Animal Microphysiological Systems (MPS): A Step Toward the 3Rs Alternative Animal Models
As science pushes for new methods to reduce in vivo animal testing, with alternative animal models like microphysiological systems (MPS), offer an ethical, effective leap forward. These engineered models mimic human and animal organ functions with remarkable accuracy, supporting crucial applications like toxicity screening and mechanistic studies. Animal MPS that can generate consistent and reproducible data are key to support the implementation of the 3Rs principles —Replacement, Reduction, and Refinement— to pre-clinical safety assessment. They can reduce, ultimately replace, live animals testing for the regulatory evaluations of drug candidates.
Bridging the Gap: Qualifying Animal MPS to build trust in Human MPS
As the drug discovery process evolves, the need for reliable MPS has never been more evident. Both industry stakeholders and regulatory bodies stress the need for qualified in vitro systems. The transition from in vivo to MPS is challenging, and animal MPS can help bridge this gap by generating comparable data in vitro and in vivo. The use of animal MPS is imperative in establishing the need to use human, physiologically relevant models in therapeutic testing as it can assess the level of overlap in predictivity between human and other preclinical animal models.
InSphero’s Leadership
InSphero together with six international drug developers and two technology providers established the XSpecies Consortium in 2021 to evaluate how spherical co-culture of primary liver cells can be used for translational, animal-to-human, toxicology applications. To this end, drug candidates with different level of hepatotoxicity in human and pre-clinical animals (i.e. Rat, dog and monkey) were tested in 3D liver microtissues derived from the respective species, and the results compared with the effect reported in vivo. The findings from this collaborative effort will be submitted for peer-reviewed publication, helping to further advance the qualification process of MPS for pre-clinical hepatotoxicity testing.
Enhancing Drug Safety with Liver Microtissues: A Case Study on Human Hepatotoxicity Prediction
A Stark Example: The Fialuridine Trial
Animal models do not always predict the human response due to species differences. The clinical trial of fialuridine is a stark example of this limitation. Despite not raising any concerns during preclinical testing in rats, dogs and monkeys, the drug caused fatal liver failure in 5 of 15 volunteers. This discrepancy stemmed from a drug transporter specifically expressed in human but not in pre-clinical animal liver. Such example underscores the importance of identifying species-specific variabilities in drug development — an area where MPS can provide significant insights.
InSphero's Innovative Approach
InSphero’s work provides a practical example of the potential of animal MPS. We developed preclinical animal MPS called 3D InSight XSpecies Liver Microtissues. These models are composed by Macaca fascicularis (Cynomolgus), Canis familiaris (Beagle), or Rattus norvegicus (Sprague Dawley) primary parenchymal and non-parenchymal liver cells. These models self-organize into spheroids, maintaining viability, morphology, and albumin production for up to 7 days. At a transcriptomic level, the liver microtissues show different global gene expression patterns across the four species, with human and monkey Liver Microtissues models being closely aligned (Figure 1).
A simple 7-day dose-response curve with ATP measurements shows that fialuridine is more cytotoxic to 3D InSight™ Human Liver Microtissues than to 3D InSight™ Animal Liver Microtissues, hereby recapitulating the in vivo observation (Figure 2). This case underscores the significant role MPS can play for pharmaceutical companies, enhancing drug safety assessment by identifying human-specific toxicities before first-in-human trials.
Applications of Liver Microtissues in Investigative Toxicology
3D InSight™ XSpecies Liver Microtissues can be used to compare the cytotoxicity of drug candidates in human and animals, and to investigate species-specific mechanism of toxicities with transcriptomic sequencing and mechanistic studies. For instance, 3D InSightTM Rat Liver Microtissues were used to evaluate the role of cytochrome P450 (CYP450) in the toxicity of Aflatoxin B1. This compound produces a toxic reactive metabolite when processed by hepatic CYP450. In 3D InSightTM Rat Liver Microtissues, the cytotoxicity of Aflatoxin B1 was significantly reduced in presence of 1-aminobenzotriazole (ABT-1), a broad spectrum CYP450 inhibitor, suggesting a mechanism of toxicity consistent with in vivo findings (Figure 3).
Alternative animal models, such as liver microtissues, can bridge the gap between preclinical studies and human responses, leading to safer pharmaceuticals.
Ready to explore how InSphero’s Animal Liver Microtissues could transform your work? Discover our full suite of 3D InSight™ Translational Services.
Join us in our commitment to the FDA Modernization Act 2.0 to reduce and replace the use of animals in research by testing hepatotoxicity against the appropriate species in vitro so as not to conduct unnecessary experiments in animals when there is no need to.
Figure 1. RNA-seq analysis of 3D InSightTM XSpecies Liver Microtissues after 7 days of culture. PCA plot shows different the global gene expression patterns. Green – human, orange – monkey, blue – dog, pink – rat.
Figure 2. 7-day dose response curve of Fialuridine treatment in human and animal liver microtissues. Graphs report IC50ATP and 95% confidence interval of the mean.
Figure 3. ATP assay and LDH assay after 7-days of Aflatoxin B1 with or w/o ABT-1 treatment in 3D InSightTM rat Liver Microtissues. Unpaired t-test, two-tailed, vehicle control vs. treatment concentration, *p<0.05; **p<0.01.
References:
1- Brown PC, Hooberman BH, Skinner BL, et al. Potential value of animal microphysiological systems. ALTEX. Published online August 7, 2024. doi:10.14573/altex.2311141
2- Pognan, F., Beilmann, M., Boonen, H.C.M. et al. The evolving role of investigative toxicology in the pharmaceutical industry. Nat Rev Drug Discov 22, 317–335 (2023). https://doi.org/10.1038/s41573-022-00633-x
3- Messner S, Agarkova I, Moritz W, Kelm JM. Multi-cell type human liver microtissues for hepatotoxicity testing. Arch Toxicol. 2013;87(1):209-213. doi:10.1007/s00204-012-0968-2
4- McKenzie R, Fried MW, Sallie R, et al. Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B. N Engl J Med. 1995;333(17):1099-1105. doi:10.1056/NEJM199510263331702
5- SILVA, J.V.B., de OLIVEIRA, C.A.F., Ramalho, L.N.Z., 2022. An overview of mycotoxins, their pathogenic effects, foods where they are found and their diagnostic biomarkers. Food Science and Technology (Brazil) 42. doi:10.1590/fst.48520
