Liver transplantation (LT) remains the only curative treatment for end-stage liver disease (ESLD) [1]. Unfortunately, biliary complications occur in 5%–35% of the recipients after LT and remain elusive to curative strategies in this transplant procedure [1–4]. Indeed, post-LT cholangiopathies such as anastomotic (AS), non-anastomotic biliary strictures (NAS), and bile leakage [4, 5] represent the major causes of morbidity and graft failure after LT [4–7]. Efforts to avoid the occurrence of cholangiopathies have been researched, as it is the case with the implementation of Dual Hypothermic Oxygenated Machine Perfusion (DHOPE), which significantly reduced the impact of the cold storage in the liver by improving the preservation of the tissue [8]. However, severe cholangiopathies can lead to ESLD, with re-LT being the only curative option for these patients [6, 9].

Due to the shortage of donor livers for transplantation, novel therapeutic approaches to treat biliary diseases so as to prevent the need for transplantation and to treat biliary complications are crucial. Newly developed technologies and methods in regenerative medicine and stem cell research potentially offer novel treatment options, which can potentially rescue diseased bile ducts and thus make a liver transplant unnecessary. However, organoids can also be used to study and could potentially even be used to treat cholangiopathies that arise after LT.

Here, we present a comprehensive review focused on cholangiocyte derived organoids, including relevant insights into biliary anatomy, as well as the development, nomenclature, and molecular characteristics of cholangiocyte organoids. Furthermore, we provide the latest developments on the potential use of cholangiocyte organoids in disease modeling and treatment modalities.

Derived from an endodermal bud, bile duct epithelium (BDE) is composed of cholangiocytes that line up to form an intricate network of branched conduits within the liver that constitute the biliary tree [10]. Closely associated with a branch of the portal vein and one or two branches of the hepatic artery, bile ducts are part of the classic view of a portal triad [11]. Stem (progenitor) cells are located intrahepatically in the canals of Hering, or extrahepatically in peribiliary glands (PBG) of the biliary tree [7]. These are called biliary tree stem cells (BTSCs), which are a set of multipotent cells that can differentiate into cholangiocytes, hepatocytes, or pancreatic islets [12].

During LT, severe damage to the biliary epithelium and PBG is associated with post-LT cholangiopathies [4, 13]. It has been shown that the PBG and its proliferating Sox9⁺ progenitor cells [14] drive bile duct regeneration. The BDE is highly vulnerable to ischemia, and although minor lesions often lead to re-epithelialization, more severe injuries can result in fibrosis and stricture formation [4, 15]. In general, injuries produced by ischemia (warm/cold ischemia, reperfusion injury, disturbed blood flow), immunological factors (ABO incompatibility, immune disease, virus infection, chronic rejection, chemokine mutations), or exposure to bile salts, can trigger post-transplant NAS cholangiopathies [16]. Damage to the PBGs and the vascular plexus strongly correlates with the development of NAS after transplantation, which is evident by poor regeneration of biliary epithelium due to progenitor cell destruction and insufficient blood supply [7]. Moreover, bile duct damage likely starts during the initial cold storage phase of the LT procedure [4]. When this damage progresses on a pathological level, poor persistent transplant function or loss is a common consequence, compared to transplanted organs that have no biliary damage [4, 14]. However, if a sufficient biliary regeneration of damaged bile ducts occurs in the early post-transplant phase, cholangiopathies can be prevented [17, 18].

To date, research on biliary injury is mostly based on two models: (i) rodent bile duct ligation, where an intrahepatic accumulation of bile leads to hepatotoxicity, cell proliferation, and fibrosis [19] to allow the study of injury repair mechanisms; and (ii) on histopathological examinations of human bile duct specimens, used to detect the molecular signatures of biliary insults [4, 5, 14]. However, both models have limitations. Animal models lack key components of human molecular mechanisms and are genetically homogenous, therefore these systems do not represent the specificity and diversity in humans [20]. The human specimen histological approach can be used to assess the morphological and molecular aspects of tissues, but this method captures only one moment in the disease course and is not amenable to dynamic therapeutic experiments [21].

The ability of cholangiocytes to adapt to new environments and the ease of culturing mature cells has recently brought bile duct organoids into focus. The concept of cultured organoids is constantly evolving, with the latest consensus defining organoids as “self-organized, three-dimensional tissues that are typically derived from stem cells and embedded in a matrix, and which mimic the key functional, structural, and biological complexity of an organ” [22]. However, as scientific knowledge rapidly expands, different variations of the term organoids arise, diversifying the already existing nomenclature. While the organoid concept is now well established, the types of organoids vary among research groups, complicating terminology overlap. In an important initiative to reduce this diversity, a consensus of terms was established to bring clarity and consistency in their use on organoids from hepatic, bile duct, and pancreas origin [23]. Three sub-classes of organoids have been defined: (i) epithelial organoids (lower complexity and higher reproducibility), (ii) multi-tissue organoids, and (iii) multi-organ organoids (higher complexity and lower reproducibility). Multi-tissue and multi-organ organoids do not harbor the capacity of self-renewal, but remain constant in a self-organizing structure; therefore, these models can be used to study, for example, cell-to-cell interactions and organ development. On the contrary, the simplicity and capacity for self-renewal of epithelial organoids, including cholangiocyte organoids, offer an excellent resource for studying bile duct biology and pathologies.

Cholangiocyte organoids derive from a variety of origins, depending on the starting biliary cell population. As previously described, these cells can be obtained from pluripotent stem cells [24] or differentiated cells [25]. Intrahepatic cholangiocyte organoids (ICOs) derive from the intrahepatic bile duct; studies have shown their ability to create branched structures, resembling smaller conduits of the biliary tree [26]. Extrahepatic cholangiocyte organoids (ECOs) derive from the common bile duct, gallbladder cholangiocyte organoids (GCOs) derive from the gallbladder [23], and finally, bile-derived organoids (BCOs) derive from extrahepatic cholangiocytes [27].

Diversity in gene expression among different biliary cell populations indicates functional variability depending on the biliary tree location, which aligns with the progression of cholangiocyte morphology through the bile ducts and their key role in bile secretion [11]. It is therefore evident that epigenetic regulations are involved, as cholangiocytes that are within or closer to the liver (as seen in intrahepatic and common bile duct organoids) are genetically more similar to each other than those derived from the gallbladder [25, 28–30]. Interestingly, these cells can modify their epigenetic imprinting to adapt to different stimuli [25]. This feature gives these cells transient and reversible characteristics that make their culturing useful for studying bile duct biology and for opening the way to potential cell therapy applications. For example, it has been shown that when subjected to specific culture methods, ICOs (but not ECOs) from human adult intrahepatic bile ducts can organize in vitro to form branching cholangiocyte organoids (BRCOs); BRCOs can maintain this architectural organization over several passages without displaying oncogenic activity. Further functional tests have confirmed the resemblance of BRCOs to those functions described for intrahepatic bile duct cells [26].

Two distinct origins define cholangiocyte derivation. Intrahepatic cholangiocytes develop from bipotent progenitor cells called hepatoblasts, which initiate differentiation by expressing the transcription factors SOX4 and SOX9, while extrahepatic cholangiocytes develop from the endoderm and express another set of transcription factors, including PDX1, PROX1, HNF6, HNF1B, HEX1, and SOX17 [31]. Once differentiated, cholangiocytes express specific biliary markers such as KRT7, KRT19, CFTR, HNF1B, and SOX9 [32]. Cholangiocytes within the liver also differ in gene expression profiles according to the close relation between their morphology and function: small cholangiocytes present a proliferative profile, while large cholangiocytes express genes associated with reabsorbing and secreting features [31]. At the same time, cholangiocytes have further differences in transcriptional patterns alongside the biliary tree, giving location specificity and functions over the chemical modification of bile [11, 28]. Despite these differences, it has been observed that small cholangiocytes can emerge from a rather dormant state to adapt to new stimuli, acquiring characteristics and functions of large cholangiocytes in response to hormones, chemical compounds, or mechanical stress such as that imposed by bile duct ligation [31]. Such plasticity has been studied in cholangiocyte organoids as well, where cultured cells stimulated to form organoids change their original genetic profile in response to specific stimuli. Moreover, these organoids can then resume functions when implanted in animal models of bile duct injury, even when placed in a region unrelated to their origin [28].

As the understanding of organoid cultures advances, the diversity of research that this model can offer becomes larger [33, 34]. Organoids can act as mini-organs and allow the study of multiple aspects including developmental diseases, infectious diseases, genetic disorders, drug screening and testing, organ replacement therapy, cancer, organ transplantation, and regenerative medicine [35]. In this section, we will focus on organoids that are currently in use to study liver diseases.

Organoid culture methods have multiple advantages. Epithelial organoids are simple to handle, have great reproducibility, very low genetic variability, and enable scientists to quickly obtain data in living human cells [36]. In particular, the use of cholangiocyte organoids derived from adult bile ducts offers rapidly established cultures that begin to approach actual in vivo conditions that are desirable to recapitulate [33, 37]. Below, more details are discussed regarding various liver conditions that can be studied with organoid techniques.

LT has been for many years the only therapeutic alternative for patients presenting with ESLD. Burden on the healthcare system is immense and the aftercare of these patients is a challenge when cholangiopathies arise. An urgent need for new approaches to reverse or even avoid damage to the liver is ever-present.

The dynamic nature of cholangiocytes together with the development of organoid systems will be an important tool in a bright future for advances in hepatic pathology and liver transplantation research. Cholangiocyte organoids have shown great repairing capacity, opening a window for the detailed study and understanding of biliary tree pathologies, potentially preventing the development of ESLD and preventing transplantation. In an earlier stage of research, the application of organoids as a regenerative approach could aid in the treatment of several cholangiopathies that remain a major issue for patients with related complications after transplantation. The establishment of cholangiocyte organoids may be a valid option for the repopulation or regeneration of an insulted transplanted liver, using cultured cells derived from the same patient as autologous transplants, or even from donated cells in biobanks as allogenic transplants. Organoids can also be used to search for new therapeutic targets in drug screenings or find early therapeutic strategies to repair damage according to the mechanistic disease cause revealed through the utilization of these cultured structures. The three-dimensional structure of organoids allows cell-cell and cell-matrix interactive studies in a system that closely recapitulates physiological and physiopathological features of the liver. Since these cells can be induced to mimic specific diseases, their transcriptional and translational profiles will be useful to better characterize and understand pathological conditions such as IRI or cholestasis. The level of manipulation that organoids can tolerate is a key to studying the course of events in a given disease, providing specific information relevant to each patient, and allowing a better-informed strategy that could benefit individuals.

Taken together, these recent advances in cholangiocyte organoid culturing are likely to contribute to personalized medicine in LT. In addition, we have seen that the implementation of cholangiocyte organoids to alleviate complications affecting the biliary tree, brings to attention the possibility of decreasing liver transplant indications by preventing ESLD in the first place. This technology sheds light on a new approach for biliary tree pathophysiology, where the implementation of these cell cultures could rescue and help resume liver and biliary physiology in a more preventive fashion with minimal intervention.