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 educational or research institution worldwide, although there is a preference for U.S. applications in order to more quickly advance alternatives here.

Expert reviewers evaluate proposals based on scientific merit and feasibility, and the potential to reduce or replace the use of animals in the near future. Proposals are considered in fields of research, testing, or education. The maximum grant is $40,000, with an average 21% funding rate from 2015 to 2024.

Since 1993, ARDF has provided over $4 million in funds for projects in 31 states and 7 countries.


2024 Grant Awardees

Congratulations to awardees:

Aitor Aguirre, PhD
Michigan State University, East Lansing, MI
New human models for the study of atrial fibrillation
ABSTRACT »
Organoids have emerged as a promising alternative to traditional animal models in biomedical and pharmaceutical sciences. Organoids are derived from human cells and closely mimic the structure and function of human organs. This makes them more relevant for studying human biology, disease mechanisms, and drug responses compared to animal models, which may not always accurately reflect human physiology. The use of animal models also raises ethical concerns regarding animal welfare and suffering. Organoids offer a cruelty-free alternative that eliminates the need for animals in research, reducing reliance on animal experimentation. Converging advances in stem cell biology and tissue engineering have recently led to the emergence of powerful human heart organoid technologies. Our team recently reported the creation of the first highly physiologically relevant human heart organoids. Our technology provides an outstanding model for the study of human cardiovascular disease that also completely bypasses the need for animal models. Our objective in this proposal is to characterize a new human heart organoid model for the study of atrial fibrillation, a common heart rhythm condition affecting 60 million people worldwide. Studies of heart rhythm conditions are classically conducted in animal models, and make extensive use of rodents, dogs, pigs, and non-human primates, among others. To meet this goal, we will create human heart organoids with disease-relevant atria and tissue-resident macrophages and trigger atrial fibrillation-like symptoms by challenging organoids with inflammatory activators of the atrial fibrillation response. We will then characterize the model structurally, functionally, and molecularly, using confocal microscopy, scRNA-seq, and electrophysiology. The successful implementation of our model could have a significant positive impact by reducing animal use and providing a more robust human model for atrial fibrillation drug development.


Matthew Brown, PhD
University of Wisconsin-Madison, Madison, WI
In Vitro Transplantation Modeling Using Induced Pluripotent Stem Cell-Derived Grafts
ABSTRACT »
The aim of this project is to identify the endothelial single cell transcriptome profile of allograft endarteritis via co-culture of donor iPSC-derived EC targets with recipient peripheral blood mononuclear cells (PBMCs). This proposal develops and validates an optimized, entirely animal-free, assay for the diagnosis of pre-clinical allograft endarteritis, an important pathological component of kidney graft rejection. Multiple clinical trials have used genomics-based approaches to identify biomarkers of renal allorejection, but often they rely upon gathering data from biopsies containing heterogeneous cell types and therefore have a high degree of inherent variability, i.e., experimental noise. Our approach uses patient-specific blood specimens modeling kidney transplant patients, incorporates classical transplant immunology principles, and applies novel iPSC technology developed and optimized by our team. We use iPSC-derived endothelial cell (EC) targets—the front line of defense against allorejection. We then culture patient PBMCs with the EC targets, to model the recipient:donor interactions that take place immediately following kidney transplantation. The gene expression profiles of the rejecting targets and immune effectors will serve as unique biomarkers of rejection. The patient-specific, and highly pure cell populations used for these novel transplantation modeling studies offer a new window into the initial stages of allorejection. This early-stage project will generate data that will enable follow-on studies in this new area of “transplantation modeling.” We anticipate that this award will provide critical support to build upon our existing pilot data, paving the way for an entirely new avenue of preclinical research that does not require animals. Ultimately, this patient-specific assay for diagnosing transplant rejection will enable lasting improvements in the health and well-being of kidney transplant patients worldwide.


Laura Crisa, MD, PhD
University of Washington, Seattle, WA
Modeling human bone marrow engraftment and hematopoiesis in a micro-fluidic platform
ABSTRACT »
Derivation of hemopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) holds great potential for clinical applications, spanning from treatment of blood disorders to control transplant rejection. Yet, yield of HSCs developed from conventional PSC cultures is limited and their ability to engraft in animal models is variable due to poorly controlled xenogenic barriers. In fact, to date, no physiologically relevant culture system exists that can model both human HSC development from PSC and engraftment. Here, we propose to integrate basic cell biology and microfluidic technologies to develop a tissue-mimetic culture system supporting the development of HSCs from human PSC-derived hemogenic endothelium as it occurs in the embryo. In this system we aim to a) develop human HSCs through induction of hemogenic programs in PSC-derived endothelial cell tubes created in a microfluidic chip mimicking early hemogenic sites of the embryonic aorta, and b) track the engraftment, expansion and multi-lineage potential of HSCs emerging from the hemogenic vessel into downstream fetal liver and bone marrow-like tissue niches. This system will allow us to model mechanisms of PSC-derived HSC development relevant to their regenerative applications and provide a valuable testing platform for therapeutics that may affect human hemopoietic progenitors' functions and engraftment.


Carlos Flores, PhD and Christoph Dehio, PhD
University of Basel, Basel, Switzerland
A human bladder model to decipher host-uropathogen interactions from tissue to single-cells
ABSTRACT »
Urinary tract infections (UTIs) are among the most common diseases worldwide, and an economical and societal burden, exacerbating the global antimicrobial resistance crisis. However, UTI is still significantly understudied, and the majority of what we know has been gleaned from mouse models or cancer cells, despite critical tissue ultrastructural and physiological differences compared with humans. This hinders the development of effective therapeutics to overcome successive rounds of antibiotic use as gold standard treatment. Alternatively, we will employ a recently developed urine-tolerant human bladder model to mimic UTI patient-like scenarios. Within the project timeframe, we are specifically aiming at studying urothelial spatial-temporal dynamics during infection with uropathogenic E. coli clinical isolates. We will combine confocal microscopy with single-cell RNA-sequencing to address: i) Which and how differently urothelial cell populations sense and respond to infection?; ii) If there is a mechanism of urothelial sensitization for cells that have previously encountered infection?; iii) What are the host innate mechanisms triggered upon infection relapse after antibiotic treatment? Among our long-term objectives, we envisage the use of spatial transcriptomics to complement scRNA-seq analysis and further mechanistic studies to understand host/bacteria molecular players involved in the different infection stages. Additionally, our setup can be used for assessing the effect of flow/stretch components on urothelial physiology and infection, the development of new treatments, and the study of bladder diseases (e.g., cancer) or tissue regeneration. Overall, this project will provide critical urothelial tissue, cell and molecular insights, difficult to obtain from animals in a far less expensive, higher through-put, more tractable and less ethically challenging way, paving the way for animal replacement in preclinical/clinical studies beyond the UTI field.


Tien-Chan Hsieh, MD and Chan Zhou, PhD
University of Massachusetts Chan Medical School, Worcester, MA
Computational discovery of long non-coding RNAs as novel relapse risk biomarkers for pediatric acute lymphoblastic leukemia
ABSTRACT »
Acute lymphoblastic leukemia (ALL) is the most prevalent pediatric cancer. It is caused by the rapid production of immature lymphocytes, a type of white blood cell, in the bone marrow. B cell ALL (B-ALL) is the most common subtype. Although current therapies have yielded a high 5-year overall survival rate, 10% of patients still suffer from relapse. The relapse risk stratification system uses several prognostic biomarkers for treatment selection. Nevertheless, outcomes for patients within the same risk group remain heterogeneous. To develop the optimal treatment strategy for B-ALL patients, it is necessary to include new prognostic biomarkers for predicting relapse risk, which is our long-term goal. Long non-coding RNAs (lncRNAs) are transcripts with more than 200 nucleotides but do not encode proteins. LncRNAs have emerged as promising biomarkers due to their highly disease- and tissue-specific expression patterns. Therefore, to achieve the long-term goal, we aim to: (1) identify lncRNAs expressed in pediatric B-ALL patients by leveraging large-scale existing transcriptomics data; and then (2) identify and characterize lncRNA biomarkers for B-ALL relapse risk by employing advanced machine learning algorithms and in silico biological function analysis. This study will identify novel lncRNA biomarkers to improve the prediction of relapse risk in pediatric B-ALL. In conjunction with the current risk stratification protocol, these biomarkers will enhance risk classification and facilitate more precise treatment decisions. These biomarkers may also shed light on new molecular mechanisms driving B-ALL progression, potentially uncovering novel therapeutic targets. Additionally, our strategy applies beyond B-ALL to various cancer types. Importantly, this bioinformatics study will avoid the use of laboratory animals or biomedical reagents. This pilot study will also serve as an educational demonstration of non-animal methods in academic and training settings.


Ramaswamy Krishnan, PhD
Harvard Medical School, Boston, MA
A human precision-cut lung slice-based platform for fibrotic drug discovery
ABSTRACT »
Idiopathic Pulmonary Fibrosis (IPF) is currently an incurable disease with a median survival of only 3-5 years following diagnosis. Thus, new IPF treatments are being pursued intensively, with a significant focus on reducing or pre-emptively slowing the debilitating IPF hallmark of progressive lung stiffening. However, in conventional drug discovery approaches using animal models, lung stiffness measurements are complex, time-consuming, and often difficult to interpret in terms of cause and effect. Furthermore, no single animal model can fully recapitulate the physiological and histopathological changes of human IPF. Animal studies can even be misleading because fibrosis naturally resolves in the widely used bleomycin mouse model. Finally, animal testing is of ethical concern. To overcome these limitations, we focus on fibrotic and fibrosis-induced precision cut lung slices (PCLS) from human lungs. Our efforts are empowered by: 1) our published methodology to measure human PCLS stiffness, 2) our preliminary studies that demonstrate the IPF-bearing and fibrosis-induced PCLS to be ~3 fold and ~2 fold stiffer, respectively, and 3) our ability to track over space and time, PCLS stiffening and concomitant structural remodeling. Building on these efforts, we hypothesize that PCLS stiffness measurements can be miniaturized in a multi-well format to enable longitudinal monitoring of drug activity to therapeutically reduce, preemptively slow, and/or stop tissue stiffening. To test this hypothesis, we propose two specific aims: 1) to enhance measurement throughput to a 6-well format, and, 2) to demonstrate proof-of-concept by testing the therapeutic and preventive ability of candidate IPF drugs to reduce human PCLS stiffening. At the completion of our project, we expect to make available to academic laboratories and biopharma companies a novel human tissue-based screening technology to target fibrotic stiffening while simultaneously reducing cost, time, and animal use.


Wonjae Lee, PhD
Duke University, Durham, NC
Developing Human Cell-Based Glioblastoma Models Free from Animal Derivatives
ABSTRACT »
Glioblastoma (GBM), known for its extreme aggressiveness, presents significant hurdles to existing treatment strategies. Research and therapy development have traditionally relied on animal models and patient-derived xenograft (PDX) models. While indispensable, these models face ethical issues, are hampered by species-specific differences, and frequently fail to replicate human diseases accurately. In response to these limitations, we propose employing the human cell-based Neurovascular Unit (NVU) chip technology from our laboratory. Thisin vitro platform closely replicates the human brain's microenvironment, allowing GBM cells to exhibit their natural behaviors within a more relevant context. This approach aims to advance cancer modeling by reducing dependence on animal models and increasing the relevance of disease models to human conditions. Our project has two aims. The first is to embed GBM cell lines within the NVU chip to replicate the complex tumor microenvironment found in GBMs. Particular emphasis will be placed on recapitulating the interactions between GBM and the NVU, specifically regarding the blood-tumor-brain barrier (BTB) dynamics, cytokine signatures, and the cellular phenotype landscape. The second aim is to perform a comparative analysis of chemosensitivity between patient-derived GBM cells cultured in our NVU chip and previously published data from PDX models. This comparison aims to validate the NVU chip's efficacy by contrasting it with established results from PDX models, thereby circumventing the use of animal models in this proposal. By advancing non-animal research methods through this project, we aim to make a significant contribution to biomedical research, product testing, and education. This endeavor supports a shift towards more ethical, accurate, and viable scientific practices, reflecting a broader movement towards reducing animal use in research and improving the translatability of preclinical findings to human patients.


Rachel Miller, PhD; Anne-Marie Malfait, MD, PhD; and Richard Miller, PhD
Rush University Medical Center, Chicago, IL
Modeling Joint Pain using an Animal-free System on a Chip
ABSTRACT »
Pain arising as a symptom of rheumatic and musculoskeletal diseases is a formidable problem worldwide. Our long-term goal is to develop novel human cell and tissue-based methods for (1) identifying new targets for pain in osteoarthritis and other types of arthritis and (2) screening drug candidates for efficacy in the context of osteoarthritis and other rheumatic diseases. To achieve these long-term goals, we need to develop methods that can model the cellular interactions within a diseased joint. Specifically, it is critical that we design microfluidic approaches for modeling interactions between nociceptors and innervated joint tissues. We have shown that neo-innervation of the diseased synovial membrane is a hallmark of osteoarthritis, and – of high interest – our preliminary data suggest that synovial fibroblasts are a major source of factors that may mediate this process. Therefore, our immediate objectives are (1) To develop human cell-based systems to model the neo-innervation (“sprouting”) of nociceptors we have observed in the synovium of osteoarthritis joints, and (2) To develop human cell-based systems to model the nociceptor hyperexcitability that occurs in osteoarthritis. We propose to use human inducible pluripotent stem cell-derived sensory neurons as our neuronal source for these experiments to be co-cultured with human synovial fibroblasts sourced from healthy or OA donors. If successful, this system will completely replace the use of animal models for developing analgesics that will be effective in treating osteoarthritis pain. We wish to note that using animal models for human pain is not only particularly ethically problematic but has been proven completely ineffective. Technological advances have now provided us with the opportunity of completely ridding ourselves of an animal model-based approach. Moreover, the success of a joint-on-chip model for joint pain will open the doors to extending similar models to other areas of pain research.


Nilda Rodriguez, PhD
University of Northern Iowa, Cedar Falls, IA
Developing a model to study lipid bodies and sex-dependent immune responses in human monocyte-derived macrophages
ABSTRACT »
My goal is to develop an animal-free model to study lipids and sex-dependent responses in macrophages. Macrophages are antimicrobial and immunomodulatory cells whose activation and functions are regulated by their lipid metabolism. Intracellular lipids are stored and metabolized in lipid bodies which are implicated in cancer, metabolic disorders, and infectious diseases. We showed that Leishmania infection induces lipid bodies in macrophages. In related work, we also showed that macrophages from males and females respond differently to Leishmania infection. Those experiments were carried out in a mouse model. However, animal models have ethical and technical challenges, particularly at primarily undergraduate institutions (PUIs) such as the university where I teach biomedical and pre-health students. I initiated studies to move away from the mouse model and instead work in human cells. In this pilot study, I will use remnant cells from anonymous blood donations to explore the lipid composition and lipid body expression of macrophages from male versus female donors. Because biological sex modulates cellular lipids and the lipids comprising lipid bodies vary according to cell type, this study will shed light on the mechanisms driving sex-dependent macrophage immune responses. In addition to its ethical advantages, using leftovers from blood donations has a lower carbon footprint, and it is more economical to generate samples for PUI projects. Around 70% of students pursuing a four-year degree attend public PUIs. Access to budget friendly biomedical models enriches the educational experience and enhances the scientific literacy of countless students. Ultimately, the goals of this work include developing an animal-free model attainable to non-research intensive institutions, sharing the protocol, as well as contributing to the understanding of how lipids and sex-dependent responses modulate macrophage functions in health and disease.


Emily Stuchfield-Denby, MD and Xavier Moisset, MD, PhD
Université Clermont Auvergne, Clermont-Ferrand, France
Regulatory T cell suppressive functions in migraine
ABSTRACT »
Migraine is a frequent and disabling disorder with significant social and economic impact worldwide. It occurs more frequently in women and patients with autoimmune or inflammatory diseases. Cytokine and immune cell dysregulations have been evidenced in the disease and inflammation seems to play an important role in migraine chronification, but the role of inflammation in migraine pathophysiology remains unclear. Regulatory T (Treg) lymphocytes are important in maintaining immune homeostasis. They regulate pro-inflammatory effector T (Teff) cells and cytokine release through different suppressive mechanisms such as the hydrolysis of adenosine triphosphate (ATP) into adenosine (ADO), by Treg surface enzymes CD39 and CD73. ATP is involved in the transduction of pain signals in migraine, and its insufficient hydrolysis can lead to pain chronification. Recent studies suggest altered Treg proportions in migraine, with decreased CD39-positive Treg cells. This further suggests altered Treg suppressive functions, but no functional assays have been led to confirm that. We aim to show that Treg suppressive functions are altered in migraine and that the ADO pathway is deficient. Through Treg/Teff coculture we will measure the ability of Tregs to inhibit Teff proliferation and cytokine secretion and assess the CD39-related hydrolytic activity of Tregs. It is important to us to reduce laboratory animal use in our experiments and in reagent production. Therefore, our study completely relies on human tissue for analysis and the reagents we intend to use are made using animal-free methods. This study, following the ‘3Rs’ principle, should lead to a better understanding of migraine pathophysiology and the development of personalized treatments according to the immune pain profile of migraine patients. This work will demonstrate the power of animal-free and human-based experimentation for immune-cell testing.

2024 GRANT GUIDELINES


The Foundation wishes to thank all applicants who submitted proposals for their interest in developing alternative methods of conducting high quality scientific research. We would also like to thank our dedicated reviewers for sharing their time and expertise.





Past Recipients

Click below to view lists of past grant recipients.
2023
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2022
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2021
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2020
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2019
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2018
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2017
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