Modelling Cardiovascular Diseases Using Human Microphysiological Systems

Pancreatic ductal adenocarcinoma (PDAC) creates complex tumor microenvironment (TME) hallmarked with a desmoplastic stroma that facilitates tumor growth/invasion, chemoresistance, and immunosuppression. It urgently needs the identification and evaluation of stromal components that can be targeted to reprogram the stroma to improve drug delivery and efficacy without making tumors more aggressive. Thus, we hypothesize that the coagulation system in the PDAC TME can be targeted to reprogram PDAC stroma to alleviate chemoresistance and drug delivery barriers. Specifically, the thrombin/protease-activated receptor 1 (PAR1) signaling axis can be targeted to suppress growth/invasion of pancreatic cancer cells (PCCs) and cancer associated fibroblast (CAF)-derived fibrosis. Our underlying rationale is based upon a leaky tumor vasculature in PDAC resulting in the release of circulating coagulation factors and subsequent activation of the coagulation system in the TME. Tissue factor expressed by PCCs initiates the conversion of prothrombin to the active serine protease thrombin, which then activates PAR1, whose signaling is thought to promote PCC growth/invasion and CAF-mediated fibrosis. We developed and employed novel microphysiological systems (MPS) of PDAC tumor-stroma, which were designed to reconstitute extravascular coagulation in the PDAC TME to specifically investigate the role of thrombin-PAR1 signaling events on PCC growth and CAF-mediated fibrosis. Our MPS was a microfluidic platform where PCC and CAF were co-cultured in the 3D extracellular matrix perfused with/without thrombin. In addition, PAR1 expression in murine and human PCCs and CAFs was genetically modified or pharmacologically inhibited. Our MPS enabled systematic and translational analyses on the therapeutic potential of blocking PAR1 signaling in PCCs, CAFs, or both. In murine MPS, genetic deletion of PAR1 drastically decreased thrombin-mediated PCC and CAF growth compared to that of MPS with wildtype cells. Human MPS with varying levels of PAR1 also suggest thrombin stimulates PCC-CAF crosstalk, including CAF growth, elevated expression of a-SMA and secreted collagen levels. Furthermore, pharmacological inhibition of PAR1 by vorapaxar decreases both PCC and CAFs in all human PCC/CAF pairs studied. Finally, we confirm the findings from our MPS using PDAC tumor-stroma xenograft models with both human PCC and CAF. A significant reduction in tumor size is observed with vorapaxar treatment, which attributes primarily to the reduction of CAFs. In summary, we validate and translate the therapeutic potential of thrombin-PAR1 signaling in reprogramming PDAC stroma using novel MPS of PDAC tumor-stroma model. Our study also demonstrates MPS as a promising system for target identification, validation, and streamlining preclinical studies for drug discovery. Citation Format: Sae Rome. Choi, Hye-ran Moon, Natalia Ospina Muñoz, Yun Chang, Xiaoping Bao, Bennett D. Elzey, Meliss L. Fishel, Matthew J. Flick, Bumsoo Han. Validation and translation of therapeutic potential of thrombin-PAR1 signaling in suppressing fibrosis using microphysiological PDAC tumor models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B037.

Oligonucleotide therapeutics (ONTs) encompass classes of medicines that selectively target and potentially ameliorate previously untreatable and often rare diseases. Several unique classes of ONTs provide versatility, enabling direct modu­lation of gene expression by virtue of Watson-Crick base pairing or modulation of cell signaling through structural mimicry or interference with protein-receptor interactions. Due to a lack of suitable in vitro models capable of recapitulating or predicting in vivo effects of ONTs, their discovery and optimization has relied heavily on animal studies for predicting efficacy and safety in humans. Since ONTs often lack cross-species activity, animal models with genetic humanization and/or species-specific surrogate ONTs are often required. Human microphysiological systems (MPS) offer an oppor­tunity to reduce the use of animals and may enable evaluation of drug mechanisms, optimization of cell and tissue targeting ligands or delivery vehicles, and characterization of pharmacokinetics (PK), pharmacodynamics (PD), and safety of candidate ONTs. The lack of published examples for MPS applications with ONT demonstrates the need for a focused effort to characterize and build confidence in their utility. The goals of this review are to summarize the current landscape of ONTs and highlight potential opportunities and challenges for application of MPS during ONT discovery and development. In addition, this review aims to raise awareness with ONT drug developers and regulatory authorities on the potential impact of MPS with respect to characterizing pharmacology, ADME, and toxicity and to educate MPS platform developers on unique design attributes needed to fully appreciate MPS advantages in ONT development.

Organ interactions resulting from drug, metabolite or xenobiotic transport between organs are key components of human metabolism that impact therapeutic action and toxic side effects. Preclinical animal testing often fails to predict adverse outcomes arising from sequential, multi-organ metabolism of drugs and xenobiotics. Human microphysiological systems (MPS) can model these interactions and are predicted to dramatically improve the efficiency of the drug development process. In this study, five human MPS models were evaluated for functional coupling, defined as the determination of organ interactions via an in vivo-like sequential, organ-to-organ transfer of media. MPS models representing the major absorption, metabolism and clearance organs (the jejunum, liver and kidney) were evaluated, along with skeletal muscle and neurovascular models. Three compounds were evaluated for organ-specific processing: terfenadine for pharmacokinetics (PK) and toxicity; trimethylamine (TMA) as a potentially toxic microbiome metabolite; and vitamin D3. We show that the organ-specific processing of these compounds was consistent with clinical data, and discovered that trimethylamine-N-oxide (TMAO) crosses the blood-brain barrier. These studies demonstrate the potential of human MPS for multi-organ toxicity and absorption, distribution, metabolism and excretion (ADME), provide guidance for physically coupling MPS, and offer an approach to coupling MPS with distinct media and perfusion requirements.

Microphysiology systems (MPS), also called organs-on-chips and tissue chips, are miniaturized functional units of organs constructed with multiple cell types under a variety of physical and biochemical environmental cues that complement animal models as part of a new paradigm of drug discovery and development. Biomimetic human liver MPS have evolved from simpler 2D cell models, spheroids and organoids to address the increasing need to understand patient-specific mechanisms of complex and rare diseases, the response to therapeutic treatments, and the absorption, distribution, metabolism, excretion and toxicity of potential therapeutics. The parallel development and application of transdisciplinary technologies, including microfluidic devices, bioprinting, engineered matrix materials, defined physiological and pathophysiological media, patient-derived primary cells, and pluripotent stem cells as well as synthetic biology to engineer cell genes and functions, have created the potential to produce patient-specific, biomimetic MPS for detailed mechanistic studies. It is projected that success in the development and maturation of patient-derived MPS with known genotypes and fully matured adult phenotypes will lead to advanced applications in precision medicine. In this Review, we examine human biomimetic liver MPS that are designed to recapitulate the liver acinus structure and functions to enhance our knowledge of the mechanisms of disease progression and of the absorption, distribution, metabolism, excretion and toxicity of therapeutic candidates and drugs as well as to evaluate their mechanisms of action and their application in precision medicine and preclinical trials.

Microphysiological systems (MPS) are designed to recapitulate aspects of tissue/organ physiology in vivo, thereby providing potential value in safety and efficacy assessments of FDA-regulated products and regulatory decision-making. While there have been significant advances in the development, use, and proposals of qualification criteria for human organ MPS, there remains a gap in the development using animal tissues. Animal MPS may be of value in many areas including the study of zoonotic diseases, assessment of the safety and efficacy of animal therapeutics, and possibly reduction of the use of animals in regulatory submissions for animal therapeutics. In addition, the development of MPS from various animal species enables comparison to animal in vivo data. This comparison, while not always critical for all contexts of use, could help gain confidence in the use and application of human MPS data for regulatory decision-making and for the potential identification of species-specific effects. The use of animal MPS is consistent with the replacement, reduction, and refinement (3Rs) principles of animal use by identifying toxic compounds before conducting in vivo studies and identifying the appropriate species for testing.

Organoids and microphysiological systems represent two current approaches to reproduce organ function in vitro. These systems can potentially provide unbiased assays of function which are needed to understand the mechanism of action of environmental toxins. Culture models that replicate organ function and interactions among cell types and tissues move beyond existing screens that target individual pathways and provide a means to assay context-dependent function. The current state of organoid cultures and microphysiological systems is reviewed and applications discussed. While few studies have examined environmental pollutants, studies with drugs demonstrate the power of these systems to assess toxicity as well as mechanism of action. Strengths and limitations of organoids and microphysiological systems are reviewed and challenges are identified to produce suitable high capacity functional assays.

Microphysiological systems (MPS) are complex in vitro tools that incorporate cells derived from various healthy or disease-state human or animal tissues and organs. While MPS have limitations, including a lack of globally harmonized guidelines for standardization, they have already proven impactful in certain areas of drug development. Further research and regulatory acceptance of MPS will contribute to making them even more effective tools in the future. This review explores the potential applications of human liver, gut, lung, and cardiac MPS in drug development, focusing on disease modeling, safety assessment, and pharmacokinetic studies. Various technical param­eters and relevant endpoints for system assessment are discussed alongside challenges such as cell sourcing, reproducibility, and the integration of multiple tissues or organs. The importance of col­laborative efforts between academia, industry, and regulatory agencies to develop standardized protocols and validation criteria is emphasized. With ongoing advancements and cooperative ini­tiatives, MPS are poised to play a significant role in enhancing the predictivity and reliability of nonclinical testing, thereby transforming drug development and regulatory processes.

A key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.

Immune responses are heavily involved in the regulation and pathogenesis of human diseases, including infectious diseases, inflammatory and autoimmune conditions, cancer, neurological disorders, and cardiometabolic syndromes. The immune system is considered a double-edged sword serving as a powerful host defense mechanism against infection and cancerous cells and causing detrimental tissue damage when the immune response is exaggerated or uncontrollable. One of the challenges in studying the efficacy and toxicity of drugs that target or modulate the immune system is the lack of suitable preclinical human models that are predictive of human response. Recent advancements in human microphysiological systems (MPS) have provided a promising in vitro platform to evaluate the response of immune organs ex vivo, to investigate the interaction of immune cells with non-lymphoid tissue cells, and to reduce the reliance on animals in preclinical studies. The development, regulation, trafficking, and responses of immune cells have been extensively studied in preclinical animal models and clinically, providing a wealth of knowledge by which to evaluate new in vitro models. Therefore, the application of immunocompetent MPS in drug discovery and development should first verify that the immune response in an MPS model recapitulates the complexity of the human immune physiology. This manuscript reviews biological functions of immune organ systems and tissue-resident immune cells and discusses contexts-of-use for commonly used immunocompetent and immune organ MPS models. Current perspective and recommendations are provided to guide the continued development of immune organ and immunocompetent MPS models and their application in drug discovery and development.

Human Microphysiological Systems (hMPS), otherwise known as organ- and tissue-on-a-chip models, are an emerging technology with the potential to replace in vivo animal studies with in vitro models that emulate human physiology at basic levels. hMPS platforms are designed to overcome limitations of two-dimensional (2D) cell culture systems by mimicking 3D tissue organization and microenvironmental cues that are physiologically and clinically relevant. Unlike animal studies, hMPS models can be configured for high content or high throughput screening in preclinical drug development. Applications in modeling acute and chronic injuries in the musculoskeletal system are slowly developing. However, the complexity and load bearing nature of musculoskeletal tissues and joints present unique challenges related to our limited understanding of disease mechanisms and the lack of consensus biomarkers to guide biological therapy development. With emphasis on examples of modeling musculoskeletal tissues, joints on chips, and organoids, this review highlights current trends of microphysiological systems technology. The review surveys state-of-the-art design and fabrication considerations inspired by lessons from bioreactors and biological variables emphasizing the role of induced pluripotent stem cells and genetic engineering in creating isogenic, patient-specific multicellular hMPS. The major challenges in modeling musculoskeletal tissues using hMPS chips are identified, including incorporating biological barriers, simulating joint compartments and heterogenous tissue interfaces, simulating immune interactions and inflammatory factors, simulating effects of in vivo loading, recording nociceptors responses as surrogates for pain outcomes, modeling the dynamic injury and healing responses by monitoring secreted proteins in real time, and creating arrayed formats for robotic high throughput screens. Overcoming these barriers will revolutionize musculoskeletal research by enabling physiologically relevant, predictive models of human tissues and joint diseases to accelerate and de-risk therapeutic discovery and translation to the clinic.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

The role of neutrophils in mediating neurovascular vulnerability has been increasingly implicated in various acute inflammatory models of neuroimmune crosstalk between the periphery and the brain. This study aimed at understanding the early phases of crosstalk following repetitive inflammation to the lung and ensuing neuropathology. Such a model of frequent inflammatory injury to the lung is pertinent to understanding the focal neurologic risk of constant exposure to aerosolized environmental hazards leading to progressive pulmonary disease. To model repeated pulmonary inflammation, we applied a three-dose regimen of intranasal (i.n.) lipopolysaccharide (LPS) in C57BL/6J mice and studied the impact on the inflammatory environment of the brain, with a specific focus on neutrophil dynamics at the neurovascular unit (NVU). Tissue and circulatory inflammatory profiles were screened via bronchoalveolar lavage (BAL) protein content and cellularity, transcript analysis of brain tissue, and flow cytometry of peripheral blood. Intravital two-photon microscopy (2PM) of the brain vasculature identified neutrophil dynamics at the NVU. Immunofluorescence validated neutrophil dynamics and identified neuroinflammatory hallmarks and peripheral immune factor interactions at the NVU. In vivo findings were corroborated and replicated in murine and human microphysiological systems (MPS) modeling the blood-brain barrier as a proxy demonstration of the translational relevance of our findings. 2PM of tdTomato-Ly6G+ neutrophils demonstrated increased levels of circulating neutrophils and corresponding engagement with cortical brain vasculature after the three-dose repeated i.n. exposure regimen. Neutrophilia at the NVU was corroborated with increased transcript levels of Ly6G and other pro-inflammatory markers. This coordination between endothelial pathophysiology and neutrophil phenotypes was recapitulated in separate murine and human MPS models. Systemic neutrophilia in the lung and circulation was found to be cotemporaneous to neutrophilia at the NVU based on the cellularity of BAL and peripheral blood samples collected at the same endpoints. Immunohistochemical analysis of brain tissue implicates temporal coordination between vascular surface adhesion molecules with changes in neutrophil dynamics from adhesion, crawling, stalling, and transmigration. Extravasation of neutrophils was accompanied by sustained paravascular deposition of fibrinogen and microgliosis up to 72 h after the final i.n. dosing. Microglia-associated effector functions for synaptic pruning and regulation of neutrophil activity demonstrated distinct temporal profiles in the hippocampus independent from transduction along the primary olfactory cortex. Our results identify systemic levels of neutrophilia accompanied by ingress and extravascular accumulation in brain parenchyma that correlated with sustained microglial activation. Pathology to the brain parenchyma is further complemented by the observation of paravascular fibrinogen deposition that alters synaptic metabolism. Thus, we highlight a key role for neutrophil signaling and ensuing neuroimmune interactions from the lung to the brain as a generalizable model of repeated respiratory exposure to inflammatory agents.

BackgroundThe role of neutrophils in mediating neurovascular vulnerability has been increasingly implicated in various acute inflammatory models of neuroimmune crosstalk between the periphery and the brain. Whether neurovascular vulnerability is similarly modulated in the context of frequent, but not acute, inflammatory activation in the periphery is the aim of our study. Such a model of frequent inflammatory irritation is pertinent to understanding the neurologic risk of constant exposure to aerosolized environmental hazards leading to progressive pulmonary disease.MethodsTo model repeated pulmonary inflammation, we applied a three-dose regimen of intranasal (i.n.) lipopolysaccharide (LPS) in C57BL/6J mice and studied the impact on the inflammatory environment of the brain, with a specific focus on neutrophil dynamics at the neurovascular unit (NVU). Tissue and circulatory inflammatory profiles were screened via bronchoalveolar lavage (BAL) protein content and cellularity, transcript analysis of brain tissue, and flow cytometry of peripheral blood. Intravital two-photon microscopy (2PM) of the brain vasculature identified neutrophil dynamics at the NVU. Immunofluorescence validated neutrophil dynamics and identified neuroinflammatory hallmarks and peripheral immune factor interactions at the NVU. In vivo findings were corroborated and replicated in murine and human microphysiological systems (MPS) modeling the blood-brain barrier as a proxy demonstration of the translational relevance of our findings.Results2PM of tdTomato-Ly6G+ neutrophils demonstrated increased levels of circulating neutrophils and corresponding engagement with the brain vasculature after the three-dose repeated i.n. exposure regimen. Neutrophilia at the NVU was corroborated with increased transcript levels of Ly6G and other pro-inflammatory markers. This coordination between endothelial physiology and neutrophil phenotypes was recapitulated in murine and human MPS models. System-wide neutrophilia in the lung and circulation was found to be cotemporaneous to neutrophilia at the NVU based on the cellularity of BAL and peripheral blood samples collected at the same endpoints. Immunohistochemical analysis of brain tissue implicates temporal coordination between vascular surface adhesion molecules with changes in neutrophil dynamics from adhesion, crawling, stalling, and transmigration. Extravasation of neutrophils was complemented by sustained paravascular deposition of fibrinogen and microgliosis up to 72 hours after the final i.n. dosing. Microglia-associated effector functions for synaptic pruning and regulation of neutrophil activity demonstrated distinct temporal profiles.ConclusionsOur results identify systemic levels of neutrophilia accompanied by ingress and extravascular accumulation in brain parenchyma that correlated with sustained microglial activation. This neutrophil-centric lung-brain interaction is complemented by the observation of paravascular fibrinogen deposition that alters synaptic metabolism. Thus, we propose a mechanistic role for neutrophilia and associated inflammatory dysregulation as essential mediators along the lung-brain neuroimmune axis in a generalizable model of repeated respiratory exposure to inflammatory agents.

Two technologies that have emerged in the last decade offer a new paradigm for modern pharmacology, as well as drug discovery and development. Quantitative systems pharmacology (QSP) is a complementary approach to traditional, target-centric pharmacology and drug discovery and is based on an iterative application of computational and systems biology methods with multiscale experimental methods, both of which include models of ADME-Tox and disease. QSP has emerged as a new approach due to the low efficiency of success in developing therapeutics based on the existing target-centric paradigm. Likewise, human microphysiology systems (MPS) are experimental models complementary to existing animal models and are based on the use of human primary cells, adult stem cells, and/or induced pluripotent stem cells (iPSCs) to mimic human tissues and organ functions/structures involved in disease and ADME-Tox. Human MPS experimental models have been developed to address the relatively low concordance of human disease and ADME-Tox with engineered, experimental animal models of disease. The integration of the QSP paradigm with the use of human MPS has the potential to enhance the process of drug discovery and development.