Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes (PHHs) as a new stable source of hepatocytes for pharmaceutical research. However, HLCs have lower hepatic functions than PHHs, require a long time for differentiation and cannot be prepared in large quantities because they do not proliferate after their terminal differentiation. To overcome these problems, we here established hepatic organoids (iHOs) from HLCs. We then showed that the iHOs could proliferate approximately 105-fold by more than 3 passages and expressed most hepatic genes more highly than HLCs. In addition, to enable their widespread use for in vitro drug discovery research, we developed a two-dimensional culture protocol for iHOs. Two-dimensionally cultured iHOs (iHO-Heps) expressed most of the major hepatocyte marker genes at much higher levels than HLCs, iHOs, and even PHHs. The iHO-Heps exhibited glycogen storage capacity, the capacity to uptake and release indocyanine green (ICG), albumin and urea secretion, and the capacity for bile canaliculi formation. Importantly, the iHO-Heps had the activity of major drug-metabolizing enzymes and responded to hepatotoxic drugs, much like PHHs. Thus, iHO-Heps overcome the limitations of the current models and promise to provide robust and reproducible pharmaceutical assays.

Induced pluripotent stem cells induced hepatocytes (iHeps) are widely used in modeling human liver diseases and as a potential cell source for replacement therapy. However, most iHeps are relatively immature and challenging to maintain for long-term in vitro culture.

We optimized the differentiation protocol by addition of a combination of small molecules to inhibit epithelial-mesenchymal transition (EMT) in iHeps (iHeps EMTi), and further characterized their function both in vitro and in vivo analyses.

Inhibition of EMT extended the in vitro culture period of iHeps EMTi from day 24 to day 60. In vitro analysis revealed that, compared to control, iHeps EMTi exhibited significantly higher expression levels of hepatic functional markers and enhanced hepatocyte functions, including lipid accumulation, glycogen storage, albumin secretion, and urea acid metabolism. Moreover, the molecular profiles of iHeps EMTi are closer to those of primary human hepatocytes. In addition, the in vivo engraftment efficiency of iHeps EMTi in the chimeric mice model was also improved as compared to iHeps alone.

We established a robust protocol to generate human iHeps with improved function and capable of long-term in vitro culturing via the suppression of EMT. Moreover, those iHeps with EMT suppression have improved engraftment in human chimeric mice.

Hepatocyte organoids (HOs) hold significant potential for constructing bioartificial liver construction, toxicology research, and liver failure therapies. However, challenges such as difficulties in induced pluripotent stem cells (iPSCs) harvest and differentiation, safety concerns of tumor-derived matrices, and limited primary cell regulation hinder clinical applications. In this study, we developed a non-tumor-derived decellularized extracellular matrix (dECM) system with tunable mechanical properties and viscoelasticity to enhance stem cell proliferation and organoid functionality using thrombospondin-1 (THBS1). Nanoindentation and transcriptomic analysis revealed that THBS1 mediates adaptation and remodeling between organoids and ECM proteins, exhibiting native tissue-like viscoelasticity and up-regulated reprogramming transcriptional factors KLF4 and SOX2 via the YAP/TAZ pathway. Transplanting HOs presenting reprogramming effects into a 70 % hepatectomy model demonstrated improved liver regeneration, underscoring the potential of the THBS1-based dynamic ECM system in organoids manipulation and liver regeneration.

An organoid is a cell-based structure that shows organ-specific properties and shares a similar spatial organization as the corresponding organ. Organoids possess powerful capability to reproduce the key functions of the associated organ structures, and their similarity to the organs makes them physiologically relevant systems. The primary challenge associated with the development of skin organoids is the complexity of the human skin architecture, which encompasses the epidermis and the dermis as well as accessory structures, including hair follicles, sweat glands, and sebaceous glands, that perform various functions such as thermoregulation. The ultimate objectives of developing skin organoids are to regenerate the complete skin structure in vitro and reconstruct the skin in vivo. Consequently, safety, reliability, and the fidelity of the tissue interfaces are key considerations in this process. For this purpose, the present article reviews the most recent advances in this field, focusing on the cell sources, culture methods, culture conditions, and biomarkers for identifying the structure and function of skin organoids developed in vitro or in vivo. The subsequent sections summarize the recent applications of skin organoids in related disease diagnosis and treatments, and discuss the future prospects of these organoids in terms of clinical applications. This review of skin organoids can provide an important foundation for studies on human skin development, disease modeling, and reconstructive surgery, with broad utility for promising future opportunities in both biomedical research and clinical practice.

Advances in organoid models, as ex vivo mini-organs, and the development of screening imaging technologies have continuously driven each other forward. A complete understanding of organoids requires detailed insights into the intertwined intraorganoid and extraorganoid activities and how they change across time and space. This study introduces a multiplexed imaging platform that integrates label-free nanoplasmonic biosensing with fluorescence microscopy to offer simultaneous monitoring of dynamics occurring within and around arrays of single spheroids with spatiotemporal resolution. The label-free module employs nanoplasmonic biosensors with extraordinary optical transmission to track biomolecular secretions into the surroundings, while concurrent fluorescence imaging enables structural analysis and viability assessment. To perform multiparametric interrogation of the data from different channels over extended periods, a deep-learning-augmented image analysis is incorporated. The platform is applied to tumor spheroids to investigate vascular endothelial growth factor A secretion alongside morphometric changes and viability, showcasing its ability to capture variations in secretion and growth dynamics between untreated and drug-treated groups. This integrated approach advances comprehensive insights into organoid models and can complement existing technologies to accelerate discoveries in disease modeling and drug development.

HBV integration is considered as the main contributor to hepatocellular carcinoma (HCC). However, whether HBV integrated sequences determine genotype pathogenicity and how to block their function during HCC progression remains unclear.

An in vitro HBV-infected PHH model and liver cancer cell lines were established to confirm the pathogenic potential of HBV-SITEs. The roles of HBV-SITE-1 in HCC development were analyzed using cellular phenotypic assays and molecular biology techniques, including the combined analysis of RNA-seq and ChIP-seq. Animal models were also used to evaluate the therapeutic effect of HBV-miR-2 inhibitors.

We identified nine fragments of HBV Sequences Integrated To Enhancer, termed as "HBV-SITEs". Particularly, a single nucleotide variation (T > G) was embedded at seed sequence of HBV-miR-2 in the highest integrated HBV-SITE-1 between genotypes B and H. Unexpectedly, B-HBV-SITE-1, not H-HBV-SITE-1, could abnormally activate oncogenic genes including TERT and accelerate HCC cell proliferation and migration. Meanwhile, HBV-miR-2 was gradually increased in HBV-infected cells and patient plasma with different HCC stages. Importantly, 227 genes upregulated by HBV, were also activated by HBV-miR-2 through triggering HBV-SITE-1 enhancer. Conversely, enhancer activities were particularly decreased by HBV-miR-2 inhibitors, and further downregulated activated oncogenic genes. Finally, HCC growth was dramatically restrained and HBV-induced transcripts were systematically reduced via injection of HBV-miR-2 inhibitors in animal models.

HBV-SITEs were identified as novel oncogenic elements for HCC, which provides an insightful perspective for the other cancers caused by oncogenic DNA viruses. We demonstrated that the integrated HBV sequence itself acted as oncogenic enhancers and nucleotide variations of HBV genotypes account for particular pathogenic progression, supporting that the viral nucleotide sequences are vital pathogenic substances beyond viral proteins. And modulation of their enhancer activities could be clinically achievable strategy for blocking DNA viruses-related cancer progression in the future.

Manufacturing sufficient quantities of high-quality hepatocytes holds significant promise for the treatment of liver diseases and drug screening. Here, we developed a chemically defined, animal-free method for the large-scale production of human gallbladder epithelial cells (hGBECs) under good manufacturing practice conditions, enabling their clinical application. The cell products were characterized for growth ability, phenotype, freeze-thaw viability, genetic stability, biological contamination, tumorigenicity, and acute toxicity to ensure quality control and biological safety. We also provide a protocol for generating functional hepatocytes from hGBECs. The derived hepatocytes demonstrated typical liver functions, including albumin secretion, urea production, and drug metabolism. In addition, these cells were used in drug toxicity testing. We conducted further functional experiments on Cu2+ transport and alcohol metabolism. Transplantation of these cells in vivo was able to rescue mice from liver failure. This large-scale, convenient strategy for manufacturing hGBECs serves as a biobank for clinical applications and provides a valuable model for studying liver diseases.

Achieving high maturity and functionality in in vitro skeletal muscle models is essential for advancing our understanding of muscle biology, disease mechanisms, and drug discovery. However, current models struggle to fully recapitulate key features such as sarcomere structure, muscle fiber composition, and contractile function while also ensuring consistency and rapid production. Adult stem cells residing in muscle tissue are known for their powerful regenerative potential, yet tissue-derived skeletal muscle organoids have not been established. In this study, we introduce droplet-engineered skeletal muscle organoids derived from primary tissue using cascade-tubing microfluidics. These droplet-engineered organoids (DEOs) exhibit high maturity, including well-developed striated sarcomeres, spontaneous and stimulated contractions, and recapitulation of parental muscle fiber types. Notably, DEOs are produced in just 8 d without the need for primary cell culture-substantially accelerating the 50- to 60-d process required by classical organoid models. Additionally, the cascade-tubing microfluidics platform enables high-throughput production of hundreds of uniform DEO replicates from a small tissue sample, providing a scalable and reproducible solution for skeletal muscle research and drug screening.

Human induced pluripotent stem cells (iPSCs) can be differentiated into hepatocyte-like cells (iPS-Heps); however, maintaining the long-term proliferation and hepatic characteristics of iPS-Heps remains a challenge. In this study, we aimed to develop a human iPSC-derived hepatic organoid (iHO) culture system that effectively retains hepatic characteristics long term. Our original culture strategy, using bile acids and their receptor (farnesoid X receptor [FXR]) agonists, yielded human iHOs capable of long-term culture with a distinctive "grape-like" structure. Comprehensive analysis showed that these iHOs maintained hepatocyte-like phenotypes, even after multiple passages, whose gene expression profiles were consistent with those of fetal hepatocytes. In addition, the overexpression of small heterodimer partner (SHP), a downstream gene of FXR, in iHOs negatively regulated genes related to the intestine and cholangiocytes. Our data demonstrated that bile acid-FXR signaling promotes both the hepatic characteristics and proliferative potential of iHOs, offering promising potential for future applications in regenerative medicine and as a disease model.

Hepatitis E virus (HEV) is a zoonotic pathogen and the main cause of acute viral hepatitis in China, resulting in a significant burden on public health. Developing a highly efficient in vitro culture system for HEV is crucial for understanding the determinants of HEV infection in humans and other animals, the pathogenic mechanisms, as well as the screening and evaluation of antiviral drugs. In this paper, the research progress on HEV in vitro culture systems is reviewed to provide a convenient reference for further research on HEV, aiding comprehensive efforts toward the widespread prevention and control of related diseases.

The novel pollutant, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) leaked out of the tire and has attracted extensive concerns due to its high lethal toxicity of salmon. However, the potential hepatotoxicity of 6-PPDQ exposure and its mechanisms are unknown. As a novel 3D cell culture, liver organoids (LOs) are more similar to real organ invitro in structure and function, which showed great potential for toxicity assessment. Herein, stable LOs were generated and their applicability on hepatotoxicity assessment was evaluated with four hepatotoxic compounds. The negative effect of 6-PPDQ was explored in LOs, live/dead staining visually demonstrated the damage to the liver, and the changes of ATP, LDH, ALT, and AST effectively reflected its hepatotoxicity. Meanwhile, machine learning-based quantitative assessments of LOs morphology changes provided objective data on area, circularity, and luminance changes, enabling sensitive detection of 6-PPDQ-induced hepatotoxicity. Furthermore, transcriptomic analysis revealed that the pathways related to DNA replication and repairment, cancers, and inflammation were significantly involved in the process of 6-PPDQ-induced liver injury; Disease enrichment analysis highlighted an increased risk of chronic liver diseases, and biliary atresia were validated by Cholyl-Lys-Fluorescein (CLF). Moreover, molecular docking analysis identified potential molecular targets of 6-PPDQ, including Slc6a9, Yes1, and Nos2. This study underscored the potential of LOs for toxicological studies and highlighted the toxic effects of 6-PPDQ on the liver, suggesting the need for further investigations to understand its long-term impact on human health.

Non-alcoholic fatty liver disease (NAFLD) is a global health concern with limited treatment options. The paucity of predictive i n v itro models in preclinical settings seems to be one of the limitations of identifying effective medicines. We therefore aimed to develop an i n v itro model that can display the key hallmarks of NAFLD, such as steatosis, inflammation, and fibrosis.

An in vitro model of steatohepatitis was developed using organoids prepared from hepatocytes of healthy individuals from a commercial source (HLOs) and the liver tissues collected from needle biopsies of NAFLD patients (HLONAFLD) using defined culture conditions. HLOs were treated with palmitic acid for 72 h to develop an i n v itro model of steatohepatitis, while HLONAFLD served as a natural model of steatohepatitis. Metformin and saroglitazar were used to validate the liver organoid model of steatohepatitis. Saroglitazar was also evaluated in the high-fat, high-fructose (HF-HF) diet-induced model of NAFLD using C57BL/6 mice to validate the findings from the i n v itro model.

HLOs and HLONAFLD exhibited bipotent properties, showing the expression of markers of hepatocytes, ductal cells, and also stem cells. Furthermore, they demonstrated the expression of nonparenchymal cell markers such as stellate cells (CD166) and Kupffer-like cells (CD68 and EMR1). The steatohepatitis models developed using these organoids displayed markers associated with steatosis, inflammation and fibrosis, which were decreased by metformin and saroglitazar.

The in vitro models developed in our lab employing HLOs and HLONAFLD display all three key hallmarks of NAFLD: steatosis, inflammation, and fibrosis without the necessity for coculture with other nonparenchymal cells. The implementation of the HLONAFLD-based model is also expected to provide a more realistic assessment of test substances to develop therapeutics for NAFLD.

Patatin-like phospholipase domain-containing 3 (PNPLA3) was the first gene identified through genome-wide association studies to be linked to hepatic fat accumulation. A missense variant, encoding the PNPLA3-148M allele, has since been shown to increase the risk for the full spectrum of steatotic liver disease (SLD), from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Despite extensive validation of this association and ongoing research into its pathogenic role, the precise mechanisms by which PNPLA3-148M contributes to the progression of SLD remain poorly understood. In this review, we evaluate preclinical in vitro and in vivo models used to investigate PNPLA3 and its involvement in SLD, with particular emphasis on metabolic dysfunction-associated steatotic liver disease. We assess the strengths and limitations of these models, as well as the challenges arising from species differences in PNPLA3 expression and function between human and murine systems.

The liver fulfils a plethora of vital functions and, due to their importance, liver dysfunction has life-threatening consequences. Liver disorders currently account for more than two million deaths annually worldwide and can be classified broadly into three groups, considering their onset and aetiology, as acute liver diseases, inherited metabolic disorders and chronic liver diseases. In the most advanced and severe forms leading to liver failure, liver transplantation is the only treatment available, which has many associated drawbacks, including a shortage of organ donors. Cell therapy via fully mature cell transplantation is an advantageous alternative that may be able to restore a damaged organ's functionality or serve as a bridge until regeneration can occur. Pioneering work has shown that transplanting adult hepatocytes can support liver recovery. However, primary hepatocytes cannot be grown extensively in vitro as they rapidly lose their metabolic activity. Therefore, different cell sources are currently being tested as alternatives to primary cells. Human pluripotent stem cell-derived cells, chemically induced liver progenitors, or 'liver' organoids, hold great promise for developing new cell therapies for acute and chronic liver diseases. This Review focuses on the advantages and drawbacks of distinct cell sources and the relative strategies to address different therapeutic needs in distinct liver diseases.

Inflammatory bowel disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), is a multifactorial, immune-mediated condition marked by chronic gastrointestinal inflammation. This condition significantly impairs patients' quality of life and represents a major public health challenge globally. Pathogenesis of IBD arises from complex interplay among genetic predisposition, environmental factors, immune dysregulation, and microbial dysbiosis. Although significant strides have been made in unraveling these mechanisms, existing therapeutic options remain inadequate in addressing the full spectrum of clinical needs, underscoring the urgent demand for innovative strategies. Regenerative medicine has emerged as a promising frontier, offering novel tools for therapeutic development. We briefly consolidated current knowledge on IBD pathogenesis and treatments, emphasized the pivotal potential of human intestinal organoids (including adult stem cell-derived organoids and pluripotent stem cell- derived organoids) as a robust platform for mechanistic studies and treatment exploration. Leveraging this technology, we aim to advance personalized and next-generation therapies for IBD.

Liver regeneration is a well-organized and phase-restricted process that involves chromatin remodeling and transcriptional alterations. However, the specific transcription factors (TFs) that act as key "switches" to initiate hepatocyte regeneration and organoid formation remain unclear. Comprehensive integration of RNA sequencing and ATAC sequencing reveals that ATF3 representing "Initiation_on" TF and ONECUT2 representing "Initiation_off" TF transiently modulate the occupancy of target promoters to license liver cells for regeneration. Knockdown of Atf3 or overexpression of Onecut2 not only reduces organoid formation but also delays tissue-damage repair after PHx or CCl4 treatment. Mechanistically, we demonstrate that ATF3 binds to the promoter of Slc7a5 to activate mTOR signals while the Hmgcs1 promoter loses ONECUT2 binding to facilitate regenerative initiation. The results identify the mechanism for initiating regeneration and reveal the remodeling of transcriptional landscapes and chromatin accessibility, thereby providing potential therapeutic targets for liver diseases with regenerative defects.

Despite the development of three-dimensional (3D) tissues that promises remarkable advances in myocardial therapies and pharmaceutical research, vascularization is required for the repair of damaged hearts using cardiac tissue engineering. In this study, we developed a method for rapid generation of a 3D cardiac tissue, with extremely high engraftment efficiency, by stacking cardiomyocyte sheets using fibrin as an adhesive. Cell sheets were created by peeling off confluent cultured cells from a culture dish grafted with a polymer that induced surface hydrophilicity in response to low temperatures. The high engraftment rate was attributed to the retention of the adhesive protein. The multistacked vascularized cell sheets prepared using fibrin, when transplanted into the subcutaneous tissue and at myocardial infarction site in rats, yielded a transplanted 3D myocardial tissue. Furthermore, multilayered cardiomyocyte sheets were transplanted twice at 1 week intervals to create a 3D myocardial tissue. Our data suggest that fibrin-based rapidly layered cell sheets can advance tissue-engineered transplantation therapy and should aid the development of next-generation tissue-engineered products in the fields of regenerative medicine and drug screening.

Three-dimensional (3D) cell cultures have gained popularity in recent years due to their ability to represent complex tissues or organs more faithfully than conventional two-dimensional (2D) cell culture. This article reviews the application of both 2D and 3D microscopy approaches for monitoring and studying 3D cell cultures. We first summarize the most popular optical microscopy methods that have been used with 3D cell cultures. We then discuss the general advantages and disadvantages of various microscopy techniques for several broad categories of investigation involving 3D cell cultures. Finally, we provide perspectives on key areas of technical need in which there are clear opportunities for innovation. Our goal is to guide microscope engineers and biomedical end users toward optimal imaging methods for specific investigational scenarios and to identify use cases in which additional innovations in high-resolution imaging could be helpful.

Proliferating hepatocytes often undergo ductal metaplasia to balance the energy trade-off between cellular functions and replication, hindering the expansion of human adult hepatocytes with functional competency1. Here we demonstrate that the combined activation of Wnt and STAT3 signalling enables long-term self-renewal of human adult hepatocyte organoids. YAP activation facilitates hepatocyte proliferation but commits it towards the biliary duct lineage. By contrast, STAT3 activation by oncostatin M induces hepatocyte proliferation while counteracting ductal metaplasia and maintaining the hepatic identity. Xenotransplanted hepatocyte organoids repopulate the recipient mouse liver and reconstitute the metabolic zonation structure. Upon niche factor removal and hormone supplementation, hepatocyte organoids form cord-like structures with bile canalicular networks and exhibit major liver metabolic functions comparable to those of in vivo hepatocytes. Hepatocyte organoids are amenable to gene editing, prompting functional modelling of inherent metabolic liver diseases. The new culture system offers a promising avenue for developing therapeutic strategies against human liver diseases.

China has a high prevalence of liver diseases, with about 300 million patients suffering from various types of liver diseases, where the incidence of severe liver diseases is 1%-3%. More than half a million people die from end-stage liver disease every year in China. In situ liver transplantation is the most effective treatment for liver failure, but only a limited number of patients undergo liver transplantation due to the shortage of donors. Hepatocyte transplantation requires only a certain number of hepatocytes rather than the whole liver, regardless of complex issues such as in vitro reconstruction of the three-dimensional structure of the liver, blood vessels and biliary ducts, enabling it to be safer and easier to promote in clinical practice than liver transplantation. This study aims to evaluate the safety and tolerability of microencapsulated hepatocyte intraperitoneal transplantation therapy in adult patients with liver failure.

This study is a single-centre, unblinded, single-arm study comprising a dose escalation phase and a preliminary assessment of efficacy. Subjects who were diagnosed with liver failure (including chronic liver failure and acute-on-chronic liver failure), who received 3 days of regular treatment with no beneficial effect and who volunteered to participate in microencapsulated hepatocyte intraperitoneal transplantation therapy will be enrolled. To minimise the number of patients receiving an unbeneficial therapeutic dosage, the accelerated titration design and '3+3' design will be used jointly for the dosage escalation method. All patients with microencapsulated hepatocyte transplantation will be monitored on the 1st, 3rd, 7th, 14th, 28th, 60th and 90th days after the treatment for safety and primary efficacy analyses.

Ethical approval has been obtained from the Shanghai Jiao Tong University School of Medicine, Ren Ji Hospital Ethics Committee. Results will be disseminated through publication in a peer-reviewed journal.

NCT05727722.

Microfluidic systems have revolutionized biological research by enabling precise control over cellular environments at microscale volumes. However, traditional pump-driven systems face challenges such as complexity, cost, cell-damaging shear stress, and limited portability. This study introduces a novel adjustable microfluidic self-perfusion chip (MSPC) that uses evaporation as a driving force, eliminating the need for external pumps. Our design offers improved metabolic waste management and simplified control over fluid dynamics. The chip features adjustable evaporation pore sizes, demonstrating a robust linear relationship (R2 = 0.95) between the pore size and fluid evaporation rate. This ensures consistent fluid flow and effective waste removal, shown by lower ammonia and lactate levels compared to conventional cultures. Its unidirectional flow system and integrated one-way valve maintain cell viability, even under complete evaporation conditions. This innovative platform facilitates the cultivation of complex tissue-like structures, providing a valuable tool for tissue and organ model development, as well as drug screening and toxicity testing. By addressing key limitations of traditional systems, our adjustable MSPC represents a significant advancement in microfluidic cell culture technology, offering improved accessibility and applicability in biological research.

End-stage liver disease represents a critical hepatic condition with high mortality, for which liver transplantation remains the only effective treatment. However, the scarcity of suitable donors results in numerous patients dying while awaiting transplantation. Novel strategies, including cell therapies and technologies mimicking liver organogenesis, offer promising alternatives for treating end-stage liver disease by potentially providing new sources of liver grafts. Recently, significant progress has been made in this field, including stem cell transplantation, hepatocyte transplantation, in vitro liver tissue generation, and liver replacement technologies. Several clinical studies have demonstrated that stem cell transplantation and hepatocyte transplantation can prolong patient survival and serve as a bridge to liver transplantation. Furthermore, in vitro liver tissue generation technologies, such as liver organoids and three-dimensional bioprinting, can generate hepatic tissues with sophisticated structures and functions, making them promising transplantation materials. Notably, liver replacement technologies hold considerable potential for producing biologically functional and transplantable liver grafts. In this review, we discuss the fundamental principles and recent advancements in cell therapies and liver organogenesis technologies while also addressing the challenges and future prospects in this rapidly evolving field.

Organoid technology has significantly transformed biomedical research by providing exceptional prospects for modeling human tissues and disorders in a laboratory setting. It has significant potential for understanding the intricate relationship between genetic mutations, cellular phenotypes, and disease pathology, especially in the field of genetic diseases. The intersection of organoid technology and genetic research offers great promise for comprehending the pathophysiology of genetic diseases and creating innovative treatment approaches customized for specific patients. This review aimed to present a thorough analysis of the current advancements in organoid technology and its biomedical applications for genetic diseases. We examined techniques for modeling genetic disorders using organoid platforms, analyze the approaches for incorporating genetic disease organoids into clinical practice, and showcase current breakthroughs in preclinical application, individualized healthcare, and transplantation. Through the integration of knowledge from several disciplines, such as genetics, regenerative medicine, and biological engineering, our aim is to enhance our comprehension of the complex connection between genetic variations and organoid models in relation to human health and disease.

Microtissues, engineered to emulate the complexity of human organs, are revolutionizing the fields of regenerative medicine, disease modelling, and drug screening. Despite the promise of traditional microtissue engineering, it has yet to achieve the precision required to fully replicate organ-like structures. Enter 3D bioprinting, a transformative approach that offers unparalleled control over the microtissue's spatial arrangement and mechanical properties. This cutting-edge technology enables the detailed layering of bioinks, crafting microtissues with tissue-like 3D structures. It allows for the direct construction of organoids and the fine-tuning of the mechanical forces vital for tissue maturation. Moreover, 3D-printed devices provide microtissues with the necessary guidance and microenvironments, facilitating sophisticated tissue interactions. The applications of 3D-printed microtissues are expanding rapidly, with successful demonstrations of their functionality in vitro and in vivo. This technology excels at replicating the intricate processes of tissue development, offering a more ethical and controlled alternative to traditional animal models. By simulating in vivo conditions, 3D-printed microtissues are emerging as powerful tools for personalized drug screening, offering new avenues for pharmaceutical development and precision medicine.

The development of high-fidelity three-dimensional (3D) tissue models can minimize the need for animal models in clinical medicine and drug development. However, physical limitations regarding the distances within which diffusion processes are effective impose limitations on the size of such constructs. That is because larger-size constructs experience necrosis, especially in their centers, due to the cells residing deep inside such constructs not receiving enough oxygen and nutrients. This hampers the sustained in vitro growth of the tissues which is required for achieving functional microtissues. To address this challenge, we used three types of 3D printing technologies to create perfusable networks at different length scales and integrate them into such constructs. Toward this aim, networks incorporating porous conduits with increasingly complex configurations were designed and fabricated using fused deposition modeling, stereolithography, and two-photon polymerization while optimizing the printing conditions for each of these technologies. Furthermore, following network embedding in hydrogels, contrast agent-enhanced micro-computed tomography and confocal fluorescence microscopy were employed to characterize one of the essential network functionalities, namely the diffusion function. The investigations revealed the effects of various design parameters on the diffusion behavior of the porous conduits over 24 h. We found that the number of pores exerts the most significant influence on the diffusion behavior of the contrast agent, followed by variations in the pore size and hydrogel concentration. The analytical approach and the findings of this study establish a solid base for a new technological platform to fabricate perfusable multiscale 3D porous networks with complex designs while enabling the customization of diffusion characteristics to meet specific requirements for sustained in vitro tissue growth. STATEMENT OF SIGNIFICANCE: This study addresses an essential limitation of current 3D tissue engineering, namely, sustaining tissue viability in larger constructs through optimized nutrient and oxygen delivery. By utilizing advanced 3D printing techniques this research proposes the fabrication of perfusable, multiscale and customizable networks that enhance diffusion and enable cell access to essential nutrients throughout the construct. The findings highlighted the role of network characteristics on the diffusion of a model compound within a hydrogel matrix. This work represents a promising technological platform for creating advanced in vitro 3D tissue models that can reduce the use of animal models in research involving tissue regeneration, disease models and drug development.

Traditional toxicological assessment relied heavily on 2D cell cultures and animal models of study, which were inadequate for the precise prediction of human response to chemicals. Researchers have now shifted focus on organoids for toxicological assessment. Organoids are 3D structures produced from stem cells that mimic the shape and functionality of human organs and have a number of advantages compared to traditional models of study. They have the capacity to replicate the intricate cellular microenvironment and in vivo interactions. They offer a physiologically pertinent platform that is useful for the researchers to monitor cellular responses in a more realistic manner and evaluate drug toxicity. Additionally, organoids can be created from cells unique to a patient, allowing for individualized toxicological research and providing understanding of the inter-individual heterogeneity in drug responses. Recent developments in the use of gut and liver organoids for assessment of the xenobiotics (environmental toxins and drugs) is reviewed in this article. Gut organoids can reveal potential damage to the digestive system and how xenobiotics affect nutrient absorption and barrier function. Liver is the primary site of detoxification and metabolism of xenobiotics, usually routed from the gut. Hence, these are linked and crucial for evaluating chemical or pollutant induced organ toxicity, forecasting their metabolism and pharmacokinetics. When incorporated into the drug development process, organoid models have the potential to improve the accuracy and efficiency of drug safety assessments, leading to safer and more effective treatments. We also discuss the limitations of using organoid-based toxicological assays, and future prospects, including the need for standardized protocols for overcoming reproducibility issues.

Drug-induced liver injury (DILI) presents a major challenge not only in new drug development but also in post-marketing withdrawals and the safety of food, cosmetics, and chemicals. Experimental model organisms such as the rodents have been widely used for preclinical toxicological testing. However, the tension exists associated with the ethical and sustainable use of animals in part because animals do not necessarily inform the human-specific ADME (adsorption, dynamics, metabolism and elimination) profiling. To establish alternative models in humans, in vitro hepatic tissue models have been proposed, ranging from primary hepatocytes, immortal hepatocytes, to the development of new cell resources such as stem cell-derived hepatocytes. Given the evolving number of novel alternative methods, understanding possible combinations of cell sources and culture methods will be crucial to develop the context-of-use assays. This review primarily focuses on 3D liver organoid models for conducting. We will review the relevant cell sources, bioengineering methods, selection of training compounds, and biomarkers towards the rationale design of in vitro toxicology testing.

Acute-on-chronic liver failure (ACLF) is a syndrome that develops in approximately 30% of patients hospitalised with cirrhosis and is characterised by an acute decompensation of liver function associated with extra-hepatic organ failures and a high short-term mortality. At present, no specific therapies are available for ACLF, and current management is limited to treatment of the precipitating event and organ support. Given the high prevalence and high mortality of this severe liver disease, there is an urgent need for targeted treatments. There is increasing evidence of the important role played by systemic inflammation and immune dysfunction in the pathophysiology of ACLF and a better understanding of these immune processes is resulting in new therapeutic targets. The aim of this review is to present an overview of ongoing studies of potentially promising therapies and how they could be utilised in the management of ACLF.

The engineering construction of the liver has attracted enormous attention. Organoids, as emerging miniature three-dimensional cultivation units, hold significant potential in the biomimetic simulation of liver structure and function. Despite notable successes, organoids still face limitations such as high variability and low maturity. To overcome these challenges, engineering strategies have been established to maintain organoid stability and enhance their efficacy, laying the groundwork for the development of advanced liver organoids. The present review comprehensively summarizes the construction of engineered liver organoids and their prospective applications in biomedicine. Initially, we briefly present the latest research progress on matrix materials that maintain the three-dimensional morphology of organoids. Next, we discuss the manipulative role of engineering technologies in organoid assembly. Additionally, we outline the impact of gene-level regulation on organoid growth and development. Further, we introduce the applications of liver organoids in disease modeling, drug screening and regenerative medicine. Lastly, we overview the current obstacles and forward-looking perspectives on the future of engineered liver organoids. We anticipate that ongoing innovations in engineered liver organoids will lead to significant advancements in medical applications.

The biliary tract is a ductal network comprising the intrahepatic (IHBDs) and extrahepatic bile duct (EHBDs). Biliary duct disorders include cholangitis, neoplasms, and injury. However, the underlying mechanisms are not fully understood. With advancements in 3D culture technology, cholangiocyte organoids (COs) derived from primary tissues or induced pluripotent stem cells (iPSCs) can accurately replicate the structural and functional properties of biliary tissues. These organoids have become powerful tools for studying the pathogenesis of biliary diseases, such as cystic fibrosis and primary sclerosing cholangitis, and for developing new therapeutic strategies for cholangiocarcinoma. Additionally, COs have the potential to repair bile duct injuries and facilitate transplantation therapies. This review also discusses the use of organoids in genetically engineered mouse models to provide mechanistic insights into tumorigenesis and cancer progression. Continued innovation and standardization of organoid technology are crucial for advancing precision medicine for biliary diseases and cancer.

Liver diseases represent a growing global health challenge, and the increasing prevalence of obesity and metabolic disorders is set to exacerbate this crisis. To meet evolving regulatory demands, patient-specific in vitro liver models are essential for understanding disease mechanisms and developing new therapeutic approaches. Organoid models, which faithfully recapitulate liver biology, can be established from both non-malignant and malignant liver tissues, offering insight into various liver conditions, from acute injuries to chronic diseases and cancer. Improved understanding of liver microenvironments, innovative biomaterials, and advanced imaging techniques now facilitate comprehensive and unbiased data analysis, paving the way for personalised medicine. In this review, we discuss state-of-the-art patient-derived liver organoid models, recent technological advancements, and strategies to enhance their clinical impact.

Hepatocellular carcinoma (HCC) represents a global health challenge, and ranks among one of the most prevalent and deadliest cancers worldwide. Therapeutic advances have expanded the treatment armamentarium for patients with advanced HCC, but obstacles remain. Precision oncology, which aims to match specific therapies to patients who have tumours with particular features, holds great promise. However, its implementation has been hindered by the existence of numerous 'HCC influencers' that contribute to the high inter-patient heterogeneity. HCC influencers include tumour-related characteristics, such as genetic alterations, immune infiltration, stromal composition and aetiology, and patient-specific factors, such as sex, age, germline variants and the microbiome. This Review delves into the intricate world of HCC, describing the most innovative model systems that can be harnessed to identify precision and/or personalized therapies. We provide examples of how different models have been used to nominate candidate biomarkers, their limitations and strategies to optimize such models. We also highlight the importance of reproducing distinct HCC influencers in a flexible and modular way, with the aim of dissecting their relative contribution to therapy response. Next-generation HCC models will pave the way for faster discovery of precision therapies for patients with advanced HCC.

Hepatic organoid-based modelling, through the elucidation of a range of in vivo biological processes and the recreation of the intricate liver microenvironment, is yielding groundbreaking insights into the pathophysiology and personalized medicine approaches for liver diseases.

This study was designed to analyse the global scientific output of hepatic organoid research and assess current achievements and future trends through bibliometric analysis.

Articles were retrieved from the Web of Science Core Collection, and CiteSpace 6.3.R1 was employed to analyse the literature, including outputs, journals, and countries, among others.

Between 2010 and 2024, a total of 991 articles pertaining to hepatic organoid research were published. The journal Hepatology published the greatest number of papers, and journals with an impact factor greater than 10 constituted 60% of the top 10 journals. The United States and Utrecht University were identified as the most prolific country and institution, respectively. Clevers H emerged as the most prolific author, whereas Huch M had the highest number of cocitations, suggesting that both are ideal candidates for academic collaboration. Research on hepatic organoids has exhibited a progressive shift in focus, evolving from initial investigations into model building, differentiation research in stem cells, bile ducts, and progenitor cells, to a broader spectrum encompassing lipid metabolism, single-cell RNA sequencing, and therapeutic applications. The phrases exhibiting citation bursts from 2022 to 2024 include "drug resistance", "disease model", and "patient-derived tumor organoids".

Research on hepatic organoids has increased over the past decade and is expected to continue to grow. Key research areas include applications for liver diseases and drug development. Future trends likely to gain focus include patient-derived tumour organoids, disease modelling, and personalized medicine.

Human organoids are a widely used tool in cell biology to study homeostatic processes, disease, and development. The term organoids covers a plethora of model systems from different cellular origins that each have unique features and applications but bring their own challenges. This review discusses the basic principles underlying organoids generated from pluripotent stem cells (PSCs) as well as those derived from tissue stem cells (TSCs). We consider how well PSC- and TSC-organoids mimic the different intended organs in terms of cellular complexity, maturity, functionality, and the ongoing efforts to constitute predictive complex models of in vivo situations. We discuss the advantages and limitations associated with each system to answer different biological questions including in the field of cancer and developmental biology, and with respect to implementing emerging advanced technologies, such as (spatial) -omics analyses, CRISPR screens, and high-content imaging screens. We postulate how the two fields may move forward together, integrating advantages of one to the other.

In the past few years, the emergence of organoids and organ-on-a-chip (OOAC) technologies, which are complementary to animal models and two-dimensional cell culture methods and can better simulate the internal environment of the human body, provides a new platform for traditional Chinese medicine (TCM) studies. Organoids and OOAC techniques have been increasingly applied in the fields of drug screening, drug assessment and development, personalized therapies, and developmental biology, and there have been some application cases in the TCM studies. In this review, we summarized the current status of using organoid and OOAC technologies in TCM research and provide key insights for future study. It is believed that organoid and OOAC technologies will play more and more important roles in research and make greater contributions to the innovative development of TCM.

Organoid culture has emerged as a forefront technology in the life sciences field. As "in vitro micro-organs", organoids can faithfully recapitulate the organogenesis process, and conserve the key structure, physiological function and pathological state of the original tissue or organ. Consequently, it is widely used in basic and clinical studies, becoming important preclinical models for studying diseases and developing therapies. Here, we introduced the definition and advantages of organoids and described the development and advances in hepatobiliary organoids research. We focus on applying hepatobiliary organoids in benign and malignant diseases of the liver and biliary tract, drug research, and regenerative medicine to provide valuable reference information for the application of hepatobiliary organoids. Despite advances in research and treatment, hepatobiliary diseases including carcinoma, viral hepatitis, fatty liver and bile duct defects have still been conundrums of the hepatobiliary field. It is necessary and crucial to study disease mechanisms, establish efficient and accurate research models and find effective treatment strategies. The organoid culture technology shed new light on solving these issues. However, the technology is not yet mature, and many hurdles still exist that need to be overcome. The combination with new technologies such as CRISPR-HOT, organ-on-a-chip may inject new vitality into future development.

To date, generating viable and functional hepatocytes in large scale remains challenge. By employing 3D suspension condition with the support of low concentration Matrigel, a novel culture system was developed to generate expandable hepatoblast organoids (HB-orgs) and mature polarised hepatocyte organoids (P-hep-orgs) from human embryonic stem cells (hESCs) in both dishes and bioreactors. scRNA-seq and functional assays were used to characterise HB-orgs and P-hep-orgs. hESC-derived HB-orgs could proliferate at least for 15 passages, leading to 1012 in total cells in 4 weeks. P-hep-orgs differentiated from HB-orgs displayed characteristics of mature hepatocytes with polarisation. Moreover, single-cell RNA sequencing exhibited that over 40% of cells in P-hep-orgs were highly fidelity with human primary hepatocytes. Eventually, large-scale production of P-hep-orgs could be generated from massively expanded HB-orgs within 1 week with similar number in bioreactors, which were achieved by the enhancements in energy metabolism contribute to the expansion of HB-orgs and maturation of P-hep-orgs in bioreactors. By providing a cost-efficient and robust platform, our study represents a significant step toward manufacturing large-scale functioning hESC-derived hepatocytes for cell-based therapeutics, disease modelling, pharmacology and toxicology studies.

Cell therapy demonstrates promising potential as a substitute therapeutic approach for liver cirrhosis. We have developed a strategy to effectively expand murine and human hepatocyte-derived liver progenitor-like cells (HepLPCs) in vitro. The primary objective of the present study was to apply HepLPCs to the treatment of liver cirrhosis and to elucidate the underlying mechanisms responsible for their therapeutic efficacy.

The effects of allogeneic or xenogeneic HepLPC transplantation were investigated in rat model of liver cirrhosis. Liver tissues were collected and subjected to immunostaining to assess changes in histology. In vitro experiments used HSCs to explore the antifibrotic properties of HepLPC-secretomes and their underlying molecular mechanisms. Additionally, proteomic analysis was conducted to characterize the protein composition of HepLPC-secretomes.

Transplantation of HepLPCs resulted in decreased active fibrogenesis and net fibrosis in cirrhosis models. Apoptosis of HSCs was observed in vivo after HepLPC treatment. HepLPC-secretomes exhibited potent inhibition of TGF-β1-induced HSC activation and promoted apoptosis through signal transducer and activator of transcription (STAT)1-mediated pathways in vitro. Furthermore, synergistic effects between amphiregulin and FGF19 within HepLPC-secretomes were identified, contributing to HSC apoptosis and exerting antifibrotic effects via activation of the janus kinase-STAT1 pathway.

HepLPCs have the potential to ameliorate liver cirrhosis by inducing STAT1-dependent apoptosis in HSCs. Amphiregulin and FGF19 are key factors responsible for STAT1 activation, representing promising novel therapeutic targets for the treatment of liver cirrhosis.