Protocol to generate a 3D atherogenesis-on-chip model for studying endothelial-macrophage crosstalk in atherogenesis

Organ Model: Blood vessel (atherogenesis)

Application: Immunology & Inflammation

  • Steps to develop an Organ-Chip model to study endothelial-macrophage interactions
  • Guidance for optimizing immunofluorescence microscopy in Organ-Chip platforms
  • Panel design for macrophage phenotyping using flow cytometry
  • Strategies for putative receptor-ligand analysis with qPCR and secretome analysis

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Multi-lineage heart-chip models drug cardiotoxicity and enhances maturation of human stem cell-derived cardiovascular cells

Organ Model: Heart

Application: Toxicology

How Organ-Chips Were Used:  Conventional models for cardiovascular drug toxicity using iPSCs lack complexity, dynamic culture conditions, and physiological relevance, resulting in immature cells. Mature cells and multi-lineage differentiation are needed for testing multi-lineage cardiotoxicities.

Here, users developed a multi-lineage cardiovascular Organ-Chip using hiPSC-ECs and hiPSC-CMs. Cells in the chip are subjected to active fluid flow and rhythmic biomechanical stretch, resulting in enhanced hiPSC-EC and hiPSC-CM functional and genetic maturity, modeling endothelial barrier permeability, and demonstrating long-term functional stability.

They also showed the utility of this model as a predictive platform for evaluating multi-lineage drug toxicity, such as cancer therapy-induced cardiotoxicity.

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A Novel 3D Dual-Cell Perfusion Model as Tool to Identify and Validate Potential Druggable Targets to Reduce Ischemia Reperfusion Injury

Featured session at Netherlands MPS Day, which took place on November 15, 2023.

Dr. Jeffrey Kroon from Amsterdam UMC discusses his team’s development of Organ-Chip models to better understand and treat cardiovascular diseases. Focusing on the role of endothelial cells in conditions such as atherosclerosis and ischemia-reperfusion injury after myocardial infarction, his group aims to identify novel therapeutic targets by recreating complex vascular environments in vitro. By manipulating endothelial metabolism, they hope to reduce inflammatory signaling and improve tissue outcomes.

This presentation highlights three distinct microphysiological systems. First, a Plaque-on-a-Chip model integrates primary endothelial cells and macrophages separated by a porous membrane. When endothelial cells are inflamed—mimicking arterial plaques—secreted cytokines alter macrophage behavior, reflecting disease progression. This model enables detailed analysis of inflammatory cross-talk and the potential to dampen plaque development by metabolically modulating endothelial cells.

Second, a Heart-on-a-Chip model uses induced pluripotent stem cell (iPSC)-derived cardiomyocytes coupled to an endothelialized vascular channel. This setup simulates blood flow, nutrient delivery, and interactions between cardiomyocytes and endothelial cells. By introducing ischemia-reperfusion conditions, they can study how endothelial metabolism affects cardiomyocyte viability, beating frequency, and tissue damage. This system can inform strategies to mitigate reperfusion injury.

Lastly, Dr. Kroon showcases an Angiogenesis-on-a-Chip model, capturing the formation of new microvessels. Such a platform helps unravel how metabolic cues, growth factors, and inflammatory signals shape new vessel formation in atherosclerotic plaques or ischemic tissue.

Altogether, these human-relevant in vitro models provide a more nuanced understanding of the endothelial cell’s central role in cardiovascular disease. The combination of metabolic and inflammatory insights opens pathways toward targeted therapies that can stabilize plaques, reduce injury post-infarction, and improve vascular health.

Key learnings from this presentation include:

  • Endothelial metabolism as a “volume control” for inflammation: Increasing endothelial glycolysis drives vascular inflammation in atherosclerosis, while reducing metabolic flux can calm inflammatory signals and prevent immune cell infiltration.
  • Plaque-on-a-Chip for macrophage-endothelium crosstalk: Incorporating macrophages in a three-dimensional collagen matrix adjacent to inflamed endothelium offers a dynamic environment to test interventions that modulate plaque stability.
  • Heart-on-a-Chip for ischemia-reperfusion injury: A model pairing iPSC-derived cardiomyocytes and endothelial cells simulates post-infarction conditions, enabling exploration of how endothelial factors influence cardiac tissue resilience, beating behavior, and stress responses.
  • Angiogenesis-on-a-Chip for vascular remodeling: Controlled growth factor gradients and flow conditions help study new vessel formation, an important aspect of plaque progression and tissue repair.
  • Toward translational impact: These advanced platforms bridge the gap between traditional cell culture and animal models, offering more predictive tools for developing therapies aimed at improving vascular health and cardiac outcomes.

Organ-on-Chip Technology Recapitulates Thrombosis Induced by an anti-CD154 Therapeutic

Abstract

Blocking of CD40L-mediated signaling represents a validated therapeutic strategy for treatment of several auto-immune disorders, however, development of therapies against this target was stalled for several years because of unexpected thrombotic and cardiovascular events during clinical development of the anti-CD40L mAb Hu5c8. These side effects were not detected during preclinical testing. Platelet activation assays have been used to test the hypothesis that thrombosis was caused by binding of Hu5c8IgG1 to FcγRIIa receptors on platelets. To provide additional confidence in the safety of new anti-CD40L mAbs that are designed not to bind FcγRIIa, a micovessel-chip (Vessel-Chip) was developed that could capture human relevant endpoints for detection of coagulopathy, providing a patient-specific platform for safety testing. The Vessel-Chip includes a vascular channel lined by human endothelial cells and perfused with human whole blood at a physiologically-relevant shear rate. Treatment with clinical-relevant concentrations of hu5c8IgG1 and sCD40L resulted in endothelial activation, platelet adhesion, platelet aggregation, fibrin clot formation, and increased secretion of thrombin anti-thrombin (TAT) complex. Conversely, these endpoints were attenuated following treatment with Hu5c8IgG2σ, a mAb that does not bind FcγRIIa receptors. Given lack of suitable preclinical models for detection of thrombosis, these data provide confidence in the potential safety of the newer generation anti-CD40L mAbs designed not to bind FcγRIIa receptors. Detection of TAT in the model confirms that important counter-regulatory mechanisms that occur during thrombosis, such as thrombin and antithrombin generation, occur de-novo in the model. This model provides a unique platform for preclinical assessment of thrombosis risk in a patient-specific manner, but can also be used for discovery of anti-thrombotic agents, and mechanism of action elucidation thus providing a useful tool for drug discovery and development.

Monoclonal Antibody

Organ‐on‐Chip Recapitulates Thrombosis Induced by an anti‐CD154 Monoclonal Antibody: Translational Potential of Advanced Microengineered Systems

Abstract

Clinical development of Hu5c8, a monoclonal antibody against CD40L intended for treatment of autoimmune disorders, was terminated due to unexpected thrombotic complications. These life-threatening side effects were not discovered during preclinical testing due to the lack of predictive models. In the present study, we describe the development of a microengineered system lined by human endothelium perfused with human whole blood, a “Vessel-Chip.” The Vessel-Chip allowed us to evaluate key parameters in thrombosis, such as endothelial activation, platelet adhesion, platelet aggregation, fibrin clot formation, and thrombin anti-thrombin complexes in the Chip-effluent in response to Hu5c8 in the presence of soluble CD40L. Importantly, the observed prothrombotic effects were not observed with Hu5c8-IgG2σ designed with an Fc domain that does not bind the FcγRIIa receptor, suggesting that this approach may have a low potential risk for thrombosis. Our results demonstrate the translational potential of Organs-on-Chips, as advanced microengineered systems to better predict human response.