Bioengineering & Translational Medicine

Human salivary stem/progenitor cell (hS/PC)-loaded hyaluronic acid (HA)-based hydrogels, termed 3D-salivary tissue constructs (3D-ST), hold great promise for restoring salivary gland function post-radiation injury. Here, we developed a next-generation 3D-ST using heparin-modified HA and bioactive peptide-modified hydrogels. This new formulation enables controlled pre-loading and localized presentation of heparin-binding growth factors prior to surgical implantation, providing opportunities to enhance in vivo hS/PC bioactivity. To model clinically relevant radiation injury, we established an athymic rat model subjected to computed tomography (CT)-guided fractionated radiation, resulting in hallmark features of radiation-induced salivary dysfunction. Over 60-days post-irradiation, glands exhibited progressive loss of acini, increased fibrosis, and disruption of endothelial, neuronal, and myoepithelial compartments. Within this injured environment, a surgical pocket was created to precisely implant 3D-STs to assess graft performance. Fluorescent labeling of the 3D-STs enabled longitudinal tracking post-implantation. Over 14 days, implanted 3D-STs remained structurally stable within irradiated glands, and hS/PCs remained viable without evidence of local inflammatory responses. Compared to non-injured glands, the irradiated microenvironment suppressed hS/PC proliferation and phenotype, indicating alterations in the irradiated local tissue negatively impact hS/PC bioactivity. In addition, host neurovascular migration into the 3D-ST was majorly restricted in irradiated glands, providing new opportunities to enhance biointegration. Overall, this work establishes a reproducible preclinical framework for assessing hydrogel biocompatibility and stability, cell bioactivity, and host-graft biointegration prior to scale up into preclinical large animal models. This study has successfully established a tractable approach for improving 3D-ST formulations to enhance hS/PC expansion, differentiation, and biointegration following implantation into radiation-injured beds.

Precision-cut lung slices (PCLS) retain the native cells and extracellular matrix that contribute to the structural and functional integrity of lung tissue. This technique enables the study of cell-matrix interactions and is particularly useful for pre-clinical pharmacological studies. More specifically, PCLS are widely used to model the complex pathophysiology of pulmonary fibrosis, an uncurable and progressive interstitial lung disease. Current ex vivo pulmonary fibrosis models expose PCLS to pro-fibrotic biochemical cues over a short timeframe (hours to days) and quickly collect samples for analysis due to viability concerns. This condensed timeline is a limitation to understanding chronic disease mechanisms. To extend the utility of ex vivo pulmonary fibrosis models, PCLS were embedded in engineered hydrogels and exposed to pro-fibrotic biochemical and biophysical cues. Hydrogel-embedded PCLS maintained greater than 80% total cell viability over 3 weeks in culture. Gene expression patterns in samples exposed to pro-fibrotic cues matched trends measured in human fibrotic lung tissue. Finally, treatment with Nintedanib, a Food and Drug Administration approved pulmonary fibrosis drug, moderately reduced fibroblast activation and influenced epithelial cell differentiation. Collectively, these results show that hydrogel-embedded PCLS models of pulmonary fibrosis extend our ability to study fibrotic processes ex vivo and, when applied to human tissues, present a new approach methodology for studying lung disease and treatment.

Non-invasive tracking of natural killer (NK) cells remains a major challenge in cancer immunotherapy, limiting our understanding of their in vivo migration and persistence. Magnetic particle imaging (MPI) offers a quantitative, real-time method for visualizing labeled cells, yet optimal labeling protocols for NK cells have not been established. Here, we evaluate commercially available iron oxide nanoparticles (IONPs) for MPI labeling of both NK92MI cells and primary human NK cells. Labeled cells retained viability and cytotoxicity, including activity against three-dimensional tumor spheroids, and were detectable by MPI. To further examine imaging performance in a biologically relevant context, we employed mouse phantoms that recapitulate organ-specific signal distributions, enabling evaluation of quantification and liver spillover effects. We identify key tradeoffs between particle colloidal stability and per-cell iron content: VivoTrax and VivoTrax Plus provided higher MPI signal but required post-labeling purification, reducing cell recovery, whereas Synomag-D and Perimag were more stable and preserved cell yield despite lower signal intensity per cell. These results provide a framework for selecting nanoparticles that balance detection sensitivity, cell viability, and workflow practicality, advancing non-invasive NK cell tracking.

Head and neck cancer (HNC) is the 6th most common malignancy worldwide. 60% of patients present with advanced disease and approximately 50% of patients recur following primary treatment. Chemoradiation remains a standard of care for most patients. However, clinicians lack functional tools to predict which patients will respond to chemoradiation prior to treatment and current models, including organoids and animal model systems, fail to capture either full complexity or patient-to-patient heterogeneity of the individual HNC tumor and microenvironment (TME). Here, we have developed, characterized, and tested a patient-specific microphysiological system (MPS) that reconstructs the HNC TME in a vascularized 3D environment. This MPS was constructed from malignant cells, fibroblasts, and immune cells from a patients surgically resected tumor, seeded within a 3D hydrogel with molded endothelial lumens. Single-cell RNA sequencing confirmed that the MPS preserved 12 transcriptionally distinct cell populations found in matched native tissue. The platform recapitulated tumor hypoxia, with a 12-fold increase in hypoxic marker expression that altered radiation response, consistent with clinical HNC biology. Compartment-resolved imaging revealed distinct treatment dynamics in tumor, stromal, and vascular regions, and individual patients exhibited divergent responses to chemoradiation in spheroid morphology, cell viability, and migration. We found the slope of spheroid area change with treatment tracked with tumor recurrence, suggesting this metric could serve as a functional predictor of therapeutic response.

Enhancing targeted delivery of biomedicines improves efficacy and can reduce off-target effects by lowering the effective dose, but achieving targeting is challenging. Extracellular vesicles (EVs) are promising biological nanovesicles which can be targeted by displaying binding proteins and are being developed as therapeutics. Currently, discovering EV targeting constructs is limited by low throughput and resource-intensive EV production and isolation. To accelerate discovery, we developed a screening pipeline to identify EV targeting constructs without requiring EV production. This approach is premised on the hypothesis that cell-cell interactions may predict some cell-EV interactions. Our cell binding assay (CELLISA) quantifies binding of a cell surface-displayed targeting protein to its cognate receptor on a target cell, employing a microscopy-based analysis pipeline. After validating the premise using existing T cell-targeting reagents, we develop CELLISA for either adherent or suspension EV producer cells. Finally, we use CELLISA to evaluate new binders and validate that hits mediate targeting and/or delivery of genetic cargo to natural killer cells and T cells. CELLISA increased throughput > 6-fold and decreased time by 40% compared to standard EV screens, and it identified a T-cell binder conferring efficient gene delivery. CELLISA is easily adaptable to other laboratories and can accelerate EV research.

The limiting step in immune-active drug development is increasingly not candidate generation but testing whether a candidate therapy is effective in a system that preserves tissue architecture, vascular exposure, multicellular interaction, and repeated pharmacodynamic sampling without patient exposure. We developed an acellular normothermic machine-perfusion platform for intact porcine spleen designed as a translational immune-organ assay. Across independent acellular perfusions, the circuit maintained physiologic parameters, preserved red- and white-pulp histology, and yielded viable effluent cells suitable for serial flow cytometry and multiomics. High-dose methylprednisolone was used as a clinically familiar perturbation to determine whether the platform could resolve steroid immunosuppression at mechanistically distinct levels. Effluent RNA-seq identified canonical glucocorticoid-responsive transcriptional programs, including DUSP1, FKBP5, PER1, DDIT4, SGK1, KLF9, ANXA1, NF-{kappa}B feedback regulators, and JAK/STAT suppressor pathways. SOCS3 was a prominent early signal in the perfusion transcriptome and was validated orthogonally at the protein level in prednisone-treated, CD3/CD28-activated primary murine splenocytes, strengthening its role as a candidate pharmacodynamic marker. In parallel, data independent acquisition (DIA) proteomics of effluent cell pellets nominated a non-transcriptional protein-level response: a Sus scrofa LGALS13-annotated, CLC/Galectin-10-like galectin detected despite absence of the corresponding effluent-cell transcript. Because this porcine LGALS13-annotated protein group is treated here as an orthologous CLC/Galectin-10-like signal rather than as canonical human placental Galectin-13/PP13, we tested recombinant human Galectin-10 in vitro. Human Galectin-10 induced marked apoptosis of CD3/CD28-stimulated Jurkat cells, prioritizing this axis for future mechanistic testing without proving causality in the perfused spleen. These data establish acellular spleen perfusion as a serial multiomic platform for translational immunopharmacology and motivate deployment with otherwise-discarded human donor spleens. One sentence summaryAn acellular intact-spleen perfusion platform enables serial cellular, transcriptomic, proteomic, and functional pharmacodynamic sampling that identifies steroid-responsive transcriptional programs, validates SOCS3 protein induction, and nominates a CLC/Galectin-10-like non-transcriptional immunosuppressive axis for translation to discarded human donor spleens.

Pancreatic ductal adenocarcinoma (PDAC) remains difficult to treat with nucleic acid therapeutics because efficient intratumoral delivery is limited and off-target liver accumulation is common. Here, we developed a structure-activity map for intraperitoneally administered mRNA lipid nanoparticles (mRNA-LNPs) to identify formulation features that improve delivery to pancreatic tumors while reducing liver expression. A full-factorial library of 48 mRNA-LNP formulations was generated by varying ionizable lipid, sterol, phospholipid, and PEG-lipid components. Formulations were characterized for size, polydispersity, zeta potential, and encapsulation, then evaluated in an orthotopic KPC8060 pancreatic tumor model after intraperitoneal administration of firefly luciferase mRNA-loaded LNPs. Biodistribution was assessed by Rhodamine B fluorescence and functional delivery by luciferase expression 12 h after dosing. Lipid composition strongly influenced both physicochemical properties and in vivo performance. G0-C14-based formulations produced the smallest and most homogeneous particles, whereas FTT5-containing formulations were generally larger. Across the 48-formulation library, mRNA expression and nanoparticle biodistribution varied significantly among tumor, pancreas, liver, and spleen. Statistical, decision-tree, and predictive modeling analyses identified composition rules associated with organ-selective delivery. High tumor expression was associated primarily with G0-C14 combined with DSPC and {beta}-sitosterol, whereas liver expression was favored by C12-200 or DLin-MC3-DMA with DOPE and DSPE-PEG. Notably, a G0-C14/DSPC/DSPE-PEG formulation emerged as a lead candidate, producing a greater than 6-fold increase in tumor luciferase signal relative to the library median while reducing liver exposure by approximately 60%. Histopathology showed no treatment-related liver or lung toxicity. These findings define actionable formulation rules for tuning intraperitoneal mRNA-LNP delivery in PDAC and support further development of tumor-selective mRNA therapeutics for pancreatic cancer.

ObjectiveEvaluate the effects of bioabsorbable magnesium wires on dermal wound healing and tissue regeneration in a murine full-thickness wound model. Approach6 mm diameter stented dorsal skin wounds were created in C57BL/6J mice and treated with implanted WE43B magnesium alloy wires or PBS control. Wound healing was evaluated on days 7 and 28 by histology, immunohistochemistry, and micro-CT. Finite element analysis modeled mechanical strain distribution due to wire degradation during healing. ResultsAt day 7, magnesium wire-treated wounds showed 100% improved granulation tissue formation, reduced inflammation (37% fewer CD45+ leukocytes and 37% fewer F4/80+ macrophages), increased neovascularization (91% more CD31+ lumens), and 74% more nerve bundles. Improved wound closure (mean difference -1.48 mm) did not reach statistical significance (d = 1.06). By day 28, magnesium-treated wounds showed improved collagen organization and normalized epidermal thickness. The increase in dermal appendages (247%), and vascular density (41%) did not reach statistical significance. Micro-CT confirmed progressive wire degradation. Modeling revealed that degrading wires dynamically altered strain gradients in healing tissue, thereby modulating the spatial mechanical cues that govern fibroblast migration and extracellular matrix (ECM) remodeling. InnovationMagnesium is an essential trace element involved in cellular processes critical to wound repair, including angiogenesis, nerve growth, inflammation modulation, and ECM remodeling. Previous magnesium delivery systems incorporated polymers or other confounding materials that degrade rapidly. We directly applied bioabsorbable pure magnesium metal to provide sustained ion release and favorable mechanical properties to support regenerative healing. ConclusionBioabsorbable magnesium wires support regenerative wound healing by reducing inflammation, enhancing neovascularization, and promoting favorable ECM remodeling without adverse inflammatory reactions. These findings provide a strong rationale to harness magnesium metal use in wound healing applications.

Intratumoral delivery of immunotherapy offers a means to enhance efficacy while limiting systemic toxicity, yet rapid diffusion from the tumor constrains dosing levels. Extracellular matrix-targeted anchoring strategies have emerged to improve tumor retention, but the influence of matrix target choice remains poorly understood. Here, we engineered a hyaluronic acid-anchoring platform and directly compared it to a well-established collagen-binding strategy for the delivery of IL-12/IL-15 combination therapy, assessing pharmacokinetic, efficacy, and toxicity endpoints. Hyaluronic acid anchoring markedly enhanced intratumoral retention and tumor loading relative to both unanchored and collagen-anchored constructs. While all anchored cytokine therapies achieved comparable curative tumor control, hyaluronic acid anchoring was associated with improved tolerability, including attenuated systemic inflammation, reduced liver toxicity, and diminished local tissue damage. Analysis of intratumoral immune signaling further indicated that the anchoring strategy modulates local cytokine exposure and immune cell infiltration, despite similar therapeutic outcomes. These findings demonstrate that extracellular matrix target selection significantly shapes the pharmacologic and safety profiles of intratumoral biologics, and identify hyaluronic acid anchoring as an alternative retention strategy with potential advantages.

In vitro models are increasingly critical for interrogating cancer biology and therapeutic response, however, accurately recapitulating the tumor microenvironment (TME) remains a persistent challenge, particularly in head and neck cancers (HNC) characterized by complex cell-matrix interactions and heterogeneity. Current models often lack independent tunability of biochemical and biophysical cues, limiting systematic investigation of microenvironmental cues in a high-throughput format. Here, we establish a 3D droplet-based bioprinting platform for the fabrication of customizable in vitro TME models using poly(ethylene glycol) (PEG) hydrogels. Human HNC cell lines (FaDu and 2A3) with differing HPV statuses were bioprinted into PEG matrices spanning physiologically relevant stiffnesses (0.7-4.8 kPa) and compositions, including non-functionalized PEG and peptide-functionalized PEG (PEGfnc: RGD, YIGSR, CNYYSNS) and cultured for 7 days. Cluster growth, cell viability, and cluster morphology were assessed across multiple time points, matrix compositions, and matrix stiffnesses. Proliferation and endpoint phenotype expression were visualized using confocal microscopy through immunofluorescence. Results indicated enhanced cell viability in PEGfnc matrices, compared to non-functionalized matrices, while effect of matrix stiffness was less prominent. Median cluster size reached 40-50 m by day 7, and linear mixed-effects modeling identified how changes in cluster surface area, volume, and tumoroid complexity varied with cell type, matrix, and stiffness. By decoupling and systematically varying key TME parameters, this approach provides a robust and scalable framework for dissecting tumor-matrix interactions and advancing physiologically relevant in vitro models for cancer research and therapeutic screening.

Intratumoral (i.t.) delivery of nanoparticles (NPs) is widely used to achieve high local NP concentrations. However, the temporal fate of i.t.-injected NPs remains poorly understood. We present a quantitative approach using whole-body magnetic particle imaging (MPI) to track magnetic NPs (MNPs) following i.t. injection. Using fiducial-calibrated imaging, we quantified MNP mass over time in subcutaneous 4T1 breast tumors. Longitudinal imaging revealed progressive loss of i.t. MNP content and heterogeneous systemic redistribution across animals despite standardized delivery conditions. Ex vivo MPI confirmed off-target accumulation primarily in the liver and spleen, consistent with reticuloendothelial clearance pathways. Histological analysis demonstrated spatially heterogeneous i.t. MNP deposition, potentially associated with local vascular features and tumor microenvironmental heterogeneity that may influence i.t. MNP retention or MNP clearance from the tumor. These findings highlight the importance of quantitative longitudinal whole-body MPI for understanding the fate of MNPs for informing localized nanotherapy.

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

Medulloblastoma (MB) is an aggressive central nervous system (CNS) malignancy that primarily affects children and frequently exhibits metastasis to the leptomeninges of the brain and spinal cord. We developed a {beta}-Cyclodextrin-poly({beta}-Amino Ester) nanoparticle system to deliver the histone deactylase inhibitor (HDACi) Panobinostat to MB by the intrathecal route. Various imaging methods were utilized to study nanoparticle and payload fate following infusion into the cerebrospinal fluid (CSF) of mice via cisterna magna or lumbar access points. Nanoparticles dramatically improved penetration of hydrophobic small molecules into distal regions of the spinal cord. Panobinostat-loaded nanoparticles were effective at treating patient-derived MB, activating pharmacodynamic targets, slowing growth of the primary tumor, decreasing incidence of metastasis at the time of death, and ultimately prolonging survival. These studies provide insight into the mechanisms mediating transport of colloids and therapeutic molecules in the subarachnoid space and highlight new approaches for treating metastatic disease in the CNS.

Cyclophosphamide (CP) is a widely used alkylating agent whose cytotoxic activity depends on hepatic CYP450-mediated bioactivation. While CP-associated neurotoxicity and cognitive impairment are recognized clinically, the mechanisms of secondary organ damage through metabolic cross-talk remain poorly understood due to limitations of conventional monoculture models. Here we employ a multi-organ microphysiological system (MPS) connecting stem cell derived liver and CNS organoids via microfluidic channels to model inter-organ drug metabolism and secondary toxicity. Liver organoids were treated with CP (0-200 {micro}M) for 48 hours, and connected CNS organoids were assessed for secondary damage by confocal Z-stack imaging of DNA damage ({gamma}H2AX), neuronal identity (NeuN), and nuclear content (DAPI). We observe dose-dependent reduction in NeuN expression and {gamma}H2AX signal in connected CNS organoids, consistent with neurotoxic metabolite transfer from liver. Critically, CNS-to-CNS control connections show no comparable damage at equivalent CP concentrations, confirming that hepatic metabolism is required for CNS toxicity. These findings validate the MPS platform for modelling multi-organ drug toxicity and provide direct evidence that liver-derived CP metabolites drive secondary neurotoxicity through inter-organ metabolic communication.

Extracellular vesicles (EVs) are versatile therapeutic candidates due to biological roles in intercellular communication and amenability to bioengineering. Compared with lipid nanoparticles (LNPs), native or surface-modified EVs may have favorable immunogenicity and biodistribution profiles. However, when administered intravenously (IV), EVs are rapidly cleared and accumulate mostly in the liver and spleen. With the goal of modifying EV biodistribution, we engineered EVs to display the human endogenous retrovirus (HERV) envelope glycoprotein Syncytin-1, an SLC1A5-binding fusogenic viral protein essential for syncytiotrophoblast formation in pregnancy. Here, we comprehensively characterize engineered Syncytin-1+ EVs, examine their interactions with cells in vitro, and assay biodistribution, immunogenicity, and pharmacokinetics ex vivo and in vivo in non-human primates. IV-administered Syncytin-1+ EVs are well tolerated, persist in the blood stream, and have altered organ biodistribution compared with unmodified EVs, suggesting therapeutic potential of Syncytin-1+ EVs at specific sites.

Sex differences influence distinct inflammatory responses after spinal cord injury (SCI), yet their impact on immune-modulating nanotherapeutics remains unclear. Here, we investigated the sex-dependent effects of drug-free poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) following SCI. Systemic NP administration enhanced locomotor recovery in both sexes and eliminated the functional gap observed in controls. Mechanistically, NPs engaged distinct immune pathways between sexes. Females accumulated more NPs in the spleen, leading to reduced monocyte-derived macrophage infiltration, whereas males showed greater NP accumulation at the lesion and attenuated microglial activation. Transcriptomic analysis showed preferential modulation of eicosanoid-related pathways in females and NF-{kappa}B-linked signaling in males. These sex-specific, yet convergent NPs-induced immunomodulatory effects reduced fibrotic scarring and enhanced remyelination, with females showing greater Schwann cell-mediated repair and males exhibiting marked suppression of microglial activation. Collectively, these findings demonstrate that NPs promote comparable functional recovery in both sexes through distinct, sex-influenced immune mechanisms and establish a translational framework for sex-informed immune targeting and nanotherapeutic design in SCI and other inflammation-mediated diseases. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/709912v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5af2ecorg.highwire.dtl.DTLVardef@1027071org.highwire.dtl.DTLVardef@12449ceorg.highwire.dtl.DTLVardef@169a327_HPS_FORMAT_FIGEXP M_FIG C_FIG

Low-pH and hypoxic conditions commonly develop in oral surgical sites and mucosal wounds, impairing cell viability and delaying healing. This study presents a simple, cell-free, and clinically translatable hydrogel patch incorporating microencapsulated calcium peroxide granules to locally deliver oxygen and buffer acidity. Calcium peroxide particles in the range of 50 to 150 micrometers, were coated with a thin PLGA shell to moderate reactivity and embedded into a GelMA-AlgMA composite membrane. In acidic artificial saliva, pH 5.2, patches containing 0.25% calcium peroxide released oxygen steadily for up to 8 hours and restored pH to physiological levels within 90 minutes. When applied to a DPSC-seeded collagen wound model exposed to lactic-acid challenge, the patches significantly improved metabolic activity and cell viability compared to acidified controls, without signs of cytotoxicity. These findings indicate that calcium peroxide-integrated hydrogels offer a low-cost, practical approach to counteract hypoxia and acidosis in oral wound environments, supporting early regenerative processes and providing a translationally viable platform for future preclinical development.

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

Background and ObjectivesCardiovascular cells differentiated from human induced pluripotent stem cells (iPSCs), including cardiomyocytes, are valuable for evaluating human cardiac pharmacology and toxicity. Early assessment of cardiotoxicity, especially for novel drugs like anticancer agents, is essential for improving drug development efficiency and reducing costs. This study aimed to develop a highly sensitive bioassay system capable of evaluating the physiological function of human cardiac tissue in vitro. MethodsHuman iPSCs were differentiated into cardiovascular cell types (cardiomyocytes, vascular endothelial cells, and vascular mural cells) and assembled into a cardiac tissue model on aligned fiber device. This tissue was cultured dynamically to induce the formation of vascular network-like structure. By combining the fiber device with our previously developed heart-on-a-chip microdevice (HMD), we created a new model of HMD (Aligned Fiber-based HMD; AF-HMD) with improved throughput and stability. Pulsatile force changes induced by drug exposure were quantified by tracking the displacement of fluorescent microbeads within the microchannels. ResultsAF-HMD demonstrated functional responses to known cardiac agonists and toxicants, such as doxorubicin. The device also replicated clinically relevant cardiotoxic events, including the synergistic effects of trastuzumab and doxorubicin, showing marked reductions in contractile force and beat rate, mirroring clinical observations. ConclusionsThe AF-HMD system provides a sensitive and reproducible platform for evaluating cardiotoxicity in drug development. It offers a promising tool for preclinical screening, with potential applications in personalized medicine and predicting cardiotoxic risk in cancer therapy.

Cardiac rhythm is a critical clinical indicator for cardiac arrhythmias and adverse events during drug toxicity studies. In vivo, cardiomyocyte responses to pharmacological agents occur within minutes and are strongly influenced by dynamic drug delivery through blood flow. However, conventional 2D and 3D static culture systems fail to replicate these fluid flow kinetics, limiting their physiological relevance for assessing beat rate responses. Here, we present Mera, an advanced microphysiological system (MPS) developed by Hooke Bio, designed for high-throughput, long-term culture and functional analysis of 3D cardiac spheroids composed of human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts. Mera enables dynamic perfusion, allowing investigation of cardiomyocyte beat rates under physiologically relevant flow conditions. The platform supports up to 640 spheroids per run and integrates automated imaging, fluid handling, and user-friendly software, operating under controlled physiological conditions (37{degrees}C, 5% CO2). Flow rates are tunable between 0 and 12.5 mL/min to mimic in vivo environments. Pharmacological testing with verapamil, isoproterenol, calcium chloride, and propranolol demonstrated real-time, reversible modulation of beat rate under flow, including recovery following drug-induced suppression. System variability was comparable to a temperature-controlled reference platform, supporting robust statistical analysis. Dose-response studies yielded IC values consistent with literature, confirming physiological relevance. Collectively, these results demonstrate that Mera provides a reproducible, scalable, and human-relevant platform for cardiac drug testing. By enabling dynamic drug exposure and automated analysis, Mera represents a powerful new approach methodology (NAM) for improving the predictive assessment of cardiac safety and beat-rate modulation drug responses.