Liver organoids: Current advances and future applications for hepatology

Abstract

The creation of self-organizing liver organoids represents a significant, although modest, step toward addressing the ongoing organ shortage crisis in allogeneic liver transplantation. However, researchers have recognized that achieving a fully functional whole liver remains a distant goal, and the original ambition of organoid-based liver generation has been temporarily put on hold. Instead, liver organoids have revolutionized the field of hepatology, extending their influence into various domains of precision and molecular medicine. These 3D cultures, capable of replicating key features of human liver function and pathology, have opened new avenues for human-relevant disease modeling, CRISPR gene editing, and high-throughput drug screening that animal models cannot accomplish. Moreover, advancements in creating more complex systems have led to the development of multicellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting technologies. These innovations enable detailed modeling of liver microenvironments and complex tissue interactions. Progress in regenerative medicine and transplantation applications continues to evolve and strives to overcome the obstacles of biocompatibility and tumorigenecity. In this review, we examine the current state of liver organoid research by offering insights into where the field currently stands, and the pivotal developments that are shaping its future.

INTRODUCTION

Liver is one of the most important and complex organs in the human body, playing crucial roles in metabolism, detoxification, protein synthesis, digestion, nutrient storage, and bile production [1-4]. Because liver performs a wide range of functions, liver injuries can lead to complex complications. Liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and liver cancer, are associated with high mortality rates and considered major health issues worldwide [5-7]. Although the liver demonstrates a high capacity for regeneration in vivo, adult primary hepatocytes cannot proliferate and change their phenotypes after a short period in vitro [8,9]. As a result, liver transplantation remains the most effective treatment option for patients with end-stage liver disease and cancer. However, donor shortages and transplant complications limit the number of patients who can receive a transplant [10,11]. Therefore, the development of accurate in vitro models is essential to study disease mechanisms and potential treatments.

To date, many researchers have relied on two-dimensional (2D) cell culture systems and animal models to study liver diseases, drug toxicity, and develop treatments for liver diseases [12-14]. While these experimental models have been actively used in liver-related research to understand complex characteristics and architectures, they have significant limitations in representing the actual human liver. 2D culture methods using primary hepatocytes, hepatoma cell lines, and stem cell-derived hepatocyte-like cells offer advantages such as high-throughput screening and ease of genetic manipulation. However, they cannot replicate the complex three-dimensional (3D) structure, multicellular interactions, and physiological characteristics of the liver. Animal models can overcome some of the limitations of 2D cell culture systems by mimicking human-like physiological characteristics. However, they have limitations such as interspecies differences and difficulties in high-throughput screening, which is one of the advantages of 2D cell culture systems [15].

Given the limitations of 2D culture systems and animal models, 3D culture systems, such as organoids, spheroids, and 3D bioprinting, have recently emerged as promising technologies to address these challenges. Spheroids represent the simplest form of 3D culture, allowing the formation of spherical 3D structures through cell aggregation without the need for extracellular matrix (ECM) components such as Matrigel [16-20]. Their simplicity enables easy culturing, and they have been widely studied in cell types that aggregate well, such as cancer cells, embryoid bodies, and hepatocytes. However, most types of spheroids form solid spherical structures, which limits their ability to replicate the specific structural characteristics of various organs. Additionally, the delivery of nutrients and oxygen to cells at the center of the spheroid is challenging, often resulting in cell necrosis and other limitations [17,20,21]. In this manuscript, we aim to focus on organoids, one of the various 3D culture systems.

In 2008, when self-organized apico-basally polarized cortical tissue were reported from embryonic stem cells [22], and Lgr5-positive stem cells isolated from adult intestinal tissue were shown to form crypt-villus structure through self-organization [23], studies on the generation and expansion methods of organoids from various organ have been published. Organoids are 3D structures formed by the self-organization of adult stem cells (ASCs), pluripotent stem cells (PSCs) or progenitor cells, which recapitulate the target organ structures and functions [24]. Organoids have recently been actively utilized and studied across various fields because of their ability to incorporate the advantages of traditional experimental models, such as high-throughput screening, genetic manipulation, and replication of physiological features [25].

ASC-derived organoids are formed by isolating primary cells from adult tissues and culturing them under the key signaling conditions of the respective organ, allowing them to maintain the structural, physiological, and functional characteristics of the organ [23,26-28]. These ASC-organoid have shown genetic stability even after long-term expansion [26] and, as they can be generated from patient-derived cells, hold potential for applications in personalized medicine, such as toxicity assessments and gene therapy. However, the requirement for invasive procedures to obtain patient tissues is considered a drawback. On the other hand, PSC-derived organoids can be generated using relatively less invasive methods and offer the advantage of generating organoids for organs, such as the brain, where tissue samples are difficult to obtain [29-32]. Brain organoid research is predominantly conducted using PSC-derived organoids, and induced-pluripotent stem cells (iPSC)-derived organoids are particularly promising in regenerative medicine, as they enable the generation of patient-specific organoids. However, the complex differentiation processes required to generate PSC-derived organoids are considered a major source of high variability among organoids [33,34]. Additionally, differences in characteristics between PSC lines are another concern. Despite these challenges, both ASC-derived and PSC-derived organoids effectively recapitulate in vivo organs in vitro and are utilized across various applications. By leveraging the strengths and addressing the limitations of each cell source, effective research tailored to specific applications can be conducted.

In this study, we focus on recent advancements in liver organoid research and their applications, including disease modeling, drug screening, and regenerative medicine. Additionally, we discuss future directions of potential applications, such as personalized medicine, gene therapy, cellular microenvironments, cell–cell/organ–organ interactions, and bioengineering technology.

CURRENT ADVANCES

Disease modeling: recapitulating chronic and acute liver pathologies

Liver organoids derived from PSCs or adult stem cells have emerged as innovative tools in disease modeling because of their ability to recapitulate the structural and functional features of the human liver (Table 1) [26-28,35-38]. Organoid-based disease modeling facilitates the investigation of liver disease progression and evaluation of potential treatments, ultimately supporting the development of personalized medicine [39]. One of the key advantages of liver organoids in disease modeling is their physiological relevance. Unlike 2D culture systems and animal models, liver organoids recapitulate the cellular microenvironment and heterogeneity of the human liver in a context that closely mimics in vivo conditions. This capability is particularly valuable in chronic liver diseases, where tissue remodeling and multicellular interactions play crucial roles in disease onset and progression. Additionally, liver organoids can be genetically modified to induce disease-specific mutations, providing a robust platform for modeling genetic liver disorders and investigating the associated molecular pathways [36,40].

Table 1.

Organoid disease modeling associated with liver

Diseases	Cellular sources	Disease inducing methods	Ref.
ALD	Human chemically-derived hepatic progenitors-derived liver organoid	100 mM EtOH treatment during hepatic differentiation.	[22]
ALD	Human ESC-derived liver organoid	100 mM EtOH treatment for 7 days in culture medium	[24]
MASLD	Human fetal hepatocyte organoid	FFA treatment for 5–7 days in culture medium	[26]
		Generation of PNPLA3 I148M variant using prime editing
		Generation of APOB or MTTP mutation using CRISPR-KO system
MASLD	Human PSC-derived liver organoid	Fatty acid treatment for 6 or 9 days in culture medium with/without HCV infection	[27]
MASLD	Human PSC-derived liver organoid	FFA treatment for 3–7 days in culture medium	[28]
MASLD	Human PSC-derived liver organoid	FFA treatment for 4 days in culture medium	[29]
MASH	Human iPSC-derived liver organoid	Characterization of patient-derived organoids harboring steatosis-associated SNPs	[21]
		FFA treatment for 3–7 days in culture medium
MASH	Human ductal organoid	Generation and characterization of MASH patients-derived organoid	[30]
HBV	Human ductal organoid	HBV infection	[31]
HEV	Human fetal and adult cholangiocyte organoid	HEV infection	[32]
HCC/CC/CHC	Human primary liver cancer-derived organoid	Generation and characterization of HCC, CC, and CHC patients-derived organoid	[20]
HCC	Human primary liver cancer-derived organoid	Generation and characterization of HCC patients-derived organoid	[34]

ALD, alcoholic liver disease; EtOH, ethanol; ESC, embryonic stem cell; MASLD, metabolic dysfunction-associated steatotic liver disease; FFA, free fatty acid; PSC, pluripotent stem cell; MASH, metabolic dysfunction-associated steatohepatitis; HBV, hepatitis B virus; HEV, hepatitis E virus; HCC, hepatocellular carcinoma; CC, cholangiocarcinoma; CHC, combined HCC/CC tumor.

Models for chronic and acute liver diseases, including alcohol-related liver disease (ALD), MASLD, MASH, viral hepatitis, liver fibrosis, and liver cancer, have been developed. In ALD organoid models generated by Salas-Silva and colleagues [28], the ALD organoids replicated the inflammatory response and depolarization of mitochondrial membrane potential, providing insights into ALD progression in hepatocytes in response to alcohol. In MASLD [40-43] and its more severe form, MASH [36,44], steatotic liver organoids have been employed to replicate lipid accumulation and inflammatory responses, offering insights into disease progression from simple steatosis to advanced fibrosis and cirrhosis. Moreover, this platform allows for the study of liver fibrosis by enabling researchers to induce collagen deposition within organoids, thereby mimicking the fibrotic process and providing an opportunity to identify potential antifibrotic agents. Viral hepatitis, particularly infections caused by hepatitis B and C viruses, is another area where liver organoids have demonstrated their utility [45-47]. By reproducing virus entry, replication, and the host immune response, liver organoids have allowed for the exploration of interactions between the viruses and hepatocytes. Furthermore, these models offer valuable platforms for investigating antiviral treatments and developing vaccines [45-47]. Organoids derived from liver tumor samples, including hepatocellular carcinoma (HCC), cholangiocarcinoma (CC), and cholangio-/HCC mixture types (CHC) or genetically engineered with oncogenic mutations, have been used to regenerate the tumor microenvironment, enabling the study of cancer progression and development of anti-cancer therapies, such as immunotherapies and targeted drugs [27,48].

Despite their many advantages, current liver organoid models have several limitations. Organoids are effective in recapitulating various disease characteristics. However, their hepatocyte-specific functions are not as advanced as those of primary hepatocytes. They exhibit functionalities akin to those or progenitor or fetal-like cells, making it challenging to consider them fully representative of mature hepatocytes. Therefore, optimizing the culture conditions and compositions to drive the differentiation of liver organoids toward more mature hepatocyte-like cells is necessary. Another primary challenge is the lack of vascularization and immune system, although several trials have been conducted to address this [41]. The above limitation restricts the study of cell-to-cell interactions between hepatocytes and the immune system, a key aspect of liver diseases. Next-generation organoid disease modeling focuses on overcoming these challenges by incorporating endothelial cells, immune cells, and perfusion systems to more accurately mimic the human liver microenvironment.

CRISPR-mediated genome editing for disease modeling

Genetics plays a role in all diseases to varying degrees, with genetic variations and environmental factors shaping disease processes. This understanding provides the foundation for creating effective therapies and preventive measures, and the genetic manipulation of isogenic patient-derived organoids offers insights that animal models cannot achieve. To fully leverage these systems, a range of genome editing tools, including RNA interference (RNAi), transposons, and CRISPR/Cas9, have been developed [49]. RNAi is confined to knockdowns [50], while transposons can enable large-scale manipulations of the genome, although their complexity limits scalability [49]. As such, CRISPR/Cas9 is the most widely used tool in engineering organoids due to its high precision, versatility, and cost-effectiveness [51].

Since CRISPR tools have mainly been developed for 2D cell lines, technical considerations are necessary for their use in 3D cultures. The delivery of CRISPR tools for genetic modulation in organoids occurs at different time points, depending on the cell source (Fig. 1). For PSC-derived organoids, gene editing is performed at the 2D PSC stage to take advantage of their immature and genetically unstable nature [52]. The modified cells are then transformed into 3D structures [49]. Gene editing of ASC-derived liver organoids is more complex because it requires the establishment of unedited organoids that can tolerate transfection and support clonal outgrowth over multiple passages [53]. Once established, organoid lines can be dissociated into single cells for genome engineering [54]. ASC-derived liver organoids reside in a specific stem cell niche. As they proliferate, transit-amplifying cells move out of this niche and differentiate into hepatic lineages [55,56]. Given the difficulty in transfecting differentiated cells [57], gene editing relies on cloning authentic stem cells as the primary target for genetic modifications. Although less common, the introduction of CRISPR tools into organoids without dissociation into single cells has been achieved by embedding human liver ductal organoids in basement membrane extract (BME) hydrogel droplets [58,59]. This method maintains cell-to-cell contact within the organoid and allows interaction with BME, thereby improving the recovery of cells after transfection. After gene editing, all organoid types follow a similar process, in which gene-edited clonal selection is performed based on transfection markers or functional outcomes at the single-cell stage or after organoid formation [51].

Figure 1.

Gene engineering process in organoids and its applications. To create genetically engineered organoids from PSCs, gene editing is performed in a 2D culture prior to organoid differentiation. By contrast, the process for generating gene-edited organoids from ASCs begins with the creation of wild-type organoids, followed by the dissociation and editing of viable ASCs before organoid formation. Both PSC- and ASC-derived organoids undergo a final selection process to isolate clones with the desired morphological and functional properties. Gene-edited organoids can be used for high-throughput drug screening, while integrated analyses of transcriptomics, proteomics, metabolomics, and in vivo studies can provide valuable insights into disease mechanisms and therapeutic strategies. PSCs, pluripotent stem cells; ASCs, adult stem cells.

Applications of CRISPR/Cas9 in liver organoids

PSC-derived liver organoids are created using an intricate fate-specialization protocol to replicate the embryonic developmental trajectory [29,60], resulting in organoids that resemble a fetal-like state [61]. This makes the use of CRISPR in organoids favorable for studying genetic disorders that manifest during early human development. A previous study investigated the role of the JAG1 genes (C829X and ALGS2) in Alagille syndrome by combining CRISPR/Cas9 with the piggyBac transposon system [62]. The absence of a disease phenotype in JAG1 knockout iPSCs-derived liver organoids indicates that a dominant-negative effect, rather than haploinsufficiency alone, may be involved in pathogenesis.

By contrast, ASC-derived liver organoids simulate the adult homeostatic state, making genome editing more suitable for cancer research. Artegiani et al. [58] reported that TP53, SMAD4, PTEN, NF1, and BAP1 knockout liver ductal organoids exhibited CC features both in vitro and after orthotopic transplantation in vivo. However, the absence of the BAP1 knockout alone failed to recapitulate the disease, indicating that its loss-of-function drives malignant transformation. The same group developed a variant of the CRISPR-Cas9 strategy utilizing non-homologous end-mediated knock-in (CRISPR-HOT) [59], achieving a 10-fold increase in efficiency for liver ductal organoids and an eight-fold increase in hepatocyte organoids. The insertion of a reporter gene (TUBB) into TP53 knockout hepatocyte organoids via CRISPR-HOT revealed a connection between p53 and mitotic spindle abnormalities during hepatocarcinogenesis.

Future applications of CRISPR technology in liver organoids could offer a powerful tool for understanding the mutation-driven pathogenesis of liver diseases with complex genetic landscapes. For example, generating a library of liver organoids with combinatorial knockouts of key cancer genes could reveal how certain mutations synergistically promote malignant transformation through transcriptomic and phenotypic changes (Fig. 2). Moreover, leveraging this CRISPR-modified organoid library for drug screening can identify mutation-specific responses to chemotherapy regimens, enabling personalized treatment strategies that improve outcomes in genetically heterogeneous patient populations. By combining organoids with gene editing, we stand at the threshold of a new era in medicine, where precision treatments tailored to individual genetic and cellular profiles may become the standard of care.

Figure 2.

Comparison of wild type hCdHO, patient-derived cholangiocarcinoma organoids, and CRISPR engineered hCdHO. (A) Wild type hCdHO. (B) patient derived cholangiocarcinoma organoids. (C) CRISPR engineered hCdHO. To model cholangiocarcinoma, we knocked out two key tumor suppressor genes BAP1 and TP53 in wild-type hCdHO (A) using CRISPR technology. The resulting double knockout organoids (C) displayed malignant morphological features characteristic of patient-derived cholangiocarcinoma organoids (B). Further analysis and comparison of various combinatorial mutations may reveal the roles of specific genetic changes in tumoriogenesis of cholangiocarcinoma. hCdHO, human chemically-derived hepatic progenitors organoids.

Drug screening and toxicology: advancing precision medicine

Organoids can be cultured on a large scale because they can form 3D structures that mimic organ-specific histological structures from a single cell and are thought to be a suitable model for drug screening based on their ability to reproduce the physiological properties of a specific organ. Organoid-generated disease models can be used to identify disease-specific drugs and novel targeted therapeutic pathways [63]. Recent studies have included treating liver organoids with free fatty acids to generate MASLD organoids to screen for drugs that inhibit lipid accumulation, and generating liver organoids with single nucleotide polymorphisms seen in MASH patients for high-throughput screening to identify potential drugs for MASH (Fig. 3A) [36,40]. In addition to chronic liver disease, studies have identified specific drugs and target genes for each type of liver tumor, including HCC, CC, and CHC [27].

Figure 3.

Drug screening and toxicity testing. (A) Liver organoids can be generated from healthy human liver tissues and used for disease modeling by treating them with various hepatotoxic agents or applying gene editing technologies. These induced liver disease organoids can be utilized in drug screening to identify disease-specific therapeutic candidates, facilitating novel drug development. In addition to induced liver disease models, liver cancer organoids can be derived from various liver cancer tissues to support the development of cancer-specific drugs. (B) In addition to generating liver disease organoids from multiple patients to develop universal drugs, single patient- derived liver organoids can also be generated and subjected to drug screening. This enables the development of patient-specific therapeutic options, paving the way for personalized medicine. (C) Liver organoids can also be employed to evaluate the hepatotoxicity of drug candidates. Unlike primary hepatocytes and animal models, which are currently used in hepatotoxicity assessments, liver organoids have shown higher sensitivity in detecting hepatotoxic effects of drugs that were released to the market but later withdrawn due to liver toxicity observed in humans. These findings suggest that liver organoids, in conjunction with primary hepatocytes, can be effectively utilized for drug toxicity evaluations. FFA, free fatty acid; SNP, single nucleotide polymorphism; MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic dysfunction-associated steatohepatitis; PHHs, primary human hepatocytes; hCdHs, human chemically-derived hepatic progenitors; hCdHO, hCdH-derived organoids.

Drug screening with organoids is more progressive than screening with other experimental models because patient-derived organoids enable personalized medicine. By generating patient-derived organoids for drug screening, we can identify not only the target drug for the disease but also the sensitivity of the patient-specific drug, allowing the application of more appropriate drugs to the patient [27,28]. The advantages of these organoid systems for drug screening including their compatibility with high-throughput screening methods are expected to significantly reduce the time and cost required for drug development (Fig. 3B).

Furthermore, organoids, particularly liver organoids, show great potential for use in drug toxicity assessments (Fig. 3B) [64,65]. One of the key functions of the liver is detoxification; while some drugs bypass significant hepatic metabolism initially depending on their route of administration, most drugs eventually pass through the liver via systemic circulation, where they may undergo metabolism or remain unchanged [66-69]. Therefore, the essential step in the drug development process is to verify the concentration of the drug and ensure that it does not exhibit toxicity to the liver [70]. Currently, primary hepatocytes and animal models are used to evaluate the toxicity of newly developed drugs [71]. In a study by Salas-Silva et al. [28], primary human hepatocytes and liver organoids were treated with drugs known to be hepatotoxic or those withdrawn from the market to assess cytotoxicity and sensitivity. Liver organoids showed higher drug sensitivity than primary human hepatocytes to several drugs, especially those withdrawn from the market (Fig. 3C). These findings suggest that liver organoids may serve as an additional cellular model for toxicity assessments, particularly in cases where hepatotoxicity cannot be detected using primary hepatocytes. Additionally, through legislation by the US and FDA that recognizes evaluation systems that can replace animal testing [72], the use of organoids in drug development and toxicity assessment has gained increasing interest from researchers and pharmaceutical companies, with evaluation systems rapidly advancing. However, there are still some drugs for which primary hepatocytes exhibit higher sensitivity than liver organoids, and organoids have certain limitations, such as the formation of 3D structures of varying sizes [28]. Therefore, it is necessary to optimize the differentiation conditions that can further mature liver organoids to more closely resemble primary hepatocytes, as well as methods for producing and distributing organoids with uniform size and cell number.

EMERGING APPLICATIONS

Functional complexity: assembloid for enhanced physiological modeling

Efforts are ongoing to refine and advance organoid culture systems to accurately and closely reproduce the structure and function of specific organs in the human body. Some approaches focus on adjusting the organoid culture conditions or recapitulating the microenvironment of the target organ. Most organoids developed for various organs are generated using only one type of parenchymal cell from each organ [9,26,28,36]. However, most organs are not composed of a single cell type; rather, parenchymal and nonparenchymal cells work in harmony to form an organ-specific cellular microenvironment and actively interact with each other to perform the specialized functions of each organ [73]. Therefore, to generate structures and microenvironments that more closely resemble actual organs, future organoid culture systems should focus on developing assembloids that incorporate parenchymal and nonparenchymal cell types (Fig. 4A).

Figure 4.

Increased complexity with multicellular and organ scale research. (A) The liver consists of a complex microenvironment that includes hepatocytes, bile duct cells, liver mesenchyme and hematopoietic cells such as hepatic stellate cells, portal fibroblasts, and others, all of which contribute to liver function. However, current organoid systems are derived from a single cell type, limiting their application to studies at the intracellular scale. As a result, many researchers are now focused on developing culture conditions that enable the co-culture of multiple cell types based on the concept of cellular assemblies. Through these assembloid systems, future studies and applications to study the cell interactions at tissue scale level are expected to become feasible. (B) The liver is interconnected with various organs, including the intestine and blood vessels, facilitating inter-organ metabolic interactions. To replicate this intra-organ environment, significant efforts have been made in the development of organoid-on-a-chip technologies. These advancements have enabled longterm studies on inter-organ interactions, which were previously difficult to investigate.

Several studies have reported the development and analysis of assemblies composed of parenchymal and nonparenchymal cells [31,32,74-76]. The approaches used in developed assembloid systems can be broadly divided into two types. The first method involves the use of PSCs to generate organoids with specific characteristics according to the developmental stage. These organoids were then aggregated and combined. This method has been used in brain-related studies, and assembloids capable of replicating the interactions between cortical and motor neurons have been reported [30,32]. Another approach involves co-culturing organoids with different cell types isolated from tissues. The main challenge of this method is that each cell type in the assemblage has different optimal culture conditions, which requires the identification of conditions that allow the formation of a self-organized tissue microenvironment. This method is used in cancer research and enables the reconstruction of the tumor microenvironment with cancer cells surrounded by cancer-associated fibroblasts (CAFs). This approach allows the analysis of the interactions between CAFs and cancer cells, facilitating the identification of novel anti-cancer drug targets [74,77]. In the case of steatohepatitis, non-parenchymal cells such as hepatic stellate cells (HSCs) and liver-specific macrophages, Kupffer cells, play critical roles in the development and progression of the disease. Therefore, there are limitations to using organoids composed solely of hepatocyte-lineage cells for research on steatohepatitis. To address this, researchers have reported the development of steatohepatitis assembloid models by assembling PSC-derived hepatocyte-, HSC-, and Kupffer-like cells [41,42]. These assembloid systems enable research on multicellular interactions, which are difficult to investigate using organoids, and allow for more precise studies of disease mechanisms, such as fibrosis. Furthermore, the scope of applied research is rapidly expanding, with assemblies incorporating the immune or vascular systems [41]. Liver sinusoidal endothelial cells (LSECs) form the unique porous vascular structure, hepatic sinusoids, in collaboration with Kupffer cells [78-80]. Unlike other blood vessels, hepatic sinusoids lack a basement membrane and are composed of fenestrated endothelial cells. These fenestrations allow the transfer of various substances into the space of Disse, the area between hepatocytes and LSECs. By assembling LSECs and Kupffer cells, it is anticipated that the spatial architecture of the liver can be more effectively recapitulated, enabling the study of the diverse roles and mechanisms of hepatic sinusoids. However, since the preferred culture conditions for each cell type involved in assembloids differ, it is essential to optimize the culture conditions for assembloid formation. Beyond simply co-culturing different cell types, it is crucial to establish self-organization conditions that can replicate the positional features of each cell type as observed in vivo.

Organoid-on-a-chip for dynamic complexity

Although assembloids capture the intricate 3D cellular architecture of tissues by increasing their internal complexity, parallel efforts have been made to replicate the dynamic external environment of living organs. Organoid-on-a-chip systems represent the next evolution, blending the strengths of organ-on-a-chip systems with those of organoid models. Despite progressing separately, these two fields offer a synergistic approach when integrated into a cutting-edge in vitro microphysiological system.

Organs-on-chip are systems that use microfabricated devices with engineered or natural tissues and dynamic fluid flow to recreate the functional units and physiological conditions of human organs in vitro [81-83]. In liver-on-a-chip models, controlled fluid dynamics can recreate metabolic zonation by mimicking in vivo oxygen and hormone gradients. These systems have been used to investigate drug metabolism [84,85], drug hepatotoxicity [86,87], drug efficacy [88], and infection.89 Furthermore, organs-on-chips can address the systemic role of the liver in inter-organ communication by fluidically linking multiple organ chips, creating “Body-onchips” (Fig. 4B) [90]. A lung/liver-on-a-chip system for aerosol toxicity testing illustrated the key functions of the liver in metabolizing drugs and molecules that affect other body regions [91]. Although organs-on-chip offer a relatively complete microenvironment, they lack the complex 3D cellular interactions necessary for accurately modeling tissue behaviors over extended periods. For instance, the absence of bile canaliculi and bile acid excretion pathways in liver-on-chip systems limits their effectiveness in drug metabolism research and restricts long-term culture [92].

However, research on organoids faces significant technical challenges in contrast to the precision offered by organon-chip systems. Organoids are typically grown in static environments that lack the dynamic features necessary to mimic vascular perfusion, interstitial flow, tissue-tissue interactions, and multiorgan interactions [81]. These limitations prevent accurate modeling of drug absorption, distribution, metabolism, and excretion.

Integration 1: Biophysical microenvironment and biosensing

Researchers have addressed these problems by merging microengineering and 3D culture techniques. One approach for solving the issue of limited microenvironmental control in organoids is the liver organoid-on-chip system introduced by Bavli et al. [93]. The platform perfuses the medium over the wells, providing the cells with physiological shear stress and a steady oxygen gradient that induces metabolic zonation. The absence of recirculation minimizes non-specific absorption, allowing hepatic organoids to grow in a stable microenvironment. With this metabolic steadiness, oxygen microprobes and electrochemical sensors can be embedded to automatically track oxygen, glucose, lactate, and glutamine in the outflow, enabling comprehensive analysis of the metabolic function [94,95].

The integration of electrochemical biosensors within chips represents a significant step forward in achieving long-term monitoring of organoids-on-chips. However, conventional affinity-based biosensors face limitations when the target biomarkers saturate the sensor. To address this issue, Aleman et al. [96] introduced a regeneratable electrochemical affinity-based biosensor chip. The system features an automatic regeneration process that clears all molecules from the biosensor, enabling automatic, continuous, and non-invasive measurement of soluble biomarkers in real time without disrupting the microenvironment. Despite these advancements, the technology still faces challenges, such as lengthy regeneration times and limited chip lifespans.

Integration 2: Multi-cellular interactions

In the liver, nonparenchymal cells such as stellate cells, Kupffer cells, and sinusoidal endothelial cells communicate with hepatocytes to aid liver growth [97] and regulate pathophysiological reactions [98]. Previous studies using 2D culture methods have demonstrated that co-culturing hepatocytes with liver sinusoidal endothelial cells can maintain albumin and hepatic urea secretion for up to 4 weeks [99,100]. Building on these initial studies, recent research has explored the creation of liver organoids with integrated vascular beds using a liver organoid-on-a-chip strategy. Jin et al. [101] utilized a decellularized liver extracellular matrix as a scaffold to create a vascularized liver organoid, which was cultured in a microfluidic device. After 3 weeks of co-culture, the 3D hepatic tissue showed enhanced hepatocyte function, including albumin secretion, urea synthesis, and CYP3A4 activity. This finding was absent in static cultures, highlighting the phenotypic evidence that complements the transcriptomic data initially provided by the 2D microfluidic systems [102].

Integration 3: Multiorgan interactions

Organs communicate via blood and lymph signals to maintain homeostasis despite being physically separated in vivo. A breakthrough innovation has recently allowed for the modeling of multiple organoids within one device [103], paving the way for multiorgan platforms to study drug metabolism [104,105], toxicity [91], pharmacokinetics [106,107], and systemic diseases [108,109].

Drug metabolism in the liver is a central factor that influences the therapeutic efficacy and safety of potential treatments. Rajan et al. [104] introduced a body-on-a-chip system featuring six organoids: the liver, heart, lung, endothelium, brain, and testes. When ifosfamide was administered, no toxicity was observed without the liver; however, in the presence of the liver, the drug was metabolized into toxic by-products, which affected the other organoids. Although the bioavailability analysis is limited, this study illustrates the importance of an integrated system for studying drug toxicity, where the function of a certain construct (i.e., liver organoid) can influence the response and toxicity in other organoids.

Cancer metastasis occurs when circulating tumor cells colonize specific niches in distant organs. A metastasis-on-a-chip model was developed using colorectal cancer organoids in a microfluidic chamber connected to liver, lung, and endothelial organoids [108]. After 2 weeks of co-culture, RFP-labeled tumor cells predominantly homed to the liver and lung organoids, mirroring the metastatic patterns of colorectal cancer seen in the clinical setting. These platforms offer the potential to model diseases in a patient- or population-specific manner, aiding the identification of personalized therapies that reduce the risk of metastasis and identifying tissues at higher risk for metastatic lesions.

Obstacles in organoid-on-a-chip

Organoid-on-a-chip technology holds great potential but is still limited by several challenges. These models are often designed in predetermined ways, which restricts their ability to mimic dynamic changes in human physiology. A key example is the absence of hierarchical vascular networks that can respond to changing metabolic needs, necessitating strategies such as 3D bioprinting, microfluidic lines, and larger vasoactive vessels [110,111]. Additionally, reducing the variability between organoid chips is vital for achieving consistency, a critical factor for industry and regulatory acceptance as a viable alternative to animal models [112]. Additionally, organoids that recapitulate different organs are generated and cultured in organ-specific environments and conditions. To implement such organ-specific organoid culture methods within chip systems, it is essential to optimize co-culture conditions for organoids. Furthermore, the high costs and low throughputs of these systems hinder their scalability. Overcoming these obstacles requires advances in automation and high-throughput systems.

Integrating 3D Bioprinting with organoids

3D bioprinting is a promising technology that combines cells and bioinks to generate artificial tissues or organs of any desired shape [113,114]. With 3D structures input into a computer, 3D bioprinting can effectively mimic complex tissue structures by placing cells wherever desired, and this can be combined with different cell types to generate multicellular organ models. This field of 3D bioprinting has also significantly advanced hepatology research [115-120]. The advantages of this bioprinting technology can be applied to organoid systems with self-organizing capacity to develop more advanced organoid technologies.

Precise positioning of the cells that constitute the structure and microenvironment of human tissue, and subsequently the generation of organoids, enables the creation of a more refined tissue structure than what current technologies can achieve (Fig. 5) [121-123]. Moreover, by integrating organoids with artificially engineered vascular networks fabricated using 3D bioprinting, this approach is expected to contribute to the development of vascularized organoid-based cell therapies suitable for transplantation [124,125]. Currently, most organoid systems are limited to generating structures on a scale of a few hundred micrometers due to challenges such as nutrient and oxygen delivery; however, it is anticipated that integrating 3D bioprinting could enable the generation of larger organoids [123]. To address the need for healing large-area skin defects, studies have reported the development of skin organoids tailored to the size and shape of the wound using a combination of skin organoid technology and 3D bioprinting [126]. These customized organoids have been successfully transplanted into mouse models, demonstrating their potential in regenerative medicine. Additionally, with the capability of mass production in 3D bioprinting, it is expected that large-scale batches of uniformly sized organoids can be applied for large-scale screening studies, such as drug development and toxicity assessment [121]. We anticipate future research at the next level through the convergence of these two promising technologies.

Figure 5.

Three-dimensional (3D) bioprinting with liver organoids. 3D bioprinting is an innovative technology that allows for the precise placement of cells mixed with hydrogels to form desired shapes and patterns. The integration of 3D bioprinting technology with organoid culture systems enables a range of advanced research applications. For example, it is possible to print organoids together with various nonparenchymal cells to create 3D constructs that mimic tissue microstructures. Additionally, bioprinting with vascular networks enables the production of vascularized organoids. It is anticipated that liver organoids traditionally cultured within Matrigel domes can be scaled up using 3D bioprinting techniques to generate larger structures. Furthermore, the labor-intensive process of manually seeding organoids into plate wells can be streamlined with bioprinting, enabling large-scale batch seeding. This approach is expected to facilitate highthroughput screening and large-scale analyses.

Organoid in regenerative medicine

Cell transplantation has made strides in multiple fields [127-129]; however, it still encounters limitations such as a lack of quality donor cells, inefficient engraftment, immune rejection, and diminishing clinical benefits over time. Organoid transplantation is based on cell transplantation and has a greater therapeutic potential. Unlike cell transplantation, in which cells are dispersed and must independently navigate their survival post-transplantation, organoids are preconditioned before implantation to self-organize into robust structures that mimic the histological and functional characteristics of real tissues. Transplantation of these self-organized tissues can offer enhanced functionality, engraftment, maintenance, survival, and purity [130]. Takahashi et al. [131] provided a proof-of-concept that vascularized islet organoid transplantation outperformed islet cell transplantation in diabetic mice by improving glucose levels, insulin secretion, and survival through enhanced cellular interactions and angiogenesis.

In this scenario, liver organoids have been utilized in attempts to partially replace organs and serve as functional units for disease treatment. In 2013, Huch et al. [132] demonstrated that transplanting mouse liver organoids derived from ASCs into tyrosinemia type I mouse models improved survival, although the enzymatic defect remained uncorrected and engraftment was not ideal. In the same year, Takebe et al. [29] reported that vascularized human liver organoids derived from PSCs could develop into vasculature within 48 h post-transplantation and successfully rescue a mouse model of drug-induced liver injury. These findings reveal the promising applications of liver organoids in regenerative medicine, marking the beginning of a new era (Fig. 6).

Figure 6.

Regenerating the liver with liver organoids. The regenerative use of liver organoids primarily focuses on direct transplantation for liver repair. Other strategies, which require further research, include gene-edited organoid transplants, therapeutic extracellular vesicles from organoids, and engraftment onto marginal donor livers with machine perfusion to confirm effective engraftment. Biobanking will enable the broader implementation of organoids in regenerative medicine.

Bringing organoid transplantation to bedside

Following a range of preclinical studies on liver organoid transplantation, the idea of transplanting organoids into patients may soon become a reality, with centers accelerating toward clinical trials [133]. In deceased-donor transplantation, organoids can be transplanted into the liver graft during the preservation period to enhance graft function and minimize recipient complications. This approach would enable the utilization of marginal organs that might otherwise be discarded, thus, addressing the organ shortage crisis as originally intended. Sampaziotis et al. [134] introduced the concept of combining a normothermic machine perfusion strategy with liver ductal organoid transplantation to assess organoid engraftment in liver grafts prior to transplantation. Genetically engineered organoids may be a viable option for individuals with genetic liver disease. Based on our experience in transplanting gene-corrected human chemically derived hepatic progenitors (hCdHs) [128] and hCdHs organoids [28], we anticipate that gene-corrected organoids could offer improved engraftment and more favorable outcomes for genetic defects. Other applications could be in patients undergoing massive surgeries such as extended hepatectomies or complex biliary reconstructions, where transplanted organoids can reduce complications and facilitate recovery.

Before introducing these exciting approaches in clinical practice, several safety issues must be considered. First, the immunogenicity of cell-based therapies must be addressed to ensure both the effectiveness and recipient safety in organoid transplantation. In vitro and in vivo transplantation assays corresponding to the cell source, differentiation protocol, maturation state, and transplantation site are needed to evaluate relevant cytokines and immune regulatory ligands [135]. Applying a personalized strategy that integrates immunosuppressive regimens, HLA matching, and genetically engineered organoids (i.e., HLA-depleted organoids [136]) tailored to patient conditions and transplantation assays is preferable. In addition, replacing non-biocompatible components with biocompatible alternatives in organoid cultures is necessary to minimize unwanted host immune responses. Most established methods for organoid generation and expansion currently rely on Matrigel, which is widely utilized in research. However, as Matrigel is derived from mouse tumor cells, it contains animal-derived proteins, posing potential risks of xenogeneic immune reactions and disease transmission when applied to patients [137]. Therefore, to advance organoid-based regenerative medicine toward clinical application, the development of biocompatible alternative ECM for organoid generation and expansion is essential. A detailed review of biocompatible scaffolds is provided elsewhere [138].

The next concern is the potential tumorigenicity of stem cells, which is a relevant issue to all applications of stem cell-based research, including organoids. Currently, PSCs are the most readily available and extensively studied cell type in the field of regenerative medicine and organoid. However, they encounter a significant limitation in translation owing to their tumorigenicity, which arises from their potential to differentiate into any type of cell. Indeed, many genes used to generate iPSCs are either known or potential oncogenes [139]. In a case report on iPSC-based cell therapy by Mandai et al., even the slightest risk of iPSC-derived retinal cells inducing cancer driver gene mutations in a patient led to the suspension of the treatment [129]. One possible solution could be to increase the purity of organoids by sorting residual undifferentiated cells according to relevant cell surface markers [140] and applying chemicals to remove them [141,142]. Alternatively, ASCs of predetermined lineages or primary hepatocytes may serve as safer cell sources. Several studies [26,143], including those by our group [28], have indicated that organoids derived from these cells maintain genetic stability over long-term culture.

FUTURE OUTLOOK

Organoid transplantation offers undeniable benefits to patients, and establishing a library of therapeutic organoid prototypes is essential to overcome concerns regarding immunogenicity and tumorigenicity. Although ASCs or primary cells may be safer alternatives, their availability is restricted by the need for invasive procedures to acquire them from patients or consenting donors. Moreover, the extended time required to culture organoids conflicts with the urgency of medical treatment, where timely availability is crucial. Biobanking organoids through advanced preservation strategies [144] could offer a solution that allows long-term storage and off-the-shelf readiness for patients in need. Beyond transplantation, organoids hold promise in regenerative medicine through the production of extracellular vesicles (EVs), which offer cell-free therapeutic applications with a higher yield and efficacy than those from 2D stem cell cultures [145]. Although organoid-derived EVs have been used to treat neurodegenerative diseases [146] and skin wounds [147], research on liver organoid-derived EVs remains unexplored and warrants attention.

CONCLUSION

A decade has passed since liver organoids were first introduced, and their impact on in vitro research has been profound with clinical trials in progress (Table 2). By replicating human liver function, these models enable studies of liver disease and drug testing without animal experiments. Innovations such as multi-cellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting are driving this field toward new levels of complexity. Researchers are now exploring the potential of organoid transplants to repopulate damaged liver tissues.

Table 2.

Ongoing clinical trials on liver organoids

Study ID	Start date	Location	Status	Patient conditions	Application of PDOs
NCT02436564*	2015	UK	Unknown	Cholangiocarcinoma, hepatocellular carcinoma, pancreatic neoplasm	Disease modeling, drug testing
NCT03307538†	2017	Netherlands	Completed	Perihilar cholangiocarcinoma	Disease modeling, radiotherapy testing
NCT04561453‡	2020	USA	Active, not recruiting	Biliary tract cancer	Disease modeling, drug testing
NCT04753996§	2021	USA	Recruiting	Cholangiocarcinoma, Primary sclerosing cholangitis	Disease modeling
NCT04755907∥	2021	China	Unknown	Colorectal cancer, liver metastases	Disease modeling, drug testing
NCT05183425**	2022	China	Recruiting	Colorectal cancer, liver metastases	Disease modeling, drug testing
NCT05634694††	2022	China	Recruiting	Cholangiocarcinoma	Disease modeling, drug testing
NCT06355700‡‡	2023	Italy	Recruiting	Hepatocellular carcinoma	Disease modeling, drug testing
NCT05913141§§	2023	China	Recruiting	Liver cancer	Disease modeling, drug testing
NCT05932836∥∥	2023	China	Recruiting	Hepatocellular carcinoma	Organoid-on-chips, drug testing
NCT05644743***	2023	n.a.	Not yet recruiting	Intrahepatic cholangiocarcinoma	Disease modeling, drug testing
NCT05720676†††	2023	Belgium	Not yet recruiting	Breast cancer, liver metastases	Disease modeling
NCT06077591‡‡‡	2024	Hong Kong	Not yet recruiting	Colorectal cancer, hepatocellular carcinoma	Disease modeling, drug testing

A total of 21 clinical trials involving liver organoids have been identified, with all utilizing patient-derived organoids (PDOs). The current use of liver organoids in clinical settings focus on non-invasive approaches such as disease modeling and drug testing, with their regenerative potential remaining unexplored.

References:

^*A study designed to develop in vitro models of liver, biliary and pancreatic cancer for the investigation of tumour biology and potential therapies. In: The Gurdon I, Ann McLaren Laboratory of Regenerative M, eds., 2015.

^†A pilot study to determine the feasibility of stereotactic body radiation therapy following chemotherapy for unresectable perihilar cholangiocarcinoma. “The STRONG Trial”. In, 2017.

^‡A prospective feasibility study of multi-platform profiling using biospecimens from patients with resected biliary tract cancer. In: Natera I, Sengine Precision Medicine I, eds., 2020.

^§Characterization of biliary cell-derived organoids from bile of PSC and non-PSC patients. In: National Institute of D, Digestive, Kidney D, eds., 2021.

^∥Validation of the three-dimensional bioprinted tumor models as a predictive method of the response to chemotherapy for colorectal cancer with or without liver metastases. In: Peking University Cancer H, Institute, Cancer I, Hospital CAoMS, China-Japan Friendship H, eds., 2021.

^**The exploratory study of patient-derived organoids for the prediction and evaluation of clinical efficiency effect of colorectal cancer liver metastasis. In, 2021.

^††Clinical study on the drug sensitivity consistency of patient-derived organoid model and actual therapeutic efficacies in cholangiocarcinoma patients receiving adjuvant chemotherapy after radical resection. In, 2022.

^‡‡Hepatocellular carcinoma organoids to explore tumor microenvironment and test treatment efficacy - hepatocellular carcinoma liver organoids (HELIO). In, 2023.

^§§Patient-derived organoids, patient-derived organoids-tumor-infiltrating lymphocyte coculture system, and patient-derived organotypic tissue spheroids for drug screen. In, 2023.

^∥∥An pancancer study on organoid-on-chips technological system based on biopsy samples and its efficacy in predicting the response to mFOLFOX6 infusion in hepatocellular carcinoma. In, 2023.

^***Clinical transformation of organoid model to predict the efficacy of GC in the treatment of intrahepatic cholangiocarcinoma. In, 2022.

^†††Clinical, histopathological, molecular and experimental characterization of liver metastases from patients with breast cancer. In: Jules Bordet I, Erasme University H, Sint-Augustinus GZAZC, eds., 2023.

^‡‡‡Prospective clinical validation of next generation sequencing (NGS) and patient-derived tumor organoids (PDO) guided therapy in patients with advanced/ inoperable solid tumors. In, 2023.

These advancements should not be interpreted as isolated achievements. Instead, they represent a growing synergy of applications that must continue to evolve and expand collectively. With novel tools such as assembloids and organoid-on-chip systems, researchers can refine disease modeling and drug testing, and the potential for grafting assembloids is also emerging. Quality control and mass production of organoids through 3D bioprinting could further support organoid transplants while allowing pharmaceutics to securely integrate organoids into their drug testing pipelines.

On the other hand, it is essential to understand the current hurdles in organoid research. Despite efforts to establish reproducible organoid models, detailed protocols such as tissue processing and medium composition often differ between laboratories. It is also crucial to comprehend organoid-to-organoid variability and utilize organoids appropriately in research. This variability includes batch issues arising from environmental factors such as Matrigel and bioreactor, as well as heterogeneity observed within the organoids themselves [33,34,148]. To recapitulate human in vivo physiological activities and broaden the application field of organoids, efforts are needed to enhance reproducibility and reduce variability in organoid generation and expansion. Matrigel, currently widely used, is derived from mouse tumor cells and is known for high batch-to-batch variability. However, the development of alternative ECMs can reduce this variability. Heterogeneity observed in organoids is attributed to various factors, including the probabilistic nature of cell fate decisions during the self-organization of cells with stem cell properties, the complex differentiation stages required for PSC-derived organoids, changes in cell characteristics during long-term expansion, and inter-line variability among PSC lines. To address these challenges, efforts such as optimizing culture medium compositions and simplifying organoid generation processes are essential. These improvements are expected to enhance reproducibility and increase the usability of organoids across various application fields. Questions remain regarding the appropriate level of complexity, as excessively large and intricate organoids may lack practicality and cost-effectiveness. For instance, immune-reactive cancer organoids may not be necessary for screening cytotoxic chemotherapeutic agents. Finally, ethical issues must be considered to ensure responsible research practices, as the interests of tissue donors, researchers, and commercial stakeholders may not align [149].

Although obstacles remain, the rapid progress made in organoid research and its promising trajectory suggest that resolving the existing gaps is within reach. The convergence of multiple disciplines and applications will accelerate progress, ultimately translating into improved therapeutic options and significant patient benefits.

Abbreviations

ALD

alcoholic liver disease

ASC

adult stem cell

BME

basement membrane extract

CAF

cancer-associated fibroblast

CC

cholangiocarcinoma

CHC

cholangio-/ hepatocellular carcinoma mixture types

EV

extracellular vesicle

HCC

hepatocellular carcinoma

hCdHs

human chemically derived hepatic progenitors

iPSC

induced pluripotent stem cell

MASH

metabolic dysfunction-associated steatohepatitis

MASLD

metabolic dysfunction-associated steatotic liver disease

PSC

pluripotent stem cell

RNAi

RNA interference

2D

two-dimensional

3D

Three-dimensional