ARDF Annual Open Grant Program
ARDF's Annual Open grant program was established to fund research projects that develop alternative methods to advance science and replace or reduce animal use. Proposals are welcome from any nonprofit, nongovernmental educational or research institution worldwide, although there is a preference for U.S. applications in order to more quickly advance alternatives here.Proposals are evaluated based on scientific merit and feasibility, and the potential to reduce or replace the use of animals in the near future. Proposals are considered across the fields of research, testing, or education, and the maximum grant is $50,000. Since 1993, ARDF has provided over $5.5 million in funds for projects in 34 states and 9 countries.
2026 Grant Awardees
Congratulations to this year’s awardees:
Kyle Amber, MD
Rush University Medical Center
Modeling epithelial-granulocyte interactions in pemphigoid in vitro
Parth Chansoria, PhD
Swiss Federal Institute of Technology in Zurich, Switzerland
Engineered tissue models of skeletal muscle disease in an automated platform for high throughput drug toxicology
Li Jin, PhD
University of Virginia
Human Multi-Organ-on-a-Chip to Define Neuro–Immune Drivers of Discogenic Pain
Tania Lopez Silva, PhD
Massachusetts Institute of Technology
Chemically Defined Peptide Hydrogels as Animal-Free Biomaterials for Human In Vitro Models
Chao Ma, PhD
Cleveland Clinic Foundation
Chip Your CAR-T: Leveraging 3D Immunocompetent Human Bone Marrow Model to Uncover CAR-T-OPENIA Mechanisms
Aditya Raghunandan, PhD
University of Michigan, Dearborn
ARIA-on-a-Chip: Engineered Human BBB Models for Assessing Alzheimer's Drug Safety
Christian Schuerch, PhD
University of Tübingen (Eberhard Karls Universität Tübingen), Germany
A 3D Bioreactor Platform to Dissect, Predict, and Optimize CAR T Cell Therapy in Lymphoma
Vivek Thacker, PhD
Universitaetsklinikum und Medizinische Fakultaet Heidelberg, Germany
Next-generation 3D in vitro granuloma models of human tuberculosis
Rush University Medical Center
Modeling epithelial-granulocyte interactions in pemphigoid in vitro
Bullous pemphigoid (BP) is the most common form of autoimmune blistering. Given its association with advanced age, BPs incidence continues to rise. It is associated with a 20-30% one year mortality. BP is caused by autoantibodies against cutaneous basement membrane zone antigens BP180 and BP230. These autoantibodies lead to keratinocyte mediated inflammation as we have recently shown and also fix complement inducing granulocyte infiltration, degranulation, and blistering.
Model systems are required to better study this interaction and develop drugs. Current models rely primarily on mouse models given the complex interplay of immune factors. In vitro models utilize cryosections of skins. A major limitation of this approach, however, is that keratinocytes are not alive. Thus, the pro-inflammatory factors released by keratinocytes cannot be studied. We propose the development of a 3-dimenesional human-skin equivalent (3D HSE) co-culture system that allows the study of epithelial and granulocyte interactions. In our first aim, we will optimize a neutrophil and eosinophil-specific co-culture system. In our second aim, we will demonstrate the potential for the 3D HSE model to study small molecular inhibitors in vitro. Our long-term aim is to develop a reproducible, fully human model that allows recapitulation of the local immune response in pemphigoid in vitro. This will not only result in a decrease in animals used to develop novel therapies, but a fully-human system that can recapitulate the human immune response in the skin.
Model systems are required to better study this interaction and develop drugs. Current models rely primarily on mouse models given the complex interplay of immune factors. In vitro models utilize cryosections of skins. A major limitation of this approach, however, is that keratinocytes are not alive. Thus, the pro-inflammatory factors released by keratinocytes cannot be studied. We propose the development of a 3-dimenesional human-skin equivalent (3D HSE) co-culture system that allows the study of epithelial and granulocyte interactions. In our first aim, we will optimize a neutrophil and eosinophil-specific co-culture system. In our second aim, we will demonstrate the potential for the 3D HSE model to study small molecular inhibitors in vitro. Our long-term aim is to develop a reproducible, fully human model that allows recapitulation of the local immune response in pemphigoid in vitro. This will not only result in a decrease in animals used to develop novel therapies, but a fully-human system that can recapitulate the human immune response in the skin.
Parth Chansoria, PhD
Swiss Federal Institute of Technology in Zurich, Switzerland
Engineered tissue models of skeletal muscle disease in an automated platform for high throughput drug toxicology
Preclinical drug development relies heavily on live-animal studies to assess skeletal muscle safety and efficacy. However, interspecies physiological differences frequently lead to late-stage clinical failures, particularly regarding human-specific liabilities like drug-induced weakness, fatigue, and myopathy. While 3D engineered human tissues offer a powerful alternative to capture integrated tissue mechanics, their potential as true regulatory replacements is currently hindered by two major bottlenecks. First, current models heavily depend on animal-derived serum, which introduces batch-to-batch variability and fundamentally conflicts with non-animal replacement mandates. Second, functional characterization remains manual, low throughput, and prone to investigator bias.
Our long-term objective is to solve these gaps by establishing a fully automated, animal-component-free screening platform based on engineered human skeletal muscle models of complex diseases exhibiting mechanical and electrical dysfunction. In this ARDF proposal, we will: Aim 1: Establish strictly defined, serum-free culture conditions to biofabricate functional, aligned 3D microtissues modeling healthy, Duchenne Muscular Dystrophy (DMD), and Myotonic Dystrophy type 1 (DM1) phenotypes; Aim 2: Quantify complex biophysical disease signatures- including contraction kinetics, peak active force generation, and mechanical fatigue – using our automated electrical pacing hardware and unbiased "StimTrace" deep-learning analysis workflow; Aim 3: Demonstrate sensitivity of the platform to pharmacological interventions by accurately evaluating myotoxic safety signals and therapeutic efficacy using a targeted small-molecule panel. By successfully recapitulating diverse electrical and mechanical disease archetypes under strictly serum-free conditions, this proposed research will deliver a robust, highly scalable, and fully human-centric assay designed to reduce and replace live-animal testing.
Our long-term objective is to solve these gaps by establishing a fully automated, animal-component-free screening platform based on engineered human skeletal muscle models of complex diseases exhibiting mechanical and electrical dysfunction. In this ARDF proposal, we will: Aim 1: Establish strictly defined, serum-free culture conditions to biofabricate functional, aligned 3D microtissues modeling healthy, Duchenne Muscular Dystrophy (DMD), and Myotonic Dystrophy type 1 (DM1) phenotypes; Aim 2: Quantify complex biophysical disease signatures- including contraction kinetics, peak active force generation, and mechanical fatigue – using our automated electrical pacing hardware and unbiased "StimTrace" deep-learning analysis workflow; Aim 3: Demonstrate sensitivity of the platform to pharmacological interventions by accurately evaluating myotoxic safety signals and therapeutic efficacy using a targeted small-molecule panel. By successfully recapitulating diverse electrical and mechanical disease archetypes under strictly serum-free conditions, this proposed research will deliver a robust, highly scalable, and fully human-centric assay designed to reduce and replace live-animal testing.
Li Jin, PhD
University of Virginia
Human Multi-Organ-on-a-Chip to Define Neuro–Immune Drivers of Discogenic Pain
Back pain is the leading cause of disability worldwide, with intervertebral disc degeneration as a major contributor, particularly in the aging population. As discs deteriorate, extracellular matrix breakdown permits immune cell infiltration and aberrant innervation of the normally a neural and avascular tissue, driving inflammation and pain. However, the mechanisms that link disc degeneration to neuro-immune interactions remain poorly defined. A major barrier is the absence of physiologically relevant human models capable of capturing the multicellular complexity of discogenic pain.
To address this gap, we have developed complementary human platforms that together recreate key features of disc degeneration and pain signaling. These include a spatially micro-patterned 3D human disc cell model that preserves annulus-nucleus organization and an innervation-on-a-chip system enabling real time neuronal activity. Integrated into a multi-organ-on-a-chip system, these platforms allow controlled, spatially organized interactions among disc cells, sensory neurons, and macrophages.
This project aims to determine how sensory neurons modulate macrophage phenotypes and inflammatory signaling during human disc degeneration. By defining how neuronal activation shapes macrophage behavior and how these immune responses feed back onto disc cells, this project will clarify mechanisms that drive or resolve inflammation and matrix breakdown.
Impact on animal replacement: This project directly replaces rodent DRG cultures, disc explants, and in vivo degeneration models by enabling mechanistic studies entirely in human tissues. The resulting multi-organ-on-a-chip system provides a scalable, modular, and fully human alternative for investigating neuro-immune mechanisms and for future therapeutic testing.
To address this gap, we have developed complementary human platforms that together recreate key features of disc degeneration and pain signaling. These include a spatially micro-patterned 3D human disc cell model that preserves annulus-nucleus organization and an innervation-on-a-chip system enabling real time neuronal activity. Integrated into a multi-organ-on-a-chip system, these platforms allow controlled, spatially organized interactions among disc cells, sensory neurons, and macrophages.
This project aims to determine how sensory neurons modulate macrophage phenotypes and inflammatory signaling during human disc degeneration. By defining how neuronal activation shapes macrophage behavior and how these immune responses feed back onto disc cells, this project will clarify mechanisms that drive or resolve inflammation and matrix breakdown.
Impact on animal replacement: This project directly replaces rodent DRG cultures, disc explants, and in vivo degeneration models by enabling mechanistic studies entirely in human tissues. The resulting multi-organ-on-a-chip system provides a scalable, modular, and fully human alternative for investigating neuro-immune mechanisms and for future therapeutic testing.
Tania Lopez Silva, PhD
Massachusetts Institute of Technology
Chemically Defined Peptide Hydrogels as Animal-Free Biomaterials for Human In Vitro Models
Animal-derived matrices (Matrigel) have become essential tools for developing 3D and organoid models that enable the study of biological mechanisms in health and disease, supporting drug discovery and helping to reduce reliance on animal experimentation. Although these in vitro models decrease animal use, they still depend on materials of animal origin because these materials provide strong support for cell viability, migration, and growth, and offer straightforward preparation protocols accessible to any researcher. However, because of their origin and complex nature, these materials exhibit high variability and lack a defined chemical composition, often containing unknown levels of growth factors or biochemical signals that may confound experimental models.
Current approaches to developing Matrigel alternatives rely on synthetic hydrogels that provide greater control over composition and mechanical properties but often consist of a single component or require biomimetic peptide functionalization to direct cell behavior, along with specialized expertise and more complex preparation, which has limited their broad use. Thus, there is a critical need to develop novel synthetic materials that offer high tunability and ease of preparation while mimicking the complexity and composition of native ECM from different tissue types. To address this need, this work aims to develop chemically defined, ECM mimetic self-assembling peptide (SAP) hydrogels that enable precise control over composition, mechanical properties, and bioactivity. SAPs comprise short synthetic peptides that spontaneously assemble into nanofiber networks upon contact with cell media, forming hydrogels through simple protocols. In this project, we propose the molecular design and development of a library of laminin-mimetic SAP hydrogels capable of supporting cell adhesion and directing cell behavior, as with Matrigel.
Current approaches to developing Matrigel alternatives rely on synthetic hydrogels that provide greater control over composition and mechanical properties but often consist of a single component or require biomimetic peptide functionalization to direct cell behavior, along with specialized expertise and more complex preparation, which has limited their broad use. Thus, there is a critical need to develop novel synthetic materials that offer high tunability and ease of preparation while mimicking the complexity and composition of native ECM from different tissue types. To address this need, this work aims to develop chemically defined, ECM mimetic self-assembling peptide (SAP) hydrogels that enable precise control over composition, mechanical properties, and bioactivity. SAPs comprise short synthetic peptides that spontaneously assemble into nanofiber networks upon contact with cell media, forming hydrogels through simple protocols. In this project, we propose the molecular design and development of a library of laminin-mimetic SAP hydrogels capable of supporting cell adhesion and directing cell behavior, as with Matrigel.
Chao Ma, PhD
Cleveland Clinic Foundation
Chip Your CAR-T: Leveraging 3D Immunocompetent Human Bone Marrow Model to Uncover CAR-T-OPENIA Mechanisms
The adoptive transfer of chimeric antigen receptor (CAR) T-cells has emerged as a promising immunotherapy for hematological malignancies. However, the therapeutic outcomes of CAR T-cell therapy differ across clinical trials. While well documented side effects of CAR T-cell therapy such as cytokine release syndrome can be managed effectively, cytopenia has become a notable adverse effect in follow-up patients and is associated with higher infection rates and poorer survival. Sadly, our understanding of the pathophysiology of cytopenia during CAR T-cell therapy (CAR-T-OPENIA) is rather limited. Previous studies attempted to model CAR-T-OPENIA using murine models yet failed due in part to the fact that the murine hematopoietic system is resilient to CAR-induced cytopenia, and more critically because it has a different immune system than that of humans. This lack of models that recapitulate the complexity of human bone marrow has not only hindered mechanistic studies of normal and malignant hematopoiesis, but also delayed the development, validation, and optimization of CAR T-cell therapy, not to mention the management of its side effects (e.g., CAR-T-OPENIA).
This proposal aims to reestablish a first-of-its-kind humanized bone marrow platform (CAR-T-OPENIA-on-a-Chip) that allows for multidimensional (molecular, cellular, and tissue-scale) and chronological (live tracking over one month) analyses to systematically model CAR-T-OPENIA in vitro and to define on-chip the causative nature of various parameters involved. We will also innovate preventive strategies to minimize cytopenia without compromising antitumor activity using a pharmacological blockade of identified biotargets or by genetically modifying CAR T-cells.
This proposal aims to reestablish a first-of-its-kind humanized bone marrow platform (CAR-T-OPENIA-on-a-Chip) that allows for multidimensional (molecular, cellular, and tissue-scale) and chronological (live tracking over one month) analyses to systematically model CAR-T-OPENIA in vitro and to define on-chip the causative nature of various parameters involved. We will also innovate preventive strategies to minimize cytopenia without compromising antitumor activity using a pharmacological blockade of identified biotargets or by genetically modifying CAR T-cells.
Aditya Raghunandan, PhD
University of Michigan, Dearborn
ARIA-on-a-Chip: Engineered Human BBB Models for Assessing Alzheimer's Drug Safety
ARIA—amyloid-related imaging abnormalities—represents the greatest barrier to widespread adoption of disease-modifying therapies for Alzheimer's disease (AD). These induced immunogenic side effects of anti-amyloid antibodies manifest as vasogenic edema (ARIA-E) or microhemorrhage (ARIA-H), affecting nearly 40% of patients homozygous for APOEe4, the highest genetic risk factor for AD. Current transgenic animal models do not develop ARIA spontaneously, lack human APOE alleles, and exhibit discordant inflammatory responses, rendering them scientifically unfit to predict human safety. With 47% of the AD drug pipeline at risk for ARIA, an urgent need exists for human-relevant preclinical models.
We propose to develop ARIA-on-a-Chip: an isogenic iPSC-derived human blood-brain barrier (BBB) model to mechanistically dissect ARIA and evaluate brain shuttle technology as a protective delivery strategy. Brain shuttles leverage receptor-mediated transcytosis to transport antibodies directly across the BBB, potentially bypassing vascular amyloid—the suspected trigger of ARIA.
While TfR-shuttled antibodies reduced ARIA-linked lesions by ~30% in mice, neither the mechanism nor genotype-specificity was reported. Our specific aims are to: (1) validate human Type 1 cerebral amyloid angiopathy pathophysiology in isogenic APOEe3 and APOEe4 neurovascular unit models, and (2) compare shuttled versus unshuttled therapeutics on ARIA-relevant endpoints—antibody transport, inflammatory response, and BBB integrity—using Lecanemab as a model antibody. We hypothesize that shuttled therapeutics will reduce inflammation and preserve barrier integrity, with the greatest benefit in APOEe4 carriers who bear the highest vascular amyloid burden. This project directly addresses ARDF's mission by replacing animal-intensive transgenic mouse breeding programs with a scalable, human-centric alternative. Our completely animal-origin-free iPSC platform enables high throughput de-risking of AD therapies
We propose to develop ARIA-on-a-Chip: an isogenic iPSC-derived human blood-brain barrier (BBB) model to mechanistically dissect ARIA and evaluate brain shuttle technology as a protective delivery strategy. Brain shuttles leverage receptor-mediated transcytosis to transport antibodies directly across the BBB, potentially bypassing vascular amyloid—the suspected trigger of ARIA.
While TfR-shuttled antibodies reduced ARIA-linked lesions by ~30% in mice, neither the mechanism nor genotype-specificity was reported. Our specific aims are to: (1) validate human Type 1 cerebral amyloid angiopathy pathophysiology in isogenic APOEe3 and APOEe4 neurovascular unit models, and (2) compare shuttled versus unshuttled therapeutics on ARIA-relevant endpoints—antibody transport, inflammatory response, and BBB integrity—using Lecanemab as a model antibody. We hypothesize that shuttled therapeutics will reduce inflammation and preserve barrier integrity, with the greatest benefit in APOEe4 carriers who bear the highest vascular amyloid burden. This project directly addresses ARDF's mission by replacing animal-intensive transgenic mouse breeding programs with a scalable, human-centric alternative. Our completely animal-origin-free iPSC platform enables high throughput de-risking of AD therapies
Christian Schuerch, PhD
University of Tübingen (Eberhard Karls Universität Tübingen), Germany
A 3D Bioreactor Platform to Dissect, Predict, and Optimize CAR T Cell Therapy in Lymphoma
Chimeric antigen receptor (CAR) T cells have improved outcomes for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL), yet many fail therapy or relapse due to antigen loss, limited CAR T cell persistence, and immunosuppressive tumor microenvironment (TME) signals. Mechanistic understanding of these resistance processes remains limited because commonly used animal models and reductionist in vitro systems fail to recapitulate the complexity of human lymphoma tissues. This project aims to develop and validate a human-relevant, animal-free functional precision medicine (FPM) platform to study CAR T cell efficacy, dynamics, and resistance directly in patient-derived DLBCL tissue.
Aim 1: Establish and validate a live tissue perfusion bioreactor model for ex vivo testing of CAR T cell responses in intact DLBCL tissues. Fresh patient samples will be cultured and treated with CD19-directed CAR T cells or controls, and cytotoxicity, infiltration, and persistence will be quantified using flow cytometry, histology, multiplexed imaging, transcriptomics, and cytokine profiling. Ex vivo responses will be correlated with matched clinical outcomes to assess predictive value for FPM.
Aim 2: Elucidate dynamic mechanisms of CAR T cell activity and resistance using live-tissue imaging. An imaging-compatible bioreactor will enable real-time two-photon microscopy of CAR T cell migration, cell-cell interactions, and killing behavior within intact human lymphoma tissue, followed by whole-tissue three-dimensional analysis.
Aim 3: Test strategies to enhance CAR T cell efficacy through T cell engineering and TME modulation, including PI3K signaling enhanced CAR T cells and immunomodulatory agents.
By replacing animal experimentation with a scalable, patient-derived human tissue platform, this project directly supports the reduction and replacement of animal use while accelerating translational cancer immunotherapy research and precision oncology.
Aim 1: Establish and validate a live tissue perfusion bioreactor model for ex vivo testing of CAR T cell responses in intact DLBCL tissues. Fresh patient samples will be cultured and treated with CD19-directed CAR T cells or controls, and cytotoxicity, infiltration, and persistence will be quantified using flow cytometry, histology, multiplexed imaging, transcriptomics, and cytokine profiling. Ex vivo responses will be correlated with matched clinical outcomes to assess predictive value for FPM.
Aim 2: Elucidate dynamic mechanisms of CAR T cell activity and resistance using live-tissue imaging. An imaging-compatible bioreactor will enable real-time two-photon microscopy of CAR T cell migration, cell-cell interactions, and killing behavior within intact human lymphoma tissue, followed by whole-tissue three-dimensional analysis.
Aim 3: Test strategies to enhance CAR T cell efficacy through T cell engineering and TME modulation, including PI3K signaling enhanced CAR T cells and immunomodulatory agents.
By replacing animal experimentation with a scalable, patient-derived human tissue platform, this project directly supports the reduction and replacement of animal use while accelerating translational cancer immunotherapy research and precision oncology.
Vivek Thacker, PhD
Universitaetsklinikum und Medizinische Fakultaet Heidelberg, Germany
Next-generation 3D in vitro granuloma models of human tuberculosis
Tuberculosis (TB) kills over 1.2 million people annually. Development of new antibiotics, host-directed therapies, and vaccines relies heavily on animal models. Preclinical efficacy studies in mice and non-human primates typically require tens to >100 animals per study, impose severe welfare burdens due to prolonged infection protocols, and translate poorly to human outcomes. No micro physiological alternatives are currently validated for TB drug or vaccine evaluation. This project develops a next generation human in vitro granuloma model to reduce and ultimately replace early-stage animal experiments in TB therapeutic development.
Our model addresses three key limitations of existing in vitro systems. First, we use alveolar macrophage-like cells (AMLs) differentiated from blood monocytes as the primary infected cell, recapitulating granuloma formation via first-responder alveolar macrophages rather than the physiologically unrealistic direct infection of PBMCs used in all current models. Second, cells are embedded in a lung-mimetic human protein hydrogel matching tissue-specific matrix composition and stiffness. Third, the model enables live-cell fluorescence imaging for real-time monitoring of bacterial growth and immune cell dynamics — a capability absent from animal experiments.
Aim 1: Characterize immunological and transcriptomic responses to M. tuberculosis infection using live-cell imaging, immunofluorescence, and bulk RNAseq, benchmarked against human TB lesion and animal model datasets to build the evidence base for replacement. Aim 2: Evaluate rifampicin monotherapy effects on bacterial clearance and granuloma inflammation, generating kinetic data directly comparable to published animal studies. The long-term objective is to establish a validated, reproducible human model that drug developers can adopt for compound screening, substantially reducing reliance on animal experimentation.
Our model addresses three key limitations of existing in vitro systems. First, we use alveolar macrophage-like cells (AMLs) differentiated from blood monocytes as the primary infected cell, recapitulating granuloma formation via first-responder alveolar macrophages rather than the physiologically unrealistic direct infection of PBMCs used in all current models. Second, cells are embedded in a lung-mimetic human protein hydrogel matching tissue-specific matrix composition and stiffness. Third, the model enables live-cell fluorescence imaging for real-time monitoring of bacterial growth and immune cell dynamics — a capability absent from animal experiments.
Aim 1: Characterize immunological and transcriptomic responses to M. tuberculosis infection using live-cell imaging, immunofluorescence, and bulk RNAseq, benchmarked against human TB lesion and animal model datasets to build the evidence base for replacement. Aim 2: Evaluate rifampicin monotherapy effects on bacterial clearance and granuloma inflammation, generating kinetic data directly comparable to published animal studies. The long-term objective is to establish a validated, reproducible human model that drug developers can adopt for compound screening, substantially reducing reliance on animal experimentation.
ARDF wishes to thank all of the applicants for their interest in advancing alternative methods and their commitment to high quality alternatives research. We would also like to thank our dedicated reviewers for sharing their time and expertise.
Information for the 2027 Annual Open Grant program will be posted at the beginning of January 2027.
Past Recipients
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