Júlia Vallverdú Ginés, PhD
Lead ScientistWhat Are Liver Organoids?
Liver organoids are three-dimensional structures derived from stem cells or primary liver tissue that self-organize through cell-cell and cell-matrix interactions to recapitulate the architecture and functional properties of the liver. Unlike spheroid cultures, which aggregate cells do not truly self-organize, organoids can be expanded long-term while maintaining genetic stability, cell polarity, and tissue-level architecture.
How Liver Organoids Are Generated
Liver organoids can be generated from two main sources:
- Pluripotent stem cell (PSC)-derived organoids — from either human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) —are produced by exposing cells to growth factors that guide them through a stepwise differentiation process that mirrors embryonic liver development. The cells are pre-differentiated in 2D culture and then fully differentiated in 3D. iPSCs have become the preferred source because they preserve the donor’s unique genetic background and avoid the ethical constraints associated with embryonic material, making them particularly well-suited for patient-specific disease modeling.
- Tissue-resident cell-derived organoids are generated from adult or fetal liver biopsies; the progenitors are enriched following enzymatic digestion of primary tissue. In both cases, an extracellular matrix scaffold, most commonly Matrigel, provides the mechanical support needed for 3D self-organization. Two subtypes of tissue-resident organoids can be distinguished based on their cell of origin: cholangiocyte-derived organoids (chol-orgs), derived from biliary epithelial cells (e.g., EPCAM⁺ or Lgr5⁺ ductal cells), and hepatocyte-derived organoids (hep-orgs), derived directly from mature hepatocytes or hepatic progenitors, consisting primarily of hepatocyte cells. Compared to chol-orgs, hep-orgs display higher expression of hepatocyte markers such as albumin, HNF4α, and CYP enzymes, and secrete 1000-fold more albumin, making them more suitable for drug metabolism and hepatotoxicity studies
Schematic representation of the generation of liver organoids. Source: Caiazza C, Parisi S, Caiazzo M. 2021.
Liver Organoids vs. 2D Hepatocyte Cultures and Animal Models
Depending on their context of use, organoids may overcome the limitations of current preclinical models. Primary human hepatocytes (PHHs) are considered the gold standard for human drug metabolism studies. Still, their 2D culture induces rapid dedifferentiation, with loss of CYP450 enzyme expression, drug transporters, albumin secretion, and urea production within days. On the other hand, immortalized cell lines, such as HepG2 or HeparG, are convenient and easy to culture on flat surfaces. Their performance can be improved by culturing them in 3D, which favors cell-cell interaction, but they still lack cytochrome P450 expression entirely. When cultured in 3D, primary human hepatocytes prolong maintenance of hepatic phenotype and function, remaining phenotypically stable for at least five weeks. Liver organoids additionally preserve bile canaliculi networks and cell polarity that are essential for modeling conditions such as cholestasis, features absent in monolayer systems.
Animal models, while more complex, are constrained by interspecies differences in drug metabolism, a gap illustrated by fialuridine. This hepatitis B drug passed every conventional preclinical test on animal species available in the early 90s, but triggered fatal hepatotoxicity in the phase II clinical trial, causing liver failure in 7 out of 15 patients. Not to mention the obvious ethical burden of using living animals in research.
Liver Organoids for Hepatotoxicity and Drug Safety Testing
The predictive limitations of conventional models have driven the development of organoid-based hepatotoxicity screening platforms, and the evidence that liver organoids outperform 2D cultures is growing. In a landmark study, Shinozawa et al. tested self-organized human iPSC-derived liver organoids against 238 pharmaceutical compounds, achieving a sensitivity of 88.7% and a specificity of 88.9% for hepatotoxicity detection. Drug-metabolizing enzyme expression remained stable after seven days of compound exposure, enabling chronic toxicity assessments that 2D cultures cannot support.
The inclusion of non-parenchymal cells further improves predictivity. Hepatic stellate cells (HSCs) regulate ECM production and sinusoidal blood flow and become activated in response to toxic stress, driving fibrogenic responses. Liver sinusoidal endothelial cells regulate the exchange of substances between blood and hepatocytes and modulate immune responses within the liver. Kupffer cells play a decisive role in hepatotoxic responses, and 3D coculture models incorporating them showed significantly greater cytotoxicity and cytokine release than monocultures exposed to the same compounds.
The regulatory environment is recognizing the potential of New Approach Methodologies (NAMs) for drug safety testing. In March 2026, the Food and Drug Administration (FDA) published a draft guidance intended to help drug developers validate new approach methodologies (NAMs) to be used instead of animal testing in drug development. Organoids were included as an example of innovative testing approaches.
FDA’s commitment to moving away from using animal testing as the default method for gaining drug safety information is strong. In June 2026, the regulatory agency accepted the first Letter of Intent under the ISTAND program for an AI-driven digital liver model designed to predict DILI, explicitly framed as a NAM to complement existing preclinical data. It is a signal that advanced in vitro tools, used together within a weight-of-evidence framework, are moving from the research bench toward regulatory acceptance.
Regulatory agencies in Europe are also embracing the movement towards animal-free, or at least animal-reduced, drug safety testing. The United Kingdom joined this commitment in 2025 and published a roadmap to phase out animal testing, starting with the use of animals to assess the potential for new treatments to cause skin and eye irritation and skin sensitisation by the end of 2026. Recently, in June 2026, the European Commission adopted the roadmap towards phasing out animal testing for chemical safety assessments. This will serve as a guiding plan for accelerating the path towards replacing, reducing, and refining animal testing for the safety assessments of chemicals.
Modeling Liver Disease with Organoids: NAFLD, Hepatitis, and Beyond
Beyond safety testing, liver organoids have demonstrated utility across a wide range of disease models. Ouchi et al. generated iPSC-derived organoids containing hepatocyte-like, stellate-like, and Kupffer-like cells and showed that exposure to free fatty acids reproduced the key stages of steatohepatitis: steatosis, inflammation, and fibrosis. Hendriks et al. used CRISPR-engineered human fetal hepatocyte organoids to identify inhibitors of ACC, FAS, and DGAT2 as effective against lipid accumulation, demonstrating the value of organoid systems for NAFLD drug target discovery.
For infectious disease, liver ductal organoids were used during the COVID-19 pandemic to show that SARS-CoV-2 directly infects the ductal epithelium, causing impaired barrier function and induction of proinflammatory cytokines. For monogenic diseases, patient-derived organoids from individuals with alpha-1-antitrypsin deficiency, Alagille syndrome, and Wilson disease have faithfully replicated the pathological phenotypes seen in patient biopsies and served as platforms for testing gene-editing correction strategies.
Challenges in Liver Organoid Workflows for High-Throughput Screening
Realizing the potential of liver organoids at scale requires solving two persistent challenges: batch-to-batch variability and limited throughput. When culture conditions fluctuate, observed differences in drug response can reflect technical noise rather than genuine biological variation, undermining assay reliability. MO:RE’s internal data reveal that automated organoid workflows consistently reduce the coefficient of variation in viability readouts across multiple 3D model types, while also increasing cell viability relative to manual handling.
Besides reproducibility, automation also means integrated quality control at early culture stages. It allows researchers to identify and discard organoids that deviate from expected morphology, avoiding the cost of carrying low-quality samples through the full workflow. Medium exchanges, compound treatments, and endpoint assays are expensive in both reagents and time; running them on organoids that will ultimately be excluded from analysis wastes resources that scale poorly in high-throughput settings.
The MO:BOT was designed with exactly these requirements in mind, integrating standardized liquid handling, gentle automated medium exchange, and real-time morphological quality control through the MO:CROSCOPE into a single platform. The MO:CROSCOPE is an imaging module that can be connected without cables or screws. Our platform automatically recognizes it and transfers the culture plate. You don’t need to remove the samples for the sterile environment.
It scans the full plate during standard handling steps, and every well is segmented during acquisition using a fast and efficient SegFormer deep learning model optimized for brightfield 3D cell culture morphology.
The plate heatmap colours every well by any selected morphology metric, enabling immediate spatial QC (e.g., edge effects, outlier wells, and treatment-group patterns) to be visible across the full plate in a single view. It is compatible with any brightfield-imageable 3D culture model, including liver organoids, and has been validated across kidney, cardiac, liver, synovial, brain organoids, and more.
Scalability will strongly depend on automation. At MO:RE, we are working hard to make it accessible.
Sources
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Caiazza C, Parisi S, Caiazzo M. Liver Organoids: Updates on Disease Modeling and Biomedical Applications. Biology (Basel). 2021 Aug 27;10(9):835. doi: 10.3390/biology10090835.
European Commission. Roadmap towards phasing out animal testing for chemical safety assessments. C(2026)3497. June 1, 2026. Available from: https://single-market-economy.ec.europa.eu/publications/roadmap-towards-phasing-out-animal-testing-chemical-safety-assessments_en
FDA. Roadmap to Reducing Animal Testing in Preclinical Safety Studies. April 2025. Available from: https://share.google/LA4d4MONFBxGtL49W
FDA CDER. FDA Releases Draft Guidance on Alternatives to Animal Testing in Drug Development. March 18, 2026. Available from: https://www.fda.gov/news-events/press-announcements/fda-releases-draft-guidance-alternatives-animal-testing-drug-development
FDA CDER. FDA Accepts First In Silico Drug Development Tool Under ISTAND Program to Help Predict Drug-Induced Liver Injury. June 3, 2026. Available from: https://www.fda.gov/drugs/drug-alerts-and-statements/fda-accepts-first-silico-drug-development-tool-under-istand-program-help-predict-drug-induced-liver
Hendriks D et al. Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis. Nat Biotechnol. 2023;41:1567–1581. doi: 10.1038/s41587-023-01680-4
Ouchi R et al. Modeling steatohepatitis in humans with pluripotent stem cell-derived Organoids. Cell Metab. 2019;30(2):374–384. doi: 10.1016/j.cmet.2019.05.007
Shao W et al. Advances in liver organoids: replicating hepatic complexity for toxicity assessment and disease modeling. Stem Cell Res Ther. 2025;16:27. doi: 10.1186/s13287-025-04139-2
Shinozawa T et al. High-Fidelity Drug-Induced Liver Injury screen using human pluripotent stem cell-derived Organoids. Gastroenterology. 2021;160(3):831–846. doi: 10.1053/j.gastro.2020.10.002
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UK Government. Replacing animals in science: A strategy to support the development, validation and uptake of alternative methods. November 11, 2025. Available from: https://www.gov.uk/government/publications/replacing-animals-in-science-strategy/replacing-animals-in-science-a-strategy-to-support-the-development-validation-and-uptake-of-alternative-methods
