Engineering liver organoids-on-a-chip

Advances in stem cell biology and microengineering have led to the emergence of liver organoids-on-a-chip systems, hybrid platforms that integrate self-organizing three-dimensional organoids with microfluidic devices. These technologies enable more physiologically relevant modeling of human liver biology by enhancing organoid maturation, incorporating dynamic cues such as flow and shear stress, and facilitating multicellular interactions across parenchymal and non-parenchymal compartments. As a result, they provide powerful opportunities to investigate liver development, interrogate mechanisms of disease progression, and assess pharmacological responses with higher fidelity than conventional models. Applications span from studying steatohepatitis and fibrosis to evaluating drug-induced liver injury and patient-specific variability in metabolism. In this Review, we highlight recent progress in liver organoids-on-a-chip systems, discuss their potential in personalized medicine and predictive toxicology, and outline current technical challenges and translational opportunities that will shape their future impact on therapeutic discovery and precision health.

1 Introduction

The liver is a highly specialized organ with remarkable structural and functional complexity. Organized into vascularized lobules, it performs a wide range of essential processes, including xenobiotic metabolism, glycogen and lipid storage, bile production, immune regulation, and the synthesis of plasma proteins (Trefts et al., 2017; Fritsche et al., 2023). These functions depend on the coordinated activity of multiple cell types, such as hepatocytes, cholangiocytes, liver sinusoidal endothelial cells (LSECs), hepatic stellate cells, and resident macrophages (Kupffer cells), embedded within a unique extracellular matrix and sinusoidal network. Together, these parenchymal and non-parenchymal populations enable zonated metabolic activity and confer the ability to adapt rapidly to physiological stress, injury, or infection (Arteel, 2024).

For decades, conventional experimental models such as two-dimensional (2D) hepatocyte monolayers (Jamshed et al., 2024), immortalized liver cell lines (Zhou et al., 2019), and animal models (Rezvani, 2025) have been indispensable for advancing our understanding of liver biology, pharmacology, and toxicology. These systems remain valuable but face well-recognized limitations, including the rapid loss of differentiated hepatocyte function in vitro (Hashemian et al., 2025), restricted cellular diversity (Harrison et al., 2021), and species-species differences (Bell et al., 2016) that may limit translational relevance. The growing need for models that faithfully recapitulate the multicellular organization, microenvironmental cues, and patient-specific features of the human liver has driven the development of more physiologically relevant platforms.

Recent advances in stem cell biology, tissue engineering, and microfabrication have transformed the field. Liver organoids, derived from pluripotent stem cells (PSCs) (He et al., 2023) or adult tissue-resident progenitors (Nantasanti et al., 2016), form three-dimensional (3D) self-organizing microtissues that recapitulate essential aspects of liver development, function, and disease. In parallel, organ-on-a-chip platforms leverage microfluidics and bioengineering principles to reproduce dynamic features of the hepatic niche, including perfusable vasculature (Dalsbecker et al., 2022), spatial gradients (Ehrlich et al., 2019), and mechanical forces (Yang et al., 2022). The integration of these approaches into liver organoids-on-a-chip systems offers a powerful strategy to overcome long-standing challenges in modeling human liver physiology, providing a platform with both cellular fidelity and microenvironmental control.

1.1 Liver organoids

Organoids are 3D, self-organizing multicellular structures derived from stem cells or tissue-resident progenitors that recapitulate aspects of organ architecture and function (Nantasanti et al., 2016). In the case of the liver, organoids reproduce key physiological processes such as albumin secretion (Sorrentino et al., 2020), urea synthesis (Sorrentino et al., 2020), bile canaliculi formation (Luce et al., 2025), and cytochrome P450 enzyme activity (Xu et al., 2022), while preserving the genetic and epigenetic background of the donor cells (Akbari et al., 2019). This combination of structural fidelity and functional activity makes liver organoids powerful systems for studying both normal hepatic physiology and disease-specific phenotypes.

Liver organoids can be generated from two main cellular sources (Figure 1A). The first approach employs tissue-derived progenitors. Foundational studies by Huch et al. (2013) and Huch et al. (2015) demonstrated that epithelial cell adhesion molecule (EpCAM)⁺ bile duct–derived cells from human liver could be expanded long-term in culture. These ductal progenitor–derived organoids retained bipotent capacity and could be directed toward either hepatocyte or cholangiocyte lineages by modulating signaling pathways such as Wnt, R-spondin, Notch, and transforming growth factor (TGF)-β. The second approach derives liver organoids from PSCs (induced pluripotent stem cells, iPSCs or embryonic stem cells, ESCs). In these systems, pluripotent cells undergo stepwise differentiation through definitive endoderm and hepatic progenitors before self-organizing into 3D liver bud–like structures. Takebe et al. (2013) showed that co-culture of PSC-derived hepatic progenitors with mesenchymal and endothelial cells yielded vascularized liver buds capable of engrafting and maturing in vivo. More recent refinements (Ramli et al., 2020) have produced PSC-derived liver organoids with improved metabolic capacity, enhanced polarity, and greater structural complexity.

Figure 1

Figure 1. Generation of liver organoids and design of liver-on-a-chip systems. (A) Liver organoids can be generated from two main sources: primary liver tissue or PSCs. In tissue-derived approaches, dissociated liver samples provide hepatic progenitor cells that, when embedded in ECM, self-organize and differentiate into either hepatocyte organoids or cholangiocyte organoids depending on lineage-specific growth factor stimulation. In PSC–based protocols, pluripotent cells are directed stepwise through definitive endoderm and hepatic progenitor stages before forming 3D liver organoids. (B) Liver-on-a-chip platforms are designed to recapitulate the native hepatic microenvironment. In the system developed by Jang et al. (Jang et al., 2019), hepatocytes are cultured in the upper channel, separated by a porous membrane and ECM layer from non-parenchymal cells, including LSECs, hepatic stellate cells, and Kupffer cells, cultured in the lower channel. Continuous perfusion through the microfluidic channels provides nutrient and oxygen delivery, waste removal, and physiological shear stress, supporting vascularized liver-like function and enabling assessment of drug metabolism and hepatotoxicity.

The versatility of liver organoids has made them a central platform for modeling both physiological and pathological states. Patient-derived organoids (PDO), in particular, retain the genetic identity of their donors (De Crignis et al., 2021) and have been used to study monogenic diseases such as alpha-1 antitrypsin deficiency (Gómez-Mariano et al., 2020) and Alagille syndrome (Ouchi and Koike, 2023), as well as acquired conditions including liver cancer (Broutier et al., 2017) and polycystic liver disease (Sampaziotis et al., 2015). Beyond disease modeling, organoids offer renewable and scalable sources of functional human liver tissue for drug screening, hepatotoxicity studies, and emerging applications in regenerative medicine.

Despite their utility, liver organoid models still face important limitations. They generally lack vascular perfusion, metabolic zonation, and the dynamic biochemical and mechanical cues that are central to liver physiology in vivo (Akbari et al., 2019). As a result, processes such as gradient-dependent metabolism, immune–parenchymal crosstalk, and long-term tissue remodeling remain incompletely represented. To overcome these shortcomings, recent efforts have focused on integrating organoids with organ-on-a-chip technologies, which provide controlled perfusion, spatial organization, and multicellular co-culture in a physiologically relevant context.

1.2 Liver-on-a-chip

Efforts to engineer physiologically relevant in vitro liver systems have progressed from hepatocyte monocultures to more structured and multicellular platforms. A key milestone was the development of micropatterned co-cultures, in which hepatocytes were spatially organized with supportive stromal cells to sustain long-term hepatic function and drug-metabolizing activity (Khetani and Bhatia, 2008). These engineered microenvironments preserved albumin secretion, urea synthesis, and cytochrome P450 enzyme activity for extended periods, setting a benchmark for in vitro hepatic functionality. Similarly, self-assembled primary hepatocyte spheroids exhibited enhanced metabolic stability and reproducible drug-induced toxicity responses that closely mirrored in vivo outcomes (Bell et al., 2016; Wang et al., 2019). Together, these systems established essential physiological reference points that continue to guide the development of iPSC-derived liver organoids and organ-on-chip technologies. By demonstrating that spatial organization and microenvironmental control can significantly extend hepatocyte viability and metabolic fidelity, these early microengineered models laid the groundwork for modern liver-on-a-chip platforms (Gough et al., 2021).

Building on these principles, liver-on-a-chip devices introduced spatial organization and controlled environments to integrate multiple hepatic cell types. Systems incorporating hepatocytes with LSECs, stellate cells, and Kupffer cells enabled modeling of inflammation, fibrogenesis, and cholestasis, processes that closely resemble human liver pathology (Vernetti et al., 2016; Jang et al., 2019). In the liver-on-a-chip developed by Jang and colleagues, hepatocytes were cultured in an upper channel, while non-parenchymal cells were positioned in a lower channel, separated by a porous membrane and extracellular matrix (ECM) (Figure 1B) (Jang et al., 2019). Continuous perfusion across the device supported nutrient exchange and waste removal while enabling dynamic paracrine signaling between compartments. This multi-channel configuration recreated key aspects of the sinusoidal microenvironment and was applied to model inflammation, fibrogenesis, and cholestasis. Importantly, the system also reproduced species-specific drug toxicities, providing improved predictive power for adverse effects in humans compared with traditional animal studies.

Technological advances have refined physiological fidelity further. Devices capable of reproducing oxygen and nutrient zonation along the sinusoidal axis revealed region-specific patterns of metabolism and toxicity that mirror in vivo physiology (Azizgolshani et al., 2021). Incorporation of ECM scaffolding and 3D organization has enhanced hepatocyte polarity and bile canaliculi formation, enabling functional outputs that approach tissue-level complexity. Such features broaden the applications of liver-on-a-chip to include nutrient sensing, metabolic disease modeling, and environmental toxicology. In parallel, stem cell–based approaches have been integrated into chip platforms to address the scarcity and variability of primary hepatocytes. The use of iPSC-derived hepatocytes provides a renewable and standardized cell source that can be maintained in engineered microenvironments for extended periods while retaining key metabolic functions. Recent studies have demonstrated that liver-on-a-chip systems incorporating iPSC-derived hepatocytes can be applied for drug hepatotoxicity screening and functional assays, establishing their utility as alternatives to donor-derived cells (Fanizza et al., 2023). These strategies broaden the accessibility of liver-on-a-chip technology and lay the groundwork for future precision medicine applications.

Despite significant progress, most liver-on-a-chip systems still depend on primary human hepatocytes. These cells remain difficult to source in sufficient numbers, display considerable donor-to-donor variability (Ehrlich et al., 2019), and progressively lose function over time in culture (Ewart et al., 2022). Such limitations constrain the reproducibility, scalability, and long-term applicability of current platforms, highlighting the need for improved strategies to maintain stable hepatic function in vitro.

1.3 Integrating liver organoids with organs-on-chips

Bringing together liver organoid and liver-on-a-chip technologies offers an opportunity to create models that combine the cellular fidelity of organoids with the microenvironmental precision of chip-based systems. Organoids capture the self-organization, lineage plasticity, and patient specificity of liver tissue, but they are typically maintained in static ECM gels that limit nutrient diffusion, vascular integration, and exposure to mechanical cues. Conversely, liver-on-a-chip devices recreate perfusion, spatial gradients, and multicellular interactions, yet they typically depend on primary hepatocytes that are already differentiated with limited proliferative capacity and a tendency to lose function over time in culture. Integrating these complementary approaches provides a strategy to overcome the limitations of each.

This review focuses on recent advances in liver organoids-on-a-chip platforms, with particular attention to how they recapitulate liver development, physiology, and disease (Table 1). We examine their applications in drug screening, hepatotoxicity testing, and precision medicine, highlighting studies that illustrate their translational relevance. Finally, we outline the key technical and conceptual challenges that remain, and discuss future directions for standardization, scalability, and validation.

Table 1

Table 1. Liver organoids-on-a-chip platforms.

2 Modeling development using liver organoids-on-a-chip

Efforts to recapitulate liver development in vitro have been limited by the inability of conventional organoid cultures to reproduce the structural, vascular, and spatial cues that underlie hepatic zonation and inter-organ communication. These missing features result in homogeneous cell populations that lack the region-specific architecture and metabolic specialization of the native liver. Recent studies are beginning to address this gap by integrating liver organoids with organ-on-a-chip technologies, which provide spatiotemporally controlled microenvironments capable of delivering mechanical, biochemical, and architectural cues essential for development.

2.1 Recreating spatial patterning in liver organoids

Conventional liver organoid cultures typically generate uniform spheroids with little spatial or metabolic heterogeneity (Zhao et al., 2022). This homogeneity stands in contrast to the highly ordered zonation of the native liver, where hepatocytes surrounding the central vein and portal triad adopt distinct transcriptional and metabolic identities (Chen et al., 2023). Without precise control over geometry and spatial orientation, conventional systems cannot reproduce this organization. Microengineered platforms now provide a way to impose spatial patterning and direct hepatocyte identity by integrating biochemical cues with defined architecture.

An emerging strategy to impose spatial heterogeneity in liver organoids is to leverage endothelial co-culture. Zhang et al. (Zhang et al., 2025) generated zonated liver organoids by combining human ESC (hESC)-derived hepatocytes with LSECs specified toward either pericentral (PC) or periportal (PP) fates. Using poly (ethylene glycol) methacrylate (PEGMA)-modified polydimethylsiloxane (PDMS) microwells that promoted low adhesion and spherical aggregation, the researchers established structured organoids in which endothelial identity dictated hepatocyte function (Figure 2A). PC LSECs, characterized by high WNT activity and venous markers, induced perivenous features in hepatocytes, whereas PP LSECs, with low WNT signaling and arterial identity, promoted periportal traits. This system recapitulated graded WNT signaling patterns essential for lobular zonation and revealed that endothelial subtype identity is a key determinant of hepatocyte function. Notably, these models reproduced zone-specific metabolic dysfunction relevant to diseases such as metabolic dysfunction–associated steatotic liver disease (MASLD).

Figure 2

Figure 2. Organoids-on-a-chip platforms for modeling liver development. (A) PEGMA-modified PDMS microwells promote low-adhesion aggregation of hESC-derived hepatocytes co-cultured with LSECs specified toward PC or PP fates, enabling zonated organoid formation. Scale bars, 200 μm. (adapted from (Zhang et al., 2025), copyright 2025 John Wiley and Sons) (B) Micropatterned agarose scaffold (mHCPCAs) containing hexagonally packed microwells direct uniform hepatic bud-like organoids from hPSC-derived foregut stem cells. Organoids show early hepatic markers (ALB, HNF4α, AFP) with VIM+ mesenchymal outer layers. Scale bars, 50 μm. (adapted from (Jiang et al., 2023), copyright 2023 the authors) (C) Multi-organ chip integrating up to nine organoid types (liver, heart, lung, kidney, adipose, pancreas, muscle, spleen, brain) under continuous perfusion demonstrates flow-dependent modulation of hepatic albumin secretion. Scale bars, 20 μm. (adapted from (Yang et al., 2025) with permission, copyright 2025 Royal Society of Chemistry) (D) Hydrogel-based liver chip with adjacent hepatic and endothelial compartments mimic lobule-like diffusion. Hepatic spheroids display tight junction (ZO–1) and canalicular (MRP2) expression. Scale bars, 100 μm. (adapted from (Meng et al., 2021) with permission, copyright 2021 John Wiley and Sons) (E) Perfusable micropillar chip supports in situ differentiation of hiPSC aggregates into liver organoids. Shear stress enhances maturation markers including AFP, ALB, HNF4α, and CYP3A4. Scale bars, 50 μm. (adapted from (Wang et al., 2018) with permission, copyright 2018 Royal Society of Chemistry) (F) High-throughput liver-on-a-chip with 29 perfused microwells enables automated matrix exchange, real-time lineage tracking, and parallelized differentiation. Organoids mature more rapidly, showing improved albumin secretion, CYP450 activity, and bile acid accumulation. Scale bars, 100 μm. (adapted from (Byeon et al., 2024), copyright 2024 the authors).

Beyond endothelial co-culture, spatial patterning can also be achieved through microengineering strategies that directly guide morphogenesis. Jiang et al. (2023) developed a high-throughput micropatterned agarose scaffold containing hexagonally packed, U-shaped microwells designed to generate uniform organoids from human PSC (hPSC)-derived foregut stem cells (Figure 2B). The agarose matrix, chosen for its biocompatibility and ultra-low cell adhesion, ensured controlled seeding and aggregation, driving the reproducible self-organization of hepatic progenitors into three-dimensional buds. Organoids formed on this platform exhibited robust expression of early hepatic markers (alpha-fetoprotein (AFP), albumin (ALB), hepatocyte nuclear factor 4 alpha (HFN4α)) and displayed functional outputs including lipid accumulation and urea synthesis. By reducing variability and enabling scalable production, this system provides a promising route to standardize hepatic morphogenesis in vitro and overcome the heterogeneity of conventional culture.

2.2 Incorporating multi-tissue interactions and systemic communication

Liver development and function depend on continuous dialogue between hepatocytes, non-parenchymal cell populations, and other organs. Non-parenchymal cells such as LSECs, stellate cells, and Kupffer cells shape hepatocyte maturation through paracrine signaling, while systemic inputs from distal tissues provide additional regulatory cues (Wang et al., 2021; Kostallari et al., 2025). Conventional organoid cultures, however, are typically isolated from these influences, limiting their ability to acquire physiologically relevant features. Organ-on-a-chip technologies are beginning to address this limitation by enabling inter-organoid signaling under flow and by recreating co-culture environments that incorporate hepatic and non-parenchymal lineages within defined spatial arrangements (Leung et al., 2022).

A key advantage of organoid-on-a-chip systems is their ability to capture communication across tissues, either through fluid-mediated signaling between different organoids or through structured co-culture of multiple cell types within the same construct. To model inter-organ communication, Yang et al. (2025) developed a linear multi-organoid chip that integrates up to nine tissue types, including liver, kidney, heart, lung, and brain, within interconnected flow chambers (Figure 2C). In this system, albumin secretion by liver organoids varied depending on both the sequence of organoids and the direction of perfusion, demonstrating that systemic context can directly modulate hepatic function. Exosomes emerged as central mediators of this crosstalk, with secretion patterns mirroring albumin levels, highlighting the role of extracellular vesicles in coordinating inter-organ communication.

Another complementary strategy is to engineer co-culture systems that organize hepatic and non-parenchymal lineages in defined spatial arrangements. Meng et al. developed a microfluidic liver-on-a-chip incorporating a di-acrylated pluronic F127 hydrogel to mimic the mass transfer and structural features of the hepatic lobule (Figure 2D) (Meng et al., 2021). In this design, hepatocyte–stellate spheroids were cultured in microwells within the outer channel, while human umbilical vein endothelial cells (HUVECs) formed monolayers in an adjacent perfused channel exposed to physiological shear. Nutrient diffusion across the hydrogel supported hepatocyte growth while shielding them from direct shear stress, more closely resembling in vivo mass transfer. Within 24 h, hepatocytes self-organized into spheroids that exhibited higher viability and enhanced liver-specific functions compared with static controls, sustained for more than a week. The presence of endothelial cells further augmented hepatocyte performance, highlighting the importance of vascular interfaces in promoting hepatic function in vitro.

2.3 Promoting hepatic maturation with mechanical and structural cues

A persistent limitation of liver organoid models is the immature phenotype of in vitro–derived hepatocytes, which restricts their utility for disease modeling and pharmacological studies (Zabulica et al., 2019). This immaturity arises in part from the absence of key microenvironmental signals, such as mechanical stimulation, matrix elasticity, and spatial organization, that are central to hepatocyte maturation in vivo. Conventional 3D cultures rarely replicate sinusoidal flow, ECM stiffness, or the dynamic deformations encountered in the native liver. Organ-on-a-chip platforms overcome these gaps by applying controlled shear stress and incorporating biomimetic scaffolds, thereby creating conditions that drive the structural and functional maturation of liver organoids.

Wang et al. developed a perfusable micropillar chip that enabled in situ differentiation of hiPSCs into functional liver organoids (Figure 2E) (Wang et al., 2018). The micropillar array supported the formation of uniform embryoid bodies, which subsequently underwent hepatic specification under continuous perfusion. This design reproduced sinusoid-like shear stress, leading to enhanced albumin secretion, elevated CYP3A4 activity, and improved long-term viability compared with static culture. These results demonstrate that dynamic mechanical forces, particularly fluid shear, are essential for promoting and sustaining hepatocyte maturation in vitro.

2.4 High-throughput platforms for liver organoid differentiation and developmental studies

Progress in liver organoid culture has expanded opportunities to model human development in vitro, yet systematic analysis remains challenging. Protocols often vary between laboratories, leading to inconsistent outcomes, and most culture methods lack the scalability needed to probe multiple variables in parallel. This poses a barrier to dissecting how combinations of extracellular matrix properties, morphogen gradients, and mechanical cues guide hepatic fate. To overcome these limitations, organoid-on-a-chip technologies are being adapted into high-throughput arrayed platforms. These systems provide precise control over microenvironments while enabling simultaneous manipulation of multiple conditions, opening the door to parallelized studies of liver differentiation and maturation trajectories.

Byeon et al. recently introduced a microphysiological system (MPS) designed as a 3D, high-throughput chip platform containing 29 microwell compartments, each capable of supporting the differentiation of liver organoids (Byeon et al., 2024). Unlike conventional Matrigel droplet cultures, this device operates under continuous perfusion with automated matrix exchange, which maintains a stable extracellular environment while reducing manual variability. Integrated imaging and analytical features further allow real-time tracking of lineage progression and quantitative assessment of hepatic marker expression (Figure 2F). The platform substantially accelerated differentiation, reducing the timeline to ∼11 days compared with the 21 days or more typically required in static culture. Importantly, organoids generated under these conditions exhibited enhanced functional maturity, including elevated albumin secretion, upregulation of cytochrome P450 enzymes, and bile acid accumulation, all of which are hallmarks of hepatocyte specialization. Beyond improving efficiency, the arrayed design enabled systematic testing of matrix stiffness and soluble factor combinations, revealing synergistic effects on hepatic fate decisions and maturation trajectories. This capacity to manipulate and monitor multiple variables in parallel represents a significant step toward standardized, reproducible, and scalable models for developmental biology and preclinical research.

3 Modeling liver disease with organoids-on-a-chip

The human liver plays a central role in nutrient metabolism, detoxification, immune regulation, and inter-organ communication. Disruption of these functions underlies a spectrum of chronic diseases, especially fatty liver disease. Traditional in vitro models, ranging from immortalized hepatocyte lines to primary hepatocyte monolayers, are limited by poor survival, rapid loss of function, and inability to recapitulate complex multicellular and microenvironmental interactions (Kim et al., 2025). Organoids derived from pluripotent or adult stem cells have addressed some of these shortcomings by self-organizing into 3D microtissues with preserved genetic backgrounds and improved physiological functions (Liu et al., 2020). However, static organoid systems often lack perfusion, immune cell components, and systemic communication, restricting their ability to model disease progression beyond early stages.

Organoids-on-a-chip platforms overcome these barriers by embedding liver organoids within microfluidic devices that enable dynamic perfusion, spatial compartmentalization, and controlled co-cultures (Park et al., 2019). The resulting systems provide a more physiologically relevant microenvironment that captures the mechanical forces, nutrient gradients, and intercellular signaling networks necessary for disease modeling. Importantly, these platforms also enhance scalability, reproducibility, and compatibility with high-throughput drug testing, making them valuable tools for both basic research and translational applications.

3.1 Modeling fatty liver disease pathology with microphysiological platforms

Liver organoids-on-a-chip provide a unique opportunity to investigate the sequential and multifactorial processes that underlie the onset and progression of metabolic dysfunction-associated steatotic liver disease (MASLD) and its more advanced form, metabolic dysfunction-associated steatohepatitis (MASH). MASLD, previously known as nonalcoholic fatty liver disease (NAFLD), was renamed in 2023 to better reflect its underlying metabolic dysfunction, move away from a “nonalcoholic” exclusion-based definition, and provide a more precise diagnostic framework aligned with current understanding of disease pathogenesis (Rinella et al., 2023). These diseases are characterized by a continuum of pathological events, including hepatocellular lipid droplet accumulation, oxidative stress, chronic inflammation, and activation of fibrogenic pathways (Brunt et al., 2015). Traditional hepatocyte cultures or static organoid models often reproduce only the earliest stages of triglyceride accumulation and fail to support the progression to steatohepatitis. By combining stem cell–derived liver organoids with microengineered perfusion systems, organoids-on-a-chip platforms provide a microenvironment where disease-associated stimuli can be introduced in a controlled and sustained manner. Importantly, such systems not only reproduce lipid overload but also capture the interplay of metabolic stress with inflammatory and fibrogenic signaling cascades, thereby generating models that are more reflective of the in vivo disease spectrum.

Wang et al. (2020) developed a microengineered platform in which human iPSC-derived liver organoids were cultured under continuous perfusion and challenged with free fatty acids (FFAs) to induce NAFLD-like pathology (Figure 3A). The microfluidic design allowed dynamic circulation of media, ensuring sustained nutrient and oxygen delivery while preventing the metabolic exhaustion that often occurs in static culture. Under these conditions, organoids exhibited hallmark NAFLD phenotypes, including the accumulation of lipid droplets within hepatocytes, elevated triglyceride storage, and generation of reactive oxygen species (ROS). Transcriptional profiling revealed upregulation of lipid metabolism–associated genes with increased expression of pro-inflammatory cytokines and fibrogenic markers, indicating activation of pathways that drive steatohepatitis. Notably, this system recapitulated the temporal evolution of disease, moving from simple steatosis to features consistent with NASH, which has been extremely challenging to achieve with conventional culture models. Beyond providing a tool for mechanistic studies, this perfused organoid-on-a-chip model establishes a foundation for testing interventions that target multiple stages of NAFLD progression.

Figure 3

Figure 3. Organoid-on-a-chip platforms for modeling liver disease. (A) Perfused hiPSC-derived liver organoids cultured under continuous flow and treated with FFAs reproduce key NAFLD features, including lipid droplet accumulation marked by Perilipin-2 (PLIN2), elevated oxidative stress (DCFH-DA), and fibrosis-related protein expression. Scale bars, 100 μm. (adapted from (Wang et al., 2020) with permission, copyright 2020 American Chemical Society). (B) The SteatoChip, a 100-well microfluidic array for HepaRG organoids, supports uniform differentiation with albumin production and bile canaliculi formation. Under FFA stimulation, organoids in perfusion show significantly higher lipid accumulation than static controls, providing a sensitive model of steatosis. Scale bars, 30 μm. (adapted from (Teng et al., 2021) with permission, copyright 2021 Elsevier). (C) A liver–islet co-culture chip integrates hiPSC-derived liver and pancreatic islet organoids in separate but connected regions. Circulatory flow supports long-term viability, and under hyperglycemic conditions both organoid types exhibit reduced GLUT1 expression, partially restored with metformin, modeling T2DM-associated dysfunction. Scale bars, 100 μm. (adapted from (Tao et al., 2022), copyright 2022 John Wiley and Sons). (D) The dual-rOoC, a pump-less recirculating chip, maintains co-culture of hPSC-derived liver and islet organoids through interconnected gravity-driven circuits. In obesogenic medium, liver organoids accumulate neutral lipids with nuclear localization of SREBP1, while islets show reduced C-peptide–positive insulin-secreting cells, reflecting metabolic stress and insulin resistance. Scale bars, 50 μm. (adapted from (Aizenshtadt et al., 2024), copyright 2024 John Wiley and Sons).

Expanding on the concept of perfused organoid cultures, Teng et al. (Teng et al., 2021) introduced the SteatoChip, a high-throughput microfluidic device consisting of 100 interconnected wells for the culture and in situ differentiation of HepaRG organoids (Figure 3B). This platform was designed not only for physiological fidelity but also for scalability, enabling long-term perfusion across a large number of independent organoid cultures simultaneously. HepaRG organoids differentiated in SteatoChip demonstrated enhanced hepatic maturity, including increased expression of albumin, improved urea metabolism, and the formation of bile canaliculi networks, features that are critical for modeling hepatic metabolic function. When exposed to FFAs, the system produced robust steatotic phenotypes with pronounced lipid accumulation and altered glucose regulation, closely mimicking the metabolic impairments seen in patients with NAFLD. Furthermore, SteatoChip proved to be highly responsive to therapeutic interventions. Treatment with antisteatotic compounds such as metformin, pioglitazone, and obeticholic acid significantly reduced lipid burden, demonstrating the platform’s utility for pharmacological screening. By integrating disease fidelity with high-throughput capability, SteatoChip represents an important advance for drug discovery pipelines, particularly in a field where clinical trial success rates for NASH therapies remain low.

3.2 Modeling systemic and inter-organ responses in liver disease

The pathogenesis of many liver-associated diseases extends beyond hepatocellular dysfunction and is shaped by continuous communication between the liver and other organs (Wang et al., 2021). In metabolic disorders such as Type 2 Diabetes Mellitus (T2DM) and MASLD, the liver interacts dynamically with the pancreas, adipose tissue, gut, and immune system through hormonal, metabolic, and inflammatory signaling (Daryabor et al., 2020). Conventional liver models, which focus on hepatocyte monocultures or liver organoids in isolation, do not capture these systemic interactions. Recent innovations in organoid-on-a-chip technology have made it possible to construct multi-organoid platforms, thereby providing systems in which the reciprocal signaling between different tissues can be studied with high fidelity. These models not only enable a better understanding of disease pathophysiology but also establish translationally relevant platforms for evaluating therapies that target systemic metabolic dysfunction.

Tao et al. (2022) developed a sophisticated liver–islet co-culture system using a microfluidic chip that supports hiPSC-derived liver and pancreatic islet organoids under continuous flow for up to 30 days (Figure 3C). The device was designed with separate but interconnected compartments, allowing controlled exchange of metabolites, hormones, and cytokines between organoid types. This dynamic interaction resulted in enhanced viability and function of both tissues. Liver organoids exhibited increased albumin secretion and metabolic activity, while islet organoids demonstrated robust glucose-stimulated insulin release. Importantly, transcriptomic analyses revealed the activation of pathways central to metabolic regulation, highlighting the functional integration of the two organ systems. Under hyperglycemic conditions, the platform reproduced T2DM-like phenotypes, including impaired glucose utilization and mitochondrial dysfunction, both of which were reversed upon treatment with metformin. By modeling the bidirectional relationship between liver and pancreas, this system provides a physiologically relevant framework for dissecting the molecular underpinnings of T2DM and MASLD and for testing candidate therapeutics that target inter-organ metabolic axes.

To extend the accessibility and scalability of multi-organoid systems, Aizenshtadt et al. (2024) introduced the dual recirculating organ-on-chip (rOoC), a pump-less, gravity-driven recirculating system that supports long-term co-culture of stem cell–derived liver and pancreatic islet organoids (Figure 3D). The design consists of two independent but interconnected fluidic circuits, separated by a permeable membrane that enables bidirectional molecular exchange without requiring external pumps. This configuration simplifies operation while preserving physiological feedback loops, allowing for prolonged co-culture with controlled metabolic interaction. The dual-rOoC successfully reproduced metabolic dysfunction induced by lipid- and fructose-rich conditions, including hepatic steatosis, insulin resistance, and secretion of pro-inflammatory cytokines. The system also demonstrated responsiveness to antidiabetic drugs, further validating its predictive capability. In addition to modeling obesity-associated metabolic disease, the dual-rOoC facilitates secretome analysis from each compartment, providing insights into organ-specific contributions to systemic dysfunction. By combining usability, scalability, and physiological relevance, the dual-rOoC represents a promising platform for both mechanistic studies and preclinical therapeutic testing.

4 Applications in drug screening and hepatotoxicity assessment

The liver is the primary site of drug metabolism and detoxification, making it central to evaluating both therapeutic efficacy and the risk of drug-induced liver injury (DILI). Accurate preclinical models are essential for assessing pharmacokinetics, predicting hepatotoxicity, and guiding the development of safer and more effective therapeutics. Liver organoids-on-a-chip provide a unique advantage for this purpose by combining physiologically relevant hepatic tissue architecture with controlled microfluidic environments. These systems enable real-time monitoring of metabolic and toxicological responses and can be tailored for high-throughput screening. As such, they represent a critical tool for modeling drug action, biodistribution, and toxicity in human-relevant contexts.

4.1 Enhancing drug screening sensitivity with perfused microenvironments

All liver organoids-on-a-chip have proven valuable for drug screening because perfused microenvironments foster hepatic maturation and enhance sensitivity to pharmacological modulation. The SteatoChip developed by Teng et al. (2021) represents one of the most advanced platforms in this space (Figure 4A). The device consists of 100 interconnected microchambers designed for long-term perfusion and in situ differentiation of HepaRG progenitors into liver organoids. This array-based format enables parallel experimentation, an essential feature for high-throughput applications.

Figure 4

Figure 4. Organoid-on-a-chip platforms for drug screening and hepatotoxicity assessment. (A) The SteatoChip, a 100-well microfluidic device, supports long-term perfusion and in situ differentiation of HepaRG progenitors into functional liver organoids. The platform enables modeling of NAFLD by inducing lipid accumulation and glucose dysregulation under FFA exposure, and demonstrates therapeutic sensitivity to anti-steatotic compounds including metformin, pioglitazone, and obeticholic acid (Teng et al., 2021). Scale bars, 30 μm. (adapted from (Teng et al., 2021) with permission, copyright 2021 Elsevier). (B) A liver–kidney multi-organ-on-a-chip integrates 3D liver organoids and kidney tubuloid monolayers under physiological flow generated by an on-chip minipump. Fluorescently labeled MSC-sEVs showed preferential accumulation and prolonged retention in injured kidney cells and concurrent uptake in liver organoids, providing a human-relevant model for biodistribution and therapeutic evaluation. Scale bars, 100 μm. (adapted from (Nguyen et al., 2022), copyright 2022 John Wiley and Sons). (C) A sensor-integrated liver-on-a-chip allows real-time monitoring of metabolic dynamics during drug-induced mitochondrial stress. Embedded ruthenium-based phosphorescent probes measure oxygen consumption, while an external sensing module quantifies glucose uptake and lactate production. This multiplexed setup revealed early metabolic shifts in response to hepatotoxic drugs such as rotenone and troglitazone, offering mechanistic insight into idiosyncratic toxicity. [adapted from (Bavli et al., 2016)]. (D) A PDO-on-a-chip platform co-cultures HCC tumor PDOs with MSCs and PBMCs under perfusion to recreate the tumor microenvironment. This system accurately predicted patient-specific responses to chemotherapy, targeted therapy, and immunotherapy, including differential sensitivity to the anti-PD-L1 antibody atezolizumab, supporting applications in precision medicine. [adapted from (Zou et al., 2023), copyright 2023 John Wiley and Sons].

When challenged with FFAs, SteatoChip organoids recapitulated pathological hallmarks of NAFLD, including significant lipid droplet accumulation and impaired glucose metabolism. The precision of the microenvironment ensured reproducibility across wells, reducing batch-to-batch variability. Importantly, pharmacological responsiveness was demonstrated by robust reductions in lipid accumulation following treatment with metformin hydrochloride, pioglitazone hydrochloride, or obeticholic acid, three agents with distinct mechanisms of action targeting insulin sensitivity, PPAR signaling, and farnesoid X receptor (FXR) activation (Clifford et al., 2021; Lange et al., 2022), respectively. These results establish SteatoChip as a sensitive tool not only for modeling drug-induced steatosis but also for screening anti-steatotic therapeutics. By offering reproducibility, scalability, and physiologic relevance in a single platform, SteatoChip provides a strong foundation for integrating liver organoids into preclinical drug development pipelines.

4.2 Modeling drug biodistribution and cross-organ toxicity

The liver’s central role in xenobiotic metabolism and systemic detoxification makes it indispensable for understanding drug biodistribution and potential off-target effects. Importantly, pharmacological interventions rarely act on the liver in isolation and renal clearance and kidney injury often modulate drug exposure and toxicity. Therefore, models that integrate hepatic and renal tissues are essential for predicting systemic responses in a human-relevant context.

To address these gaps, Nguyen et al. (2022) developed a liver–kidney multi-organ-on-a-chip (MOC) model that combines human adult stem cell-derived kidney tubuloids with 3D human liver organoids in a perfused microfluidic circuit (Figure 4B). The device features two culture chambers interconnected by microchannels, with physiological fluid flow maintained by an integrated on-chip minipump. Within the system, kidney tubuloids were grown as polarized epithelial monolayers on semi-permeable membranes, mimicking the renal tubular barrier, while liver organoids were maintained in parallel under 3D conditions. This configuration supported long-term viability and function of both organoid types and enabled dynamic cross-talk between hepatic and renal compartments.

The model was applied to investigate the therapeutic efficacy and biodistribution of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) in the context of acute kidney injury (AKI) (Kellum et al., 2021). In the injury model, kidney tubuloids exhibited compromised barrier integrity and impaired transporter activity, as demonstrated by decreased accumulation and transport of the fluorescent reporter substrate 5 (6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA). Importantly, administration of MSC-sEVs restored tubuloid barrier function within 24 h, and CDFDA transport significantly improved, highlighting the therapeutic potential of vesicle-based interventions.

Furthermore, biodistribution was evaluated using fluorescent labeling of MSC-sEVs. Tracking studies revealed preferential uptake and prolonged retention of vesicles in injured kidney tubuloids compared with healthy controls, consistent with their role in repair processes. Interestingly, significant accumulation was also observed in liver organoids, reflecting systemic biodistribution and hepatic clearance. This dual-organ readout provided mechanistic insights into the trafficking and tissue targeting of extracellular vesicles, revealing that organ injury can alter vesicle distribution patterns and therapeutic efficacy.

By recapitulating both therapeutic outcomes and biodistribution dynamics in a human-relevant in vitro setting, this platform provides a powerful tool for drug development. The ability to simultaneously study efficacy, biodistribution, and off-target accumulation reduces dependence on animal models, improves translational accuracy, and allows mechanistic exploration of compound trafficking under health and disease conditions. This study demonstrates how multi-organ organoid-on-a-chip systems can bridge a critical gap between single-organ models and in vivo pharmacology, offering a scalable strategy for evaluating regenerative therapies and drug safety in complex physiological contexts.

4.3 Real-time monitoring of hepatotoxic responses

Unanticipated hepatotoxicity remains a leading cause of late-stage drug attrition and post-market drug withdrawal. One of the primary challenges in predicting DILI lies in the limitations of traditional screening platforms, which often rely on short-term, endpoint measurements and fail to capture the dynamic metabolic responses underlying toxicity. Moreover, many hepatotoxic mechanisms, particularly those involving mitochondrial dysfunction and metabolic adaptation, manifest only after prolonged or repeated drug exposure. To address these gaps, liver organoids-on-a-chip equipped with integrated sensing technologies have been developed to enable continuous, multiparametric monitoring of hepatocyte physiology in real time.

Bavli et al. (2016) introduced a sensor-integrated liver-on-a-chip system specifically designed to model mitochondrial dysfunction, a major driver of DILI (Figure 4C). The platform consists of a microfluidic bioreactor fabricated from polymethyl methacrylate (PMMA) and outfitted with removable PDMS microwell inserts to support the growth of HepG2/C3A spheroids. These spheroids were maintained under physiological shear forces and oxygen gradients, conditions that preserve hepatocyte metabolic activity more effectively than static culture. A central innovation of this system is the incorporation of ruthenium-based phosphorescent microprobes within the bioreactor, enabling non-invasive optical oxygen measurements through two-frequency phase modulation. These probes provided stable, calibration-free oxygen monitoring for over 28 days, ensuring long-term assessment of metabolic activity. To complement oxygen sensing, the device was coupled to a computer-controlled modular sensing unit via a microfluidic switchboard. This configuration allowed automated, real-time amperometric measurement of glucose consumption and lactate production using medical-grade sensors. Together, the multiplexed system tracked oxygen uptake, glycolysis, and glutaminolysis, providing a comprehensive metabolic profile of hepatocyte function.

The system revealed early, subtle shifts in energy metabolism in response to hepatotoxic agents. For example, exposure to rotenone, a mitochondrial complex I inhibitor, and troglitazone, a withdrawn antidiabetic drug linked to idiosyncratic hepatotoxicity, induced measurable alterations in oxygen consumption and lactate production at concentrations previously considered safe. Such findings demonstrate the sensitivity of the platform for detecting mitochondrial stress and predicting toxicity that eludes conventional assays. Importantly, by supporting long-term monitoring, the system captured adaptive responses and cumulative effects, features critical for modeling chronic hepatotoxicity.

By combining physiologically relevant liver organoid cultures with integrated, high-resolution sensing, Bavli et al.’s liver-on-a-chip represents a significant advance in hepatotoxicity prediction. The ability to continuously monitor mitochondrial function, energy metabolism, and cellular adaptation establishes this technology as a valuable tool for uncovering mechanisms of DILI, particularly in drugs with delayed, cumulative, or patient-specific toxic effects.

4.4 Personalized drug testing and inter-individual variability

Interpatient variability in drug response presents one of the most significant barriers in clinical pharmacology and oncology (Wilkinson, 2005). Differences in genetic background, tumor heterogeneity, and immune interactions often dictate therapeutic success or failure (Marusyk and Polyak, 2010), yet conventional preclinical models typically lack the complexity required to predict these individualized outcomes. PDOs have addressed some of these limitations by preserving tumor-specific features (Tong et al., 2024), but their predictive capacity is still constrained by static culture conditions that fail to replicate dynamic immune and stromal interactions. Integrating PDOs with organ-on-a-chip technology offers a path toward more accurate and personalized drug screening.

Zou et al. (2023) developed a microengineered PDO-on-a-chip platform designed specifically to predict therapeutic responses in hepatocellular carcinoma (HCC) (Figure 4D). In this model, patient-derived liver tumor organoids were co-cultured with MSCs and peripheral blood mononuclear cells (PBMCs) inside a microfluidic device. This tripartite system recreated essential components of the tumor microenvironment, including stromal support and immune modulation, while continuous perfusion maintained nutrient and oxygen delivery. This configuration allowed long-term culture of PDOs with preservation of cellular heterogeneity and viability, overcoming challenges commonly encountered in static PDO assays.

The platform demonstrated strong predictive capacity across multiple therapeutic classes. For chemotherapy and targeted therapies, drug sensitivity profiles of PDOs-on-a-chip closely mirrored those obtained in conventional PDO assays, confirming its reliability for standard drug screening. However, the greatest advantage of the system emerged in the context of immunotherapy. When tested with the anti-PD-L1 antibody Atezolizumab, the device accurately distinguished resistance versus sensitive PDOs: resistant patient-derived tumors maintained resistance in the chip model, while sensitive PDOs showed restored responsiveness. This level of predictive fidelity was not achieved in standard PDO cultures lacking immune co-culture, highlighting the importance of incorporating immune components and dynamic flow for immunotherapy testing.

Beyond predictive accuracy, the system provided practical benefits. The microfluidic configuration allowed parallel, high-content drug testing on limited patient material, supporting faster turnaround times for personalized decision-making. Moreover, the reproducibility of immune–tumor interactions within the platform reduced variability and improved confidence in therapeutic predictions. By faithfully modeling the tumor microenvironment, including immune checkpoint interactions, the PDO-on-a-chip system advances the potential for individualized treatment planning in HCC. Together, these findings highlight the promise of PDO-on-a-chip platforms for bridging the gap between precision oncology and preclinical testing. By integrating perfusion, stromal and immune co-culture, and patient-specific tumor biology, this technology provides a physiologically relevant and scalable tool for predicting therapeutic efficacy and resistance, particularly in immunotherapy.

5 Discussion

The integration of liver organoids with microengineered chip platforms has created a foundation for advanced in vitro modeling of liver biology, disease progression, and therapeutic responses. While early studies have demonstrated feasibility and generated proof-of-concept data, the field is now entering a stage where the emphasis must shift toward reproducibility, scalability, and translational relevance (Fang et al., 2023). The challenge moving forward is to ensure that liver organoids-on-a-chip systems move beyond specialized academic demonstrations to become standardized, widely adopted tools in pharmaceutical research, toxicology, and regenerative medicine.

Drug development is one of the most immediate areas where these systems are expected to have substantial impact. Liver organoid chips have been shown to replicate key hepatic processes such as metabolic activity (Kizawa et al., 2017), tissue organization (Meng et al., 2021), and lobule-like zonation (Weng et al., 2017) more faithfully than conventional cell cultures. The incorporation of dynamic microfluidic flow helps sustain long-term function and supports the formation of perfusable vascular compartments, which allows for more physiologically relevant modeling of absorption, distribution, metabolism, and excretion (Jang et al., 2015; Fritschen et al., 2024). By providing a more accurate reflection of human liver responses, organoids-on-a-chip models address a persistent gap between preclinical predictions and clinical outcomes. Comparative studies have already shown that these systems provide improved predictive data on drug efficacy and toxicity relative to static cultures or animal models (Ehrlich et al., 2018; Jiao et al., 2024). Their adoption by industry and regulatory agencies could accelerate drug development pipelines while reducing reliance on animal testing.

Patient-derived liver organoids-on-a-chip systems also hold promise for precision medicine. Organoids generated from iPSCs or patient biopsies (Man et al., 2024) can be integrated into microfluidic devices to create individualized liver models. These patient-specific platforms could be used to evaluate risk of drug-induced liver injury or to screen treatment regimens before administration, effectively functioning as “clinical trials in a dish.” (Strauss and Blinova, 2017) Such approaches would capture patient-to-patient variability that is often missed by conventional models (Palasantzas et al., 2023). Although technical challenges related to scale and regulatory acceptance remain, the potential to guide therapy selection and reduce trial-and-error prescribing highlights the clinical relevance of this direction.

Beyond pharmaceutical applications, liver organoid chips are increasingly recognized as valuable tools in toxicology and environmental health science. They provide human-relevant models for assessing xenobiotic metabolism (Harrison et al., 2023), chemical toxicity (Yang et al., 2023), and pollutant exposures (Bridgeman et al., 2025), with the ability to monitor injury markers such as oxidative stress and enzyme release in real time (Liu et al., 2022). These platforms maintain metabolic competence under controlled conditions and can be linked with other organ systems to capture multi-organ interactions (Shao et al., 2025). Their improved predictive value compared with animal models has attracted attention in regulatory toxicology (Meyer et al., 2024), and their adoption for safety testing in pharmaceuticals, cosmetics, and environmental risk assessment would provide both ethical and scientific advantages.

Another emerging application is in regenerative medicine, where liver organoids are being investigated as potential therapeutics for liver failure (Tadokoro et al., 2024; Reza et al., 2025). Organoids-on-a-chip system provides a setting to evaluate the engraftment potential of liver organoid constructs under physiologic flow and mechanical stress (Lam et al., 2021). These platforms allow systematic testing of factors that influence transplant success, including oxygenation (Gallinat et al., 2019), vascular integration (Koffler et al., 2011), and immune interactions (Li and Lan, 2023). They are also being used to assess biomaterials such as hydrogels and scaffolds that support organoid survival and function (Sorrentino et al., 2020). Integration with 3D bioprinting technologies has led to hybrid constructs that combine liver organoids with engineered matrices, creating prototypes of bioartificial liver tissue (Yang et al., 2021). In this context, organoids-on-a-chip devices act as preclinical simulators to refine transplantation protocols before moving into animal or clinical studies.

Despite substantial advances, challenges remain. Most current systems often struggle to achieve robust vascularization, which limits nutrient and oxygen delivery and leads to heterogeneity in organoid maturation. Within dense 3D structures, steep oxygen gradients often give rise to hypoxic cores, necrotic regions, and uneven metabolic zonation, particularly problematic in hepatic tissues where oxygen-dependent enzymatic processes are spatially regulated (Kietzmann, 2017). The incorporation of multiple cell types and extracellular matrices improves physiological relevance but introduces variability and technical complexity. Standardization is also lacking, as protocols for organoid derivation, chip fabrication, and culture conditions vary across laboratories. Without harmonized workflows resembling good manufacturing practices, reproducibility will remain a barrier to adoption. Scalability is another pressing concern, particularly for pharmaceutical applications that require high-throughput screening (Zhang et al., 2024). Recent advances in multiplexed and automated liver chip systems are promising (Wang et al., 2024a), but further optimization is needed to replace animal-derived matrices with defined synthetic scaffolds and to overcome technical issues such as bubble formation and channel clogging that undermine long-term reliability.

Several emerging technological innovations are enhancing the functionality and analytical power of liver organoids-on-a-chip systems. The integration of embedded sensors allows continuous monitoring of oxygen levels (Izadifar et al., 2024), nutrient consumption (Saorin et al., 2023), and barrier integrity (Wang J. et al., 2024), providing real-time readouts of organoid physiology. For instance, optical and electrochemical microsensors incorporated within microfluidic channels allow precise tracking and regulation of dissolved oxygen, thereby improving the viability and metabolic performance of 3D liver tissues (Azimzadeh et al., 2021; Kaufman et al., 2025). Real-time measurement of nutrient uptake, pH, and barrier function creates a dynamic feedback loop that facilitates adaptive control of flow rates, medium composition, and cell density. Furthermore, modular sensor architectures built on printed circuit board technology now support multiplexed detection of oxygen, pH, flow, and secreted biomarkers in scalable and reproducible formats (Carvalho et al., 2024; Kanioura et al., 2025). Collectively, these sensor-integrated organ-on-chip platforms provide unprecedented temporal resolution of microenvironmental dynamics, which is an essential capability for modeling the complex metabolic and immune interactions that underlie hepatic physiology.

Beyond sensor-based feedback systems, functional hydrogels and synthetic extracellular matrices have emerged as powerful strategies to address diffusion limitations, reduce variability, and enhance long-term culture stability (Gan et al., 2023). In contrast to animal-derived matrices such as Matrigel, which exhibit batch-to-batch variability and undefined composition, synthetic hydrogels can be precisely engineered with tunable stiffness, porosity, biochemical ligand density, and even oxygen-releasing functionalities. For example, hydrogel bioinks optimized for 3D bioprinting enable fine control over matrix architecture and mechanical properties tailored for organ-on-a-chip applications (Unagolla and Jayasuriya, 2020). Incorporating microchannels or oxygen-generating particles within these scaffolds further improves mass transport, supporting more uniform nutrient and oxygen distribution and reducing the formation of necrotic cores in larger organoids (Hsiung et al., 2025). The chemical definability and reproducibility of synthetic hydrogels also facilitate scalability and parallelization of organoid-chip systems, a critical step toward higher-throughput and standardized pharmaceutical testing.

Artificial intelligence and machine learning are increasingly being leveraged to interpret the complex, high-dimensional datasets generated by organoid-on-a-chip platforms and to uncover subtle indicators of tissue maturation or toxicity (Bai et al., 2024). Deep learning models have demonstrated strong performance in pattern recognition tasks using image-based readouts, such as cell morphology, viability staining, and functional reporter dynamics, across liver-, lung-, and other organ-on-a-chip systems (Wang et al., 2024a; Qin et al., 2025). In parallel, computational modeling frameworks are being developed to inform experimental design by predicting optimal flow rates, nutrient formulations, and co-culture configurations (Norfleet et al., 2020). Integrating these predictive tools with real-time feedback from embedded sensors enables adaptive control of microenvironmental parameters, minimizing empirical trial-and-error and enhancing system responsiveness (Miedel et al., 2025a). Taken together, such data-driven approaches are poised to improve reproducibility, reduce human bias, and accelerate the standardization of organoid-on-a-chip platforms (Miedel et al., 2025b).

Parallel advances in vascularization strategies are pivotal for overcoming the persistent challenges of nutrient and oxygen transport, as well as waste clearance, in 3D tissues and organoids-on-a-chip (Takebe et al., 2013; Werschler et al., 2024). Recent efforts to engineer perfusable microvascular networks have accelerated rapidly, encompassing co-culture of endothelial and pericyte-like cells within microfluidic channels (Wang et al., 2023), 3D bioprinting of branching vascular architectures (Duan et al., 2025), and templated hydrogel microchannels that replicate native capillary geometry. Advances in biofabrication now allow precise recreation of liver lobule-like zonation and hierarchical vascular branching within chip platforms (Lekkala et al., 2025). Together, these strategies are elevating the physiological fidelity and reproducibility of organoid-on-a-chip models while facilitating their broader adoption for standardized and scalable applications.

The field is moving toward the development of integrated and adaptive liver organoids-on-a-chip platforms capable of replicating human physiology with high accuracy and throughput. These systems have the potential to become essential tools for drug development, precision medicine, toxicology, and regenerative therapies. Continued progress in reproducibility, scalability, and standardization will determine whether the technology matures into a reliable and widely adopted platform that bridges the gap between basic research and clinical translation.

Author contributions

JZ: Writing – original draft, Writing – review and editing. AD: Writing – original draft. KG: Writing – original draft. SP: Writing – original draft, Writing – review and editing.

Funding

The authors declare that financial support was received for the research and/or publication of this article. This work was supported by the Riley Children’s Foundation (grant no. 41001400) and the Purdue Engineering Initiative in Engineering Medicine and Purdue University Women’s Global Health Institute.

Acknowledgements

We thank the Purdue Office of Research for their support.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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