Organoids as preclinical platforms for metabolic dysfunction-associated steatotic liver disease: Model construction, mechanistic dissection, and therapeutic translation

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as a global public health challenge, necessitating reliable in vitro models to unravel its complex pathophysiology and accelerate therapeutic development. Organoid systems have revolutionized MASLD research by recapitulating key hepatic structural features, cellular heterogeneity, and pathological processes. This review systematically summarizes liver organoids derived from human pluripotent stem cells, primary tissues, and immortalized cell lines in MASLD research, highlighting their distinct advantages in modeling MASLD pathogenesis, elucidating mechanisms, screening biomarkers, and validating therapeutics. In addition, we elaborate on the progress gained from multi-tissue organoid systems in deciphering inter-organ crosstalk and systemic metabolic perturbations associated with MASLD. We further discuss the inherent limitations of current organoid models and propose future directions for their development. Collectively, this review highlights the indispensable role of organoid models in unraveling the complex molecular mechanisms of MASLD, in advancing drug discovery, and in identifying diagnostic biomarkers, while also providing insights to accelerate their clinical translation toward precision medicine for MASLD.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Metabolic dysfunction-associated steatohepatitis; Organoid; Disease modeling; Mechanism

Core Tip: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease worldwide, yet targeted therapeutic options are limited. This review systematically summarizes liver organoids derived from human pluripotent stem cells, primary tissues, and immortalized cell lines in MASLD research, highlighting their distinct advantages in modeling MASLD pathogenesis, elucidating mechanisms, screening biomarkers, and validating therapeutics. It further discusses multi-tissue organoid systems, alongside current challenges and future directions to accelerate clinical translation and advance personalized medicine for MASLD.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly termed non-alcoholic fatty liver disease, is the most prevalent chronic liver disease worldwide. It arises from nutrient excess and insulin resistance in genetically susceptible individuals and affects an estimated 33% of adults worldwide, rendering it a major public health concern[1]. The disease encompasses a spectrum of liver conditions from simple steatosis to severe metabolic dysfunction-associated steatohepatitis (MASH) and further progressing to fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC)[2]. The complexity of MASLD pathogenesis, coupled with limited therapeutic approaches and a lack of efficient noninvasive early diagnostic biomarkers, highlights the requirement for novel research tools and models to deepen the understanding of pathogenic mechanisms and to expedite the identification and validation of novel therapeutic strategies and biomarkers[3].

In vivo animal models have contributed partially to our understanding of MASLD pathogenesis, yet interspecies differences in genetics and physiology severely compromise their translational relevance, frequently leading to clinical trial failures of therapeutic candidates validated in animals[4]. Meanwhile, conventional in vitro two-dimensional cell models lack the native tissue architecture, intact cell-cell crosstalk and physiological microenvironments, and thus fail to faithfully recapitulate the complex progression of MASLD[5]. To overcome the critical drawbacks of conventional in vivo animal models and traditional in vitro two-dimensional cell cultures, organoids, defined as in vitro three-dimensional (3D) cellular clusters derived from tissue-resident stem/progenitor cells or human pluripotent stem cells (hPSCs) with capacities for self-renewal and self-organization have emerged as a powerful research tool. Organoids exhibit tissue-specific structural and physiological functions, faithfully recapitulate core MASLD pathological phenotypes (including steatosis, inflammation and fibrosis), and enable the development of personalized disease models and multi-organ co-culture systems, thereby emerging as an ideal research tool for MASLD mechanistic exploration and therapeutic screening[6].

In this review, we comprehensively summarize the recent advances achieved with organoid technology in MASLD research over the past six years. Categorizing organoids by cellular origin, we elaborate on liver organoids (LOs) derived from hPSCs, primary tissues, or immortalized cell lines, focusing on their applications in MASLD model construction, exploration of pathogenic mechanisms, and therapeutic research (Figure 1). We also outline the research progress of multi-organoid systems in MASLD research. Finally, we analyze the current limitations of existing organoid models and explore future development directions, aiming to provide a reliable reference for basic research on MASLD and the clinical translation of potential therapeutics.

Figure 1 Schematic of liver organoids in metabolic dysfunction-associated steatotic liver disease research. MASLD: Metabolic dysfunction-associated steatotic liver disease; hPSCs: Human pluripotent stem cells; FFA: Free fatty acids; LPS: Lipopolysaccharide; TGF-β: Transforming growth factor-β.

LOS IN MASLD RESEARCH

hPSC-derived LOs in MASLD research

MASLD model construction and phenotypic recapitulation: hPSCs, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), serve as a robust cellular source for generating physiologically relevant LOs that faithfully recapitulate the core pathological spectrum of MASLD. Notably, hPSC-derived LOs originate from two distinct stem cell types with markedly different biological properties. hiPSCs are generated by reprogramming adult somatic cells. hiPSC-derived organoids retain the donor’s genetic background for patient-specific MASLD modeling, but the reprogramming process may introduce epigenetic aberrations and genomic instability[7]. In contrast, hESCs are isolated from the inner cell mass of blastocyst-stage embryos. hESC-derived organoids are devoid of reprogramming-associated defects and exhibit high batch-to-batch consistency, yet their broader application remains constrained by ethical considerations[8]. Accordingly, hiPSC-derived organoids are optimally suited for individualized disease modeling and precision drug screening, while hESC-derived organoids are more appropriate for standardized mechanistic investigations[9]. Over the past few years, continuous advances in hPSC-derived LO culture strategies have enabled the stepwise in vitro modeling of MASLD, covering the full pathological spectrum from simple steatosis to severe steatohepatitis and fibrosis[10].

Early studies aimed to establish foundational organoid models that could recapitulate the core pathological features of MASLD, thereby laying a methodological foundation for subsequent research. Ramli et al[11] established a protocol to generate human LOs (HLOs) comprising hepatocytes and cholangiocytes from hESCs. Incubation of HLOs with oleic acid (OA) and palmitic acid (PA) results in structural changes relevant to MASLD, including the disruption of the bile canalicular network and ductular reactions. In a related study, Qin et al[12] established an in vitro MASLD model by treating hESC-derived mature polarized hepatocyte organoids with 450 μmol/L free fatty acids (FFA) (OA:PA = 2:1), and this model recapitulates core pathological progression hallmarks of MASLD. Transcriptomic analysis revealed that this model partially recapitulates the molecular signatures of human MASH livers at the transcriptomic level. Using this platform, the therapeutic effects of several established antioxidants and lipid-lowering agents were evaluated against the key pathological phenotypes of MASLD using this model.

However, a major limitation shared by the aforementioned HLO models is that they contain only hepatocytes and lack hepatic stellate cells (HSCs) and Kupffer cells (KCs), two cell types that play indispensable roles in mediating inflammation and fibrosis during MASLD progression[13]. To overcome this critical cellular deficiency, Ouchi et al[14] established a repeatable method to generate HLOs comprising hepatocyte-, stellate-, and Kupffer-like cells from hiPSCs. After OA treatment, these LOs reproduce the core characteristics of steatohepatitis, exhibiting successive phenotypes of steatosis, inflammation, and fibrosis. Despite such remarkable advances in cellular complexity, existing organoid models still suffer from another key limitation: The absence of liver zonation, a structural feature essential for maintaining hepatic physiological heterogeneity and metabolic function. To fill this structural gap, Zhang et al[15] co-cultured hESC-derived hepatocytes separately with pericentral liver sinusoidal endothelial cells (LSECs) and periportal LSECs from hESC-derived liver lobules on poly (ethylene glycol) methyl ether acrylate grafted polydimethylsiloxane microchips to generate pericentral organoids and periportal organoids, and observed significant differences in lipid metabolism between pericentral organoids and periportal organoids. After incubation with 450 μmol/L OA and 150 μmol/L PA, lipid droplets accumulated in pericentral organoids in greater number and at a faster rate. Moreover, glucagon-like peptide-1 receptor (GLP-1R) agonist was shown to indirectly alleviate MASLD by activating GLP-1R on LSECs, rather than exerting direct impacts on hepatocytes.

To further improve the pathological fidelity of HLOs and simulate the heterogeneity of MASLD progression, researchers have optimized induction protocols and expanded model validation dimensions. Hess et al[16] differentiated hPSCs into HLOs, which were composed of hepatocyte-, HSC-, and cholangiocyte-like cells. Subsequently, single-cell analyses were performed to evaluate MASLD induction under three conditions: OA, PA, and transforming growth factor-β (TGF-β). The results displayed that OA induces gene expression patterns that are more reflective of benign steatosis, while PA triggers inflammatory alterations characteristic of steatohepatitis. TGF-β treatment leads to the most severe combined pathology of steatohepatitis and fibrosis. Building on these findings, Wu et al[17] designed a stage-specific induction protocol: hPSC-derived HLOs were first incubated with 500 μmol/L OA for 4 days, followed by treatment with 500 μmol/L OA combined with 25 ng/mL TGF-β for another 3 days, which successfully recapitulated the progressive course of MASH. RNA-sequencing (RNA-seq) and single-cell RNA-seq analyses unveiled that this MASH-HLO model not only exhibits characteristic pathological features including steatosis, oxidative stress, mitochondrial dysfunction, inflammation, and fibrosis, but also shares partial similarities with human MASH regarding transcriptomic profiles and cell communication patterns, thus strengthening the pathological relevance of the model.

Beyond biochemical induction and cellular/structural optimization, recent studies have expanded MASLD organoid modeling in two new critical dimensions: The hepatic mechanical microenvironment and organ-on-chip technology. Liu et al[18] cultured induced pluripotent stem cell (iPSC)-derived HLOs in their pioneering dynamic stiffening hydrogels and incubated the HLOs with 500 μmol/L OA for 3 days to establish a MASLD model. The HLOs in matrices with higher stiffness showed more lipid droplet accumulation as well as higher expression of fibrosis and lipid metabolism markers, which could be prevented by Rho kinase-inhibitor. This study further revealed the contribution of mechanosignaling to MASLD progression. Organ-on-a-chip refers to an innovative microfluidic culture system that recapitulates the physiological environment and function of human organs in vitro, enabling disease modeling and drug testing[19]. To overcome the inherent limitations of static culture (e.g., uneven nutrient diffusion, lack of physiological fluid shear stress), hPSC-derived HLOs were further integrated within microfluidic organ-on-chip systems to enhance model physiological relevance and scalability. Wang et al[20] put forward a novel strategy to construct an MASLD disease model using a hiPSC-derived HLOs-on-a-chip system. After incubation with 600 μmol/L FFA (OA:PA = 2:1), HLOs-on-a-chip exhibit characteristics of lipid metabolism dysfunction associated with MASLD progression, such as lipid droplet formation and abnormal triglyceride accumulation, along with typical pathological features characteristic of MASH, including increased reactive oxygen species (ROS) production and enhanced expression of inflammatory and fibrotic markers. Further optimizing the chip-based multicellular system, Balachander et al[21] established a functional liver tissue-on-a-chip by co-culturing human adult liver stem cell-derived hepatobiliary organoids with iPSC-derived KCs and HSCs. The MASH model was successfully induced by incubating the cells on the chip with 450 μmol/L OA, 150 μmol/L PA, 100 mmol/L fructose, 10 ng/mL lipopolysaccharide (LPS), and 10 mmol/L phenylacetic acid. Subsequently, anti-MASH drugs were tested, and the model was proven to recapitulate the responses observed in clinical trials. Building on microengineered chip technology, You et al[22] developed MASLD-specific HLO models derived from hiPSCs on a microengineered array chip. HLOs were incubated with 400 μmol/L OA and 200 μmol/L PA for 48 hours to successfully establish a MASLD model characterized by dysregulated lipid metabolism, elevated inflammatory cytokine secretion, and fibrosis. Furthermore, semaglutide, a GLP-1R agonist, was shown to exert significant biological activity in mitigating the pathological hallmarks of MASLD in this model. The distinct strategies for MASLD model construction and phenotypic recapitulation in LOs derived from hPSCs, along with their pathological characteristics, are summarized in Table 1.

Table 1 Metabolic dysfunction-associated steatotic liver disease model construction and phenotypic recapitulation in liver organoids derived from human pluripotent stem cells.

Cellular origin	Cell types	Model induction method	Phenotype reproduced	Ref.
hESCs	Hepatocyte-like, cholangiocyte-like	200/500 μM OA and PA for 4-14 days	Time- and dose-dependent elevation of lipid accumulation, ROS, and lipid peroxidation, accompanied by bile canaliculi network decay and ductular reactions	Ramli et al[11], 2020
hESCs	Hepatocyte-like	450 μM FFA (OA:PA = 2:1) for 2 days	Disrupted glucose/Lipid metabolism, oxidative stress, apoptosis, loss of polarization, impaired liver function, and ductular reaction	Qin et al[12], 2025
hiPSCs	Hepatocyte-like, KC-like, HSC-like	200/400/800 μM OA for 3-7 days	Steatosis, inflammation, and fibrosis	Ouchi et al[14], 2019
hESCs	Hepatocyte-like, liver sinusoidal endothelial cell-like	450 μM OA and 150 μM PA for 24/48 hours	Pericentral organoids from liver lobules exhibit faster and greater lipid droplet accumulation than periportal organoids	Zhang et al[15], 2025
hPSCs	Hepatocyte-like, cholangiocyte-like, HSC-like	10 ng/mL and 25 ng/mL TGF-β1/500 μM OA/500 μM PA for 4 days	OA induces benign steatosis; PA induces inflammatory changes of steatohepatitis; TGF-β1 induces steatohepatitis and fibrosis most prominently	Hess et al[16], 2023
hiPSCs	Hepatocyte-like, HSC-like, biliary-like	500 μM OA for 4 days, followed by treatment with 500 μM OA combined with 25 ng/mL TGF-β for another 3 days	Severe steatosis, oxidative stress, mitochondrial dysfunction, inflammation, and fibrosis as progressive characteristics of human MASH	Wu et al[17], 2024
hiPSCs	Hepatocyte-like, cholangiocyte-like	500 μM OA for 3 days	Increased lipid droplet accumulation, upregulated expression of fibrosis and lipid metabolism markers in higher stiffness matrices	Liu et al[18], 2025
hiPSCs	Hepatocyte-like, cholangiocyte-like	600 μM FFA (OA:PA = 2:1) for 7 days	Lipid accumulation, ROS production and up-regulated expression of inflammatory and fibrotic markers	Wang et al[20], 2020
hiPSCs and liver stem cells	Hepatocytes, cholangiocyte-like, KC-like, HSC-like	450 μM OA, 150 μM PA, 100 mmol/L fructose for 3 days, followed by addition of 10 ng/mL lipopolysaccharide and 10 mmol/L phenylacetic acid another 3/5 days	Steatosis, hepatocellular ballooning, inflammatory and fibrogenic microenvironment	Balachander et al[21], 2025
hiPSCs	Hepatocyte-like, cholangiocyte-like	400 μM OA and 200 μM PA for 48 hours	Dysregulated lipid metabolism, increased inflammatory factor secretion, and fibrosis	You et al[22], 2026

hESCs: Human embryonic stem cells; OA: Oleic acid; PA: Palmitic acid; ROS: Reactive oxygen species; FFA: Free fatty acids; hiPSCs: Human induced pluripotent stem cells; KC: Kupffer cell; HSC: Hepatic stellate cell; TGF-β: Transforming growth factor-β; MASH: Metabolic dysfunction-associated steatohepatitis; hPSCs: Human pluripotent stem cells.

Mechanistic exploration, therapeutic target identification, and drug screening: With the continuous optimization and maturation of hPSC-derived HLO models, these systems have become irreplaceable platforms for in-depth mechanistic research and therapeutic exploration of MASLD. Organoid technology has demonstrated unique value in dissecting the genetic mechanisms of MASLD and evaluating targeted interventions. Kimura et al[23] developed a pooled population organoid strategy, representing an en masse approach for generating genetically diverse HLOs from 24 donor-derived iPSCs with distinct genotypes. Phenotypic and genotypic analyses of this HLO panel identified the GCKR-rs1260326:C>T variant as a key genetic factor strongly associated with MASH-relevant traits and showed that the GCKR-rs1260326 TT genotype increases susceptibility to de novo lipid accumulation in HLOs. Furthermore, multi-omics analyses revealed that mitochondrial dysregulation is linked to GCKR^TT-related metabolic assaults. Nicotinamide riboside and nitazoxanide mitigate the mitochondrial dysfunction of the GCKR^TT HLO steatohepatitis-like model and reduce inflammatory gene expression. Building on this, the team further demonstrated that inhibition of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase, a modulator of de novo nicotinamide adenine dinucleotide synthesis, corrects fibrosis, inflammation, and DNA damage in MASLD by elevating hepatic nicotinamide adenine dinucleotide levels and enabling genomic protection. Using iPSC-derived HLOs harboring the GCKR-rs1260326:C>T variant, they found that pharmacological inhibition of only α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase ameliorates OA-induced steatosis as well as PA- and doxorubicin-induced DNA damage in GCKR^TT HLOs, which may be attributed to the fact that the GCKR^TT variant is associated with mitochondrial oxidative phosphorylation impairment[24].

In addition to genetic variant-mediated metabolic disorders, post-translational modification mechanisms also serve as critical regulators of MASLD pathogenesis, which have been effectively elucidated using HLO models. Yang et al[25] transplanted liver bud organoids derived from CDK5RAP3-null hESCs into immunodeficient mice and found that loss of CDK5RAP3 severely impairs hepatic marker expression, liver bud maturation, vascularization, and structural formation, demonstrating that UFMylation is essential for human liver development. Subsequent studies verified the critical role of CDK5RAP3-mediated ribosomal protein L26 UFMylation in ribosome dissociation, hepatic differentiation, and lipid homeostasis, suggesting that ribosomal UFMylation might serve as a potential therapeutic target for MASLD. In addition to dissecting genetic susceptibility and post-translational modification events in MASLD pathogenesis, hPSC-derived HLOs can also be assembled into multicellular models to simulate intercellular communication and viral comorbidity in MASLD. Lee et al[26] established a multicellular HLO model comprising hepatocytes, Kupffer-like cells, and HSCs using a co-culture system of macrophages and HLOs, both differentiated from the same hPSCs. Further results proved that hepatitis C virus infection results in increased lipogenesis in macrophage-co-cultured HLOs. Meanwhile, exposure of HLOs to FFA increases hepatitis C virus amplification and exacerbates inflammation and fibrosis. This model faithfully recapitulates host-virus and intercellular interactions, as well as patient-specific immune properties.

Beyond the association studies between genetic variations and targeted therapies, HLO models also serve as a powerful tool for the screening of MASLD biomarkers, the characterization of MASLD subtypes, and the identification of key regulatory molecules. Kømurcu et al[27] incubated hiPSC-derived HLOs with 250 μmol/L OA, 250 μmol/L PA, and 10 mmol/L fructose for 48 hours and observed elevated levels of 26-hydroxycholesterol, which supports the possibility of oxysterols acting as MASLD indicators. Correia de Sousa et al[28] demonstrated that miR-149-5p upregulation exacerbates MASLD development by affecting energy metabolism, which was further proven using hESC-derived HLOs. Specifically, HLOs overexpressing miR-149-5p exhibit markedly elevated lipid accumulation, along with mild inflammation and fibrosis, after 7 days of exposure to 400 μmol/L OA and 200 μmol/L PA. Pirola et al[29] identified adhesion G-protein-coupled receptor G1 as a key proteomic biomarker for MASLD via multi-cohort high-throughput targeted circulating proteomics combined with histological and transcriptomic analyses, finding its circulating levels to be elevated in patients with progressive MASH and significant liver fibrosis and to be associated with increased risks of end-stage liver disease, extrahepatic complications, and mortality, with the liver as the main tissue with high expression in the hepatic fibrotic niche. Single-cell RNA-seq data from hPSC-derived HLOs mimicking human MASH inflammatory and fibrotic injury were used to demonstrate that adhesion G-protein-coupled receptor G1 is transcriptionally upregulated in a cell type-specific manner in fibrotic niche cells upon fatty acid and TGF-β1 stimulation, and its expression correlates with pathways driving MASLD-related hepatic fibrosis and systemic organ damage.

Furthermore, HLO models have been extensively employed in evaluating the therapeutic potential of natural products and immunomodulatory molecules for MASLD. Zuo et al[30] found that Fu brick tea can maintain intestinal homeostasis and remodel the hepatic immune microenvironment to protect against MASLD and associated endotoxemia. hiPSC-derived HLOs were further employed to demonstrate that Fu brick tea alleviated liver immune-inflammatory responses induced by OA or LPS via inhibiting KC activation. Huang et al[31] discovered that hepatic interleukin 22 (IL-22) receptor alpha 1 deficiency promotes hepatic steatosis by modulating lipid homeostasis. hiPSC-derived HLOs were used to reveal that the pro-lipogenic effect induced by 3 beta-hydroxy-5-cholestenoic acid is effectively attenuated by IL-22, which highlights the importance of hepatic oxysterols, especially 3 beta-hydroxy-5-cholestenoic acid, in regulating hepatic lipids, as well as the therapeutic potential of IL-22/IL-22 receptor alpha 1 in MASLD.

LOs from primary tissues in MASLD research

MASLD model construction and phenotypic recapitulation: Although hPSC-derived LOs have enabled robust in vitro modeling of MASLD, they still face inherent limitations such as insufficient hepatic maturation and potential genetic deviation. In contrast, LOs directly derived from primary hepatic tissues, including mouse liver tissues, normal human liver specimens, and MASLD patient-derived hepatic samples, retain the native genetic background, cellular composition, and physiological functions of the original tissue, making them a more clinically faithful model for MASLD research[32].

Primary LOs derived from mouse liver tissues were first established to preliminarily recapitulate MASLD pathological processes. Elbadawy et al[33] generated LOs from each stage of MASH in a mouse model after feeding with a methionine- and choline-deficient diet for 4 weeks, 8 weeks, or 12 weeks to produce mild, moderate, or severe degrees of MASH, respectively. The organoids from mice with moderate and severe MASH recapitulated several histological features of MASH liver tissues, such as hepatic fibrosis. Additionally, RNA-seq profiling demonstrated that the expression of several genes was upregulated in all MASH LOs, indicating their promise as an in vitro tool for studying MASH pathogenesis and discovering innovative biomarkers for early MASH diagnosis. Subsequently, Zhou et al[34] incubated LOs directly derived from normal mouse liver tissues with FFA (OA: 245 μM, PA: 122.5 μM) for 2 days to induce lipid accumulation and secretion of pro-inflammatory factors. RNA-seq results indicated that nucleotide-binding oligomerization domain-like receptors and peroxisome proliferator-activated receptor (PPAR) signaling pathways contribute to the MASLD process, which was further verified using nucleotide-binding oligomerization domain pathway inhibitor and PPAR agonist.

To translate findings from murine models to human pathophysiology and establish mature, reliable human primary LOs, researchers have optimized culture systems and generated patient-derived LOs. Liu et al[35] developed innovative culture conditions that integrate optimal concentrations of triiodothyronine with growth factor depletion, enabling the production of mature LOs from both murine and human hepatocytes, which provide a powerful in vitro model for studies of human liver physiology and diseases. Additionally, compared with the mouse LOs, their HLOs exhibit greater sensitivity upon FFA treatment, highlighting species-specific differences in MASLD modeling. Building on this optimized human organoid system, Zhu et al[36] established a methodology to derive cholangiocyte organoids from adult stem cells of normal liver tissue that differentiate into HLOs. When these HLOs are further incubated with OA, they faithfully recapitulate the steatosis and inflammation characteristic of MASLD in vitro after exposure to 600 μM OA for 3 days. Subsequently, anti-steatosis drug testing was performed, and the drug sensitivity of this liver steatosis model was validated, providing the basis for subsequent application of the model. To further expand the sources and applications of human primary LOs, Wang et al[37] incubated human liver-derived intrahepatic cholangiocyte organoids and their hepatocyte-differentiated LOs with two concentrations of lactate, pyruvate, and octanoic acid for 96 hours. This treatment induces lipid accumulation and metabolic dysregulation in the LOs, providing a novel tool for exploring pathogenesis and developing therapeutics for MASLD. To fully recapitulate the core pathological features of MASLD, Goswami et al[38] established an in vitro model of steatohepatitis using co-cultures of HLOs derived from primary hepatocytes of healthy individuals with primary human stellate cells treated with 750 μM PA for 72 hours and HLOs derived from liver tissues collected from needle biopsies of MASLD patients as a natural model of steatohepatitis. This HLO model exhibits steatosis, inflammation, and fibrosis characteristic of MASLD, and thus, is expected to be a valuable tool for exploring therapeutics for MASLD. Furthermore, McCarron et al[39] developed a protocol that enables reliable derivation, propagation, and hepatic differentiation of bipotent ductal organoids from irreversibly injured MASH patient liver tissues. These organoid transcriptomes display marked upregulation of proinflammatory and cytochrome P450-related pathways, along with well-characterized markers of hepatic fibrosis and tumor. Additionally, the MASH LOs exhibit markedly enhanced permissiveness to severe acute respiratory syndrome-coronavirus 2 vesicular stomatitis pseudovirus, which might be related to the upregulation of ubiquitin D. This study demonstrated the feasibility of deriving, culturing, and functionally analyzing organoids from irreversibly injured human MASH liver tissue. The distinct strategies for MASLD model construction and phenotypic recapitulation in LOs derived from primary tissues, together with their pathological characteristics, are summarized in Table 2.

Table 2 Metabolic dysfunction-associated steatotic liver disease model construction and phenotypic recapitulation in liver organoids derived from primary tissues.

Cellular origin	Cell types	Model induction method	Phenotype reproduced	Ref.
Mice liver tissue	Mixture of cells from liver tissue	Mice fed with methionine-and choline-deficient diet for 4, 8, and 12 weeks	Liver fibrosis without lipid accumulation	Elbadawy et al[33], 2020
Mice liver tissue	Mixture of cells from liver tissue	245 μM OA and 122.5 μM PA for 48 hours	Lipid accumulation, increased liver fibrosis and inflammation	Zhou et al[34], 2024
Mice liver tissue and human fetal liver tissue	Mixture of cells from liver tissue	200 μM OA/150 μM OA and 150 μM PA for 3 days	Greater lipid accumulation in human vs mouse organoids	Liu et al[35], 2024
Adult stem cells from human liver tissue	Hepatocyte-like	600 μM OA for 3 days	Lipid accumulation, ROS production and inflammation	Zhu et al[36], 2025
Harbor residential bipotent progenitor cells from human liver tissue	Cholangiocyte-like, hepatocyte-like	Lactate/pyruvate/octanoic acid (10:1:2 mM; 15:1.5:3 mM) for 96 hours	Lipid accumulation, suppressed organoid growth, metabolic dysregulation, and mitochondrial impairment	Wang et al[37], 2022
Liver tissue from healthy individuals and MASLD patients and HLO: Primary human liver stellate cells = 5:1	Hepatocytes, cholangiocytes, stem cells, KC, HSC	750 μM PA for 72 hours	Steatosis, inflammation, and fibrosis	Goswami et al[38], 2025
Liver tissue from MASH patients	Hepatocytes, cholangiocytes	1.2 mmol/L and 2 mmol/L PA for 6 hours/2 mmol/L OA for 24 hours	MASH liver-derived organoids exhibit significantly reduced regenerative capacity, upregulated activity of multiple pathogenic pathways (inflammation, fibrosis, tumorigenesis), and greater susceptibility to PA-induced apoptosis and OA-induced lipid accumulation	McCarron et al[39], 2021

OA: Oleic acid; PA: Palmitic acid; ROS: Reactive oxygen species; MASLD: Metabolic dysfunction-associated steatotic liver disease; HLO: Human liver organoid; KC: Kupffer cell; HSC: Hepatic stellate cell; MASH: Metabolic dysfunction-associated steatohepatitis.

Mechanistic exploration, therapeutic target identification, and drug screening: Benefiting from the native genetic background and superior pathological fidelity of primary tissue-derived LOs, these models constitute irreplaceable platforms for dissecting the sophisticated molecular mechanisms of MASLD and developing precision targeted therapies. Leveraging mouse-derived, healthy human-derived, and MASLD patient-derived primary LOs, researchers have systematically delineated the core regulatory networks of MASLD spanning genetic predisposition, metabolic signaling, epigenetic modification, mitochondrial homeostasis, immune crosstalk, and gut-liver axis communication, while achieving pivotal advances in drug screening, biomarker identification, and personalized treatment.

Genetic predisposition and variant-driven MASLD susceptibility: Primary LOs retain the donor’s genetic background, making them ideal for clarifying genetic variant-specific susceptibility to MASLD. Toward these goals, gene mutation-related modeling and targeted screening have become core research directions. Hendriks et al[40] established engineered hepatocyte organoids from human fetal livers that recapitulate hepatic steatosis induced by FFA overload, the patatin-like phospholipase domain-containing 3 (PNPLA3) mutation, and monogenic lipid disorders (apolipoprotein B and microsomal triglyceride transfer protein mutations). Based on this organoid platform, the authors screened 17 candidate MASLD drugs and indicated that suppression of de novo lipogenesis (DNL) is a common and highly effective mechanism to reduce steatosis. Additionally, the PNPLA3 I148M variant was found to attenuate organoid responses to steatosis-reducing drugs, suggesting the potential value of personalized medicine for MASLD. Finally, apolipoprotein B^-/- and microsomal triglyceride transfer protein^-/- HLOs were used to establish the FatTracer platform, which was designed for clustered regularly interspaced short palindromic repeats-based loss-of-function genetic screening to uncover steatosis regulators and therapeutic targets. Through this FatTracer platform, fatty acid desaturase 2 was identified as a critical regulator of hepatic steatosis, the enhancement of which elevates polyunsaturated fatty acid levels and suppresses DNL, supporting its potential as a therapeutic target for MASLD.

Hormonal regulation of MASLD susceptibility: Beyond genetic mutations, hormonal effects also contribute to MASLD susceptibility, and this has been verified using primary LOs. Cherubini et al[41] identified via population studies, mouse models, human hepatocyte experiments, and molecular assays that a multiplicative interaction exists between female sex and the PNPLA3 p.I148M variant, and estrogen receptor-α binding to the PNPLA3 enhancer drives upregulation of this mutant, which is a key cause of MASLD in women. Primary HLOs were used to demonstrate that estrogen receptor-α agonists induce PNPLA3 expression in human in vitro hepatic models. Additionally, Yu et al[42] established an in vitro model for follicle-stimulating hormone-induced lipid metabolism abnormalities using primary biliary duct-derived LOs. High-throughput sequencing and bioinformatics analysis were further carried out to screen long non-coding RNAs that act as pivotal regulatory factors in follicle-stimulating hormone-induced abnormal lipid metabolism, and the results showed promise for early screening of lipid metabolism abnormalities.

Metabolic signaling and epigenetic regulatory mechanisms: In addition to genetic and hormonal-related factors, metabolic disorders and epigenetic dysregulation act as core drivers of MASLD[43], and primary LOs provide a reliable model for clarifying these regulatory axes. Sano et al[44] discovered that deficiency of methionine and tyrosine impairs the biosynthesis of fumarate, thereby suppressing the nuclear translocation of nuclear factor erythroid 2-related factor 2, which in turn impairs the assembly of mature very low-density lipoprotein in steatotic hepatocytes and induces hepatic steatosis. Results from LOs derived from hepatocyte-specific Kelch-like ECH-associated protein 1 knockout mice demonstrated that hepatic deficiency of this gene markedly reduces lipid accumulation and ROS levels in LOs under methionine and tyrosine deprivation. Beyond amino acid metabolism, glycogen-lipid metabolism crosstalk also constitutes a pivotal regulatory node in MASLD. Chen et al[45] identified a novel molecular mechanism in which uridine diphosphate glucose (UDPG), an intermediate metabolite of glycogenesis, directly binds to site-1 protease and induces its degradation via the ubiquitin-proteasome pathway, which further inhibits site-1 protease-mediated cleavage of sterol regulatory element-binding proteins, thereby suppressing lipogenesis in hepatocytes. Organoids derived from patient cells were further pretreated with 300 μmol/L OA for 24 hours followed by exposure to UDPG for another 24 hours. The results revealed that UDPG treatment significantly restored glycogen content and attenuated lipid droplet accumulation in steatotic HLOs, thereby making UDPG-mediated regulation of lipogenesis a novel therapeutic avenue for treating abnormal lipid metabolism. Moreover, dysregulated metabolite metabolism also participates in pediatric MASLD progression, which has been verified using organoid models. Zhan et al[46] characterized metabolic alterations in children and adolescents with obesity-related MASLD, identified significantly elevated circulating tryptophan metabolism as a key metabolic change, and developed and validated a machine learning-based non-invasive diagnostic model using tryptophan-derived metabolites for the early diagnosis of obesity-related MASLD. LOs derived from primary ductal organoids isolated from mouse liver tissues were incubated with 200 μmol/L OA and 100 μmol/L PA, along with tryptophan-derived metabolites, and the results revealed that elevated tryptophan-derived metabolites contribute to the pathogenesis of MASLD by promoting lipid droplet formation and enhancing the inflammatory response. In parallel with metabolic signaling, epigenetic modifications are also critical for MASLD progression. Qiao et al[47] further revealed an epigenetic mechanism underlying MASLD progression using clinical liver tissue samples and mouse models in which nuclear receptor binding SET domain protein 2 (NSD2) exacerbates MASLD progression by epigenetically repressing transcription factor EB transcription via the promotion of histone H4 lysine 20 trimethylation, thereby impairing the transcription factor EB-mediated autophagy-lysosomal pathway. Furthermore, pharmacological inhibition of NSD2 in PA-stimulated primary HLOs was found to reduce hepatic steatosis and restore autophagic flux, thereby supporting NSD2 as a potential therapeutic target for MASLD.

Mitochondrial and endoplasmic reticulum homeostasis: Mitochondrial dysfunction and endoplasmic reticulum-mitochondria crosstalk play important roles in MASLD pathogenesis[48], which have been precisely dissected using primary LOs. Elbadawy et al[49] generated LOs from MASH model mice fed a methionine- and choline-deficient diet for 12 weeks and observed abnormal mitochondrial morphology, accompanied by elevated dynamin-related protein 1 expression and enhanced ROS generation. In these LOs, treatment with the dynamin-related protein 1 inhibitor improves organoid morphology, reduces the expression of fibrosis-related genes, and decreases lipid accumulation, identifying Mdivi-1 as a promising therapeutic agent for MASH. In addition to mitochondrial fission, mitochondrial functional regulation is also a key target for MASLD intervention. Li et al[50] identified using primary rat hepatocytes that growth differentiation factor 15 mitigates lipid accumulation by inhibiting extracellular signal-related kinases 1 and 2 to restore mitochondrial morphodynamic homeostasis and enhance fatty acid oxidation under lipotoxic PA exposure, whereas it primarily downregulates lipogenic pathways to ameliorate lipid accumulation under non-lipotoxic FFA stimulation. These findings were further validated in primary HLOs, providing a theoretical basis for growth differentiation factor 15 as a promising therapeutic target for MASLD. Furthermore, endoplasmic reticulum-mitochondria crosstalk was shown to play a crucial role in environmental risk factor-driven MASH progression. Zhou et al[51] identified bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) as an environmental risk factor for MASH progression that promotes MASH pathogenesis by disrupting mitofusin 2-mediated endoplasmic reticulum-mitochondria contacts. When murine-derived LOs with MASH phenotypes were constructed and incubated with TBPH and mitofusin 2 agonists, the mitofusin 2 agonists significantly ameliorated TBPH-induced reduction in endoplasmic reticulum-mitochondria contacts. However, the MASH phenotype in organoids described in this study was induced by incubation with 20 mmol/L acetaminophen for 48 hours, which may not recapitulate the full spectrum of MASH characteristics. Further studies using conventional stimulation methods (e.g., FFA stimulation) to model the MASH phenotype in organoids are therefore warranted.

Cellular crosstalk, immune inflammation, and biomarker discovery: Primary LOs effectively simulate the hepatic immune microenvironment and cell-cell interactions, supporting the identification of key inflammatory factors and diagnostic biomarkers. Shen et al[52] proved that hepatocyte-derived vascular endothelial growth factor A (VEGFA) accelerates the progression of MASLD to HCC by activating HSCs. Organoids derived from MASLD patients were used to demonstrate elevated VEGFA levels in the medium. LX2 cells were further proven to be activated by incubation with conditioned medium from MASLD patient-derived hepatocyte organoids, an effect which was blocked by a VEGFA antibody. Macrophage-hepatocyte crosstalk forms a positive feedback loop that further amplifies inflammatory injury. Jin et al[53] provided new mechanistic insight into MASH progression by identifying a positive feedback loop in which macrophage tumor necrosis factor alpha (TNFα)-mediated degradation of hepatocyte Myc-interacting zinc-finger protein 1 results in peroxiredoxin 6-mediated suppression of hepatocyte mitophagy, aggravated mitochondrial injury, and elevated macrophage TNFα production. Organoids derived from patient cells were further used to verify these molecular mechanisms, which supports the TNFα/Myc-interacting zinc-finger protein 1 loop as a key pathogenic mechanism and promising therapeutic target for human MASH. In addition to mediating inflammatory crosstalk among hepatic cell types, lipid accumulation also modulates the secretion of soluble factors that shape local immune responses and disease progression. Bitterer et al[54] proved that lipid accumulation in human hepatocytes reduces cell-surface CD46 via matrix metalloproteinases, thereby releasing soluble CD46 and promoting the differentiation of IL-4⁺ invariant natural killer T cells. Primary HLOs were used to verify that soluble CD46 levels are significantly higher in fat-loaded cultures than in control cultures, and soluble CD46 was further identified as a novel, accurate diagnostic marker for hepatic steatosis using two independent clinical cohorts, with predictive performance superior to established clinical scores and allowing stratification by steatosis severity. Population proteomics combined with organoid models further identified key circulating biomarkers for MASLD. Abozaid et al[55] performed a population-based proteomic study within the Rotterdam study cohort and identified 27 plasma proteins that are significantly correlated with fatty liver disease, of which 15 are also significantly related to liver fibrosis. Human liver-derived organoids were used to establish a fatty liver disease model, and the results showed that, in line with plasma proteomic profiling, the expression levels of IL-18 receptor 1 and carboxylesterase 1 are upregulated, which suggests their important roles in the pathogenesis of MASLD and its progression to fibrosis as well as their potential as disease biomarkers. The key findings regarding the identification of potential MASLD biomarkers based on LO models are summarized in Table 3.

Table 3 Identification of potential metabolic dysfunction-associated steatotic liver disease biomarkers using liver organoid models.

Biomarker	Cellular origin	Model	Expression	Function/Description	Ref.
26-hydroxycholesterol	hiPSCs	250 μM OA, 250 μM PA, 10 mmol/L fructose for 48 hours	Upregulated	26-hydroxycholesterol exerts an anti-inflammatory signal on quiescent HSCs	Kømurcu et al[27], 2023
ADGRG1	hPSCs	500 μM OA/500 μM PA/10 ng/mL TGF-β1 for 4 days	Upregulated	The transcriptional profile of ADGRG1 is upregulated in cells of the fibrotic and inflammatory niche in response to injury	Pirola et al[29], 2026
lncRNA ENSMUST00000244884	Primary biliary duct-derived LOs from mice liver tissue	2/10 ng/mL FSH for 48 hours	Upregulated	Knocking down this long non-coding RNA promotes liver cholesterol excretion and accelerating lipid metabolism	Yu et al[42], 2026
Tryptophan-derived metabolites	Primary ductal organoids-derived LOs from mice liver tissue	200 μM OA and 100 μM PA for 3 days	Upregulated	Tryptophan-derived metabolites promote lipid droplet formation and enhance the inflammatory response	Zhan et al[46], 2025
sCD46	Human liver tissue	2 mmol/L FFA (OA:PA = 2:1) for 24 hours	Upregulated	Lipid accumulation in human hepatocytes reduces cell-surface CD46 via matrix metalloproteinases, thereby releasing sCD46 and promoting the differentiation of IL-4+ iNKT cells	Bitterer et al[54], 2024
CES1/IL18R1	Harbor residential bipotent progenitor cells from human liver tissue	Lactate/pyruvate/octanoic acid (10:1:2 mM; 15:1.5:3 mM) for 96 hours	Upregulated	CES1, IL-18R1: Population-based proteomics biomarkers validated in human LOs for MASLD/fibrosis	Abozaid et al[55], 2023
HAMP1	Human liver tissue	400 μM OA and 200 μM PA	Upregulated	Elevated pre-treatment HAMP1 predicts better response to saponin therapy in MASLD, and reduced HAMP1 post-treatment links to histological improvement	Kim et al[62], 2025

hiPSCs: Human induced pluripotent stem cells; OA: Oleic acid; PA: Palmitic acid; HSC: Hepatic stellate cell; ADGRG1: Adhesion G-protein-coupled receptor G1; hPSCs: Human pluripotent stem cells; TGF-β: Transforming growth factor-β; LO: Liver organoid; sCD46: Soluble CD46; FSH: Follicle-stimulating hormone; FFA: Free fatty acids; iNKT: Invariant natural killer T; CES1: Carboxylesterase 1; IL18R1: Interleukin-18 receptor 1; MASLD: Metabolic dysfunction-associated steatotic liver disease; HAMP1: Hepcidin antimicrobial peptide 1.

Gut-liver axis and microbial metabolite regulation: The gut-liver axis is a core regulatory system for MASLD, and gut microbial metabolites act as key messengers to modulate hepatic pathophysiology[56]. Primary LO models have been widely used to verify the regulatory effects of beneficial gut bacteria and their metabolites on MASLD. Kim et al[57] demonstrated that Lactobacillus plantarum (a species of beneficial gut lactic acid bacteria) treatment enhances L-arginine, thereby ameliorating MASH-related phenotypes. An experiment in which mouse LOs and KCs were co-cultured in a Transwell system and exposed to LPS showed that L-arginine alone is sufficient to attenuate liver inflammation. Lau et al[58] further demonstrated that gut Lactobacillus acidophilus secretes short-chain fatty acids, especially valeric acid, which translocates to the liver and exerts protective and anti-tumorigenic effects in vitro and in vivo by binding to hepatocyte surface G protein-coupled receptors 41/43 and inactivating oncogenic pathways, thereby suppressing MASLD-HCC development. Mouse organoids derived from MASLD-HCC tumor tissues were further used to demonstrate the inhibitory effect of Lactobacillus acidophilus and valeric acid on tumor organoid growth, suggesting that Lactobacillus acidophilus supplementation might be a promising prophylactic against MASLD-HCC development.

Drug validation and phytotherapies: Primary LOs possess high clinical relevance, making them high-confidence models for screening and validating conventional drugs, natural products, and phytochemicals against MASLD. Given that numerous food and medicinal homologous resources have been demonstrated to exert lipid-regulating effects through diverse mechanisms[59], natural plant extracts have gained increasing attention as potential candidates for MASLD treatment. Based on their primary mechanisms of action, the drugs and natural products discussed below can be categorized into three groups: Lipid-lowering, anti-inflammatory, and anti-fibrotic agents.

First, statins and bergamot polyphenol extract are characterized by their predominant lipid-lowering effects. Ayada et al[60] demonstrated significant inverse associations of statin use with MASH and hepatic fibrosis through population cohort studies and meta-analysis. Further in vitro experiments in primary HLOs and THP-1 macrophages demonstrated that statins markedly decrease lipid droplet accumulation in HLOs and downregulate pro-inflammatory cytokine expression in macrophages, further validating the protective role of statins against MASLD. In addition to clinical conventional drugs, natural plant extracts have become potential candidates for MASLD treatment. Mirarchi et al[61] revealed that bergamot polyphenol extract reduces neutral lipid content potentially by enhancing beta-oxidation. Primary human hepatocytes-derived HLOs were further utilized to verify that bergamot polyphenol extract incubation reduces intracellular neutral lipid content, making it a promising therapeutic approach for MASLD.

Second, distinct from the above lipid-lowering agents, red ginseng saponins and fired black sesame pigment primarily exert their protective effects through anti-inflammatory and antioxidant pathways. Kim et al[62] showed that red ginseng saponins attenuate MASLD by dampening inflammation, which in turn reduces fibrosis, lipogenesis, and oxidative stress, and identified hepcidin antimicrobial peptide 1 as a potential exploratory biomarker for evaluating saponin responses in preclinical settings. Organoids derived from patient liver tissue were further used, and the results demonstrated that red ginseng saponins mitigate lipotoxic injury in LOs by decreasing intracellular lipid accumulation and inhibiting lipogenesis through alleviation of oxidative stress induced by 400 μmol/L OA and 200 μmol/L PA. Huang et al[63] revealed that fired black sesame pigment ameliorates MASLD through bidirectional regulation of gut microbiota and the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Primary HLOs were incubated with 600 μM OA and PA, and the results displayed that fired black sesame pigment may regulate levels of genes upstream of the HIF-1 signaling pathway, thereby suppressing oxidative stress, lipid accumulation, and secretion of inflammatory factors during MASLD development in vitro.

Finally, in contrast to the aforementioned agents, moronic acid stands out for its anti-fibrotic properties, although it also exhibits lipid-lowering and anti-inflammatory activities. Han et al[64] revealed that moronic acid exerts therapeutic effects against MASLD and liver fibrosis by activating PPARs and mediating lipidome reprogramming. A 3D liver physiological model was constructed using human primary hepatic parenchymal cells and human primary nonparenchymal cells, and the results demonstrated that moronic acid exerts lipid-lowering, anti-inflammatory, and anti-fibrotic effects, which are mediated via PPAR activation, supporting the potential of moronic acid as an innovative phytotherapeutic agent for MASLD. Core drugs and phytotherapies for MASLD validated in LO models are summarized and illustrated in Figure 2.

Figure 2 Core drugs and phytotherapies for metabolic dysfunction-associated steatotic liver disease validated in liver organoid models. HIF-1: Hypoxia-inducible factor 1; hPSCs: Human pluripotent stem cells; KC: Kupffer cell; PPARs: Peroxisome proliferator-activated receptors.

Biliary epithelial cell reprogramming and comorbidity-associated mechanisms: During the progression of simple MASLD, lipid overload also induces the activation and phenotypic reprogramming of biliary epithelial cells (BECs). Yildiz et al[65] focused on the activation of BECs during MASLD progression. BEC organoids isolated from high-fat diet-fed mouse livers were generated to reveal the novel mechanism that lipid overload-induced E2F activation controls the reprogramming of BECs into progenitor cells by driving cell cycle progression while promoting glycolytic metabolism.

Furthermore, primary LOs are also applied to clarify the synergistic pathogenic mechanism of MASLD comorbid with other liver diseases. Nguyen et al[66] generated clonal intrahepatic cholangiocyte organoid cultures from human liver biopsies obtained from healthy liver donors and patients with MASH or other precancerous liver diseases. Whole-genome sequencing results revealed that these precancerous liver disease conditions do not lead to increased accumulation of mutations, which challenges the assumption that liver disease promotes oncogenesis through a direct mechanism of induced mutagenesis. Wu et al[67] demonstrated using clinical tissue samples and mouse models that in chronic hepatitis B-MASLD comorbidity, hepatitis B virus and lipotoxicity synergistically inhibit macrophage Rab7 to block lysosomal degradation of the stimulator of interferon genes (STING), resulting in the vesicular transfer of undegraded STING to hepatocytes, inducing endoplasmic reticulum stress and exacerbating liver injury. These findings were further validated in primary HLOs, thereby rendering targeting Rab7 for excessive STING degradation a novel therapeutic approach.

LOs from immortalized cell lines in MASLD research

MASLD model construction and phenotypic recapitulation: Compared with hPSC-derived and primary tissuederived LOs, LOs established from immortalized hepatic cell lines feature easier accessibility, higher stability, and better scalability, enabling their application in high-throughput drug screening and preliminary mechanistic studies. Early cell line-derived models focused on constructing multicellular 3D spheroids to mimic the core pathological features of MASLD. Pingitore et al[68] created an organoid system genetically predisposed to MASLD using multilineage 3D spheroids composed of HepG2 cells and LX-2 cells with a physiological ratio (24:1), which are homozygous for the PNPLA3 I148M sequence variant. Incubation of these organoids with 500 μM FFA (OA:PA = 2:1) leads to the accumulation of lipids and collagen in the organoids. Subsequent drug tests demonstrated that this organoid model is applicable for evaluating the effects of drugs on hepatic steatosis and liver fibrosis. To improve hepatic function and physiological relevance, researchers further optimized cell sources and culture platforms. Teng et al[69] developed SteatoChip, a 100-well perfusable microfluidic chip, for the in situ differentiation of HepaRG progenitor cells into uniformly sized HepaRG organoids with a well-controlled microenvironment. Compared with the in situ differentiated HepaRG organoids in static conditions and the pre-differentiated HepaRG spheroids, the in situ differentiated HepaRG organoids exhibit significantly enhanced hepatic functions, improved cellular polarity, and better-formed bile canaliculi. A human hepatic steatosis model was successfully induced by incubating the in situ differentiated HepaRG organoids (under perfusion condition) on the chip with 500 μM FFA (OA:PA = 1:1), after which they exhibited prominent lipid accumulation, impaired glucose regulation, and reduced Akt phosphorylation. Subsequently, three anti-steatosis compounds were tested, and the chip model was proven to detect steatosis reduction with high sensitivity and scalability, showing high sensitivity and scalability for drug screening and serving as a robust drug testing platform for MASLD. Subsequent studies further enriched cellular components to simulate inflammatory and fibrotic microenvironments. Bronsard et al[70] and Duclos et al[71] generated 3D multi-cell-type LOs from HepaRG cells, primary human macrophages, and LX-2 cells at a physiological ratio (80:10:10) and observed typical features of steatosis and upregulation of fibrosis markers after incubation with 150 μM stearic acid and 300 μM OA for 9 days. Subsequently, the cytotoxicity of hepatotoxic drugs was evaluated in these MASLD organoids. This model was shown to enable assessment of drug-induced liver injury risks in the context of MASLD via half-maximal drug inhibitory concentration and benchmark dose calculations, supporting its value for in vitro drug safety assessments and mechanistic studies of adverse outcome pathways in MASLD. The distinct strategies for MASLD model construction and phenotypic recapitulation in LOs derived from immortalized cell lines, together with their pathological characteristics, are summarized in Table 4.

Table 4 Metabolic dysfunction-associated steatotic liver disease model construction and phenotypic recapitulation in liver organoids derived from immortalized cell lines.

Cellular origin	Cell types	Model induction method	Phenotype reproduced	Ref.
HepG2 (PNPLA3 I148M homozygous):LX-2 = 24:1	Hepatocytes, HSC	500 μM FFA (OA:PA = 2:1) for 48 hours	Lipid accumulation and fibrosis	Pingitore et al[68], 2019
HepaRG	Hepatocytes, cholangiocytes	500 μM FFA (OA:PA = 1:1) for 48 hours	Lipid accumulation, impaired glucose regulation, and reduced Akt phosphorylation	Teng et al[69], 2021
HepaRG:primary human macrophages:LX-2 = 8:1:1	Hepatocytes, cholangiocytes, HSC, KC	150 μM stearic acid and 300 μM OA for 9 days	Steatosis, increased expression of fibrosis markers and pro-inflammatory cytokines	Bronsard et al[70], 2024; Duclos et al[71], 2026

HSC: Hepatic stellate cell; FFA: Free fatty acids; OA: Oleic acid; PA: Palmitic acid; KC: Kupffer cell.

Therapeutic target identification: Immortalized cell line-based 3D models also provide a convenient and reliable tool for investigating immune-related mechanisms and therapeutic targets in MASLD. Gwag et al[72] revealed that anti-CD47 antibody treatment attenuates liver inflammation and fibrosis in experimental steatohepatitis models. A 3D human in vitro MASH model was established by co-culturing human hepatocytes, THP1-derived macrophages, and HSCs at a ratio of 4:1:1, followed by induction with 0.5 mmol/L PA, 30 mmol/L glucose, and 2 μg/mL LPS. Anti-CD47 antibody treatment of this model attenuates MASH media-stimulated inflammation and fibrosis in the HLOs, validating the human relevance of anti-CD47 antibody treatment in MASH. This well-established 3D model was further applied to explore platelet-associated immune regulatory mechanisms. In a follow-up study, Gwag et al[73] identified that thrombospondin 1-deficient platelets significantly attenuate inflammation and fibrosis in the same in vitro MASH model, suggesting that platelet-derived thrombospondin 1 may serve as an innovative therapeutic target for MASLD/MASH intervention.

Despite their advantages in accessibility, stability, and scalability, LOs derived from immortalized cell lines have inherent biological limitations owing to their tumor or preneoplastic origins: Tumor-associated metabolic reprogramming disrupts core lipid metabolism pathways, dysregulates redox homeostasis[74,75], and is accompanied by attenuated cytochrome P450 enzyme activity[76], which may reduce the fidelity of MASLD phenotype recapitulation. Accordingly, these models are optimally applied to high-throughput preliminary drug screening and early mechanistic validation.

MULTI-TISSUE ORGANOID SYSTEMS IN MASLD RESEARCH

In addition to LOs, intestinal and pancreatic organoids, as crucial components of the gut-liver axis and liver-pancreas axis, have emerged as indispensable models for unraveling the systemic regulatory mechanisms of MASLD. Combined with organ-on-chip engineering, these models enable the simulation of interorgan crosstalk, barrier dysfunction, metabolic crosstalk, and immune-microbial interactions, greatly expanding the physiological relevance of in vitro MASLD research.

Intestinal organoids in MASLD research

The gut-liver axis plays a crucial role in MASLD initiation and progression[77], and intestinal organoids have become a core tool for elucidating intestinal epithelial metabolism, barrier function, and microbial signal transduction.

Intestinal PPARα has emerged as a central regulator in gut-liver axis crosstalk during MASLD, yet its exact role appears paradoxical as revealed by several organoid-based studies. Yan et al[78] demonstrated that intestinal PPARα directly regulates its target gene fatty acid-binding protein 1 (FABP1) to promote fatty acid uptake, thereby driving the progression of MASH. Intestinal organoids isolated from mice were used to prove that fatty acids activate intestinal PPARα and upregulate Fabp1 mRNA expression, and functional validation confirmed that intestinal PPARα modulates fatty acid uptake via its target gene FABP1. Specifically, both PPARα inhibition and FABP1 disruption reduce fatty acid uptake in the intestinal organoids, with PPARα inhibition exerting no additional effect in FABP1-knockout organoids. Moreover, the impaired fatty acid uptake caused by PPARα deletion can be rescued by FABP1 overexpression. Finally, intestinal organoids isolated from PPARα-humanized mice were used to support a direct role for human PPARα in facilitating intestinal fatty acid uptake. Dixon et al[79] further corroborated the pro-absorptive role of intestinal PPARα from another perspective, while expanding the understanding of PPARα signaling in the gut-liver axis. Their study demonstrated that pharmacological inhibition of adipose triglyceride lipase attenuates PPARα signaling in both the liver and intestine, which induces a shift in bile acid composition toward hydrophilic forms, thereby interfering with dietary lipid absorption and alleviating MASH. Using human primary ileum-derived organoids, a novel human adipose triglyceride lipase inhibitor was validated to reduce intestinal PPARα activity and delay lipid absorption, further confirming the regulatory role of intestinal PPARα in lipid absorption. However, Na et al[80] reported a contrasting finding regarding the role of intestinal PPARα, revealing its protective effect in maintaining intestinal homeostasis and alleviating MASLD. Their study showed that intestinal epithelial PPARα deficiency upregulates necroptotic signaling, disrupts the gut vascular barrier, and promotes LPS translocation to the liver, thus aggravating hepatic steatosis or fibrosis induced by high-fat diet. Mouse-derived ileal organoids were utilized to avoid the impact of gut microbiota on cell death pathways, and the results displayed that suppression of PPARα upregulates necroptosis signals in the intestinal organoids triggered by TNFα and LPS stimuli. These findings indicate that intestinal PPARα exerts dual context-dependent roles in MASLD progression: Its pro-absorptive effect promotes MASLD by enhancing fatty acid uptake, while its role in maintaining intestinal barrier integrity and suppressing necroptosis alleviates MASLD, reflecting the complexity of PPARα signaling regulation in the gut-liver axis. Several factors may underlie these seemingly contradictory observations: (1) The functional divergence of intestinal PPARα may arise from the cellular heterogeneity of the intestinal epithelium, with its pro-absorptive effect presumably predominating in enterocytes and its barrier-protective role likely involving Paneth and goblet cells, a hypothesis worth testing with single-cell transcriptomic analyses; (2) The relative contribution of these two effects may be stage-dependent, with the pro-absorptive action potentially dominating in the steatotic stage and the protective effect becoming paramount during MASH when the gut barrier fails; (3) The chemical nature of fatty acid ligands may influence PPARα conformation and co-factor recruitment, leading to differential transcriptional outcomes depending on ligand composition (e.g., saturated vs unsaturated fatty acids)[81]; and (4) The compositional and functional shifts in gut microbiota during MASLD may regulate intestinal PPARα signaling via microbial metabolites, potentially influencing the balance of its dual effects[82]. The specific functions and underlying mechanisms of intestinal PPARα in MASLD merit further exploration.

Beyond the PPARα regulatory network, intestinal organoids have become core tools to elucidate additional signaling pathways, gut microbiota-immune crosstalk, and epithelial barrier mechanisms driving MASLD. Xu et al[83] demonstrated that activation of intestinal HIF-1α ameliorates MASLD by enhancing glycolysis, which in turn promotes intestinal epithelial cell proliferation and strengthens intestinal barrier function. Mouse-derived ileal organoids were further utilized to confirm that pharmacological activation of HIF-1α significantly enhances glycolysis, indicating that intestinal HIF-1α activation might be a promising therapeutic strategy for MASLD. In terms of fungal microbiota regulation, Pandyarajan et al[84] revealed that caspase recruitment domain protein 9 (CARD9) mitigates MASLD-related liver injury and fibrosis by maintaining gut barrier function and modulating gut mycobiota (notably the beneficial Penicillium genus). Primary mouse intestinal organoids were used to demonstrate that CARD9 regulates the expression of key gut barrier-related molecules to preserve gut barrier integrity. These findings provide new insights into the gut-liver axis mechanism of MASLD and identify CARD9 as a potential therapeutic target. Focusing on probiotic-mediated immune and metabolic regulation, Lopez-Escalera et al[85] screened six functional bacterial strains capable of enhancing intestinal barrier integrity and inducing GLP-1 secretion. Co-cultivation with human intestinal organoids verified their immunomodulatory effects via upregulated IL-17C and chemokine transcripts, and their derived indole metabolites were shown to strongly inhibit hepatic DNL, providing valuable probiotic candidates for MASLD prevention. Clinically derived intestinal organoids further complemented mechanistic research by reflecting disease progression. For example, Filippello et al[86] established intestinal organoids from non-fibrotic MASLD and MASLD-cirrhotic patients and identified extensive regenerative, metabolic, and inflammatory abnormalities in organoids from cirrhotic individuals, revealing progressive intestinal epithelial dysfunction closely correlated with advanced MASLD. Core protective mechanisms against MASLD uncovered using organoid models are summarized and illustrated in Figure 3.

Figure 3 Main key protective mechanisms against metabolic dysfunction-associated steatotic liver disease revealed in liver organoid studies. IL-22: Interleukin-22; IL-22RA1: Interleukin-22 receptor subunit alpha-1; UDPG: Uridine diphosphate glucose; S1P: Site-1 protease; SREBP: Sterol regulatory element-binding protein; 3β HCA: 3 beta-hydroxy-5-cholestenoic acid; DRP1: Dynamin-related protein 1; FAO: Fatty acid oxidation; ERK: Extracellular signal-regulated kinase; GDF15: Growth differentiation factor 15; TSP1: Thrombospondin 1; HSC: Hepatic stellate cell; ROS: Reactive oxygen species; HIF-1α: Hypoxia-inducible factor 1 alpha.

Furthermore, intestinal organoids serve as reliable preclinical platforms for evaluating the therapeutic potential of natural bioactive compounds and microbial metabolites against MASLD. Beisner et al[87] demonstrated that inulin and sodium butyrate alleviate MASLD by restoring α-defensin expression and repairing intestinal antimicrobial barriers. Research in mouse small intestinal organoids confirmed that short chain fatty acids, fermentation products of inulin, directly induce Paneth cell α-defensin expression, supporting prebiotic supplementation as an effective strategy to improve diet-induced metabolic disorders. In addition, natural active ingredients and their microbial metabolites also exert protective effects on the gut-liver axis. Liu et al[88] found that colonic delivery of torularhodin enriches Akkermansia muciniphila and promotes adenosylcobalamin synthesis, which suppresses colonic ceramide accumulation and ultimately relieves hepatic steatosis. A mouse colonic organoid co-culture system with fecal lipids further validated that adenosylcobalamin enhances intestinal epithelial and stem cell proliferation, strengthens colonic barrier function, inhibits HIF-2α signaling, and reduces long-chain ceramide levels, demonstrating the great potential of natural carotenoids and their microbial metabolites for MASLD treatment.

Pancreatic organoids in MASLD research

To simulate systemic metabolic disorders such as obesity and type 2 diabetes mellitus (T2DM) complicated by MASLD, multi-organoid models and organ-on-chip systems have been developed to recapitulate liver-pancreas crosstalk. Aizenshtadt et al[89] developed a dual pump-less recirculating liver-islet organ-on-chip platform using hPSC-derived liver and islet organoids. Under 250 μM FFA (OA:PA = 1:1) and 10 mmol/L fructose stimulation, their LOs develop steatosis and insulin resistance, while the islet organoids secrete pro-inflammatory cytokines. Their platform reproduces known effects of anti-diabetic drugs, supporting its value for studying obesity, T2DM, and MASLD. Further optimized multi-organoid devices enable directional convective transport and separate medium systems. Kim et al[90] designed a multi-organoid device with convective flow and separated media for mouse tissue-derived liver and pancreatic organoids, overcoming the limitations of passive diffusion and shared media of existing systems. Treatment of the LOs with 0.3 mmol/L PA triggers β-cell apoptosis and impairs insulin secretion via Fetuin-A crosstalk. The system enabled physiological glucose regulation and metformin response evaluation, representing a robust tool for studying metabolic crosstalk and drug screening in MASLD and T2DM.

CONCLUSION

Unresolved issues

MASLD-related organoid models have several notable limitations: (1) hPSC-derived organoids present inherent limitations, including genomic instability, epigenetic abnormalities, as well as potential tumorigenicity and immunogenicity risks that restrict their further clinical transformation and safe application in MASLD research[91]. Additionally, iPSC-derived organoid models fail to achieve precise regulation of the proportional composition of distinct hepatocyte subtypes, further limiting the accuracy of pathological simulation. Meanwhile, primary human liver tissue-derived organoids are restricted by dependence on scarce surgical specimens and inherent donor heterogeneity, resulting in inconsistent experimental outcomes and poor reproducibility[92], which impede standardized mechanistic exploration and drug screening for MASLD. In addition, immortalized cell lines present tumor phenotypes and altered metabolic functions (e.g., insufficient cytochrome P450 activity)[93], prohibiting their derived organoids from faithfully recapitulating physiological hepatic features in MASLD research; (2) Hepatic 3D organoid cultures suffer from prominent experimental variability, owing to a lack of unified standard protocols not only for LO generation but also for pathological modeling of MASLD and MASH, resulting in poor reproducibility and inconsistent phenotypic outcomes across different laboratories even with the use of similar induction strategies; (3) Consistent with other types of organoids, LOs inevitably exhibit prominent batch-to-batch and even individual heterogeneity, which greatly hinders the preclinical advancement and translational application of LO technology in MASLD research; (4) A notable limitation of most current hepatic organoid protocols is their failure to recapitulate liver zonation and functional biliary network structures, coupled with the lack of vascularization. In addition, existing models do not incorporate key immune cell populations closely involved in MASH progression, such as T cells and B cells, which further compromises the simulation of inflammatory infiltration and immune-driven pathological progression during disease development. These structural, vascular, and immunological deficiencies prevent organoid systems from fully mirroring the complex multicellular microenvironment of diseased liver tissue, limiting their reliability for deciphering MASH-related immune mechanisms and evaluating targeted therapeutic interventions. Nevertheless, multiple advanced culture strategies have been developed recently to address this immune deficiency: Air-liquid interface culture systems offer a powerful strategy to preserve endogenous tissue-resident immune cells within organoid constructs[94], while co-culture of LOs with autologous immune cells provides a robust engineering approach to reconstitute immune-competent liver models[95]. Future integration of these technical innovations into MASLD organoid construction is expected to greatly improve the recapitulation of immune-mediated inflammatory pathogenesis and facilitate more accurate evaluation of immunomodulatory therapeutic strategies; and (5) Emerging studies have increasingly recognized the critical role of inter-tissue crosstalk in the onset and progression of MASLD. Future research should prioritize the development of advanced organoid models to reconstruct physiological communication between the liver and other peripheral organs. Ultimately, it is essential to achieve integrated assembly of the liver with multiple organ components and even establish sophisticated whole-body-on-a-chip systems, so as to fully recapitulate the complex interorgan regulatory networks driving MASLD pathogenesis and support more in-depth mechanistic studies and reliable preclinical therapeutic validation.

Conclusion and outlook

In summary, LOs from different sources have distinct advantages and limitations (Figure 4). LOs derived from hPSCs enable the construction of personalized models and recapitulate the genetic heterogeneity of MASLD, making them ideal platforms for mechanistic exploration as well as genetic research and personalized medicine. LOs established from primary tissues faithfully recapitulate the physiological functions of the in vivo liver, conferring unique strengths for preclinical drug validation and translational research. In contrast, LOs derived from immortalized cell lines offer the advantages of straightforward construction and scalable preparation, serving as efficient models for preliminary MASLD-related studies and high-throughput compound screening. Furthermore, the development of multi-organoid systems, including intestinal and pancreatic organoids, enables the simulation of multi-organ regulatory mechanisms underlying the gut-liver axis and liver-pancreas axis in MASLD, overcoming the limitations of single hepatic organoid models. Overall, diverse organoid systems have shown remarkable value in recapitulating MASLD pathological phenotypes, unraveling core pathogenic mechanisms, identifying key protective mechanisms, discovering novel biomarkers, screening therapeutic targets, and validating the efficacy of candidate drugs. In conclusion, organoid models serve as powerful preclinical tools to drive progress in MASLD research. They facilitate in-depth exploration of pathogenic mechanisms, accelerate therapeutic drug screening and development, support the identification of reliable disease biomarkers, and advance the research framework of personalized medicine for MASLD. Nevertheless, the widespread application of such organoid systems still encounters considerable technical hurdles and translational limitations. Resolving these core challenges is essential to fully unleash the potential of LOs, so as to deepen the understanding of MASLD pathogenesis and optimize targeted therapeutic strategies for clinical translation.

Figure 4 Main advantages and limitations of liver organoids from different sources. hPSC: Human pluripotent stem cell.