeutic discovery platform for post-transplant ischemic cholangiopathy and other cholestatic liver diseases.

bioengineering↗

bioRxiv2025

16p11.2 duplication shows early male-biased impacts on reward learning, but NMDA receptor antagonism reduces optimal choice selection in both wildtypes and 16p11.2 duplication

Rationale16p11.2 duplication is associated with numerous neuropsychiatric conditions at a genome-wide level, including psychosis. Mice modeling 16p11.2 duplication may provide important insights into cognitive risk factors, in particular in reward-guided decision making. NMDAR function has also been implicated in psychosis phenotypes, but whether these phenotypes differ by genetic risk factor is unknown. ObjectivesWe aimed to: 1) identify sex and genotype differences in early operant training and two-arm spatial restless bandit task performance; 2) examine the effects of an NMDAR antagonist on task performance and strategy across genotypes. Methods16p11.2 duplication and wildtype mice completed a series of training schedules of escalating difficulty followed by bandit tasks. MK-801 and saline were administered in alternating sessions prior to later bandit task performance. ResultsLarge sex differences in early operant training revealed some male-biased impacts of 16p11.2 duplication, contingent on training schedule difficulty. Once on the two-arm spatial restless bandit task, 16p11.2 duplication was no longer a strong contributor to decision making. However, MK-801 decreased the tendency to stay with a rewarded choice, lowered the probability of selecting the highest rewarded option, and decreased the influence of prior outcomes on choice. ConclusionsThe male-biased vulnerability in early operant training suggests that strategies for learning early schemas or in novel environments may be impacted by 16p11.2 duplication in males. In contrast, NMDAR are influential in the ability to flexibly switch between choices, and disrupting this function significantly impairs decision making in all animals.

animal behavior and cognition↗

bioRxiv2025

Sex-biased computations underlying differential set shift performance in mice

Cognitive flexibility can be defined as the ability to adaptively shift between choices or strategies based on environmental feedback and it is disrupted in numerous neuropsychiatric conditions. Individual differences in the computations supporting cognitive flexibility are poised to reveal mechanisms of neuropsychiatric risk and resilience. One critical variable well known to influence individual differences in neuropsychiatric risk is sex. While previous research has identified sex differences in value based decision making in mice, whether sex reflects a major source of variation in cognitive flexibility remains unknown. To directly assess sex-biased individual differences in cognitive flexibility, we developed a novel touchscreen Set Shift task that permits robust and continuous testing in mice. Using this task, we discovered that female mice completed significantly more rule shifts with fewer errors than males. We next employed a suite of computational models that revealed sex-biased individual differences in the computations underlying cognitive flexibility. Overall, our results suggest that following rule shifts, female mice learn the new rule faster and commit to exploiting rule choices sooner compared to males - sometimes because they commit to multiple rules simultaneously. This suggests that increased choice stability in female rodents enhances commitment to a strategy during periods of uncertainty and directly contributes to increased rule shifting. This supports the counterintuitive conclusion that a high degree of stable choice is a strong requirement for enhanced cognitive flexibility in the Set Shift task, one of the gold standard cognitive flexibility tasks.

animal behavior and cognition↗

bioRxiv2024

Expandable, Functional Hepatocytes Derived from Primary Cells Enable Liver Therapeutics

Liver disease affects millions annually in the United States, with orthotopic transplantation as the only curative option for many patients. However, the scarcity of donor organs highlights a need for alternative cell-based therapies. Hepatocyte-based approaches are promising due to the cells inherent synthetic, metabolic, and detoxifying functions, but they face critical barriers, including the lack of a scalable source of functional hepatocytes and poor engraftment. In this study, we developed a scalable process for expanding primary human hepatocytes (PHHs) while preserving their identity and function. By leveraging heterocellular aggregation with stromal cells, we generated cryopreserved "seed" constructs that maintained viability and function post-thaw. Seeds demonstrated enhanced metabolic and detoxification functions and robust engraftment across multiple anatomic sites outside of the liver. Our approach addresses key limitations of hepatocyte-based therapies, offering a stable, scalable, and clinically viable platform for liver cell therapy applications.

bioengineering↗

bioRxiv2024

Touchscreen response precision is sensitive to the explore/exploit tradeoff

The explore/exploit tradeoff is a fundamental property of choice selection during reward-guided decision making. In perceptual decision making, higher certainty decisions are more motorically precise, even when the decision does not require motor accuracy. However, while we can parametrically control uncertainty in perceptual tasks, we do not know what variables - if any - shape motor precision and reflect subjective certainty during reward-guided decision making. Touchscreens are increasingly used across species to measure choice, but provide no tactile feedback on whether an action is precise or not, and therefore provide a valuable opportunity to determine whether actions differ in precision due to explore/exploit state, reward, or individual variables. We find all three of these factors exert independent drives towards increased precision. During exploit states, successive touches to the same choice are closer together than those made in an explore state, consistent with exploit states reflecting higher certainty and/or motor stereotypy in responding. However, exploit decisions might be expected to be rewarded more frequently than explore decisions. We find that exploit choice precision is increased independently of a separate increase in precision due to immediate past reward, suggesting multiple mechanisms regulating choice precision. Finally, we see evidence that male mice in general are less precise in their interactions with the touchscreen than females, even when exploiting a choice. These results suggest that as exploit behavior emerges in reward-guided decision making, individuals become more motorically precise reflecting increased certainty, even when decision choice does not require additional motor accuracy, but this is influenced by individual differences and prior reward. These data uncover the hidden potential for touchscreen tasks in any species to uncover the latent neural states that unite cognition and movement.

animal behavior and cognition↗

bioRxiv2024

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o-nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

synthetic biology↗

bioRxiv2024

Persistent Decision-Making in Mice, Monkeys, and Humans

Humans have the capacity to persist in behavioural policies, even in challenging environments that lack immediate reward. Persistence is the scaffold on which many higher executive functions are built. However, it remains unclear whether humans are uniquely persistent or, instead, if this capacity is widely conserved across species. To address this question, we compared humans with mice and monkeys in harmonised versions of a dynamic decision-making task. The task encouraged all species to strike a balance between persistently exploiting one policy and exploring alternative policies that could become better at any moment. Although all three species had similar strategies, we found that both primate species--humans and monkeys--were able to persist in exploitation for longer than the mice. The similarities in persistence patterns in humans and monkeys, as opposed to mice, may be related to the various ecological, neurobiological, or cognitive factors that differ systematically between these species.

animal behavior and cognition↗

bioRxiv2024

Three-dimensional flow assessment of microvascular beds with interstitial space

The health and function of microvascular beds are dramatically impacted by the mechanical forces that they experience due to fluid flow. These fluid flow-generated forces are challenging to measure directly and are typically calculated from experimental flow data. However, current computational fluid dynamics (CFD) models either employ truncated 2D models or overlook the presence of extraluminal flows within the interstitial space between vessels that result from the permeability of the endothelium lining the vessels, which are crucial components affecting flow dynamics. To address this, we present a bottom-up modeling approach that assesses fluid flow in 3D-engineered vessel networks featuring an endothelial lining and interstitial space. Using image processing algorithms to segment 3D confocal image stacks from engineered capillary networks, we reconstructed a 3D computational model of the networks. We incorporated vascular permeability and matrix porosity values to model the contributions of the endothelial lining and interstitial spaces to the flow dynamics in the networks. Simulations suggest that including the endothelial monolayer and the interstitium significantly affects the predicted flow magnitude in the vessels and flow profiles in the interstitium. To demonstrate the importance of these factors, we showed experimentally and computationally that while cytokine (IL-1{beta}) treatment did not affect the network architecture, it significantly increased vessel permeability and resulted in a dramatic decrease in wall shear stresses and flow velocities intraluminally within the networks. In conclusion, this framework offers a robust methodology for studying flow dynamics in 3D in vitro vessel networks, enhancing our understanding of vascular physiology and pathology. TRANSLATIONAL IMPACT STATEMENTThis study introduces a new approach to modeling and flow assessment in 3D microvascular beds and surrounding interstitial spaces. Modeling interstitial space and endothelial monolayer thickness is essential for capturing fluid leakage from the microvascular network into the interstitial space and vice versa when the endothelial monolayer permeability is significantly affected in pathological conditions. Our approach to modeling 3D vascular networks can be used in vivo and in clinical settings to understand flow in tissue microvasculature and its surroundings under disease and healthy conditions.

bioengineering↗

bioRxiv2024

Perivascular cells function as mechano-structural sensors of vascular capillaries

A wide range of conditions, including chronic inflammatory diseases and cancer, are characterized by the fibrotic microarchitecture and increased stiffness of collagen type I extracellular matrix. These conditions are typically accompanied by altered vascular function, including vessel leakiness, abnormal capillary morphology and stability. The dynamic cell-matrix interactions that regulate vascular function in healthy tissues have been well documented. However, our understanding of how the gradual mechanical and structural alterations in collagen type I affect vascular homeostasis remains elusive, especially as a function of the interactions between endothelial and perivascular cell with the altered matrix. Here we hypothesized that perivascular cells might function as mechano-structural sensors of the microvasculature by mediating the interaction between endothelial cells and altered collagen type I. To test that, we utilized an organotypic model of perivascular cell-supported vascular capillaries in collagen scaffolds of controlled microarchitecture and mechanics. Our results demonstrate that capillaries cultured in soft reticular collagen exhibited consistent pericyte differentiation, endothelial cell-cell junctions, and barrier function. In contrast, capillaries embedded in stiff and bundled collagen fibrils to mimic a more fibrotic matrix induced abluminal migration of perivascular cells, increased leakage, and marked expression of vascular remodeling and inflammatory markers. These patterns, however, were only observed when endothelial capillaries were engineered with perivascular cells. Silencing of NOTCH3, a mediator of endothelial-perivascular cell communication, largely re-established normal vascular morphology and function. In summary, our findings point to a novel mechanism of perivascular regulation of vascular dysfunction in fibrotic tissues which may have important implications for anti-angiogenic and anti-fibrotic therapies in cancer, chronic inflammatory diseases and regenerative medicine. Significance StatementThe fibrotic alterations in extracellular matrix structure and mechanics that are common to many chronic and inflammatory conditions are often associated with a decrease in vascular homeostasis. The mechanisms regulating these abnormalities remain poorly understood. Here, we demonstrate that perivascular cells play a critical role in sensing progressive microarchitectural and mechanical changes occurring in the ECM, drastically altering vascular capillary morphology and barrier function, and exacerbating the production of inflammatory and remodeling markers. These results point to a previously unknown mechano-structural sensory mechanisms mediated by perivascular cells in vascular capillaries that may help elucidate the progression of many profibrotic conditions, and point to possible new targets for antiangiogenic and antifibrotic therapies in cancer, chronic inflammatory conditions and regenerative medicine.

bioengineering↗

bioRxiv2023

Matrix architecture and mechanics regulate myofibril organization, costamere assembly, and contractility of engineered myocardial microtissues

The mechanical function of the myocardium is defined by cardiomyocyte contractility and the biomechanics of the extracellular matrix (ECM). Understanding this relationship remains an important unmet challenge due to limitations in existing approaches for engineering myocardial tissue. Here, we established arrays of cardiac microtissues with tunable mechanics and architecture by integrating ECM-mimetic synthetic, fiber matrices and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), enabling real-time contractility readouts, in-depth structural assessment, and tissue-specific computational modeling. We find that the stiffness and alignment of matrix fibers distinctly affect the structural development and contractile function of pure iPSC-CM tissues. Further examination into the impact of fibrous matrix stiffness enabled by computational models and quantitative immunofluorescence implicates cell-ECM interactions in myofibril assembly and notably costamere assembly, which correlates with improved contractile function of tissues. These results highlight how iPSC-CM tissue models with controllable architecture and mechanics can inform the design of translatable regenerative cardiac therapies.

bioengineering↗