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Background: The eradication of Plasmodium vivax malaria is complicated due to the presence of hypnozoites, the hidden dormant form of the parasite that is present in the liver. Currently available drug regimens are effective at killing hypnozoites but cause side effects and are difficult to administer. Studies testing drugs for liver-stage malaria remain rare and mainly rely on the use of cancerous or immortalized hepatic cells and primary hepatocytes. Methods: Organoids were used as platform to model liver-stage vivax malaria. Hepatic endoderm cells, endothelial progenitor cells and mesenchymal cells were generated from human induced pluripotent stem cells and self-assembled into liver organoids on top of Matrigel layer. Liver characteristic and maturity were examined through genes and proteins expression of liver markers, and liver functional tests before infected with Plasmodium vivax sporozoites. The infection was then verified by the detection of parasitophorous vacuole membrane proteins, Upregulated in Infectious Sporozoite 4 (UIS4), and blood-stage infection following co-culture with human reticulocytes. Results: Generated liver organoids showed upregulation of liver specific transcripts including hepatic nuclear factor 4A (HNF4A), alpha-fetoprotein (AFP), and albumin (ALB) which also confirmed by the protein expression. Furthermore, those organoids resembled mature hepatocytes in terms of albumin secretion, fat and glycogen storage and cytochrome activity. Following invasion of P. vivax sporozoites, PvUIS4 was detected and the hepatic merozoites could develop into ring-stage and early trophozoites in human reticulocytes. Moreover, differential expression patterns of genes involved in lipid and cholesterol synthesis were also detected. Conclusions: Stem cell-derived liver organoids resemble mature liver cells in terms of liver functions and are susceptible to infection with P. vivax sporozoites, paving the way for studies on the mechanism of hypnozoite formation and testing of possible hypnozoitocidal drugs. [ABSTRACT FROM AUTHOR]

Organoids are expected to function as effective human organ models for precision cancer studies and drug development. Currently, primary tissue-derived organoids, termed non-engineered organoids (NEOs), are produced by manual pipetting or liquid handling that compromises organoid-organoid homogeneity and organoid-tissue consistency. Droplet-based microfluidics enables automated organoid production with high organoid-organoid homogeneity, organoid-tissue consistency, and a significantly improved production spectrum. It takes advantage of droplet-encapsulation of defined populations of cells and droplet-rendered microstructures that guide cell selforganization. Herein, we studied the droplet-engineered organoids (DEOs), derived from mouse liver tissues and human liver tumors, by using transcriptional analysis and cellular deconvolution on bulk RNA-seq data. The characteristics of DEOs are compared with the parental liver tissues (or tumors) and NEOs. The DEOs are proven higher reproducibility and consistency with the parental tissues, have a high production spectrum and shortened modeling time, and possess inter-organoid homogeneity and inter-tumor cell heterogeneity. [ABSTRACT FROM AUTHOR]

Liver cancer a leading cause of cancer-related deaths worldwide, yet understanding of its development mechanism remains limited, and treatment barriers present substantial challenges. Owing to the heterogeneity of tumors, traditional 2D culture models are inadequate for capturing the complexity and diversity of tumor biology and understanding of the disease. Organoids have garnered considerable attention because of their ability to self-renew and develop functional structures in vitro that closely resemble those of human organs. This review explores the history of liver organoids, their cellular origins, techniques of constructing tumor microenvironments that recapitulate liver cancer organoids, and the biological and clinical applications of liver and liver cancer organoids and explores the current challenges related to liver cancer organoid applications and potentially valuable solutions, with the aim of facilitating the construction of in vitro clinical models of liver cancer therapeutic research. [ABSTRACT FROM AUTHOR]

Abstract Background The eradication of Plasmodium vivax malaria is complicated due to the presence of hypnozoites, the hidden dormant form of the parasite that is present in the liver. Currently available drug regimens are effective at killing hypnozoites but cause side effects and are difficult to administer. Studies testing drugs for liver-stage malaria remain rare and mainly rely on the use of cancerous or immortalized hepatic cells and primary hepatocytes. Methods Organoids were used as platform to model liver-stage vivax malaria. Hepatic endoderm cells, endothelial progenitor cells and mesenchymal cells were generated from human induced pluripotent stem cells and self-assembled into liver organoids on top of Matrigel layer. Liver characteristic and maturity were examined through genes and proteins expression of liver markers, and liver functional tests before infected with Plasmodium vivax sporozoites. The infection was then verified by the detection of parasitophorous vacuole membrane proteins, Upregulated in Infectious Sporozoite 4 (UIS4), and blood-stage infection following co-culture with human reticulocytes. Results Generated liver organoids showed upregulation of liver specific transcripts including hepatic nuclear factor 4A (HNF4A), alpha-fetoprotein (AFP), and albumin (ALB) which also confirmed by the protein expression. Furthermore, those organoids resembled mature hepatocytes in terms of albumin secretion, fat and glycogen storage and cytochrome activity. Following invasion of P. vivax sporozoites, PvUIS4 was detected and the hepatic merozoites could develop into ring-stage and early trophozoites in human reticulocytes. Moreover, differential expression patterns of genes involved in lipid and cholesterol synthesis were also detected. Conclusions Stem cell-derived liver organoids resemble mature liver cells in terms of liver functions and are susceptible to infection with P. vivax sporozoites, paving the way for studies on the mechanism of hypnozoite formation and testing of possible hypnozoitocidal drugs.

Antifibrotic drug screening requires evaluating the inhibitory effects of drug candidates on fibrotic cells while minimizing any adverse effects on normal cells. It is challenging to create organ-specific vascularized organoids that accurately model fibrotic and normal tissues for drug screening. Our previous studies have established methods for culturing primary microvessels and epithelial cells from adult tissues. In this proof-of-concept study, we used rats as a model organism to create a two-dimensional vascularized liver organoid model that comprised primary microvessels, epithelia, and stellate cells from adult livers. To provide appropriate substrates for cell culture, we engineered ECMs with defined stiffness to mimic the different stages of fibrotic tissues and normal tissues. We examined the effects of two TGFβ signaling inhibitors, A83-01 and pirfenidone, on the vascularized liver organoids on the stiff and soft ECMs. We found that A83-01 inhibited fibrotic markers while promoting epithelial genes of hepatocytes and cholangiocytes. However, it inhibited microvascular genes on soft ECM, indicating a detrimental effect on normal tissues. Furthermore, A83-01 significantly promoted the expression of markers of stem cells and cancers, increasing the potential risk of it being a carcinogen. In contrast, pirfenidone, an FDA-approved compound for antifibrotic treatments, did not significantly affect all the genes examined on soft ECM. Although pirfenidone had minor effects on most genes, it did reduce the expression of collagens, the major components of fibrotic tissues. These results explain why pirfenidone can slow fibrosis progression with minor side effects in clinical trials. In conclusion, our study presents a method for creating vascularized liver organoids that can accurately mimic fibrotic and normal tissues for drug screening. Our findings provide valuable insights into the potential risks and benefits of using A83-01 and pirfenidone as antifibrotic drugs. This method can be applied to other organs to create organ-specific vascularized organoids for drug development. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease (ALD) poses a significant health challenge, so comprehensive research efforts to improve our understanding and treatment strategies are needed. However, the development of effective treatments is hindered by the limitation of existing liver disease models. Liver organoids, characterized by their cellular complexity and three-dimensional (3D) tissue structure closely resembling the human liver, hold promise as ideal models for liver disease research. In this study, we use a meticulously designed protocol involving the differentiation of human induced pluripotent stem cells (hiPSCs) into liver organoids. This process incorporates a precise combination of cytokines and small molecule compounds within a 3D culture system to guide the differentiation process. Subsequently, these differentiated liver organoids are subject to ethanol treatment to induce ALD, thus establishing a disease model. A rigorous assessment through a series of experiments reveals that this model partially recapitulates key pathological features observed in clinical ALD, including cellular mitochondrial damage, elevated cellular reactive oxygen species (ROS) levels, fatty liver, and hepatocyte necrosis. In addition, this model offers potential use in screening drugs for ALD treatment. Overall, the liver organoid model of ALD, which is derived from hiPSC differentiation, has emerged as an invaluable platform for advancing our understanding and management of ALD in clinical settings.

"Organoids", three-dimensional self-organized organ-like miniature tissues, are proposed as intermediary models that bridge the gap between animal and human studies in drug development. Despite recent advancements in organoid model development, studies on toxicity using these models are limited. Therefore, in this study, we aimed to analyze the functionality and gene expression of pre- and post-differentiated human hepatic organoids derived from induced pluripotent stem cells and utilize them for toxicity assessment. First, we confirmed the functional similarity of this hepatic organoid model to the human liver through various functional assessments, such as glycogen storage, albumin and bile acid secretion, and cytochrome P450 (CYP) activity. Subsequently, utilizing these functionally validated hepatic organoids, we conducted toxicity evaluations with three hepatotoxic substances (ketoconazole, troglitazone, and tolcapone), which are well known for causing drug-induced liver injury, and three non-hepatotoxic substances (sucrose, ascorbic acid, and biotin). The organoids effectively distinguished between the toxicity levels of substances with and without hepatic toxicity. We demonstrated the potential of hepatic organoids with validated functionalities and genetic characteristics as promising models for toxicity evaluation by analyzing toxicological changes occurring in hepatoxic drug-treated organoids. [ABSTRACT FROM AUTHOR]

Abstract Organoids, three-dimensional structures cultured in vitro, can recapitulate the microenvironment, complex architecture, and cellular functions of in vivo organs or tissues. In recent decades, liver organoids have been developed rapidly, and their applications in biomedicine, such as drug screening, disease modeling, and regenerative medicine, have been widely recognized. However, the lack of repeatability and consistency, including the lack of standardized culture conditions, has been a major obstacle to the development and clinical application of liver organoids. It is time-consuming for researchers to identify an appropriate medium component scheme, and the usage of some ingredients remains controversial. In this review, we summarized and compared different methods for liver organoid cultivation that have been published in recent years, focusing on controversial medium components and discussing their advantages and drawbacks. We aimed to provide an effective reference for the development and standardization of liver organoid cultivation.

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

Liver organoids generated with single or multiple cell types have been used to investigate liver fibrosis development, toxicity, pathogenesis, and drug screening. However, organoid generation is limited by the availability of cells isolated from primary tissues or differentiated from various stem cells. To ensure cell availability for organoid formation, we investigated whether liver organoids could be generated with cell-line-based Huh-7 hepatocellular carcinoma cells, macrophages differentiated from THP-1 monocytes, and LX-2 hepatic stellate cells (HSCs) and primary liver sinusoidal endothelial cells (LSECs). In liver organoids, hepatocyte-, LSEC-, macrophage-, and HSC-related gene expression increased relative to that in two-dimensional (2D)-cultured Huh-7/LSEC/THP-1/LX-2 cells without Matrigel. Thioacetamide (TAA) increased α-smooth muscle actin expression in liver organoids but not in 2D-cultured cells, whereas in TAA-treated organoids, the expression of hepatic and LSEC markers decreased and that of macrophage and HSC markers increased. TAA-induced fibrosis was suppressed by treatment with N-acetyl-L-cysteine or tumor-necrosis-factor-stimulated gene 6 protein. The results showed that liver toxicants could induce fibrotic and inflammatory responses in liver organoids comprising Huh-7/LSEC/macrophages/LX-2 cells, resulting in fibrotic liver organoids. We propose that cell-line-based organoids can be used for disease modeling and drug screening to improve liver fibrosis treatment. [ABSTRACT FROM AUTHOR]

Organoids, three-dimensional structures cultured in vitro, can recapitulate the microenvironment, complex architecture, and cellular functions of in vivo organs or tissues. In recent decades, liver organoids have been developed rapidly, and their applications in biomedicine, such as drug screening, disease modeling, and regenerative medicine, have been widely recognized. However, the lack of repeatability and consistency, including the lack of standardized culture conditions, has been a major obstacle to the development and clinical application of liver organoids. It is time-consuming for researchers to identify an appropriate medium component scheme, and the usage of some ingredients remains controversial. In this review, we summarized and compared different methods for liver organoid cultivation that have been published in recent years, focusing on controversial medium components and discussing their advantages and drawbacks. We aimed to provide an effective reference for the development and standardization of liver organoid cultivation. [ABSTRACT FROM AUTHOR]

“Organoids”, three-dimensional self-organized organ-like miniature tissues, are proposed as intermediary models that bridge the gap between animal and human studies in drug development. Despite recent advancements in organoid model development, studies on toxicity using these models are limited. Therefore, in this study, we aimed to analyze the functionality and gene expression of pre- and post-differentiated human hepatic organoids derived from induced pluripotent stem cells and utilize them for toxicity assessment. First, we confirmed the functional similarity of this hepatic organoid model to the human liver through various functional assessments, such as glycogen storage, albumin and bile acid secretion, and cytochrome P450 (CYP) activity. Subsequently, utilizing these functionally validated hepatic organoids, we conducted toxicity evaluations with three hepatotoxic substances (ketoconazole, troglitazone, and tolcapone), which are well known for causing drug-induced liver injury, and three non-hepatotoxic substances (sucrose, ascorbic acid, and biotin). The organoids effectively distinguished between the toxicity levels of substances with and without hepatic toxicity. We demonstrated the potential of hepatic organoids with validated functionalities and genetic characteristics as promising models for toxicity evaluation by analyzing toxicological changes occurring in hepatoxic drug-treated organoids.

Non-alcoholic fatty liver disease (NAFLD) has become the world’s most common chronic liver disease. However, due to the lack of reliable in vitro NAFLD models, drug development studies have faced many limitations, and there is no food and drug administration-approved medicine for NAFLD treatment. A functional biomimetic in vitro human liver model requires an optimized natural microenvironment using appropriate cellular composition, to provide constructive cell-cell interactions, and niche-specific bio-molecules to supply crucial cues as cell-matrix interplay. Such a suitable liver model could employ appropriate and desired biochemical, mechanical, and physical properties similar to native tissue. Moreover, bioengineered three-dimensional tissues, specially microtissues and organoids, and more recently using infusion-based cultivation systems such as microfluidics can mimic natural tissue conditions and facilitate the exchange of nutrients and soluble factors to improve physiological function in the in vitro generated constructs. This review highlights the key players involved in NAFLD initiation and progression and discussed the available cells and matrices for in vitro NAFLD modeling. The strategies for optimizing the liver microenvironment to generate a powerful and biomimetic in vitro NAFLD model were described as well. Finally, the current challenges and future perospective for promotion in this subject were discussed.

Abstract Background The generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves. Methods The mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines. Results We found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage. Conclusion Our results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.

Liver cancer remains one of the most common cancers worldwide with limited therapy options. The main risk factors for hepatocellular carcinoma (HCC), the most common form of liver cancer, include chronic infection with hepatitis B or hepatitis C viruses, alcohol abuse, and metabolic disease. Current systemic therapies for advanced HCCs have greatly improved in the last decade, but there is still a need to develop more targeted drug therapy for HCCs. The development of liver organoids, a self-organising and self-renewal three-dimensional cell culture model, has greatly improved cancer research, including liver cancer. The generation of liver organoids provides a physiologically relevant model to study cancer drug screening and development, personalized medicine, liver disease modeling, and liver regeneration. However, the advent of organoid development also comes with few shortcomings that must be overcome, including the high cost of the model, the availability of origin tissues, and the need for multilineage liver organoids to replicate the true cellular heterogeneity of the liver. Despite all the limitations, liver organoids provide a reliable in vitro model for translational applications to develop more effective HCC therapy and to understand the underlying pathogenic mechanism in various liver diseases. [ABSTRACT FROM AUTHOR]

Abstract Primary liver cancer (PLC) is the second leading cause of cancer mortality worldwide, and its morbidity unceasingly increases these years. Hepatitis B virus (HBV) infection accounted for approximately 50% of hepatocellular carcinoma (HCC) cases globally in 2015. Due to the lack of an effective model to study HBV-associated liver carcinogenesis, research has made slow progress. Organoid, an in vitro 3D model which maintains self-organization, has recently emerged as a powerful tool to investigate human diseases. In this review, we first summarize the categories and development of liver organoids. Then, we mainly focus on the functions of culture medium components and applications of organoids for HBV infection and HBV-associated liver cancer studies. Finally, we provide insights into a potential patient-derived organoid model from those infected with HBV based on our study, as well as the limitations and future applications of organoids in liver cancer research.

Primary liver cancers (PLC), including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are among the leading causes of cancer-related mortality worldwide. Bi-dimensional in vitro models are unable to recapitulate the key features of PLC; consequently, recent advancements in three-dimensional in vitro systems, such as organoids, opened up new avenues for the development of innovative models for studying tumour's pathological mechanisms. Liver organoids show self-assembly and self-renewal capabilities, retaining essential aspects of their respective in vivo tissue and allowing modelling diseases and personalized treatment development. In this review, we will discuss the current advances in the field of liver organoids focusing on existing development protocols and possible applications in regenerative medicine and drug discovery. [ABSTRACT FROM AUTHOR]

Background: The generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves. Methods: The mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines. Results: We found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage. Conclusion: Our results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) has become the world’s most common chronic liver disease. However, due to the lack of reliable in vitro NAFLD models, drug development studies have faced many limitations, and there is no food and drug administration-approved medicine for NAFLD treatment. A functional biomimetic in vitro human liver model requires an optimized natural microenvironment using appropriate cellular composition, to provide constructive cell-cell interactions, and niche-specific bio-molecules to supply crucial cues as cell-matrix interplay. Such a suitable liver model could employ appropriate and desired biochemical, mechanical, and physical properties similar to native tissue. Moreover, bioengineered three-dimensional tissues, specially microtissues and organoids, and more recently using infusion-based cultivation systems such as microfluidics can mimic natural tissue conditions and facilitate the exchange of nutrients and soluble factors to improve physiological function in the in vitro generated constructs. This review highlights the key players involved in NAFLD initiation and progression and discussed the available cells and matrices for in vitro NAFLD modeling. The strategies for optimizing the liver microenvironment to generate a powerful and biomimetic in vitro NAFLD model were described as well. Finally, the current challenges and future perospective for promotion in this subject were discussed. [ABSTRACT FROM AUTHOR]

Liver organoids generated with single or multiple cell types have been used to investigate liver fibrosis development, toxicity, pathogenesis, and drug screening. However, organoid generation is limited by the availability of cells isolated from primary tissues or differentiated from various stem cells. To ensure cell availability for organoid formation, we investigated whether liver organoids could be generated with cell-line-based Huh-7 hepatocellular carcinoma cells, macrophages differentiated from THP-1 monocytes, and LX-2 hepatic stellate cells (HSCs) and primary liver sinusoidal endothelial cells (LSECs). In liver organoids, hepatocyte-, LSEC-, macrophage-, and HSC-related gene expression increased relative to that in two-dimensional (2D)-cultured Huh-7/LSEC/THP-1/LX-2 cells without Matrigel. Thioacetamide (TAA) increased α-smooth muscle actin expression in liver organoids but not in 2D-cultured cells, whereas in TAA-treated organoids, the expression of hepatic and LSEC markers decreased and that of macrophage and HSC markers increased. TAA-induced fibrosis was suppressed by treatment with N-acetyl-L-cysteine or tumor-necrosis-factor-stimulated gene 6 protein. The results showed that liver toxicants could induce fibrotic and inflammatory responses in liver organoids comprising Huh-7/LSEC/macrophages/LX-2 cells, resulting in fibrotic liver organoids. We propose that cell-line-based organoids can be used for disease modeling and drug screening to improve liver fibrosis treatment.

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., Competing Interests: Yuanjin Zhao is an editorial board member for Bioactive Materials and was not involved in the editorial review or the decision to publish this review. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Primary liver cancer (PLC) is the second leading cause of cancer mortality worldwide, and its morbidity unceasingly increases these years. Hepatitis B virus (HBV) infection accounted for approximately 50% of hepatocellular carcinoma (HCC) cases globally in 2015. Due to the lack of an effective model to study HBV-associated liver carcinogenesis, research has made slow progress. Organoid, an in vitro 3D model which maintains self-organization, has recently emerged as a powerful tool to investigate human diseases. In this review, we first summarize the categories and development of liver organoids. Then, we mainly focus on the functions of culture medium components and applications of organoids for HBV infection and HBV-associated liver cancer studies. Finally, we provide insights into a potential patient-derived organoid model from those infected with HBV based on our study, as well as the limitations and future applications of organoids in liver cancer research. [ABSTRACT FROM AUTHOR]

End-stage liver disease (ESLD) is a term used clinically in reference to a group of liver diseases with liver transplantation as the choice of treatment. Due to the limitations of liver transplantation, alternative treatments are needed. The use of primary human hepatocytes represents a valid alternative treatment, but the limitations related to hepatocyte quality, viability, function, conservation, and storage need to be overcome. Transplanted hepatocytes have only been followed for 6–9 months. Therefore, long-term causes of failures are not yet established, including rejection, apoptosis, or other causes. Other alternative therapies to replace liver transplantation include plasmapheresis, hemodiafiltration, and artificial livers. Unfortunately, these methods are highly limited due to availability, high cost, anaphylaxis reaction, development-deposition of immune-complexes, and restricted functionality. Liver organoids, which utilize stem cells instead of 'impractical' adult hepatocytes, may be a solution for the development of a complex bioartificial liver. Recent studies have explored the benefits of differentiating mature hepatocytes from stem cells inside a bioreactor. When the use of human-induced Hepatocytes (hiHeps) was investigated in mouse and pig models of liver failure, liver failure markers were decreased, hepatocyte function indicated by albumin synthesis improved, and survival time increased. Bioartificial liver treatment may decrease the infiltration of inflammatory cells into liver tissue by down-regulating pro-inflammatory cytokines. [ABSTRACT FROM AUTHOR]

As the largest internal organ, the liver is the key hub for many physiological processes. Previous research on the liver has been mainly conducted on animal models and cell lines, in which not only there are deficiencies in species variability and retention of heritable material, but it is also difficult for primary hepatocytes to maintain their metabolic functions after in vitro expansion. Because of the increased burden of liver disease worldwide, there is a growing demand for 3D in vitro liver models—Liver Organoids. Based on the type of initiation cells, the liver organoid can be classified as PSC-derived or ASC-derived. Liver organoids originated from ASC or primary sclerosing cholangitis, which are co-cultured in matrix gel with components such as stromal cells or immune cells, and eventually form three-dimensional structures in the presence of cytokines. Liver organoids have already made progress in drug screening, individual medicine and disease modeling with hereditary liver diseases, alcoholic or non-alcoholic liver diseases and primary liver cancer. In this review, we summarize the generation process of liver organoids and the current clinical applications, including disease modeling, drug screening and individual medical treatment, which provide new perspectives for liver physiology and disease research. [ABSTRACT FROM AUTHOR]

A liver organoid is an in vitro reconstruction of the liver that mimics the in vivo liver microstructure and performs liver functions. Liver organoids can be used for drug testing, as a model of liver disease pathogenesis, and as a bioartificial liver prototype material to develop promising alternative therapies for liver failure. In this study, we reconstructed liver organoids using primary rat hepatocytes, a hepatic stellate cell line (LX2), human umbilical cord-mesenchymal stem cells (UC-MSCs), and human umbilical cord blood (UCB)-CD34+ hematopoietic stem/progenitor cells. Suspensions of primary rat hepatocytes, LX2 cells, UC-MSCs, and UCB-CD34+ cells were co-cultured using 11 ratio sets, and spheroid formation was evaluated for 2 days. Ratio sets with a positive liver organoid appearance were cultured in four different culture media, and after they were harvested, their viability was compared with that of a hepatocyte monoculture. Liver organoids were further analyzed for 14 days to assess albumin and urea production as well as relative gene expression. We found that a 5:1:2:2 cellular density ratio of hepatocytes:LX2 cells:UC-MSCs:UCB-CD34+ cells, respectively, and Williams E medium supplemented with platelet lysate, ITS, and dexamethasone were the most suitable conditions for liver organoid reconstruction. Expression of the albumin and GPT1 genes and CD31 in the liver organoid increased until day 14, while urea secretion increased until day 5. Liver organoids reconstructed through the 3D co-culture of primary rat hepatocytes, LX2 cells, UC-MSCs, and UCB-CD34+ cells at a specific cellular ratio using an economical medium with a simple composition maintained their functions until day 14. As a future direction, these organoids can be used to develop a bioartificial liver.

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

The liver is the most important metabolic hub of endo and xenobiotic compounds. Pre-clinical studies using rodents to evaluate the toxicity of new drugs and cosmetics may produce inconclusive results for predicting clinical outcomes in humans, moreover being banned in the European Union. Human liver modeling using primary hepatocytes presents low reproducibility due to batch-to-batch variability, while iPSC-derived hepatocytes in monolayer cultures (2D) show reduced cellular functionality. Here we review the current status of the two most robust in vitro approaches in improving hepatocyte phenotype and metabolism while mimicking the hepatic physiological microenvironment: organoids and liver-on-chip. Both technologies are reviewed in design and manufacturing techniques, following cellular composition and functionality. Furthermore, drug screening and liver diseases modeling efficiencies are summarized. Finally, organoid and liver-on-chip technologies are compared regarding advantages and limitations, aiming to guide the selection of appropriate models for translational research and the development of such technologies.

Summary: Organoid technology is a state‐of‐the‐art cell culture tool that has revolutionized study of development, regeneration, and diseases. Human liver organoids (HLOs) are now derived from either adult stem/progenitors or pluripotent stem cells (PSCs), emulating cellular diversity and structural symphony akin to the human liver. With the rapid rise in decompensated liver disease conditions only treated by liver transplant therapy, HLOs represent an alternate source for transplantation to address the ongoing shortage of grafts. Although ongoing advancements in bioengineering technology have moved the organoid transplant approach to the next level, sustained survival of the transplanted tissue still eludes us toward functional organ replacement. Herein, we review the development of HLOs and discuss promises and challenges on organoid transplant approaches. Organoid based therapy potentially revolutionize future of transplant medicine. We herein review state‐of‐art development of liver organoid transplantation approaches and discuss their promise and challenges towards clinical use. [ABSTRACT FROM AUTHOR]

Organoids are three-dimensional structures fabricated in vitro from pluripotent stem cells or adult tissue stem cells via a process of self-organization that results in the formation of organ-specific cell types. Human organoids are expected to mimic complex microenvironments and many of the in vivo physiological functions of relevant tissues, thus filling the translational gap between animals and humans and increasing our understanding of the mechanisms underlying disease and developmental processes. In the last decade, organoid research has attracted increasing attention in areas such as disease modeling, drug development, regenerative medicine, toxicology research, and personalized medicine. In particular, in the field of toxicology, where there are various traditional models, human organoids are expected to blaze a new path in future research by overcoming the current limitations, such as those related to differences in drug responses among species. Here, we discuss the potential usefulness, limitations, and future prospects of human liver, heart, kidney, gut, and brain organoids from the viewpoints of predictive toxicology research and drug development, providing cutting edge information on their fabrication methods and functional characteristics. [ABSTRACT FROM AUTHOR]

Organoids are three-dimensional structures fabricated in vitro from pluripotent stem cells or adult tissue stem cells via a process of self-organization that results in the formation of organ-specific cell types. Human organoids are expected to mimic complex microenvironments and many of the in vivo physiological functions of relevant tissues, thus filling the translational gap between animals and humans and increasing our understanding of the mechanisms underlying disease and developmental processes. In the last decade, organoid research has attracted increasing attention in areas such as disease modeling, drug development, regenerative medicine, toxicology research, and personalized medicine. In particular, in the field of toxicology, where there are various traditional models, human organoids are expected to blaze a new path in future research by overcoming the current limitations, such as those related to differences in drug responses among species. Here, we discuss the potential usefulness, limitations, and future prospects of human liver, heart, kidney, gut, and brain organoids from the viewpoints of predictive toxicology research and drug development, providing cutting edge information on their fabrication methods and functional characteristics.

The current organoid culture systems allow pluripotent and adult stem cells to self-organize to form three-dimensional (3D) structures that provide a faithful recapitulation of the architecture and function of in vivo organs. In particular, human pluripotent stem cell-derived liver organoids (PSC-LOs) can be used in regenerative medicine and preclinical applications, such as disease modeling and drug discovery. New bioengineering tools, such as microfluidics, biomaterial scaffolds, and 3D bioprinting, are combined with organoid technologies to increase the efficiency of hepatic differentiation and enhance the functional maturity of human PSC-LOs by precise control of cellular microenvironment. Long-term stabilization of hepatocellular functions of in vitro liver organoids requires the combination of hepatic endodermal, endothelial, and mesenchymal cells. To improve the biological function and scalability of human PSC-LOs, bioengineering methods have been used to identify diverse and zonal hepatocyte populations in liver organoids for capturing heterogeneous pathologies. Therefore, constructing engineered liver organoids generated from human PSCs will be an extremely versatile tool in in vitro disease models and regenerative medicine in future. In this review, we aim to discuss the recent advances in bioengineering technologies in liver organoid culture systems that provide a timely and necessary study to model disease pathology and support drug discovery in vitro and to generate cell therapy products for transplantation.

Alcohol-associated liver disease (ALD) is a prevalent liver disease, yet research is hampered by the lack of suitable and reliable human ALD models. Herein, we generated human adipose stromal/stem cell (hASC)-derived hepatocellular organoids (hAHOs) and hASC-derived liver organoids (hALOs) in a three-dimensional system using hASC-derived hepatocyte-like cells and endodermal progenitor cells, respectively. The hAHOs were composed of major hepatocytes and cholangiocytes. The hALOs contained hepatocytes and nonparenchymal cells and possessed a more mature liver function than hAHOs. Upon ethanol treatment, both steatosis and inflammation were present in hAHOs and hALOs. The incubation of hALOs with ethanol resulted in increases in the levels of oxidative stress, the endoplasmic reticulum protein thioredoxin domain-containing protein 5 (TXNDC5), the alcohol-metabolizing enzymes ADH1B and ALDH1B1, and extracellular matrix accumulation, similar to those of liver tissues from patients with ALD. These results present a useful approach for understanding the pathogenesis of ALD in humans, thus facilitating the discovery of effective treatments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background The use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells. Methods The peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12–17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy. Results CD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

Understanding the development, regeneration, and disorders of the liver is the major goal in liver biology. Current mechanistic knowledge of human livers has been largely derived from mouse models and cell lines, which fall short in recapitulating the features of human liver cells or the structures and functions of human livers. Organoids as an in vitro system hold the promise to generate organ-like tissues in a dish. Recent advances in human liver organoids also facilitate the understanding of the biology and diseases in this complex organ. Here we review the progress in human liver organoids, mainly focusing on the methods to generate liver organoids, their applications, and possible future directions. [ABSTRACT FROM AUTHOR]

Liver organoids (LOs) are receiving considerable attention for their potential use in drug screening, disease modeling, and transplantable constructs. Hepatocytes, as the key component of LOs, are isolated from the liver or differentiated from pluripotent stem cells (PSCs). PSC-derived hepatocytes are preferable because of their availability and scalability. However, efficient maturation of the PSC-derived hepatocytes towards functional units in LOs remains a challenging subject. The incorporation of cell-sized microparticles (MPs) derived from liver extracellular matrix (ECM), could provide an enriched tissue-specific microenvironment for further maturation of hepatocytes inside the LOs. In the present study, the MPs were fabricated by chemical cross-linking of a water-in-oil dispersion of digested decellularized sheep liver. These MPs were mixed with human PSC-derived hepatic endoderm, human umbilical vein endothelial cells, and mesenchymal stromal cells to produce homogenous bioengineered LOs (BLOs). This approach led to the improvement of hepatocyte-like cells in terms of gene expression and function, CYP activities, albumin secretion, and metabolism of xenobiotics. The intraperitoneal transplantation of BLOs in an acute liver injury mouse model led to an enhancement in survival rate. Furthermore, efficient hepatic maturation was demonstrated after ex ovo transplantation. In conclusion, the incorporation of cell-sized tissue-specific MPs in BLOs improved the maturation of human PSC-derived hepatocyte-like cells compared to LOs. This approach provides a versatile strategy to produce functional organoids from different tissues and offers a novel tool for biomedical applications.

Several types of human stem cells from embryonic (ESCs) and induced pluripotent (iPSCs) to adult tissue-specific stem cells are commonly used to generate 3D liver organoids for modeling tissue physiology and disease. We have recently established a protocol for direct conversion of primary human hepatocytes (hPHs) from healthy donor livers into bipotent progenitor cells (hCdHs). Here we extended this culture system to generate hCdH-derived liver organoids for diverse biomedical applications. To obtain hCdHs, hPHs were cultured in reprogramming medium containing A83-01 and CHIR99021 for 7 days. Liver organoids were established from hCdHs (hCdHOs) and human liver cells (hLOs) using the same donor livers for direct comparison, as well as from hiPSCs. Organoid properties were analyzed by standard in vitro assays. Molecular changes were determined by RT-qPCR and RNA-seq. Clinical relevance was evaluated by transplantation into FRG mice, modeling of alcohol-related liver disease (ARLD), and in vitro drug-toxicity tests. hCdHs were clonally expanded as organoid cultures with low variability between starting hCdH lines. Similar to the hLOs, hCdHOs stably maintained stem cell phenotype based on accepted criteria. However, hCdHOs had an advantage over hLOs in terms of EpCAM expression, efficiency of organoid generation and capacity for directed hepatic differentiation as judged by molecular profiling, albumin secretion, glycogen accumulation, and CYP450 activities. Accordingly, FRG mice transplanted with hCdHOs survived longer than mice injected with hLOs. When exposed to ethanol, hCdHOs developed stronger ARLD phenotype than hLOs as evidenced by transcriptional profiling, lipid accumulation and mitochondrial dysfunction. In drug-induced injury assays in vitro , hCdHOs showed a similar or higher sensitivity response than hPHs. hCdHOs provide a novel patient-specific stem cell-based platform for regenerative medicine, toxicology testing and modeling liver diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

UGT1A1 (UDP glucuronosyltransferase family 1 member A1) is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic anion transporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Dyskeratosis congenita (DC) is a telomere biology disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction experience multiorgan failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear., Methods: We used isogenic human induced pluripotent stem cells (iPSCs) harboring a causal DC mutation in DKC1 or a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-corrected control allele to model DC liver pathologies. We differentiated these iPSCs into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single-cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships., Results: Directed differentiation of iPSCs into HEPs and stellate cells and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, with DC HEPs becoming hyperplastic and also eliciting a pathogenic hyperplastic, proinflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant HEPs and hepatostellate organoids could be rescued via suppression of serine/threonine kinase AKT (protein kinase B) activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation., Conclusions: Isogenic iPSC-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Liver organoids are primary or pluripotent stem cell-derived three-dimensional structures that recapitulate regenerative or ontogenetic processes in vitro towards biomedical applications including disease modelling and diagnostics, drug safety and efficacy prediction, and therapeutic use. The cellular composition and structural organization of liver organoids may vary depending on the goal at hand, and the key challenge in general is to direct their development in a rational and controlled fashion for gaining targeted maturity, reproducibility, and scalability. Such endeavor begins with a detailed understanding of the biological processes in space and time behind hepatogenesis, followed by precise translation of these narrative processes through a bioengineering approach. Here, we discuss advancements in liver organoid technology through the lens of 'narrative engineering' in an attempt to synergize evolving understanding around molecular and cellular landscape governing hepatogenesis with engineering-inspired approaches for organoidgenesis. [ABSTRACT FROM AUTHOR]