Liver Organoids Research Articles

Rotating culture regulates the formation of HepaRG-derived liver organoids via YAP translocation.

Liver organoid serves as an alternative model for liver pathophysiology in carbohydrate or lipid metabolism and xenobiotic metabolism transformation. Biomechanical cues including spaceflight mission can affect liver organoid construction and their related functions, but their underlying mechanisms are not fully understood yet. Here, a rotating cell culture device, namely Rotating Flat Chamber (RFC), was specifically designed for adhering cells or cell aggregated to elucidate the effects of altered gravity vector on HepaRG-derived liver organoids construction. The organoids so formed under RFC presented the fast growth rate and large projection area. Meanwhile, the expressions of two pluripotency markers of SOX9 and CD44 were enhanced. This finding was positively correlated with the increased YAP expression and nuclear translocation as well as the elevated α4β6-integrin expression. Inhibition of YAP expression and nuclear translocation decreased the expression of SOX9 and CD44 under RFC, thereby attenuating the pluripotency of HepaRG-derived liver organoids. In conclusion, we proposed a novel liver organoid construction method using rotating culture, by which the pluripotency of liver organoids so constructed is mediated by α4β6-integrin and YAP translocation. This work furthered the understanding in how the gravity vector orientation affects the construction of liver organoids and the related mechanotransductive pathways.

Ciliopathy Organoid Models - a Comprehensive Review.

Cilia are membrane-bound organelles found on the surface of most mammalian cell types and play numerous roles in human physiology and development, including osmo- and mechanosensation, as well as signal transduction. Ciliopathies are a large group of - usually rare - genetic disorders resulting from abnormal ciliary structure or ciliary dysfunction that have a high collective prevalence. Autosomal dominant or recessive polycystic kidney disease (ADPKD/ARPKD), Bardet-Biedl-Syndrome and primary ciliary dyskinesia (PCD) are the most frequent etiologies. Rodent and zebrafish models have improved the understanding of ciliopathy pathophysiology. Yet, the limitations of these genetically modified animal strains include the inability to fully replicate the phenotypic heterogeneity found in humans, including variable multi-organ involvement. Organoids, self-assembled 3D-cell-based models derived from human induced pluripotent stem cells (iPSCs) or primary tissues, can recapitulate certain aspects of the development, architecture, and function of the target organ in the dish. The potential of organoids to model patient-specific genotype-phenotype correlations has increased their popularity in ciliopathy research and led to the first preclinical organoid-based ciliopathy drug screens. This review comprehensively summarizes and evaluates current ciliopathy organoid models, focusing on kidney, airway, liver, and retinal organoids, as well as the specific methodologies used for their cultivation and for interrogating ciliary dysfunction.

Insight to Biofabrication of Liver Microtissues for Disease Modeling: Challenges and Opportunities.

In the last decade, liver diseases with high mortality rates have become one of the most important health problems in the world. Organ transplantation is currently considered the most effective treatment for compensatory liver failure. An increasing number of patients and shortage of donors has led to the attention of reconstructive medicine methods researchers. The biggest challenge in the development of drugs effective in chronic liver disease is the lack of a suitable preclinical model that can mimic the microenvironment of liver problems. Organoid technology is a rapidly evolving field that enables researchers to reconstruct, evaluate, and manipulate intricate biological processes in vitro. These systems provide a biomimetic model for studying the intercellular interactions necessary for proper organ function and architecture in vivo. Liver organoids, formed by the self-assembly of hepatocytes, are microtissues and can exhibit specific liver characteristics for a long time in vitro. Hepatic organoids are identified as an impressive tool for evaluating potential cures and modeling liver diseases. Modeling various liver diseases, including tumors, fibrosis, non-alcoholic fatty liver, etc., allows the study of the effects of various drugs on these diseases in personalized medicine. Here, we summarize the literature relating to the hepatic stem cell microenvironment and the formation of liver Organoids.

Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells.

The fetal liver is a hematopoietic organ, hosting a diverse and evolving progenitor population. While human liver organoids derived from pluripotent stem cells (PSCs) mimic aspects of embryonic and fetal development, they typically lack the complex hematopoietic niche and the interaction between hepatic and hematopoietic development. We describe the generation of human Fetal Liver-like Organoids (FLOs), that model human hepato-hematopoietic interactions previously characterized in mouse models. Developing FLOs first integrate a yolk sac-like hemogenic endothelium into hepatic endoderm and mesoderm specification. As the hepatic and hematopoietic lineages differentiate, the FLO culture model establishes an autonomous niche capable of driving subsequent progenitor differentiation without exogenous factors. Consistent with yolk sac-derived waves, hematopoietic progenitor cells (HPCs) within FLOs exhibit multipotency with a preference for myeloid lineage commitment, while retaining fetal B and T cell differentiation potential. We reconstruct in FLOs the embryonic monocyte-to-macrophage and granulocyte immune trajectories within the FLO microenvironment and assess their functional responses in the liver niche. In vivo, FLOs demonstrate a liver engraftment bias of hematopoietic cells, recapitulating a key phenomenon of human hematopoietic ontogeny. Our findings highlight the intrinsic capacity of liver organoids to support hematopoietic development, establishing FLOs as a platform for modeling and manipulating human blood-liver niche interactions during critical stages of development and disease.

Protein absorption alters the cellular targeting of glycopolymeric nanoparticles

Glycopolymeric nanoparticles are widely employed in biomedical applications due to their high targeting ability, biocompatibility, and biodegradability. The pendant saccharides serve as targeting ligands to be explicitly coordinated to receptors on cell membrane surfaces. However, proteins in the blood often absorb onto the nanoparticle surface, potentially impacting the exposure and effectiveness of the saccharide ligands for targeted delivery. To elucidate the protein absorption effects, we prepared micelles decorated with different percentages of fructose moieties and evaluated the bioactivity of nanoparticles using 2D cell culture, tumor spheroid, liver organoid, and coculture models. The 2D cell culture model did not show a significant difference in activity between protein-coated and non-coated micelles. However, a dramatic decrease in cellular uptake and penetration ability of micelles was observed in 3D tumor spheroids after protein absorption. Additionally, protein absorption notably increased micelle uptake in liver organoids. In the coculture model, protein absorption reduced micelle uptake in tumor spheroids while favoring uptake in liver organoids. Our work suggests that decreasing non-specific protein absorption of glycopolymeric nanoparticles could enhance their delivery efficiency. These findings underscore the importance of understanding protein interactions in nanoparticle applications for drug delivery.

Optimization of culture conditions to generate vascularized multi-lineage liver organoids with structural complexity and functionality

Hepatic organoids (HOs), primarily composed of hepatobiliary cells, do not represent the pathogenesis of liver diseases due to the lack of non-parenchymal cells. Multi-lineage liver organoids (mLOs) containing various cell types found in the liver offer a promising in vitro disease model. However, their structural complexity remains challenging to achieve due to the difficulty in optimizing culture conditions that meet the growth need of all component cell types. Here, we demonstrate that cystic HOs generated from hPSCs can be expanded long-term and serve as a continuous source for generating complex mLOs. Assembling cystic HOs with hPSC-derived endothelial and hepatic stellate cell-like cells under conventional HO culture conditions failed to support the development of multiple cell types within mLOs, resulting in biased differentiation towards specific cell types. In contrast, modulating the cAMP/Wnt/Hippo signaling pathways with small molecules during assembly and differentiation phases efficiently generate mLOs containing both hepatic parenchymal and non-parenchymal cells. These mLOs exhibited structural complexity and functional maturity, including vascular network formation between parenchymal lobular structures, cell polarity for bile secretion, and the capacity to respond to fibrotic stimuli. Our study underscores the importance of modulating signaling pathways to enhance mLO structural complexity for applications in modeling liver pathologies.

Human Multi-Lineage Liver Organoid Model Reveals Impairment of CYP3A4 Expression upon Repeated Exposure to Graphene Oxide.

Three-dimensional hepatic cell cultures can provide an important advancement in the toxicity assessment of nanomaterials with respect to 2D models. Here, we describe liver organoids (LOs) obtained by assembling multiple cell lineages in a fixed ratio 1:1:0.2. These are upcyte® human hepatocytes, UHHs, upcyte® liver sinusoidal endothelial cells, LSECs, and human bone marrow-derived mesenchymal stromal cells, hbmMSCs. The structural and functional analyses indicated that LOs reached size stability upon ca. 10 days of cultivation (organoid maturation), showing a surface area of approximately 10 mm2 and the hepatic cellular lineages, UHHs and LSECs, arranged to form both primitive biliary networks and sinusoid structures, alike in vivo. LOs did not show signs of cellular apoptosis, senescence, or alteration of hepatocellular functions (e.g., dis-regulation of CYP3A4 or aberrant production of Albumin) for the entire culture period (19 days since organoid maturation). After that, LOs were repeatedly exposed for 19 days to a single or repeated dose of graphene oxide (GO: 2-40 µg/mL). We observed that the treatment did not induce any macroscopic signs of tissue damage, apoptosis activation, and alteration of cell viability. However, in the repeated dose regimen, we observed a down-regulation of CYP3A4 gene expression. Notably, these findings are in line with recent in vivo data, which report a similar impact on CYP3A4 when mice were repeatedly exposed to GO. Taken together, these findings warn of the potential detrimental effects of GO in real-life exposure (e.g., occupational scenario), where its progressive accumulation is likely expected. More in general, this study highlights that LOs formed by many cell lineages can enable repeated exposure regimens (suitable to mimic accumulation); thus, they can be suitably considered alternative or complementary in vitro systems to animal models.

LP-29 Development of human pluripotent stem cell-derived liver organoids for liver toxicity testing

LP-29 Development of human pluripotent stem cell-derived liver organoids for liver toxicity testing

Drug evaluation platform based on non-destructive and real-time in situ organoid fate state monitoring by graphene field-effect transistor

3D organoid models have been widely used in drug evaluation because they effectively mimic physiological conditions and cell–cell interactions. However, current organoid-based drug evaluation heavily relies on invasive methods like qRT-PCR and immunofluorescence quantification, which disrupts the organoid’s structure and physiological function during the evaluation process. Therefore, it is urgent to develop a non-destructive drug evaluation platform to continuously monitor organoid fate. Here, a non-destructive and real-time in situ platform based on field effect transistor sensor (NDRS-FET) was developed to monitor organoid fate and obtained accurate drug evaluation by conducting the dynamic models. Using liver organoid as the model, the transition kinetics of liver organoid fate in response to candidate drug compounds were recorded by the NDRS-FET platform. OSM, FH1 and BG45 were successfully identified as effective regulators of liver organoid fate. Compared to traditional methods, the NDRS-FET platform could not only accurately capture the drug effect on organoid in real time, but also reduce the intergroup deviation among samples, providing more accurate and reliable tool for drug development and screening.

Self-Assembled Generation of Multi-zonal Liver Organoids from Human Pluripotent Stem Cells.

Distinct hepatocyte subpopulations are spatially segregated along the portal-central axis and critical to understanding metabolic homeostasis and liver injury. While several bioactive molecules have been described to play a role in directing zonal fates, including ascorbate and bilirubin, in vitro replication of zonal liver architecture has not been achieved to date. In order to evaluate hepatic zonal polarity, we developed a self-assembling zone-specific liver organoid culture by co-culturing ascorbate and bilirubin enriched hepatic progenitors derived from human induced pluripotent stem cells. We found that preconditioned hepatocyte-like cells exhibited zone-specific functions associated with urea cycle, glutathione synthesis and glutamate synthesis. Single nucleus RNA sequencing analysis of these zonally patterned organoids identifies hepatoblast differentiation trajectory that mimics periportal-, interzonal-, and pericentral human hepatocytes. Epigenetic and transcriptomic analysis showed that zonal identity is orchestrated by ascorbate or bilirubin dependent binding of histone acetyltransferase p300 (EP300) to methylcytosine dioxygenase TET1 or hypoxia-inducible factor 1-alpha (HIF1α). Transplantation of the self-assembled zonally patterned human organoids improved survival of immunodeficient rats who underwent bile duct ligation by ameliorating the hyperammonemia and hyperbilirubinemia. Overall, this multi-zonal organoid system serves as an in vitro human model to better recapitulate hepatic architecture relevant to liver development and disease.

Generation of human iPSC-derived 3D bile duct within liver organoid by incorporating human iPSC-derived blood vessel

In fetal development, tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis. Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC). We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells, both derived from hiPSCs. After 3 weeks, liver progenitors within hiPSC-BV-incorporated liver organoids (BVLO) differentiate to cholangiocytes and acquire epithelial characteristics, including intercellular junctions, microvilli on the apical membrane, and secretory functions. Furthermore, liver surface transplanted-BVLO temporarily attenuates cholestatic injury symptoms. Single cell RNA sequence analysis suggests that BD interact with the BV in BVLO through TGFβ and Notch pathways. Knocking out JAG1 in hiPSC-BV significantly attenuates bile duct formation, highlighting BVLO potential as a model for Alagille syndrome, a congenital biliary disease. Overall, we develop a novel 3D co-culture method that successfully establishes functional human BDs by emulating liver epithelial-BV interaction.

Framework nucleic Acid-MicroRNA mediated hepatic differentiation and functional hepatic spheroid development for treating acute liver failure

The specific induction of hepatic differentiation presents a significant challenge in developing alternative liver cell sources and viable strategies for clinical therapy of acute liver failure (ALF). The past decade has witnessed the blossom of microRNAs in regenerative medicine. Herein, microRNA 122-functionalized tetrahedral framework nucleic acid (FNA-miR-122) has emerged as an unprecedented and potential platform for directing the hepatic differentiation of adipose-derived mesenchymal stem cells (ADMSCs), which offers a straightforward and cost-effective method for generating functional hepatocyte-like cells (FNA-miR-122-iHep). Additionally, we have successfully established a liver organoid synthesis strategy by optimizing the co-culture of FNA-miR-122-iHep with endothelial cells (HUVECs), resulting in functional Hep:HUE-liver spheroids. Transcriptome analysis not only uncovered the potential molecular mechanisms through which miR-122 influences hepatic differentiation in ADMSCs, but also clarified that Hep:HUE-liver spheroids could further facilitate hepatocyte maturation and improved tissue-specific functions, which may provide new hints to be used to develop a hepatic organoid platform. Notably, compared to transplanted ADMSCs and Hep-liver spheroid, respectively, both FNA-miR-122-iHep-based single cell therapy and Hep:HUE-liver spheroid-based therapy showed high efficacy in treating ALF in vivo. Collectively, this research establishes a robust system using microRNA to induce ADMSCs into functional hepatocyte-like cells and to generate hepatic organoids in vitro, promising a highly efficient therapeutic approach for ALF.

Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture.

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.

Human Liver Organoids to Predict the Outcome of Kasai Portoenterostomy

BackgroundKasai portoenterostomy (KPE) remains the primary intervention for biliary atresia (BA), but its outcomes are highly variable. Reliable prognostic biomarkers remain elusive, complicating the management and prediction of postoperative progression. MethodLiver biopsies from BA patients taken at and after KPE (post-KPE) were used to generate organoids for RNA-sequencing analysis. Control organoids were derived from non-BA livers. Differential gene expression and enrichment analyses were performed to assess post-KPE transcriptomic changes between native liver survivors (NLS) and patients who eventually became liver transplant recipients (LTR). ResultsOrganoid datasets: 70 from liver biopsies at KPE (10 patients), 112 from post-KPE livers (13 livers; 12 patients), and 47 from control livers (9 patients). At KPE, BA organoids displayed mainly hepatocyte expression, a trait notably reduced in control organoids. Similarly, post-KPE organoids from NLS revealed a significant decrease in hepatocyte expression features and an overall increase in cholangiocyte expression features. A similar hepatocyte-to-cholangiocyte expression transition was evidenced in paired liver organoids (at- and post-KPE) generated from an NLS. In contrast, post-KPE organoids from LTR maintained a high level of hepatocyte expression features. ConclusionOur study demonstrated that an elevated expression of hepatocyte features in KPE organoids may indicate aberrant cholangiocyte development in BA livers. In contrast, a post-KPE hepatocyte-to-cholangiocyte expression transition in NLS may imply effective biliary recovery. The lack of this transition in LTR organoids indicates ongoing disease progression, highlighting the potential for organoid-based transcriptomic profiling to inform KPE success and guide BA management. Level of EvidenceLevel III.

Aged fragmented-polypropylene microplastics induced ageing statues-dependent bioenergetic imbalance and reductive stress: In vivo and liver organoids-based in vitro study

Ageing is a nature process of microplastics that occurrs daily, and human beings are inevitably exposed to aged microplastics. However, a systematic understanding of ageing status and its toxic effect is currently still lacking. In this study, plastic cup lids-originated polypropylene (PP) microplastics were UV-photoaged until the carbonyl index (CI), a canonical indicator for plastic ageing, achieved 0.08, 0.17, 0.22 and 0.28. The adverse hepatic effect of these aged PPs (aPPs) was evaluated in Balb/c mice (75 ng/mL water, about 200 particles/day) and human-originated liver organoids (LOs, 50 particles/mL, ranged from 5.94 to 13.15 ng/mL) at low-dose equivalent to human exposure level. Low-dose of aged PP could induce hepatic reductive stress both in vitro and in vivo, by elevating the NADH/NAD+ratio in a CI-dependent manner, together with hepatoxicity (indicated by increased AST secretion and cytotoxicity), and disrupted the genes encoding the nutrients transporters and NADH subunits accompanied by the restricted ATP supply, declined mitochondrial membrane potential and mitochondrial complexI/IV activities, without significant increase in MDA levels in the liver. These changes in the liver disrupted systematic metabolism, representing a circulatory panel of increases in the lactate, triglyceride, Fgf21 levels, and decreases in the pyruvate level, linked the reductive stress to the declined body weight gain but elevated hepatic NADH contents following aPPs exposure. Additionally, assessing by the LOs, it was found that digestion drastically accelerated the ageing of aPPs and worsen the energy supply upon mitochondria, representing a “scattergun effect” induced by the formation of micro- and nano-plastics mixture toward NADH/NAD+imbalance.

Fast formation and maturation enhancement of human liver organoids using a liver-organoid-on-a-chip.

Background: Spatial and functional hepatic zonation, established by the heterogeneous tissue along the portal-central axis of the liver, is important for ensuring optimal liver function. Researchers have attempted to develop reliable hepatic models to mimic the liver microenvironment and analyze liver function using hepatocytes cultured in the developed systems. However, mimicking the liver microenvironment in vitro remains a great challenge owing to the lack of perfusable vascular networks in the model systems and the limitation in maintaining hepatocyte function over time. Methods: In this study, we established a microphysiological system that operated under continuous flush medium flow, thereby allowing the supply of nutrients and oxygen to liver organoids and the removal of waste and release of cytokines therefrom, similar to the function of blood vessels. Results: The application of microphysiological system to organoid culture was advantageous for reducing the differentiation time and enhancing the functional maturity of human liver organoid. Conclusion: Hence, our microphysiological culture system might open the possibility of the miniaturized liver model system into a single device to enable more rational in vitro assays of liver response.

The Construction of Stem Cell-Induced Hepatocyte Model and Its Application in Evaluation of Developmental Hepatotoxicity of Environmental Pollutants.

Stem cells, with their ability to self-renew and differentiate into various cell types, are a unique and valuable resource for medical research and toxicological studies. The liver is the most crucial metabolic organ in the human body and serves as the primary site for the accumulation of environmental pollutants. Enrichment with environmental pollutants can disrupt the early developmental processes of the liver and have a significant impact on liver function. The liver comprises a complex array of cell types, and different environmental pollutants have varying effects on these cells. Currently, there is a lack of well-established research models that can effectively demonstrate the mechanisms by which environmental pollutants affect human liver development. The emergence of liver cells and organoids derived from stem cells offers a promising tool for investigating the impact of environmental pollutants on human health. Therefore, this study systematically reviewed the developmental processes of different types of liver cells and provided an overview of studies on the developmental toxicity of various environmental pollutants using stem cell models.

The liver organoid's past, present and future: a personalized medicine strategy

Organoids are three-dimensional (3D) cell culture systems derived from human pluripotent stem cells or organotypic differentiation, replicating the complex interactions and functionalities of actual organs. These systems offer significant advantages for studying human tissue and organ biology, addressing limitations of animal models related to sample accessibility and ethical concerns. Liver organoids, in particular, are advanced models developed to study hepatic phenotypes, encompassing various cell types and enabling detailed investigation of cellular, molecular, and genetic aspects of liver diseases, drug metabolism, and protein secretion. They hold promise for fundamental research, drug discovery, and regenerative medicine applications. Despite their potential, organoids face limitations such as simplicity, lack of high-fidelity cell types, flexibility, and atypical physiology. Enhancements in organotypic liver-like surrogates, incorporating in vivo-like cell interactions and architecture, along with advancements in microfluidic chip technology, are expected to improve models for disease, toxicity, and drug discovery, paving the way for new treatments. This review will provide an overview of the history and development of liver organoids, their current progress, challenges, applications, and future prospects in the field of personalized medicine.

Animal and organoid models to elucidate the anti-fibrotic effect of steroid on biliary atresia

PurposeWe performed animal and organoid study to evaluate the anti-fibrotic effect of steroid on biliary atresia (BA) and the underlying patho-mechanism.MethodsBA animal models were created by inoculation of mice on post-natal day 1 with rhesus rotavirus (RRV). They received either 20 µl phosphate-buffered saline (PBS) or steroid from day 21 to day 34. On day 34, their serum samples were collected for hormonal markers. Necrosis, fibrosis and CK 19 expression in the liver were evaluated. Liver organoids were developed and their morphology as well as bulk RNA sequencing data were analyzed.ResultsTwenty-four mice developed BA features after RRV injection and were equally divided into steroid and PBS groups. On day 34, the weight gain of steroid group increased significantly than PBS group (p < 0.0001). All mice in the PBS group developed liver fibrosis but only one mouse in the steroid group did. Serum bilirubin and liver parenchymal enzymes were significantly lower in steroid group. The morphology of liver organoids were different between the two groups. A total of 6359 differentially expressed genes were found between steroid group and PBS group.ConclusionBased on our findings obtained from RRV-induced BA animal and organoid models, steroid has the potential to mitigate liver fibrosis in BA.

Generation of Multicellular 3D Liver Organoids From Induced Pluripotent Stem Cells as a Tool for Modelling Liver Diseases.

The liver is an essential organ that is involved in the metabolism, synthesis, and secretion of serum proteins and detoxification of xenobiotic compounds and alcohol. Studies on liver diseases have largely relied on cancer-derived cell lines that have proven to be inferior due to the lack of drug-metabolising enzymes. Primary human hepatocytes are considered the gold-standard for evaluating drug metabolism. However, several factors such as lack of donors, high cost of cells, and loss of polarity of the cells have limited their widescale adoption and utility. Stem cells have emerged as an alternative source for liver cells that could be utilised for studying liver diseases, developmental biology, toxicology testing, and regenerative medicine. In this article, we describe in detail an optimised protocol for the generation of multicellular 3D liver organoids composed of hepatocytes, stellate cells, and Kupffer cells as a tractable robust model of the liver. Key features • Optimising a protocol for generating multicellular 3D liver organoids from induced pluripotent stem cells. Graphical overview.