RESOURCES

Abstract
Abstract There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88–107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21–51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.

Abstract
Abstract Liver parenchymal microtissues (LPMTs) are three-dimensional (3D) aggregates of hepatocytes that recapitulate in vivo-like cellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural–functional integrity over the long term. Here, we present a high-throughput methodology to produce LPMTs in a novel, economic, and reusable Hanging-drop Culture Chamber (HdCC). A drop-on-demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200–300 μm. Tissue size, cell viability, cellular arrangement and polarity, and insulin-mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate-like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver-specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ-specific functions of LPMTs in vitro.

Abstract
Abstract 3D bioprinting enables the fabrication of human organ models that can be used for various fields of biomedical research, including oncology and infection biology. An important challenge, however, remains the generation of vascularized, perfusable 3D models that closely simulate natural physiology. Here, a novel direct ink writing (DIW) approach is described that can produce vascularized organ models without using sacrificial materials during fabrication. The high resolution of the method allows the one-step generation of various sophisticated hollow geometries. This sacrificial-free DIW (SF-DIW) approach is used to fabricate hepatic metastasis models of various cancer types and different formats for investigating the cytostatic activity of anti-cancer drugs. To this end, the models are incorporated into a newly developed perfusion system with integrated micropumps and an agar casting step that improves the physiological features of the bioprinted tissues. It is shown that the hepatic environment of the tumor models is capable of activating a prodrug, which inhibits breast cancer growth. This versatile SF-DIW approach is able to fabricate complicated perfusable constructs or microfluidic chips in a straightforward and cost-efficient manner. It can also be easily adapted to other cell types for generating vascularized organ tissues or cancer models that may support the development of new therapeutics.

Abstract
This study discusses the potential for mending portions or entire hepatic to cure persistent liver disease. Fabricating functioning organs is the ultimate objective of tissue science, and this study highlights the possibility of being accomplished. The liver, the most significant gland in the human body, serves as accountable for a wide range of metabolism-related processes and activities. Persistent liver failure is a prevalent cause of mortality worldwide, and the currently prevalent technique of organ transplantation has challenges. Therefore, it is necessary to develop an artificial liver model in the laboratory that accurately replicates the natural surroundings. The generated model should be dependable in comprehending the etiology, evaluating medications, and aiding in the restoration and substitution of the impaired liver. 3-D bioprinting is a promising method that develops an in vitro model that closely resembles the in vivo environment, with the ultimate aim of being used for transplantation by tissue engineers. The technique has significant promise as a result of its exact manipulation and its capacity to uniformly disperse cells across all levels inside an intricate framework. This study provides a comprehensive examination of liver tissue engineering, specifically highlighting the use of three-dimensional bioprinting and bio-inks for liver disease models and pharmaceutical screening.

Abstract
The spheroid culture system has gained significant attention as an effective in vitro model to mimic the in vivo microenvironment. Even though numerous studies were focused on developing spheroids, the structural organization of encapsulated cells within hydrogels remains a challenge. Allylated gelatin or GelAGE is used as a bioink due to its excellent physicochemical properties. In this study, GelAGE was evaluated for its capacity to induce spontaneous spheroid formation in encapsulated HepG2 cells. GelAGE was synthesized and characterized using 1HNMR spectroscopy and ninhydrin assay. Then the physicochemical and biological attributes of GelAGE hydrogel was examined. The results demonstrate that GelAGE has remarkable ability to induce the encapsulated cells to self-organize into spheroids.

Abstract
Three-dimensional liver bioprinting is an emerging technology in the field of regenerative medicine that aids in the creation of functional tissue constructs that can be used as transplantable organ substitutes. During transplantation, the bioprinted donor liver must be protected from the oxidative stress environment created by various factors during the transplantation procedure, as well as from drug-induced damage from medications taken as part of the post-surgery medication regimen following the procedure. In this study, Silymarin, a flavonoid with the hepatoprotective properties were introduced into the GelMA bioink formulation to protect the bioprinted liver against hepatotoxicity. The concentration of silymarin to be added in GelMA was optimised, bioink properties were evaluated, and HepG2 cells were used to bioprint liver tissue. Carbon tetrachloride (CCl4) was used to induce hepatotoxicity in bioprinted liver, and the effect of this chemical on the metabolic activities of HepG2 cells was studied. The results showed that Silymarin helps with albumin synthesis and shields liver tissue from the damaging effects of CCl4. According to gene expression analysis, CCl4 treatment increased TNF-α and the antioxidant enzyme SOD expression in HepG2 cells while the presence of silymarin protected the bioprinted construct from CCl4-induced damage. Thus, the outcomes demonstrate that the addition of silymarin in GelMA formulation protects liver function in toxic environments.

Abstract
Research within the hepato-biliary system and hepatic function is currently experiencing heightened interest, this is due to the high frequency of relapse rates observed in chronic conditions, as well as the imperative for the development of innovative therapeutic strategies to address both inherited and acquired diseases within this domain. The most commonly used sources for studying hepatocytes include primary human hepatocytes, human hepatic cancer cell lines, and hepatic-like cells derived from induced pluripotent stem cells. However, a significant challenge in primary hepatic cell culture is the rapid decline in their phenotypic characteristics, dedifferentiation and short cultivation time. This limitation creates various problems, including the inability to maintain long-term cell cultures, which can lead to failed experiments in drug development and the creation of relevant disease models for researchers' purposes. To address these issues, the creation of a powerful 3D cell model could play a pivotal role as a personalized disease model and help reduce the use of animal models during certain stages of research. Such a cell model could be used for disease modelling, genome editing, and drug discovery purposes. This review provides an overview of the main methods of 3D-culturing liver cells, including a discussion of their characteristics, advantages, and disadvantages.

Abstract
Methacrylated gelatin (GelMA) in the form of methacryloyl, methacrylate, and methacrylamide is an established and widely accepted photocrosslinkable bioink, for three dimensional bioprinting of various tissues. One of the limitations of photocrosslinkable bioinks is the inability to control the free radicals generated by photoinitiators and ultraviolet (UV) rays. The presence of excess free radicals compromises the viability and functionality of cells during crosslinking. In this study, ascorbic acid, a known free radical scavenger (FRS) molecule, was introduced into the GelMA bioink formulation to protect the cell viability, proliferation, and tissue functions of 3D bioprinted parenchymal liver constructs. The concentration of FRS in the bioink was optimized and used for 3D bioprinting of HepG2 cells. The results confirmed that the inclusion of 3.4 mM FRS in the GelMA bioink formulation nullified the excess ROS formed inside the cells. Furthermore, the optimized GelMA formulation containing FRS preserved and improved the cell activity, albumin, and urea synthesis in the 3D construct over 7 days in culture. In the future, this concept could be implemented in the biofabrication of large liver constructs that require multiple or longer durations of UV irradiation.

Abstract
Despite the exciting properties and wide-reaching applications of nanobiomaterials (NBMs) in human health and medicine, their translation from bench to bedside is slow, with a predominant issue being liver accumulation and toxicity following systemic administration. In vitro 2D cell-based assays and in vivo testing are the most popular and widely used methods for assessing liver toxicity at pre-clinical stages; however, these fall short in predicting toxicity for NBMs. Focusing on in vitro and in vivo assessment, the accurate prediction of human-specific hepatotoxicity is still a significant challenge to researchers. This review describes the relationship between NBMs and the liver, and the methods for assessing toxicity, focusing on the limitations they bring in the assessment of NBM hepatotoxicity as one of the reasons defining the poor translation for NBMs. We will then present some of the most recent advances towards the development of more biologically relevant in vitro liver methods based on tissue-mimetic 3D cell models and how these could facilitate the translation of NBMs going forward. Finally, we also discuss the low public acceptance and limited uptake of tissue-mimetic 3D models in pre-clinical assessment, despite the demonstrated technical and ethical advantages associated with them.

Abstract
AbstractWhile alternative methods for toxicity testing using re-constructed human skin and cornea have been written into guidelines and adopted by regulatory authorities, three-dimensional (3D) liver models are currently applied in the industrial settings for hepatotoxicity screening and prediction. These 3D liver models can recapitulate the architecture, functionality and toxicity response of the native liver, demonstrated by a set of related hallmarks. In this comprehensive review, non-scaffold and scaffold-based methods available for 3D liver model formation are introduced, with an emphasis on their advantages and drawbacks. We then focus on the characteristics of primary human hepatocytes, stem cell derived hepatocyte like cells, and immortalized hepatic cell lines as cell resources for model reconstruction. Primary hepatocytes are generally regarded to be superior to other cell types due to their comparable metabolic profiles to the native liver. Additionally, the application of 3D liver models (mostly liver spheroids) on the evaluation of drug induced liver injury and chronic liver diseases (steatosis, cirrhosis, cholestasis), as well as the potential of nanomaterials to introduce hepatotoxicity are summarized. Finally, the global 3D cell market from 3D liver model manufacturing to the contract service of in vitro hepatotoxicity testing using the models is extensively explored. However, 3D liver models face cultural and regulatory barriers in different countries, and therefore the business development of 3D liver models is not easy. Toxicologists, material scientists, engineers should work together to develop, validate and apply 3D liver models for hepatotoxicity testing under the support from industrial organizations and governmental agencies.