The liver is a complex organ that is vital for regulating metabolism, energy storage, and detoxification in the human body [1]. The liver is the largest internal organ, and it mainly comprises hepatocytes that are central to the performance of most of its functions [2,3]. Other cell types in liver tissue include parenchymal cholangiocytes and nonparenchymal liver cells (NPCs): liver sinusoidal endothelial cells, hepatic stellate cells (HSCs), Kupffer cells (KCs), and other immune cell types. Each cell type plays different roles, and together, they maintain the structural integrity of liver tissue during homeostasis and make repairs when needed due to injury or disease [4,5]. These cells are spatially organized into lobules and form channels, including the sinusoid and bile canaliculi networks, that are essential for transporting nutrients into the liver and exporting metabolites and bile into the digestive system. In addition to the loss of hepatocytes, disruption of these liver structures and cellular interactions often occurs during drug-induced liver injury (DILI) and liver disease, resulting in the loss of liver function.

Chronic liver disease (CLD) remains a leading cause of death and morbidity worldwide [6]. CLD is the major contributor to liver injury, fibrosis development, and the eventual progression to cirrhosis and primary liver cancer (PLC). Historically, the main etiology for CLD is viral hepatitis. However, with the development of antiviral treatments and vaccinations, a pandemic of obesity, and increased alcohol consumption, the primary emerging CLD etiologies are nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), and DILI [6,7]. Due to the complex interplay among the cellular components of the liver in varying disease conditions, it has been challenging to construct a robust human model for understanding disease manifestations and treatment responses [8,9]. Most human liver disease models have focused on the ways in which diseases and chemical agents induce hepatocyte injury and cell death. However, apart from those cellular changes, remodeling of the hepatic extracellular matrix (ECM) also plays an integral role in disease progression. Fibrogenesis is a common pathophysiological feature in injured and diseased livers, and it involves extensive ECM remodeling by activated HSCs [10]. These cells secrete fibril-associated collagens and matrix metalloproteinases that gradually remodel the soft and perforative endothelial/epithelial basal lamina, making it rigid and obscure. To accurately reflect disease progression, it is essential for research models to capture such ECM changes as they are driven by interactions among various cell-types.

Scientists have long used three-dimensional (3D) culture methods to create human organ models that mimic in vivo niches and promote multidimensional cell–cell and cell–environment interactions [11,12]. In recent years, two cell culture platforms for generating such 3D cellular models have been increasingly adopted by laboratories across various research fields: pluripotent stem cell (PSC)-derived organoids and tissue-derived organoids. Since their discovery, PSCs have been an invaluable cell source for creating almost all cell types in the human body [13,14]. PSCs have a limitless self-renewing capacity and the ability to mimic embryogenesis and give rise to adult tissue cell types, and they are highly amenable to gene editing, so they are a preferred starting cell source for generating human organ models. One of the first 3D approaches to initiate differentiation into various germ layers is embryoid body formation, in which cells of all three germ layers begin to form [15]. It remains an initiation step in many differentiation protocols for PSC-derived organoids. To date, organoids resembling the brain [16], lung [17], kidney [18], stomach [19], intestines [20], pancreas [21], and liver [22,23] have been generated. The advent of human induced pluripotent stem cell (iPSC) reprogramming technology, which enables the generation of patient-specific PSC cells, has further accelerated the adoption and utility of the PSC-derived organoid platform [24].

The ability of isolated adult mammalian cells to reassemble themselves into functional tissues when embedded in a culture matrix was first demonstrated using a 3D culture of epithelial cells derived from mammary glands and lung alveoli [25,26]. Both studies highlighted the ability of basement membrane matrix secreted from Engelbreth-Holm-Swarm sarcoma (commonly known as Matrigel) to support 3D human cell culture and the marked improvements in functioning shown by the 3D cultured cells compared with their twodimensional (2D) counterparts. In a landmark study by the Hans Clevers group, a single adult crypt stem cell was shown to generate functional intestine crypt organoids in Matrigel culture [27]. Specifically, those authors showed that by supplementing the cultures with growth factors and small molecules that modulate essential signaling pathways supported by the tissue niche, adult stem cells marked by Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) could be cultured and propagated for an extended period. Clevers’s group and others quickly showed that by manipulating culture media to activate essential signaling pathways that support progenitor cells in other tissue types, the platform could be adopted to derive organoid cultures from other organs, including the liver [28-30]. Remarkably, the tissue-derived organoid platform is also highly suitable for deriving cancer cells from tumors of various indications. These 3D cancer cell cultures, commonly called tumoroids, have facilitated a breakthrough in deriving patient-specific cancer cells for colon, pancreatic, liver, ovarian, and nasopharyngeal cancer [31-35].

Human models, including immortalized hepatic cell lines, PSC-derived hepatocyte-like cells, and primary human hepatocytes (PHHs), have been widely used to study the pathogenesis of liver disease and to screen and evaluate treatment strategies [36,37]. Each model has its limitations and advantages. For instance, PHHs can preserve the genetic information and metabolic function of their in vivo counterparts. They are suitable for studying toxicity, metabolism, viral infection, and congenital disease. However, their adoption is significantly constrained by a lack of donors and limited proliferation capacity in vitro. 38 On the other hand, immortalized cell lines derived from hepatoblastoma (hepG2) or hepatocellular carcinoma (HCC) (Hep3B) provide a renewable cell source, but they lack multiple hepatocyte functions. Their cancerous features and genomic instability also remain a concern for many applications [39]. During the past decade, hepatic organoids have emerged as a new human in vitro option for modeling multiple liver diseases, including hepatitis, NAFLD, ALD, monogenic disorder, and primary liver cancer (PLC) (Tables 1–4). In general, liver organoids have significantly increased the feasibility of establishing patient-specific models. Additionally, they incorporate multiple liver cell types to model disease pathophysiology and enable the recapitulation of structural changes observed during tissue injury and disease progression. Liver organoids of different cellular compositions, sizes, structures, and functions can be derived from PSCs and human liver tissue (Fig. 1) [40,41]. In this review, we follow the consensus nomenclature of Marsee et al. [42],which reflects the cellular origins of tissues and the cellular composition and function of the organoid. Several reviews have provided in-depth discussions about how various liver organoids (Fig. 1) can be generated using different cell culture systems [40,41]. In this review, we focus on how PSC-derived and tissue-derived liver organoid systems are being used to advance the study of major liver diseases.

Monogenic liver diseases are caused by single-gene mutations. These diseases include conditions that directly induce injury in liver parenchymal cells and diseases in which the liver is intact and the phenotype develops in other parts of the body [43]. The prevalence of individual diseases caused by a single-gene mutation is low, with such diseases occurring in approximately 1% of newborns [43]. Current pharmacological interventions include enzyme replacement therapy and chelation therapy to enhance toxin excretion [44,45]. Unfortunately, those therapies are usually expensive and have poor cost-effectiveness. Gene therapy has emerged as a promising therapeutic option [46]. However, the application of such therapy is limited by the efficacy of the delivery system and the expression of the therapeutic gene. In addition, adverse immune responses and safety concerns about the delivery vectors pose significant difficulties to clinical translation. Liver transplantation remains the standard intervention for many hereditary liver diseases.

The successful derivation of organoids from diseased tissue provides a new resource for modeling and understanding hereditary disease and searching for novel therapeutic targets (Table 1). Organoids derived from diseased tissues recapitulate the genomic backgrounds of individual patients and retain the functional dysregulations of monogenetic disorders [28,47-49]. In addition, genome editing technology enables the precise insertion and correction of mutated sequences for further mechanistic study. Clevers’s group reported the first platform for isolating bipotent intrahepatic cholangiocyte organoids (ICOs) from healthy and diseased tissues [47]. Bipotent ICOs can be further differentiated in vitro into functional hepatocytes that secrete albumin, exhibit CYP3A4 activity, and give rise to new hepatocytes upon engraftment in mice. This platform thus makes it possible to model adult liver functions and mimic disease pathophysiology in vitro. Those authors first demonstrated that possibility by isolating diseased organoids from patients with a1-antitrypsin (A1AT) deficiency or Alagille syndrome (ALGS). A1AT deficiency is a genetic disorder caused by the defective production of the A1AT protein in hepatocytes [47]. The A1AT protein is secreted in the lungs to protect the lungs against damage caused by the neutrophil elastase. A1AT deficiency is characterized by an excessive accumulation of abnormal A1AT protein in the liver and reduced A1AT levels in the lung, resulting in the injury to the alveoli [49,50]. Differentiated organoids from A1AT patients displayed normal albumin secretion and low-density lipoprotein (LDL) uptake ability, and the cells acquired the hallmarks of A1AT-deficient patients [47,49]. The patient-derived organoids exhibited reduced A1AT secretion compared with the healthy control organoids, and aggregates of abnormal A1AT increased. These phenotypes are similar to the phenotypes observed in diseased tissue. ALGS is a common monogenic liver disease resulting from a loss-of-function mutation in the JAG1 gene; it affects various organs, particularly the heart, liver, eyes, and vertebrae. The JAG-NOTCH signaling pathway is highly conserved and critical in the development of multiple organs and determination of cellular fates. The clinical manifestation of ALGS in the liver includes bile duct malformation [51]. Patients can present with impaired biliary differentiation, duct paucity, or chronic cholestasis. Isolated patient bipotent organoids are indistinguishable from healthy organoids. However, upon differentiation, the patient organoids failed to upregulate cholangiocyte markers such as cytokeratin 19 and cytokeratin 7 [47]. Subsequently, the organoids lost their morphology and underwent apoptosis. In summary, that pioneering study offered a novel approach for deriving renewable, patient-specific, primary cell models. The rapid adoption of that technique by other research teams for similar genetic liver disease studies supports the robustness of the ICO platform. Gómez‐Mariano’s team isolated diseased ICOs from patients with heterozygous and homozygous mutations in the A1AT-encoding gene SERPINA1, and the organoids with corresponding genotypes exhibited different protein deficiency levels [49]. Those authors further demonstrated that the organoids could respond to external stimuli, such as oncostatin M, which is a well-known inducer of A1AT expression. Other teams have further extended the application of this technique to animal models, including genetically engineered mouse models (GEMMs) [51] and large animal models such as canines (Table 1) [52,53].

iPSC technology was a landmark breakthrough that enabled the efficient establishment of patient-specific cell models [54]. Models of genetic liver diseases can be established using iPSCs generated from patients with the disease or via gene editing of healthy iPSC cell lines. Liver organoid models of those diseases can subsequently be established from the iPSCs using various differentiation strategies (Fig. 1). PSC-derived organoids range from simple single-cell-type hepatocyte or cholangiocyte organoids with cystic structures [55-57] to more complex multicellular organoids such as parenchymal hepatobiliary organoids and multicellular liver organoids containing both parenchymal and nonparenchymal cell types [23,58,59]. The structural features present in the organoids allow researchers to recapitulate disease phenotypes in a dish. Harnessing the cystic structure formed by cholangiocyte-like cell (CLC) organoids, Sampazio et al. [56] were able to model the disruption of molecules and fluid transport that occur in cholangiopathy-inducing genetic diseases such as ALGS, polycystic liver disease, and cystic fibrosis (CF). CF is one of the most common autosomal-recessive genetic diseases in the Western population and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [60]. The most frequent CF mutation seen in patients is the deletion of phenylalanine 508 (ΔF508), which results in rapid endoplasmic reticulum degradation of misfolded CFTR. The loss of CFTR results in the disruption of chloride transport in cells. CLC organoids derived from the iPSCs of CF patients exhibited disrupted intracellular chloride transport and chloride transport into the organoid lumen. Given the close relationship between chloride transport and fluid secretion in cholangiocytes, those authors further demonstrated that chloride transport dysfunction also affected fluid transport into the organoid lumen. Notably, they demonstrated that the organoids could be used to validate the efficacy of the experimental drug VX809, which is currently in phase III clinical trials. Using a similar strategy, other researchers have reported iPSC-derived liver organoid models of ALGS [61], citrullinemia type 1 [62], and Wolman disease [58]. All those liver organoids exhibited the hallmarks of their respective genetic disease, and subsequent correction with gene-editing approaches reversed the phenotypes [61]. Although many of those proof-of-concept studies demonstrated the potential of generating liver organoids with patient-specific genotypes, how those organoids could advance the study of monogenic liver diseases or replacement therapy remains to be explored. For example, future studies could examine how the different cell types within the liver interact to modulate disease development and progression. In addition, many of the iPSC-derived organoids described herein face the inherent challenge that the hepatocytes generated are immature [63]. These PSC-derived cells are often referred to as hepatocyte-like cells (HLCs) because they closely resemble fetal liver hepatocytes and express markers such as alpha-fetoprotein (AFP) that are absent from adult liver hepatocytes. Of concern, these immature cells might also lack adult hepatocyte functions, such as selected drug-induced cytochrome p450 activity [64]. Significant progress has been achieved in identifying the molecular pathways necessary to induce maturity in fetal hepatocytes, and subsequent optimized culture conditions have enabled considerable progress in PSC-derived HLCs [65]. Therefore, culture conditions for liver organoid models could similarly be optimized in the future to enhance the functional maturity of the cells.

PLCs are some of the most malignant tumors and the fourth-leading cause of cancer-related death worldwide [66]. The types of liver cancer include HCC, which accounts for the majority (70–85%) of PLCs, cholangiocarcinoma (CCC), and combined hepatocellular cholangiocarcinoma (cHCC-CCC), which exhibits features of both HCC and CCC [66]. Hepatoblastoma is a rare liver tumor that predominantly originates in early childhood; it is the most common pediatric liver malignancy [66,67]. The main etiologies of PLC include hepatitis virus infections, alcohol abuse, NAFLD, and aflatoxins [66,68]. With the development of vaccinations and antiviral treatments, the major cause for PLC has gradually shifted from viral infections to NASH, in correlation with the growing global obesity pandemic [69,70].

Conventional treatment options for liver cancer patients include surgical resection, radiofrequency ablation, liver transplantation and multiple-kinase inhibitors such as, Sorafenib and Lenvatinib [71]. Unfortunately, the median progression-free survival time remains less than six months for most of these treatments [72]. With the emerging success of immunotherapy in targeting advanced-stage lung cancer and melanoma, immune checkpoint inhibitors have also been explored to treat liver cancer [73]. Currently, the combination of antibodies blocking PD-L1 and VEGF is the 1st line of treatment for HCC patients with unresectable tumours. However, the available data from clinical trials showed that less than half of the patients responded to the treatment [74].

Liver cancer manifestation is highly complex and involves the dysregulation of multiple genes and signaling pathways as the disease progresses [75]. Comprehensive genome and transcriptome profiling studies have been conducted to reveal the genetic landscapes of cancer cells and unravel the mechanisms of HCC initiation and progression [76-78]. Mutations in genes such as TP53, CTNNB1, CDKN2A, and TERT have been commonly identified in different patient cohorts [78-80]. Various underlying diseases and etiologies can induce tumorigenesis in the liver. Postinfection, the HBV genome potentially integrates with host cells and activates genes relevant to the cell cycle and proliferation such as TERT [81-83], stabilizing the telomeric ends of chromosomes to support unlimited cell expansion. Diseases such as NAFLD and long-term drug and alcohol abuse induce chronic inflammation in the hepatic niche [84]. Constant exposure to inflammatory cytokines promotes genetic insults that lead to unique gene mutations and epigenetic profile changes that favor cell survival under harsh conditions [85,86]. The interplay among those different disease etiologies creates an environment that promotes oncogenic transformation, clonal evolution, and the expansion of diverse cancer-initiating cells. Thus, liver cancers, such as HCC, are reported to exhibit significant interpatient and intratumoral heterogeneity.

For decades, researchers have derived 2D monolayer cancer cell lines from tumors and created mouse models to understand the disease and search for therapeutic targets. These immortalized cell lines are highly expandable at low cost, enabling high-throughput drug screening applications. However, a single immortalized cancer cell line only partially captures the tumor profile and constantly acquires new mutations during culture [8,40]. The lack of intratumor heterogeneity produces an inaccurate reflection of patient responses when such cell lines are used for drug screening. Moreover, a monolayer cell culture limits cell–cell interactions to a horizontal plane, and the nutrient and oxygen gradients observed in tumors are absent [8]. A study by Takai et al. [87] demonstrated that immortalized HCC cell lines cultured in 3D produce structural features resembling the glandular epithelium observed in vivo, and exhibit they increased resistance to chemotherapeutic agents. The cells in 3D also showed increased sensitivity to the TGF/β-induced epithelial–mesenchymal transition. Correspondingly, after engraftment in mice, the cells from the spheroids formed tumors more efficiently that metastasized more frequently than the cells cultured in 2D. Such studies highlight the advantages of alternative 3D in vitro models of PLCs. The advent of organoid culture thus presents a long-sought alternative strategy for creating human PLC models (Fig. 2). Commonly used immortalized cell lines have been derived and grown with undefined serum-supported media in monolayer cultures [88]. Although such serum-based culture conditions have enabled the creation of widely used immortalized cell lines, they fail to recapitulate the interpatient and intratumor heterogeneity observed in HCC tumors [89]. In contrast, organoid culture platforms use a chemically defined nutrient- and growth factor (cytokines and small molecules)–enriched medium that potentially supports the growth of various cancer cell clones. Another critical advantage of the organoid platform is the ability to culture both cancerous and healthy tissue from the same patient in parallel, which creates opportunities to study tumor development without interfering with individual genetic backgrounds (Fig. 2) [35]. The liver cancer tumoroid platform has contributed significantly toward understanding cancer cell biology, and it opens new possibilities for precision treatment in the clinic (Table 2) [8,90,91].

The human liver is a highly vascularized organ that is exposed to many external pathogens in the blood [1]. Common pathogens that infect hepatocytes include malaria-causing Plasmodium parasites and the hepatitis virus [108]. In contrast to Plasmodium parasites, the hepatitis virus affects host cell function and integrity, inducing liver inflammation and injury. Chronic hepatitis can progress to end-stage liver cirrhosis or liver cancer, and viral hepatitis was estimated to cause 1.34 million deaths globally and HBV and HCV infections accounted for 96% of mortality reported [109].

Understanding how these viral proteins and host–virus protein interactions function in each essential step of the viral life cycle has enabled the discovery of therapeutics. In HBV therapy, nucleoside reverse transcriptase inhibitors (NRTIs), which target HBV polymerase to disrupt viral genome replication, are currently prescribed to suppress viral levels in the liver [110,111]. Drugs targeting the interaction between L-HBs and NTCP, such as the myristoylated peptide Myrcludex B, have also been shown to reduce viral infection in preclinical models and early clinical trials [112]. Although targeting many of those viral pathways has enabled the effective management of the disease in patients with few liver complications, a cure is still unavailable because viral covalently closed circular DNA (cccDNA) persists in the host cell as a mini-chromosome or is integrated into the host genome [113]. In stark comparison, HCV is now widely considered to be a curable disease thanks to a range of direct-acting antiviral (DAA) drugs that target various viral nonstructural proteins [114]. Combinations of DAA drugs now enable 80–95% of HCV patients to be cured.

In a previous review of HCV research breakthroughs that enabled the rapid development of therapies, in vitro modeling for HCV replication was demonstrated to play a crucial role [114]. The discovery that the overexpression of cDNA encoding various viral genotypes induces full viral replication in hepatic cell lines enabled the rapid investigation and screening of therapeutics targeting different parts of the viral life cycle [115,115]. In vitro models continue to offer researchers mechanistic insights into how the HCV virus infects the host [117]. Human hepatic cell models, each with advantages and limitations, have also been used for HBV infection studies. Immortalized cancer cell lines, such as HepG2 and Huh7, are easy to culture and proliferate rapidly. However, they do not support HBV infection due to a lack of endogenous NTCP expression, so exogenous expression of NTCP is used in those cell lines to solve that problem. Although the ectopic expression of NTCP in those cells has proved useful [118-120], immortalized and mutating cancer cells might have lost cellular components that regulate viral entry. In light of that concern, HepaRG, a tumor-derived hepatic progenitor cell line capable of generating NTCP-expressing HLCs, is increasingly being adopted for HBV modeling [121-125]. However, the limitations of HepaRG in HBV modeling include low infection efficiency (requiring a high infection dose of >1,000 GE/cell) and low levels of cccDNA [126].

NAFLD is a CLD with a high clinical incidence, and it is closely associated with other metabolic disorders such as obesity, diabetes mellitus, and metabolic syndrome [146,147]. Chronic inflammation and hepatic injury promote the progression of NAFLD to NASH and eventually cirrhosis and HCC [148]. The global prevalence of NAFLD in 2018 was ~24.4% [149], and correspondingly, the number of patients with end-stage cirrhosis and HCC due to NASH continues to rise [147]. Although NAFLD patients are clinically asymptomatic, and the condition is relatively benign, NASH is rapidly becoming the leading cause of end-stage liver disease and liver transplantation [150,151]. The high incidence of NAFLD is primarily attributed to diet and lifestyle changes. Irregular lifestyles, such as insufficient sleep and exercise, and the long-term intake of foods high in sugar and fats contribute to excessive fat accumulation in the liver [152]. Common metabolic diseases such as hypertension, hyperglycemia, obesity, and metabolic syndrome are risk factors for NAFLD and NASH development [153-155]. In parallel, genetics also likely play a role in NAFLD development. Familial studies have shown that children of parents with a high liver-fat content are more likely than others to develop NAFLD and have an increased risk of progression to cirrhosis [156]. Genome-wide association studies in multiple NAFLD cohorts have identified enriched single-nucleotide polymorphisms (SNPs) in various genes in NAFLD patients, including apolipoprotein C3, glucokinase regulator (GCKR), membrane-bound O-acyltransferase domain-containing 7, patatin-like phospholipase domain containing 3 (PNPLA3), and transmembrane 6 superfamily member 2 [157]. Furthermore, a protective allele that reduces the risk of NAFLD progression to NASH has been reported in the HSD17B13 gene [158].

NAFLD is a continuum of disease ranging from benign hepatic steatosis to NASH and cirrhosis [159], with 43–44% of patients with steatosis progressing to NASH, and 7–30% of patients with NASH progressing to cirrhosis [160,161]. The mechanisms underlying NAFLD progression have yet to be fully elucidated. A two-hit theory was first proposed for the pathogenesis of NAFLD [162]. The first hit involves lipid metabolism disorders and manifests as hepatic steatosis. That results in insulin resistance and leads to increased serum levels of free fatty acids (FFAs) and sensitizes the hepatocytes for the second hit, which comprises endoplasmic reticulum stress, oxidative stress, and mitochondrial dysfunction. Together, the two hits result in hepatocyte damage and the secretion of large amounts of proinflammatory factors that recruit and activate immune cells into a chronic inflammatory response [163]. Accumulating evidence has shifted that traditional two-hit model into a model of multiple, parallel pathogenic influences in which fatty acids, insulin resistance, inflammation, oxidative stress, mitochondrial dysfunction, lipid peroxidation, and endoplasmic reticulum stress combine to induce the onset and development of NAFLD [164]. In addition, gut microbes have been reported to be involved in the development of NAFLD via the regulation of bile acid metabolism, lipid handling, and endotoxin and metabolite production in the gut [165,166]. Chronic inflammation is central to NAFLD disease progression and involves both residential and circulating immune cells, including macrophages and lymphocytes [167,168]. Interactions among the different cell types also involve a slew of inflammatory chemokines and cytokines, such as chemokine ligand 2, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1β, IL-17, and IL-18 [169-172].

Models of NAFLD have been critical for elucidating the mechanisms underlying this disease and identifying potential therapeutic targets. Multiple diet–induced models and GEMMs have been developed for NAFLD, and most of them recapitulate the histological features of NAFLD [173]. However, only a few studies have captured the metabolic syndromes commonly observed in patients [174], and there have been discrepancies in the NAFLD phenotypes observed in mouse and human models. In follow-up studies about PNPLA3 (the most widely reported NAFLD-associated gene), PNPLA3 depletion in mice did not affect hepatocyte lipid accumulation, which is in contrast to the findings in human in vitro models [175,176]. Human PNPLA3 levels are also much higher in liver tissue than adipose tissue, which is in contrast to rodent PNPLA3, which is more highly expressed in adipose tissue [177,178]. Given the increasing prevalence and lack of therapeutic options for NAFLD, many human models of NAFLD have been created using different hepatic cell lines and 2D and 3D culture platforms, including sophisticated microfluidics platforms [179] and PSC and tissue-derived organoid models (Table 4).

DILI is a common cause of liver injury and a significant challenge for drug development. Statistically, nearly one-third of drug candidates are withdrawn from the clinic due to adverse drug reactions [193]. Therefore, preclinical drug safety studies are critical to drug development. Drug toxicology studies rely heavily on animal testing in Investigational New Drug applications. However, animal model results have limitations due to significant species-specific physiological differences and the long latency period of some drug-induced injuries [194]. PHHs remain the preferred human in vitro model for DILI testing. The methods used to assess DILI with PHHs are currently limited to the measurement of hepatic enzyme activity, cell structural changes, and cell redox status [195,196]. Therefore, more sophisticated liver models are needed to allow researchers to test for changes in human liver tissue features. Shinozawa et al. [57] reported an iPSC-derived hepatocyte organoid model for high-throughput toxicity screening. The hepatocytes in the cyst-like organoid exhibited polarity and unidirectional transport of bile into the lumen. Using those organoids, those authors set up a screening platform that allows the real-time imaging of bile transport and parallel assays for cell viability. They validated 206 clinically available drugs with reported DILI and showed a high predictive value of ~90% sensitivity and specificity. By treating the organoids with FFA, those authors also demonstrated that the system could be used to investigate DILI under various liver disease backgrounds. In the abovementioned hepatobiliary organoid generated by Ramli et al. [59], the authors demonstrated how it could be used to model cholestatic events in DILI. They performed live-imaging of the organoids with 5(6)-carboxyfluorescein diacetate (CDFDA), which is a fluorescent probe that is transported in the bile system. Treatment of the organoids with the cholestasis-inducing troglitazone resulted in a gradual loss of signals in the bile canaliculi network and the accumulation of CDF (de-esterified CDFDA) in the enlarged cells. Overall, both studies highlight how researchers can harness the structural features observed in organoids to model DILI. We expect that toxicological screening applications will increase in the future with the generation of organoid models that can recapitulate more complex structural features of the liver.

In addition to DILI, alcohol abuse is a common cause of liver morbidity and mortality. Globally, approximately 3 million people die each year from alcohol consumption, and 8–20% of heavy drinkers develop cirrhosis [197]. Similar to NAFLD, ALD phenotypes include steatosis, fibrosis, and cirrhosis [223]. A study by Wang et al. [182] explored the use of PSC-derived hepatic organoids to recapitulate the pathogenesis of ALD. They first derived an expandable hepatic progenitor organoid that the enabled long-term expansion and generation of functional cholangiocyte or hepatocyte organoids. Subsequently, they achieved a co-culture of fetal mesenchymal cells and hepatocyte organoids. The organoid expressed ethanol (EtOH) metabolism–associated enzymes such as alcohol dehydrogenase and CYP2E1. When they were treated with EtOH, the cells exhibited increased cellular stress, steatosis, and a proinflammatory response. In addition, the authors detected elevated expression levels of fibrogenesis-related proteins, such as alpha-smooth muscle actin, type 1 collagen, and desmin. In summary, PSC-derived hepatic organoids can also be used to model ALD, but the utility of this model for mechanistic studies and the identification of novel therapeutics remains to be explored.

Liver organoids have provided researchers with an alternative human in vitro model that harbors features of in vivo tissue. Although these models encompass more physiological characteristics of the human organ than monolayer hepatocyte models, organoids should not be considered as a replacement for animal models, at least not in their current state. Organoids still lack many structural features of the tissue, a functional vascular system, and circulating hemopoietic cells. Future improvements to cell culture reagents and tools and the integration of bioengineering platforms will likely bring significant breakthroughs. Nonetheless, the current organoid culture platforms have already produced several advances in modeling human liver diseases. In particular, the ICO platform has enabled the generation of patient-specific liver organoid models for all the conditions discussed. This breakthrough will allow the generation of patient-specific models for various applications, from evaluating host susceptibility to viral infection to liver regenerative therapy (Fig. 3). In the next decade, we expect to see the establishment of sizeable organoid biobanks for various liver diseases. These biobanks will greatly facilitate pharmacogenetic studies by correlating patient genotypes to disease phenotypes and patient drug responses (Fig. 3), laying the foundations for precision therapeutics for treating liver disease.