Hepatitis B virus (HBV) is a hepatotropic DNA virus that belongs to the Hepadnaviridae family and is known to cause both acute and chronic liver infections in humans [1]. Despite the availability of a highly effective vaccine, HBV remains a major global health concern [2]. It is estimated that over 400 million people worldwide are living with chronic HBV infection, making it one of the leading causes of liver-related morbidity and mortality [3]. Chronic HBV infection is a primary contributor to several liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), which together cause nearly 820,000 deaths annually [4]. The risk of developing HCC is significantly higher in individuals with chronic HBV infection, particularly those with cirrhosis [5]. Moreover, HBV can be vertically transmitted from mother to child during childbirth, contributing to the high prevalence of chronic HBV infection [6]. Antiviral treatments, such as nucleoside analogues (e.g., tenofovir and entecavir) and interferon-based therapies, can suppress viral replication and reduce liver damage [7]. However, they do not completely cure chronic HBV infection; specifically, the currently available antiviral drugs do not fully eliminate HBV DNA, particularly in the form of covalently closed circular DNA (cccDNA) [8]. In addition, the long-term use of antiviral drugs can lead to drug resistance and potential side effects. Therefore, when antiviral treatment is stopped, viral replication begins again with HBV cccDNA remaining in the nucleus of the host cell [9]. Consequently, there is a pressing need for the development of more effective treatment strategies to completely eradicate HBV.

HBV contains a small, partially double-stranded DNA genome of approximately 3.2 kilobases, referred to as relaxed circular DNA (rcDNA) (Fig. 1) [1]. The virus exhibits strong tropism for human liver hepatocytes. It initiates infection by binding to the sodium taurocholate co-transporting polypeptide (NTCP) receptor on the surface of hepatocytes, mediated by the epidermal growth factor (EGF) receptor as a co-receptor [10,11]. After attachment, the virus is internalized into the cell, and its rcDNA is transported into the nucleus, where host factors and viral polymerase convert it into cccDNA [12,13]. This cccDNA serves as a transcriptional template to produce viral RNAs, including pregenomic RNA (pgRNA) and several subgenomic RNAs [1]. HBV pgRNA is a template for synthesizing rcDNA. The viral polymerase interacts with pgRNA 5' epsilon structure and promotes nucleocapsid assembly [14,15]. Within the capsid, the negative strand of rcDNA is synthesized from pgRNA by the reverse-transcription activity of the polymerase [16]. Then, the positive strand is produced from the negative strands as a template. The HBV life cycle also involves the production of several viral proteins, including the surface antigen (HBsAg), the core antigen (HBcAg), and the X protein (HBx), which play various roles in viral replication and pathogenesis [1]. HBV cccDNA persistence within hepatocyte nuclei plays a central role in chronic HBV infection, enabling immune evasion and functioning as a stable reservoir for ongoing viral replication [17]. This persistence is a primary reason why current antiviral therapies, which target viral reverse transcription, are unable to completely eradicate the virus in infected individuals. The transition from acute to chronic HBV infection is influenced by several factors, including the age at which infection occurs, the individual’s immune response, and viral characteristics [18,19]. In adults, the immune system often clears acute HBV infection, with only 5% to 10% of cases progressing to chronic infection [20]. However, in neonates and young children, the risk of chronic infection is much higher, with up to 90% of infants born to HBV-infected mothers developing chronic HBV if not properly vaccinated [21].

Despite advancements in HBV research, there remains a significant need for infection models that effectively replicate the virus’s life cycle and interactions with human liver cells. To date, several models have been employed to study HBV infection, replication, and pathogenesis. Hepatoma cell lines, such as HepG2 and Huh7, have been widely used due to their ease of maintenance and ability to support HBV replication when transfected with the viral genome [22]. However, these cell lines lack the NTCP receptor necessary for HBV entry, meaning they cannot be naturally infected by the virus. Primary human hepatocytes (PHHs), which are considered the gold standard for liver research, offer a more authentic cellular environment for HBV studies and do allow viral entry, but they are difficult to obtain, exhibit rapid dedifferentiation in culture, and cannot sustain long-term infection studies due to their short lifespan [23]. Additionally, animal models, such as transgenic mice expressing HBV components, provide insights into viral replication and immune responses but present challenges due to species-specific differences; for example, mouse hepatocytes lack NTCP expression, requiring genetic modifications that bypass natural infection pathways and limit their translational relevance [24]. These limitations of traditional models highlight the need for an alternative approach to accurately study HBV infection dynamics, chronic disease progression, and host-virus interactions. Three-dimensional (3D) liver organoids, derived from hepatic stem cells or patient liver tissue, offer a promising solution by closely mimicking human liver architecture and supporting the full HBV life cycle, from viral entry to cccDNA formation and replication [25-27]. These organoids provide a stable, long-term platform that captures liver-specific functions and cellular diversity, including hepatocytes and cholangiocytes [28]. As such, they serve as an invaluable tool for examining chronic HBV infection, fibrosis, and HCC development [29]. Additionally, liver organoids can be genetically manipulated to study specific viral-host interactions. They are amenable to drug testing, positioning them as a powerful platform for both basic research and translational applications, potentially accelerating the development of more effective HBV therapies [30,31]. This review presents insights into the potential of 3D liver organoids, which contain human stem cells and patient-derived primary hepatocytes, as a novel platform for studying HBV infection and pathogenesis.

Ethics statement: This study was a literature review of previously published studies and was therefore exempt from institutional review board approval.

Research on HBV infection and the progression of HBV-associated diseases has historically relied on a variety of in vitro and in vivo models, including immortalized human hepatoma cell lines, PHHs, and animal models such as chimpanzees, woodchucks, and mice [32-34]. Each model has provided valuable insights into various aspects of the HBV life cycle, including viral entry, replication, and host immune responses. However, these models exhibit significant limitations that restrict their ability to fully replicate HBV infection and pathogenesis in humans. These limitations hinder the comprehensive understanding of HBV pathogenesis and viral-host interactions.

Chimpanzees are non-human primates that can support the full HBV life cycle, making them one of the most historically important models for studying HBV infection, replication, and immune responses [35,36]. Their close genetic similarity to humans facilitates relatively accurate modeling of HBV behavior, immune responses, and disease progression [37]. However, the use of chimpanzees in research has been severely restricted due to ethical concerns, high costs, and the limited availability of this endangered species [38]. Additionally, despite their ability to be infected with HBV, chimpanzees exhibit different immune responses and disease progression compared to humans [39,40]. These differences limit their utility for studying the long-term pathogenesis of HBV, particularly in terms of chronic infection and HCC progression, which are critical components of HBV disease in humans. Researchers have explored the use of other non-human primates, particularly rhesus macaques (RMs), for HBV research [41,42]. RMs, a species of monkey, are easier to access than chimpanzees and are often used in biomedical research due to their greater availability and lower costs [43]. However, RMs have shown only partial susceptibility to HBV infection under conditions of immunosuppression [44]. Although they can be experimentally infected with HBV, the infection is typically transient and does not become chronic. Furthermore, RMs do not reliably support the full replication cycle of HBV, as seen in humans or chimpanzees [44]. Even though RMs exhibit only partial susceptibility to HBV, they are still helpful for investigating aspects of immune responses and acute infection. However, due to their inability to develop chronic HBV infection or mimic long-term disease progression, they are not ideal models for researching the chronic stages of HBV or for the development of antiviral therapies aimed at curing chronic HBV infection.

Mouse models have been widely developed for HBV research because of their accessibility, ease of genetic manipulation, and relatively low cost [45]. Transgenic mouse models with integrated HBV genomes have been developed to study viral replication and immune responses [46,47]. Specifically, HBx and HBsAg transgenic mice have been utilized to explore the functional roles of these viral proteins in viral replication and pathogenesis [48,49]. Additionally, the entire HBV genome has been integrated into mouse chromosomes to mimic chronic HBV infection [50,51]. This integration facilitates viral protein expression, replication, and virion production. Although these models are useful for studying HBV replication and HBV-related liver disease, they do not fully replicate the natural HBV life cycle [52]. A significant limitation of transgenic mouse models is the absence of viral entry and cccDNA formation, as mouse hepatocytes do not express the human NTCP receptor, which is necessary for HBV entry [53]. Therefore, these models skip the natural infection process by integrating HBV DNA directly into mouse chromosomes, which prevents the study of the initial stages of HBV infection and the role of cccDNA in viral persistence. Furthermore, immune responses in mice significantly differ from those in humans, complicating the translation of findings related to HBV immunopathogenesis from mouse models to human disease [54]. For instance, cytokine and T-cell responses in mice do not accurately mirror those in human HBV infection, limiting the utility of these models in studying immune-mediated liver damage and viral clearance [55,56]. Researchers developed a humanized-liver mouse model transplanted with PHHs to overcome these obstacles [57,58]. In these models, human hepatocytes are engrafted into immunocompromised mice lacking mature T, B, and NK cells. The humanized-liver mouse supports replication of the entire HBV life cycle, including viral entry, replication, and cccDNA formation [59,60]. However, humanized mouse models also have several limitations that reduce their relevance for studying HBV. First, these models rely on the engraftment of human hepatocytes, which is a complex and technically challenging procedure. The degree of engraftment can vary among individual mice, leading to variability in experimental outcomes. Additionally, these mice lack a functional immune system, as they are immunodeficient to prevent rejection of the human hepatocytes [61]. Due to this limitation, they cannot be used to study immune responses to HBV infection, which are critical for understanding the pathogenesis of chronic HBV infection and the development of liver disease. Moreover, the cost of generating and maintaining humanized mice is high, and the availability of human hepatocytes for engraftment remains limited.

Immortalized human hepatoma cell lines, such as HepG2, Huh7, and Hep3B, have been the most commonly used in vitro models for HBV research for almost 30 years [23,32]. These cell lines offer several advantages, including their ease of maintenance, high reproducibility, and ability to support long-term cultures. However, these cell lines can only be used for HBV research when artificially transfected with HBV DNA. To address this limitation, the HepAD38 cell line was developed [62]. HepAD38 is a HepG2-derived cell line that contains an integrated HBV genome under the control of a tetracycline-inducible promoter. In this system, HBV replication is tightly regulated and can be switched on or off depending on the presence of tetracycline. This allows researchers to study the HBV replication process, including the production of viral RNA and the synthesis of viral proteins and virions [63]. Despite the utility of these immortalized human hepatoma cell lines for studying HBV replication, they have critical limitations that restrict their utility in studying HBV infection and pathogenesis. One of the primary drawbacks of these cell lines is their lack of susceptibility to natural HBV infection. These cell lines do not express the necessary host receptors, such as NTCP, which is essential for HBV entry into hepatocytes. This limitation prevents the study of the early stages of HBV infection, such as viral attachment, entry, and cccDNA formation. Furthermore, these cell lines often harbor significant genomic and epigenetic alterations due to their cancerous origins [64]. For example, HepG2 cells have altered expression of genes involved in cell cycle regulation and tumor suppression, which affects their response to the HBV life cycle [65]. Their inability to accurately model the physiological conditions of a normal liver limits their relevance in studying HBV-mediated liver diseases. Another major issue with hepatoma cell lines is the absence of key liver-specific functions, such as metabolic activity and immune responses [66,67]. Although these cell lines can support HBV replication to some extent, they do not replicate the complex liver microenvironment, which includes interactions with non-parenchymal cells like Kupffer cells, hepatic stellate cells, and liver sinusoidal endothelial cells. These interactions are crucial for understanding the immune responses to HBV infection and the development of chronic liver disease. In addition to hepatoma cell lines, primary hepatocytes (i.e., PHHs) are frequently used in HBV research [68,69]. PHHs express HBV entry receptors, allowing the study of the early stages of HBV infection [70]. However, their limited availability, rapid phenotypic dedifferentiation, and finite lifespan make it difficult to model persistent HBV infection and liver disease progression [71]. One of the most significant challenges is the limited availability and variability of PHHs. Human hepatocytes are typically obtained from liver resections or biopsies, which are invasive and result in a limited supply of cells. Additionally, the quality and susceptibility of PHHs to HBV infection can vary greatly depending on the donor’s health status and genetic background, as well as the conditions under which the cells are isolated and cultured [72]. This variability makes it difficult to standardize experiments and obtain reproducible results. PHHs also experience a rapid loss of phenotype and functionality in culture [73,74]. When cultured in 2-dimensional (2D) monolayers, PHHs quickly lose their differentiated hepatocyte markers, including albumin and cytochrome P450 enzymes, which are essential for liver function. This dedifferentiation process occurs within days of culture, limiting the time window during which PHHs can be used to study HBV infection and replication. Moreover, PHHs are not easily expandable, meaning that large-scale experiments requiring high cell numbers are often unfeasible. Another drawback is the short-term survival of PHHs in culture. Unlike immortalized cell lines, PHHs have a finite lifespan and cannot be cultured for extended periods. This limitation poses a challenge for studying long-term HBV infection and the chronic disease processes associated with HBV, such as liver fibrosis and cirrhosis.

Liver organoids, which are derived from hepatic stem cells that represent the heterogeneity of liver cells, have revolutionized the field of HBV research by providing more physiologically relevant in vitro models for studying HBV infection and HBV-mediated liver diseases (Fig. 2) [75-77]. These 3D cell cultures closely mimic the structure and functionality of the human liver, offering a platform to understand the molecular mechanisms of the initiation and progression of HBV infection and pathogenesis. Several types of stem cells, such as adult stem cells (ASCs) and induced pluripotent stem cells (iPSCs), are used to establish liver organoids [78,79]. These organoids can be genetically manipulated using tools such as CRISPR-Cas9, enabling researchers to model specific genetic mutations associated with liver diseases, including HBV-related liver dysfunction and cancer [30].

ASC-derived liver organoids are generated using liver stem cells or liver progenitor cells residing in the adult liver (Fig. 2) [78]. These cells can differentiate into both hepatocytes and cholangiocytes, mimicking the architectural organization and function of liver tissue [80]. The development of ASC-derived liver organoids begins with the isolation of progenitor cells from adult liver tissue, followed by embedding these cells in a 3D extracellular matrix such as Matrigel [25]. Isolated progenitor cells self-organize into 3D liver-like structures resembling liver lobules. Under specific culture conditions, with an optimized expansion medium (EM) with growth factors such as Wnt agonists and EGF, liver organoids expand for long-term culture without the need for genetic manipulation [75]. These liver organoids express HNF4α, a key hepatocyte marker, but maintain low levels of mature hepatocyte markers that are important for studying liver-specific functions, including albumin and CYP3A4. To drive further maturation, the EM is replaced with a differentiation medium, which includes Notch signaling inhibitors and hormones promoting hepatocyte-specific differentiation [75]. This shift halts organoid expansion and steers cell differentiation toward a mature phenotype, marked by the expression of albumin, cytochrome P450 enzymes, apolipoproteins, and complement factors. One of the key features of ASC-derived liver organoids for HBV research is that they can support the full HBV life cycle, including viral entry, cccDNA formation, replication, and particle production [75]. ASC-derived liver organoids can support viral replication for up to 2 weeks post-infection. When EM is used for intermittent recovery, HBV viral particles are released for up to 1 month in ASC-derived liver organoids. In addition, HBV infection can induce markers of the development of hepatocellular damage, fibrosis, and even tumor formation in ASC-derived liver organoids [75]. This ability to model HBV pathogenesis, including liver fibrosis and HCC, makes them invaluable for investigating how HBV causes liver damage and cancer. Although ASC-derived liver organoids have many advantages for HBV research, there are several limitations. One of the main challenges for ASC-derived organoids is the heterogeneity of progenitor cell populations, leading to variability in organoid formation and function [81]. This heterogeneity makes it difficult to standardize experiments across different samples. Additionally, while these organoids recapitulate basic liver functions, they may lack the full complexity of the liver microenvironment, including the interaction with non-parenchymal cells like Kupffer cells and endothelial cells [82].

iPSCs are reprogrammed from somatic cells, such as fibroblasts, into a pluripotent state, which allows them to differentiate into any cell type, including hepatocytes (Fig. 2) [83,84]. Generating iPSC-derived liver organoids involves several steps, starting with reprogramming somatic cells into iPSCs using factors such as Oct4, Sox2, Klf4, and c-Myc. These iPSCs are then differentiated into hepatocyte-like cells by sequentially adding growth factors and signaling molecules that mimic liver development [85,86]. When cultured in 3D matrices, these hepatocyte-like cells self-organize into organoid structures resembling liver tissue. iPSC-derived liver organoids are generated by co-culturing iPSC-derived endodermal, mesenchymal, and endothelial cells [87]. These liver organoids can also replicate the complete HBV life cycle and even support the maintenance of significantly higher levels of pgRNA, cccDNA, and virion production than is possible with iPSC-derived hepatocyte-like cells and PHHs, because iPSC-derived liver organoids can express more viral infection-promoting factors, including GPC5, PPARα, and CEBPA, than iPSC-derived hepatocyte-like cells [87]. In iPSC-derived liver organoids, HBV infection upregulates markers of the epithelial-to-mesenchymal transition, such as SNAI2 and TWIST1, which are often associated with liver cancer progression [87]. This indicates that HBV-infected liver organoids can effectively model virus-induced hepatic dysfunction. Furthermore, high viral titers in iPSC-derived liver organoids induced abnormal gene expression and the release of early markers of acute liver failure, underscoring their utility in studying HBV-mediated liver damage and dysfunction [87]. While iPSC-derived liver organoids offer numerous advantages, they also have some limitations. One of the main challenges is the time and complexity involved in generating iPSC-derived liver organoids, which typically require several weeks to months of differentiation and culturing [84]. Additionally, iPSC-derived hepatocyte-like cells may not fully mature in vitro, resulting in immature hepatocytes that do not completely replicate the functional properties of adult liver cells. This can limit the utility of iPSC-derived liver organoids in studying certain aspects of liver function, such as drug metabolism. Another limitation is the variability in differentiation efficiency among iPSC lines [88]. Different iPSC lines can exhibit varying capacities to differentiate into hepatocyte-like cells, leading to inconsistencies in organoid formation and function. Furthermore, iPSC-derived organoids, like ASC-derived organoids, often lack the non-parenchymal cells needed to recapitulate the full spectrum of liver biology, limiting their ability to model immune responses to HBV infection.

In contrast, patient-derived liver organoids represent a personalized approach, as they are directly generated from liver biopsy samples taken from individuals with specific liver conditions, including chronic HBV infection [75]. Healthy liver biopsy or poorly differentiated liver tumors can be used to generate patient-derived liver organoids with exogenous HBV infection [89]. Importantly, patient-derived liver organoids from primary liver malignancies reflect the histological architecture and intratumoral heterogeneity of individual liver cancer patients, enabling examinations of cancers in the advanced stages and recapitulating the features of primary tumors. These organoids retain the donor liver's genetic, epigenetic, and histological characteristics, making them an ideal model for studying patient-specific liver disease progression. Patient-derived liver organoids are also cultured using similar 3D matrix systems as ASC-and iPSC-derived liver organoids, but offer the added advantage of preserving the unique genetic background of the patient, including any mutations or disease-specific characteristics that influence liver function and response to viral infections such as HBV. One of the most promising applications of patient-derived liver organoids in HBV research is in the field of personalized medicine, because they can be used to test antiviral therapies and assess drug responses in a patient-specific manner. This enables the development of personalized treatment regimens, where therapies can be tailored to the individual’s viral load, genetic predispositions, and liver disease stage.

Despite their transformative potential in HBV research, ASC, iPSC, and patient-derived liver organoids share several common limitations that impact their effectiveness as comprehensive HBV models. One major limitation across these organoid types is the absence or limited presence of non-parenchymal cells, such as Kupffer cells (liver-resident macrophages), hepatic stellate cells (critical for fibrosis), and sinusoidal endothelial cells [82]. These cells are integral to immune responses, fibrosis development, and liver microvascular dynamics, all of which are key to understanding the full pathogenesis of HBV. Without these non-parenchymal components, liver organoids cannot fully recapitulate the immune responses or fibrotic processes that occur in vivo during HBV infection and progression to liver disease. Another common challenge is the heterogeneity in differentiation efficiency and functional maturity, particularly in iPSC-derived liver organoid models, where the extent of hepatocyte maturation is often limited, leading to incomplete replication of adult liver cell functions such as drug metabolism [27,29]. This immaturity is also observed, albeit to varying extents, in ASC- and patient-derived liver organoids, which may not achieve full hepatocyte function in vitro, thereby constraining long-term studies of chronic HBV infection and its sequelae, such as cirrhosis and HCC. Furthermore, the variability in cellular composition and functional properties across individual organoid batches adds additional complexity, resulting in inconsistent experimental outcomes [88]. This variability is especially pronounced in patient-derived organoids due to donor-specific genetic and epigenetic differences, which, while offering insights into personalized HBV pathology, make it challenging to standardize findings across studies. Together, these limitations highlight the need for further advancements in organoid engineering and co-culturing techniques to develop more representative and stable liver models for HBV research.

Although liver organoids have several limitations for HBV research, these models hold significant promise for advancing our understanding of HBV pathogenesis and developing personalized antiviral therapies compared to conventional models. One of the most significant applications of liver organoids in HBV research is their ability to model HBV infection and replication [87]. Unlike traditional hepatoma cell lines, which often lack the full repertoire of functional liver characteristics, liver organoids express essential HBV entry receptors, such as NTCP, facilitating natural infection modeling that enables comprehensive exploration of the HBV life cycle from entry to virion release within a physiologically relevant context [32]. This makes them a powerful tool for studying viral-host interactions at various stages of HBV infection. Moreover, liver organoids have been used to investigate the mechanisms of HBV-mediated liver damage because, in liver organoids, HBV infection induces the development of hepatocellular damage and fibrosis, both of which are hallmarks of chronic HBV infection [87]. Studies have also shown that these organoids can be used to screen for novel antiviral therapies and assess the hepatotoxicity of drugs in a more physiologically relevant context than is possible with 2D hepatoma cell cultures [89,90]. By using liver organoids, HBV researchers can investigate the mechanisms of drug resistance and identify novel therapeutic targets to overcome this challenge.

In conclusion, the emergence of 3D liver organoid models represents a significant leap forward in HBV research, providing more physiologically relevant systems than traditional in vitro and in vivo models [75,78,85,87,89]. ASC, iPSC, and patient-derived liver organoids each offer distinct advantages for studying various aspects of HBV infection, from viral replication and immune responses to liver fibrosis and tumorigenesis. These models provide deeper insights into HBV pathogenesis, highlighting the complexity of viral-host interactions and the challenges of developing curative therapies. However, despite these advancements, liver organoids are still evolving, and several limitations need to be addressed. The absence of immune cells, the challenges of replicating the complex liver microenvironment, and the variability in organoid maturity are all hurdles to overcome. Moving forward, integrating immune components, advancing organoid maturation, and leveraging gene-editing technologies will be key to unlocking the full potential of liver organoids in HBV research. The potential applications of liver organoids extend beyond basic research, offering exciting opportunities in drug discovery, personalized medicine, and the development of targeted therapies for chronic HBV and HBV-associated liver cancer. The ongoing refinement of liver organoid models positions them as pivotal tools in HBV research, bridging the gap between conventional in vitro systems and clinical applications by providing platforms for high-throughput drug screening, personalized medicine, and mechanistic studies into HBV-associated liver disease and carcinogenesis.