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. The maximum award amount is $50,000, and the historical funding rate is approximately 20%.
Since 1993, ARDF has provided over $5.3 million in funds for projects in 34 states and 9 countries.
2025 Grant Awardees
Congratulations to this year’s awardees:
Aitor Aguirre, PhD
Michigan State University, East Lansing, MI
Engineered Human Heart Models for Advancing Drug Cardiac Safety Assessment
Jian Gu, PhD
University of Arizona, Tucson, AZ
In Vitro Modelling of Bubble-Assisted Focused Ultrasound Blood-Brain Barrier Opening
James Hagood, MD
University of North Carolina at Chapel Hill, Chapel Hill, NC
An Advanced ex vivo Model for Idiopathic Pulmonary Fibrosis
Maria Karlgren, PhD
Uppsala University, Uppsala, Sweden
Replacing Animal-Derived Products in Drug Development In Vitro Models
Fenna Sille, PhD
Johns Hopkins University, Baltimore, MD
Immune Disruptors and the Developing Brain: Unraveling how Immunotoxicants Shape Neurodevelopment
Lena Smirnova, PhD
Johns Hopkins University, Baltimore, MD
Advancing Organoid Intelligence with a Hippocampal-Cortical System
Jason Tchieu, PhD
Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Generation of the Olfactory Epithelium from Human Pluripotent Stem Cells to Study Neurodegeneration
Aranzazu Villasante, PhD
Fundacio Institut de Bioenginyeria de Catalunya (Institute for Bioengineering of Catalonia), Barcelona, Spain
A Human-Relevant Model for Real-Time Monitoring of MYCN-Driven Neuroblastoma Spreading and Drug Testing
Michigan State University, East Lansing, MI
Engineered Human Heart Models for Advancing Drug Cardiac Safety Assessment
Single ventricle defects (SVDs) are a type of grave congenital heart defect associated with genetic and environmental factors. Maternal exposure to certain drugs, including FDA-approved medications taken during early pregnancy, are potential risk factors, yet their impact remains understudied due to a lack of suitable human models. Human pluripotent stem cell (hPSC)-derived heart organoids (hHOs) offer a powerful alternative for studying human cardiac development and congenital defects independent of animal use. Our lab has developed an advanced hHO system that mimics key aspects of cardiogenesis and closely resembles human embryonic hearts at 6-10 weeks, a critical period for SVD formation. We found that hHOs exposed to clinical doses of ondansetron (Zofran), a 5-HT3 receptor antagonist used during pregnancy, exhibit hypoplastic left ventricular (LV) cardiomyocytes. At the same time, the right ventricle and atria remained unaffected, suggesting a potential link between serotonin receptor modulators (SRMs) and SVDs such as hypoplastic left heart syndrome (HLHS). Ondansetron is part of a broader family of SRMs, including psychotropics and gastroprokinetics, raising the question of whether SRMs are an unrecognized but preventable cause of SVDs. We will use hHOs to screen the effects of 5-HT3 and 5-HT4 receptor antagonists/agonists on LV formation and investigate the molecular mechanisms underlying SRM-induced LV hypoplasia. Our proposal is significant because it will enhance drug safety assessments during pregnancy, improve understanding of SVD mechanisms, and establish a physiologically relevant human heart model for future research and cardiac toxicity testing while also reducing reliance and use of animal models.
Jian Gu, PhD
University of Arizona, Tucson, AZ
In Vitro Modelling of Bubble-Assisted Focused Ultrasound Blood-Brain Barrier Opening
Bubble-assisted focused ultrasound (BaFUS) blood-brain barrier opening (BBBO) has emerged as a promising technology to deliver drugs to targeted brain locations non-invasively. This can have a profound impact on treating numerous neurological diseases, which have been identified as the top contributor to the global disease burden. However, the mechanisms of BaFUS BBBO are only partially understood, which limits its fast, safe, and effective translation to the clinics. In this project, we plan to use an ultrasound-transparent (UST) organ-on-chip (OoC) platform to generate an in vitro model for BaFUS BBBO. The ultrasound acoustic pressure effects on paracellular pathways of the BBBO will be illustrated. The BBBO acoustic pressure threshold will also be compared with that reported in vivo. Results from this study could be the first critical step in generating predictive in vitro models for BaFUS BBBO, which could reduce or eliminate the existing need for the use of animals in developing BaFUS BBBO techniques to treat various neurological diseases.
James Hagood, MD
University of North Carolina at Chapel Hill, Chapel Hill, NC
An Advanced ex vivo Model for Idiopathic Pulmonary Fibrosis
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease marked by unexplained scarring, with lung transplantation as the only curative option. Its pathogenesis involves disrupted epithelial cell homeostasis driven by genetic, environmental, and aging factors, leading to overproduction of profibrotic mediators such as TGF-ß and heightened endoplasmic reticulum (ER) stress. Pathologically, IPF manifests as usual interstitial pneumonia (UIP) with hallmark fibroblastic foci and honeycombing, and emerging evidence implicates aberrant basaloid cells (KRT17+/KRT5-) in disease progression. Despite extensive use of bleomycin-induced mouse models, these systems fail to fully capture human IPF pathology and raise ethical concerns. To address these limitations, we propose a human precision-cut lung slice (PCLS) model maintained under air-liquid interface (ALI) conditions. Prolonged ALI culture (3–4 weeks or more) enhances viability compared to submerged PCLS, enabling deeper investigation of fibrotic processes. A fibrotic cocktail (TGF-ß, PDGF-AB, TNF-a, LPA) induces collagen and fibronectin production, while low-titer Sendai virus (SeV) infection triggers ER stress, mirroring key components of IPF pathophysiology. Culturing PCLS on recombinant gelatin-coated supports avoids animal-based additives and facilitates long-term maintenance. This approach seeks to optimize and validate a more physiologically relevant ex vivo IPF model, capturing both fibrotic and ER stress-driven events. By leveraging human tissue, it directly reduces reliance on animal models, supports improved translational research, and accelerates preclinical evaluation of new antifibrotic therapies—ultimately advancing precision medicine in IPF.
Maria Karlgren, PhD
Uppsala University, Uppsala, Sweden
Replacing Animal-Derived Products in Drug Development In Vitro Models
In vitro models are promising tools for reducing animal use in drug development by mimicking human-specific biological processes more accurately. Our laboratory has pioneered 3Rs-compliant approaches by developing many of the cell models currently used as gold standards in drug development. However, many of these systems rely on fetal bovine serum (FBS), which raises ethical concerns, introduces variability, and limits reproducibility. To address these challenges, we are transitioning widely used in vitro drug development models to animal product-free conditions and validating their performance. This project focuses on cell models for studying drug metabolism, toxicity, permeability, and bioavailability—key properties in drug development. Preliminary findings show that animal serum-free media enhance the phenotype and functionality of these models compared to traditional FBS-based methods, supporting their potential to fully achieve 3R compliance by replacing animal testing and eliminating the use of animal products in the in vitro systems.
Currently, we are finalizing the validation of two gold-standard cell models for metabolism and permeability under serum-free conditions and expanding this approach to additional models. Comprehensive standard operating procedures for media preparation and serum-free culturing are being established to ensure reproducibility. We will benchmark each cell system against FBS-based protocols using global proteomics and functional performance metrics. Additionally, we are exploring new applications for validated models, such as long-term and dynamic culture studies, to enhance physiological relevance. Through collaborations with academia, industry, and the national 3Rs agency (Swedish 3Rs Center), we aim to disseminate these advancements broadly. Our goal is to promote the adoption of animal product-free in vitro approaches, contributing to the replacement, refinement, and reduction of animal use in biomedical research.
Currently, we are finalizing the validation of two gold-standard cell models for metabolism and permeability under serum-free conditions and expanding this approach to additional models. Comprehensive standard operating procedures for media preparation and serum-free culturing are being established to ensure reproducibility. We will benchmark each cell system against FBS-based protocols using global proteomics and functional performance metrics. Additionally, we are exploring new applications for validated models, such as long-term and dynamic culture studies, to enhance physiological relevance. Through collaborations with academia, industry, and the national 3Rs agency (Swedish 3Rs Center), we aim to disseminate these advancements broadly. Our goal is to promote the adoption of animal product-free in vitro approaches, contributing to the replacement, refinement, and reduction of animal use in biomedical research.
Fenna Sille, PhD
Johns Hopkins University, Baltimore, MD
Immune Disruptors and the Developing Brain: Unraveling how Immunotoxicants Shape Neurodevelopment
Neurodevelopmental disorders (NDDs) are rising at an alarming rate, with autism spectrum disorder (ASD) increasing by over 317% in the last two decades. While both genetic and environmental factors contribute to NDDs, immune dysregulation has emerged as a key but understudied pathway in their development. Immune effectors play a dual role in neurodevelopment, with both protective and harmful influences. Maternal infections and gut microbiome changes impact fetal brain development through neuroimmune cross-talk, while environmental toxicants such as lead (Pb), arsenic, and pesticides may disrupt both immune and neurodevelopmental processes. Despite strong associations between immune dysregulation and NDD risk, the underlying mechanisms and the role of environmental risk factors remain unclear.
This study aims to explore the cross-reactivity between developmental immunotoxicity (DIT) and neurotoxicity using a human-relevant approach. We will expose human monocyte-derived macrophages (hMDMs) and iPSC-derived microglia—representing peripheral and brain-resident immune responses—to selected DIT compounds (Pb, Tributyltin oxide [TBTO], Chlorpyrifos, and dexamethasone [DEX]). The microenvironment created by these immune cells, including cytokines, chemokines, metabolites, and lipid signaling molecules, will be assessed for its impact on neurodevelopment using iPSC-derived brain organoids.
Aim 1: Investigate how systemic inflammation induced by immunotoxicants affects brain organoid development by culturing them in macrophage-conditioned media.
Aim 2: Examine how local neuroinflammation influences neurodevelopment by exposing organoids to microglia-conditioned media following chemical exposures.
By integrating human-relevant microphysiological brain systems (MPS) with human immune cell models, this study will advance hazard identification and provide critical insights into the mechanisms by which developmental immunotoxicants contribute to NDDs.
This study aims to explore the cross-reactivity between developmental immunotoxicity (DIT) and neurotoxicity using a human-relevant approach. We will expose human monocyte-derived macrophages (hMDMs) and iPSC-derived microglia—representing peripheral and brain-resident immune responses—to selected DIT compounds (Pb, Tributyltin oxide [TBTO], Chlorpyrifos, and dexamethasone [DEX]). The microenvironment created by these immune cells, including cytokines, chemokines, metabolites, and lipid signaling molecules, will be assessed for its impact on neurodevelopment using iPSC-derived brain organoids.
Aim 1: Investigate how systemic inflammation induced by immunotoxicants affects brain organoid development by culturing them in macrophage-conditioned media.
Aim 2: Examine how local neuroinflammation influences neurodevelopment by exposing organoids to microglia-conditioned media following chemical exposures.
By integrating human-relevant microphysiological brain systems (MPS) with human immune cell models, this study will advance hazard identification and provide critical insights into the mechanisms by which developmental immunotoxicants contribute to NDDs.
Lena Smirnova, PhD
Johns Hopkins University, Baltimore, MD
Advancing Organoid Intelligence with a Hippocampal-Cortical System
Current neurocognitive research relies heavily on animal models that fail to fully replicate human-specific brain functions, limiting the translational success of therapeutic interventions. Organoid Intelligence (OI) is an initiative to develop alternative human brain models by leveraging neural organoids in combination with a computational interface (biocomputing). This project aims to significantly advance OI by developing a hippocampus-cortical organoid system derived from human induced pluripotent stem cells. By integrating hippocampal and cortical organoids, we seek to establish a physiologically relevant in vitro model capable of mimicking key neural circuits involved in learning and memory. The system will be validated using single-cell RNA sequencing to confirm region-specificity and electrophysiological assays with high-density multi-electrode arrays to assess network formation and synaptic plasticity. This entirely animal-free approach eliminates the ethical and scientific limitations of rodent and primate models, replacing traditional cognitive function assessments, such as the Morris water maze, with high-throughput electrophysiological readouts including network connectivity and burst frequency. Furthermore, our use of human patient-derived extracellular matrices and serum-free media formulations ensures full alignment with non-animal-based methodologies. By providing a scalable, reproducible, and human-relevant model for learning and memory, this work not only advances fundamental neuroscience but also contributes to replacing animal testing in neurodevelopmental and neurotoxicological research.
Jason Tchieu, PhD
Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Generation of the Olfactory Epithelium from Human Pluripotent Stem Cells to Study Neurodegeneration
Olfactory dysfunction is increasingly recognized as an early biomarker for neurodegenerative diseases such as Alzheimer’s and Parkinson’s, yet its underlying mechanisms remain poorly understood. Current models rely heavily on rodents, which differ from humans in olfactory receptor gene expression, regenerative capacity, and immune responses. These limitations hinder translational research and highlight the need for a human-specific model to study olfactory neurobiology, disease susceptibility, and regeneration.
This project aims to replace animal models by establishing a human pluripotent stem cell (hPSC)-derived model of the olfactory epithelium (OE) and olfactory sensory neurons (OSNs). Our approach builds on preliminary data demonstrating that WNT inhibition promotes anterior cranial placode identity, a key step in OE differentiation. Specific Aim 1 will optimize directed differentiation of olfactory epithelial cells from anterior placodes by testing the role of FGF8 and BMP4 in OE fate specification. We will also assess the ability of OE cells to secrete cytokines in response to infection, modeling human-specific neuroimmune interactions. Specific Aim 2 will generate functional olfactory sensory neurons (OSNs) from OE progenitors. We will identify inducers of OSN differentiation, determine the timing of neurogenesis, and evaluate functionality using odorant exposure and calcium imaging.
This project will significantly reduce reliance on animal models by offering a scalable, human-based system to study olfactory development, neurodegeneration, and therapeutic interventions. Our long-term objective is to establish a patient-derived model to assess olfactory dysfunction in neurodegenerative diseases (e.g., combining olfactory bulb with the epithelium) and addressing whether the loss of smell is a critical biomarker for neurodegeneration, bridging a critical gap in translational research.
This project aims to replace animal models by establishing a human pluripotent stem cell (hPSC)-derived model of the olfactory epithelium (OE) and olfactory sensory neurons (OSNs). Our approach builds on preliminary data demonstrating that WNT inhibition promotes anterior cranial placode identity, a key step in OE differentiation. Specific Aim 1 will optimize directed differentiation of olfactory epithelial cells from anterior placodes by testing the role of FGF8 and BMP4 in OE fate specification. We will also assess the ability of OE cells to secrete cytokines in response to infection, modeling human-specific neuroimmune interactions. Specific Aim 2 will generate functional olfactory sensory neurons (OSNs) from OE progenitors. We will identify inducers of OSN differentiation, determine the timing of neurogenesis, and evaluate functionality using odorant exposure and calcium imaging.
This project will significantly reduce reliance on animal models by offering a scalable, human-based system to study olfactory development, neurodegeneration, and therapeutic interventions. Our long-term objective is to establish a patient-derived model to assess olfactory dysfunction in neurodegenerative diseases (e.g., combining olfactory bulb with the epithelium) and addressing whether the loss of smell is a critical biomarker for neurodegeneration, bridging a critical gap in translational research.
Aranzazu Villasante, PhD
Fundacio Institut de Bioenginyeria de Catalunya (Institute for Bioengineering of Catalonia), Barcelona, Spain
A Human-Relevant Model for Real-Time Monitoring of MYCN-Driven Neuroblastoma Spreading and Drug Testing
Neuroblastoma (NB) is the most common extracranial solid tumor in children, with high-risk cases frequently driven by MYCN amplification, which promotes aggressive tumor progression and metastasis. Despite advances in therapy, survival rates for high-risk NB remain below 50%, emphasizing the need for human-relevant, predictive models to study early metastatic dissemination and improve drug development. Current preclinical models, including murine xenografts and transgenic mouse models, fail to accurately replicate early NB metastasis and introduce significant species-specific limitations, delaying clinical translation. To address these challenges, we propose the development of an NB-on-a-chip platform, integrating microfluidics, biomimetic scaffolds, and real-time biosensors to model NB metastasis in the bone marrow niche. This system will allow precise control and monitoring of tumor invasion while providing a non-animal alternative for preclinical drug testing. Our specific aims are to: (1) Develop and validate a microengineered NB-on-a-chip platform mimicking the bone marrow microenvironment, (2) Integrate impedance biosensors for real-time, label-free monitoring of tumor migration and invasion, and (3) Evaluate the efficacy of three anti-metastatic therapies, including LEM3, a novel MYCN-targeting drug.
The long-term objective of this work is to establish a scalable, reproducible, and physiologically relevant alternative to animal models for studying NB metastasis and testing anti-metastatic drugs. By bridging the gap between traditional 2D cultures and in vivo models, this system will enhance predictive preclinical screening, reduce reliance on murine models, and accelerate the development of targeted therapies for pediatric cancers.
The long-term objective of this work is to establish a scalable, reproducible, and physiologically relevant alternative to animal models for studying NB metastasis and testing anti-metastatic drugs. By bridging the gap between traditional 2D cultures and in vivo models, this system will enhance predictive preclinical screening, reduce reliance on murine models, and accelerate the development of targeted therapies for pediatric cancers.
The Foundation wishes to thank all of the applicants who submitted proposals for their interest in advancing alternative methods for conducting high quality scientific research. We would also like to thank our dedicated reviewers for sharing their time and expertise.
Information for the 2026 Annual Open Grant program will be posted at the beginning of January 2026.
Past Recipients
Click below to view lists of past grant recipients.
2024
Grantees
Grantees
2023
Grantees
Grantees
2022
Grantees
Grantees
2021
Grantees
Grantees
2020
Grantees
Grantees
2019
Grantees
Grantees
2018
Grantees
Grantees
2017
Grantees
Grantees
