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![Structures of the liver lobule and liver sinusoids. Hepatocytes are aligned radially to form the liver plate along with the sinusoids. The portal veins and hepatic artery branches terminate in the sinusoids, draining blood into the sinusoids and through the acinus to the central vein. The sinusoids are lined by fenestrated liver sinusoidal endothelial cells with Kupffer cells interspersed onto the endothelium. Between the liver plate and the sinusoids is the space of Disse, containing extracellular matrix components and hepatic stellate cells [5].](publication/356217234/figure/fig1/AS:1091807777689603@1637318667646/Structures-of-the-liver-lobule-and-liver-sinusoids-Hepatocytes-are-aligned-radially-to.png)
Structures of the liver lobule and liver sinusoids. Hepatocytes are aligned radially to form the liver plate along with the sinusoids. The portal veins and hepatic artery branches terminate in the sinusoids, draining blood into the sinusoids and through the acinus to the central vein. The sinusoids are lined by fenestrated liver sinusoidal endothelial cells with Kupffer cells interspersed onto the endothelium. Between the liver plate and the sinusoids is the space of Disse, containing extracellular matrix components and hepatic stellate cells [5].
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![Figure 2. Functions of activated HSCs in diseased livers [19].](publication/356217234/figure/fig2/AS:1091807781896192@1637318668392/Functions-of-activated-HSCs-in-diseased-livers-19_Q320.jpg)
Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help...
Contexts in source publication
Context 1
... sinusoids are 10-15 µm cavities with physiological microenvironments localized among liver plates as shown in Figure 1. LSs demonstrate strong permeability for the material exchange between liver cells and bloodstream due to the presence of fenestrated liver sinusoidal endothelial cells. ...
Context 2
... are polygonal in shape and are polarized. Inside the liver, they are located either facing sinusoids (Sinusoidal face/sinusoidal domain) or facing neighboring HEPs (Lateral face/canalicular domain) as shown in Figure 1. Some canalicular domain HEPs are modified to form the bile canaliculi and are exposed to highly concentrated bile salts for bile formation, whereas sinusoidal HEPs have microvilli on the sinusoidal face for better absorption from the plasma [8]. ...
Context 3
... are spindle-shaped cells with an oval or elongated nucleus. They are present in the subendothelial space between hepatic plates and the anti-luminal face of endothelial cells as shown in Figure 1. They include one-third of the total non-parenchymal cells and 5-8% of total cells present in the liver. ...
Context 4
... are elongated endothelial cells and are present at the interface of blood cells, hepatic stellate cells, and hepatocytes. Lack of diaphragm and basement membrane, along with the association of 'fenestrae' makes them the most permeable cells in the mammalian body, as shown in Figure 1. They are highly specialized cells and represent a major fraction of non-parenchymal cells (48%). ...
Context 5
... are elongated endothelial cells and are present at the interface of blood cells, hepatic stellate cells, and hepatocytes. Lack of diaphragm and basement membrane, along with the association of 'fenestrae' makes them the most permeable cells in the mammalian body, as shown in Figure 1. They are highly specialized cells and represent a major fraction of non-parenchymal cells (48%). ...
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Recent advancements in 3D in vitro culture have allowed for the development of cancer tissue models which accurately recapitulate the tumour microenvironment. Consequently, there has been increased innovation in therapeutic drug screening. While organoid cultures show great potential, they are limited by the time scale of their growth in vitro and...
Citations
... However, 2D monocultures of hepatocytes or of hepatic cell lines suffer from several disadvantages associated with the loss of tissuespecific architecture, mechanical and biomechanical cues, and cellecell and cellematrix interactions. Consequently, these models fail to recapitulate the complexity of the in vivo physiological environment, show limited prediction capacity for xenobiotics, and cells are prone to dedifferentiation within 48e72 h (1,12,13). ...
The liver is one of the main organs involved in the metabolism of xenobiotics and a key organ in toxicity studies. Prior to accessing the hepatocytes, xenobiotics pass through the hepatic sinusoid formed by liver sinusoidal endothelial cells (LSECs). The LSECs barrier regulates the kinetics and concentrations of the xenobiotics before their metabolic processing by the hepatocytes. To mimic this physiological situation, we developed an in vitro model reproducing an LSECs barrier in coculture with a hepatocyte biochip, using a fluidic platform. This technology made dynamic coculture and tissue crosstalk possible. SK-HEP-1 and HepG2/C3a cells were used as LSECs and as hepatocyte models, respectively. We confirmed the LSECs phenotype by measuring PECAM-1 and stabilin-2 expression levels and the barrier's permeability/transport properties with various molecules. The tightness of the SK-HEP-1 barrier was enhanced in the dynamic coculture. The morphology, albumin secretion, and gene expression levels of markers of HepG2/C3a were not modified by coculture with the LSECs barrier. Using acetaminophen, a well-known hepatotoxic drug, to study tissue crosstalk, there was a reduction in the expression levels of the LSECs markers stabilin-2 and PECAM-1, and a modification of those of CLEC4M and KDR. No HepG2/C3a toxicity was observed. The metabolisation of acetaminophen by HepG2/C3a monocultures and cocultures was confirmed. Although primary cells are required to propose a fully relevant model, the present approach highlights the potential of our system for investigating xenobiotic metabolism and toxicity.
... Of course, a future direction of liver organoid technology should aim to fully reproduce the inter-cellular, inter-tissue, and even inter-organ communications that occur in the human body. Microfluidic-based organ-on-chip technology, together with standardized liver organoid production technology, may facilitate this concept (210,211). However, current liver organoid technology is able to recapitulate only relatively limited crosstalk across distinct cell types, tissues, and organs. ...
Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) have presented a major and common health concern worldwide due to their increasing prevalence and progressive development of severe pathological conditions such as cirrhosis and liver cancer. Although a large number of drug candidates for the treatment of NASH have entered clinical trial testing, all have not been released to market due to their limited efficacy, and there remains no approved treatment for NASH available to this day. Recently, organoid technology that produces 3D multicellular aggregates with a liver tissue-like cytoarchitecture and improved functionality has been suggested as a novel platform for modeling the human-specific complex pathophysiology of NAFLD and NASH. In this review, we describe the cellular crosstalk between each cellular compartment in the liver during the pathogenesis of NAFLD and NASH. We also summarize the current state of liver organoid technology, describing the cellular diversity that could be recapitulated in liver organoids and proposing a future direction for liver organoid technology as an in vitro platform for disease modeling and drug discovery for NAFLD and NASH.
... An explanation of these discordant results can be found in the fact that disease onset and progression implicate the interaction between various cell lineages within the same organ. Therefore, the presence in the same organoid of all these cell types that participate in the development of a specific disease becomes of utmost importance to fully recapitulate any aspect of the diseased organ [148][149][150][151][152][153][154][155]. Organoids provide a better representation than primary cells in culture, as they recapitulate a phenotype closer to the in vivo condition, allowing for a higher disease model fidelity. ...
Over 40,000 patients in the United States are estimated to suffer from end-stage liver disease and acute hepatic failure, for which liver transplantation is the only available therapy. Human primary hepatocytes (HPH) have not been employed as a therapeutic tool due to the difficulty in growing and expanding them in vitro, their sensitivity to cold temperatures, and tendency to dedifferentiate following two-dimensional culture. The differentiation of human-induced pluripotent stem cells (hiPSCs) into liver organoids (LO) has emerged as a potential alternative to orthotropic liver transplantation (OLT). However, several factors limit the efficiency of liver differentiation from hiPSCs, including a low proportion of differentiated cells capable of reaching a mature phenotype, the poor reproducibility of existing differentiation protocols, and insufficient long-term viability in vitro and in vivo. This review will analyze various methodologies being developed to improve hepatic differentiation from hiPSCs into liver organoids, paying particular attention to the use of endothelial cells as supportive cells for their further maturation. Here, we demonstrate why differentiated liver organoids can be used as a research tool for drug testing and disease modeling, or employed as a bridge for liver transplantation following liver failure.
... However, 2D monocultures of hepatocytes or of hepatic cell lines suffer from several disadvantages associated with the loss of tissuespecific architecture, mechanical and biomechanical cues, and cellecell and cellematrix interactions. Consequently, these models fail to recapitulate the complexity of the in vivo physiological environment, show limited prediction capacity for xenobiotics, and cells are prone to dedifferentiation within 48e72 h (1,12,13). ...
The liver is one of the main organs involved in the metabolism of xenobiotics and a key organ in toxicity studies. Prior to accessing the hepatocytes, xenobiotics pass through the hepatic sinusoid formed by liver sinusoidal endothelial cells (LSECs). The LSECs barrier regulates the kinetics and concentrations of the xenobiotics before their metabolic processing by the hepatocytes. To mimic this physiological situation, we developed an in vitro model reproducing an LSECs barrier in coculture with a hepatocyte biochip, using a fluidic platform. This technology made dynamic coculture and tissue crosstalk possible. SK-HEP-1 and HepG2/C3a cells were used as LSECs and as hepatocyte models, respectively. We confirmed the LSECs phenotype by measuring PECAM-1 and stabilin-2 expression levels and the barrier’s permeability/transport properties with various molecules. The tightness of the SK-HEP-1 barrier was enhanced in the dynamic coculture. The morphology, albumin secretion, and gene expression levels of markers of HepG2/C3a were not modified by coculture with the LSECs barrier. Using paracetamol, a well-known hepatotoxic drug, to study tissue crosstalk, there was a reduction in the expression levels of the LSECs markers stabilin-2 and PECAM-1, and a modification of those of CLEC4M and KDR. No HepG2/C3a toxicity was observed. The metabolisation of paracetamol by HepG2/C3a monocultures and cocultures was confirmed. Although primary cells are required to propose a fully relevant model, the present approach highlights the potential of our system for investigating xenobiotic metabolism and toxicity.
... In fact, hepatocytes are highly polarized cells that require their 3D environment to remain functional throughout the time span of the toxicity assay. 78 The research area of 3D models is expanding at a rapid pace. 3D models usually consist of cells cultured in 3D conditions, in the presence 79 or absence 80 of a supporting material. ...
Drug induced liver injury (DILI) is one of the major reasons of drug withdrawal during the different phases of drug development. The later in the drug development a drug is discovered to be toxic, the higher the economical as well as the ethical impact will be. In vitro models for early detection of drug liver toxicity are under constant development, however to date a superior model of the liver is still lacking. Ideally, a highly reliable model should be established to maintain the different hepatic cell functionalities to the greatest extent possible, during a period of time long enough to allow for tracking of the toxicity of compounds. In the case of DILI, toxicity can appear even after months of exposure. To reach this goal, an in vitro model should be developed that mimics the in vivo liver environment, function and response to external stimuli. The different approaches for the development of liver models currently used in the field of tissue engineering will be described in this review. Combining different technologies, leading to optimal materials, cells and 3D-constructs will ultimately lead to an ideal superior model that fully recapitulates the liver.
... Optimizing decellularization methods has become an interesting issue for researchers in order to reduce the destructive impact of the current techniques by selecting suitable material and conditions regarding cellular density, specific ECM structure, physiological function, and anatomy of aimed tissue (7). The optimized decellularization method is a rapid and effective procedure for cell destruction and removal of the cell membrane, cytoplasmic organelles, and nuclear [35][36][37][38][39][40][41][42][43][44] Liver Decellularization through Different Perfusing Routes content debris from the tissue in the shortest time. Simultaneously, maintaining the structural components of tissue scaffold such as various collagen types, laminin, fibronectin, and glycosaminoglycans (GAGs) and preventing washing of growth factors, cytokines, and other signaling molecules out of the ECM are essential parameters in an optimized decellularization protocol (8). ...
... As well, human umbilical vein endothelial cells (HUVECs) are one of the most popular cell line used as an in vitro model for endothelial cells (39). We used these two different cell lines to assess cell proliferation on decellularized liver scaffolds because the liver is composed of many different cell types in which, hepatocytes and endothelial cells have some essential performances as two types of primary cells (non-parenchymal cells) (40). ...
Objective:
Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack ofsufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical applicationpotential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellularcomponents and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy throughthree anatomical routes.
Materials and Methods:
In this experimental study, rat liver decellularization was performed through biliary duct (BD),portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy wasevaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs).ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electronmicroscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines.
Results:
Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BDroute produced an opaque scaffold with incomplete decellularization. H&E staining confirmed these results. MaximumDNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNAcontent decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups weresignificantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV andHV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEMimages showed that the ECM structure was preserved and cellular components were entirely removed. MTT assayshowed the biocompatibility of the decellularized scaffold.
Conclusion:
The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.
... The development of 3D in vitro models for the detection of the chronic hepatotoxicity of drugs has shown greater effectiveness than 2D cultures [73,74]. To establish the ability of our gelatin-based cryostructurates to be the basis for creating a 3D model for pharmacological studies, we used HepG2 cells since they perform liver cell-specific functions [75,76]. ...
Various gelatin-containing gel materials are used as scaffolds for animal and human cell culturing within the fields of cell technologies and tissue engineering. Cryostructuring is a promising technique for the preparation of efficient macroporous scaffolds in biomedical applications. In the current study, two new gelatin-based cryostructurates were synthesized, their physicochemical properties and microstructure were evaluated, and their ability to serve as biocompatible scaffolds for mammalian cells culturing was tested. The preparation procedure included the dissolution of Type A gelatin in water, the addition of urea to inhibit self-gelation, the freezing of such a solution, ice sublimation in vacuo, and urea extraction with ethanol from the freeze-dried matter followed by its cross-linking in an ethanol medium with either carbodiimide or glyoxal. It was shown that in the former case, a denser cross-linked polymer phase was formed, while in the latter case, the macropores in the resultant biopolymer material were wider. The subsequent biotesting of these scaffolds demonstrated their biocompatibility for human mesenchymal stromal cells and HepG2 cells during subcutaneous implantation in rats. Albumin secretion and urea synthesis by HepG2 cells confirmed the possibility of using gelatin cryostructurates for liver tissue engineering.
... HepaRG cells appeared to be more sensitive and more suitable toward FIAU toxicity prediction than HepG2 cells. In addition, 3D culture models are more conducive to detection of chronic drug hepatotoxicity over 2D culture systems owing to in vivo-like features [54,70]. Overall, our protein-based cryogels made of mainly albumin methacryloyl as a building block featured a highly porous interconnected structure, had liver ECM coating capability, supported 3D cell culture, and exhibited drug screening potential. ...
Drug-induced liver injury (DILI) is a leading cause of attrition in drug development or withdrawal; current animal experiments and traditional 2D cell culture systems fail to precisely predict the liver toxicity of drug candidates. Hence, there is an urgent need for an alternative in vitro model that can mimic the liver microenvironments and accurately detect human-specific drug hepa-totoxicity. Here, for the first time we propose the fabrication of an albumin methacryloyl cryogel platform inspired by the liver's microarchitecture via emulating the mechanical properties and ex-tracellular matrix (ECM) cues of liver. Engineered crosslinkable albumin methacryloyl is used as a protein-based building block for fabrication of albumin cryogel in vitro models that can have potential applications in 3D cell culture and drug screening. In this work, protein modification, cryo-gelation, and liver ECM coating were employed to engineer highly porous three-dimensional cryo-gels with high interconnectivity, liver-like stiffness, and liver ECM as artificial liver constructs. The resulting albumin-based cryogel in vitro model provided improved cell-cell and cell-material interactions and consequently displayed excellent liver functional gene expression, being conducive to detection of fialuridine (FIAU) hepatotoxicity.
... The liver comprises several cell types including hepatocytes, stellate cells, Kupffer cells, and liver sinusoidal endothelial cells. Any damage to these cells caused by drug toxicity, excessive alcohol consumption, infectious agents (hepatitis virus), or physical trauma, can result in hepatic fibrosis and even cirrhosis, which leads to one million annual deaths globally [80,81]. Notably, the generation of liver or hepatic organoids is motivating mechanistic studies of liver diseases and efforts to identify potential drugs for organ therapies using in vitro models. ...
Conventional 2-dimensional cell culture poorly mimics human-relevant models, which is considered a major challenge in biological research. Organoids are a recent breakthrough in 3-dimensional (3D) in vitro tissue engineering that better reflect the physiological, morphological, and functional properties of in vivo organs (e.g., brain, heart, kidney, lung, and liver). Consequently, organoids are extensively used in various impactful biomedical applications including organ development, disease modeling, and clinical drug testing. However, organoid technology still has several limitations, including low reproducibility, vascularization, limited nutrient uptake and distribution (affecting the level of organoid maturation), lack of standardization, and intra-clonal variability. Efforts have been made to overcome these shortcomings of organoid culture. Microfluidic technology has successfully facilitated the establishment of organoid-on-a-chip systems, which effectively improve the structural and physiological features of organoids in a controlled manner. This review discusses the recent advances and developments in organoid-on-a-chip technology. We hope that this study will motivate researchers to explore the possible engagement between microfluidic devices and self-assembled 3D cell cultures to leverage the enhanced quality of organoids, which will have favorable impacts on future tissue regeneration and regenerative therapies.
... The vast majority of liver modeling in vitro is still carried out by the use of static culture platforms (Panwar et al., 2021), resulting in the maintenance of metabolic competence for short periods. However, these models lead to accumulation of metabolites, lacking relevant tissue-tissue interfaces and spatial organization, key drivers of cell function. ...
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.
