The effects of simvastatin-loaded nanoliposomes on human multilineage liver fibrosis microtissue.

In this in vitro study, for the first time, we evaluate the effects of simvastatin-loaded liposome nanoparticles (SIM-LipoNPs) treatment on fibrosis-induced liver microtissues, as simvastatin (SIM) has shown potential benefits in the non-alcoholic fatty liver disease process. We developed multicellular liver microtissues composed of hepatic stellate cells, hepatoblastoma cells and human umbilical vein endothelial cells. The microtissues were supplemented with a combination of palmitic acid and oleic acid to develop fibrosis models. Subsequently, various groups of microtissues were exposed to SIM and SIM-LipoNPs at doses of 5 and 10 mg/mL. The effectiveness of the treatments was evaluated by analysing cell viability, production of reactive oxygen species (ROS) and nitric oxide (NO), the expression of Kruppel-like factor (KLF) 2, and pro-inflammatory cytokines (interleukin(IL)-1 α, IL-1 ß, IL-6 and tumour necrosis factor-α), and the expression of collagen I. Our results indicated that SIM-LipoNPs application showed promising results. SIM-LipoNPs effectively amplified the SIM-klf2-NO pathway at a lower dosage compatible with a high dosage of free SIM, which also led to reduced oxidative stress by decreasing ROS levels. SIM-LipoNPs administration also resulted in a significant reduction in pro-inflammatory cytokines and Collagen I mRNA levels, as a marker of fibrosis. In conclusion, our study highlights the considerable therapeutic potential of using SIM-LipoNPs to prevent liver fibrosis progress, underscoring the remarkable properties of SIM-LipoNPs in activating the KLF2-NO pathway and anti-oxidative and anti-inflammatory response.

In vitro modeling of liver fibrosis in 3D microtissues using scalable micropatterning system.

Liver fibrosis is the late consequence of chronic liver inflammation which could eventually lead to cirrhosis, and liver failure. Among various etiological factors, activated hepatic stellate cells (aHSCs) are the major players in liver fibrosis. To date, various in vitro liver fibrosis models have been introduced to address biological and medical questions. Availability of traditional in vitro models could not fully recapitulate complicated pathology of liver fibrosis. The purpose of this study was to develop a simple and robust model to investigate the role of aHSCs on the progression of epithelial to mesenchymal transition (EMT) in hepatocytes during liver fibrogenesis. Therefore, we applied a micropatterning approach to generate 3D co-culture microtissues consisted of HepaRG and human umbilical cord endothelial cells (HUVEC) which co-cultured with inactivated LX-2 cells or activated LX-2 cells, respectively, as normal or fibrotic liver models in vitro. The result indicated that the activated LX-2 cells could induce EMT in HepaRG cells through activation of TGF-ß/SMAD signaling pathway. Besides, in the fibrotic microtissue, physiologic function of HepaRG cells attenuated compared to the control group, e.g., metabolic activity and albumin secretion. Moreover, our results showed that after treatment with Galunisertib, the fibrogenic properties decreased, in the term of gene and protein expression. In conclusion, it is proposed that aHSCs could lead to EMT in hepatocytes during liver fibrogenesis. Furthermore, the scalable micropatterning approach could provide enough required liver microtissues to prosper our understanding of the mechanisms involved in the progression of liver fibrosis as well as high throughput (HT) drug screening.

Particulate and drug-induced toxicity assessed in novel quadruple cell human primary hepatic disease models of steatosis and pre-fibrotic NASH.

In an effort to replace, reduce and refine animal experimentation, there is an unmet need to advance current in vitro models that offer features with physiological relevance and enhanced predictivity of in vivo toxicological output. Hepatic toxicology is key following chemical, drug and nanomaterials (NMs) exposure, as the liver is vital in metabolic detoxification of chemicals as well as being a major site of xenobiotic accumulation (i.e., low solubility particulates). With the ever-increasing production of NMs, there is a necessity to evaluate the probability of consequential adverse effects, not only in health but also in clinically asymptomatic liver, as part of risk stratification strategies. In this study, two unique disease initiation and maintenance protocols were developed and utilised to mimic steatosis and pre-fibrotic NASH in scaffold-free 3D liver microtissues (MT) composed of primary human hepatocytes, hepatic stellate cells, Kupffer cells and sinusoidal endothelial cells. The characterized diseased MT were utilized for the toxicological assessment of a panel of xenobiotics. Highlights from the study included: 1. Clear experimental evidence for the pre-existing liver disease is important in the augmentation of xenobiotic-induced hepatotoxicity and 2. NMs are able to activate stellate cells. The data demonstrated that pre-existing disease is vital in the intensification of xenobiotic-induced liver damage. Therefore, it is imperative that all stages of the wide spectrum of liver disease are incorporated in risk assessment strategies. This is of significant consequence, as a substantial number of the general population suffer from sub-clinical liver injury without any apparent or diagnosed manifestations.

[Study on potential hepatotoxicity of main monomers of Polygonum multiflorum based on liver micro-tissue].

In this study, we aimed to establish a rat liver micro-tissue evaluation system to evaluate the hepatotoxicity of the main monomers in Polygonum multiflorum. Rat primary hepatocytes were isolated and purified by two-step in situ perfusion method to prepare hepatic parenchymal cells. The ultra-low adsorption plate and the inverted model were used to establish an in vitro hepatotoxicity evaluation system. After the system was established, the main monomer components(monanthone with emodin type, rhein, emodin, emodin-8-O-ß-D-glucopyranoside, physcion) of P. multiflorum were selected for in vitro hepatotoxicity evaluation. This study showed that the primary cells of the liver can form liver micro-tissues in the low adsorption plate method and the mold perfusion method, with good liver structure and function, which can be used to evaluate the hepatotoxicity of the drug to be tested after long-term administration. The five monomers to be tested in P. multiflorum can significantly affect the proliferation of primary liver micro-tissues in rats in a dose-and time-dependent manner. The hepatotoxic effects were as follows: monanthone with emodin type > rhein > emodin > emodin-8-O-ß-D-glucopyranoside > physcion. The results suggested that the emodin-type monoterpene and rhein might be the potential hepatotoxic components, while the metabolites of emodin-8-O-ß-D-glucoside and emodin methyl ether showed more toxic risks. The rat primary hepatocyte micro-tissue model system established in this experiment could be used to achieve long-term drug administration in vitro, which was consistent with the clinical features of liver injury caused by long-term use of P. multiflorum. The experimental results provided important information and reference on the clinical application and toxic component of P. multiflorum.

Assessment of nanomaterial-induced hepatotoxicity using a 3D human primary multi-cellular microtissue exposed repeatedly over 21 days - the suitability of the in vitro system as an in vivo surrogate.

BACKGROUND: With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver's recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. RESULTS: The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. CONCLUSION: In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

Bioengineering scalable and drug-responsive in vitro human multicellular non-alcoholic fatty liver disease microtissues encapsulated in the liver extracellular matrix-derived hydrogel.

Non-alcoholic fatty liver disease (NAFLD) is a high-prevalence and progressive disorder. Due to lack of reliable in vitro models to recapitulate the consecutive phases, the exact pathogenesis mechanism of this disease and approved therapeutic medications have not been revealed yet. It has been proven that the interplay between multiple hepatic cell types and liver extracellular matrix (ECM) are critical in NAFLD initiation and progression. Herein, a liver microtissue (LMT) consisting of Huh-7, THP-1, and LX-2 cell lines and human umbilical vein endothelial cells (HUVEC), which could be substituted for the main hepatic cells (hepatocyte, Kupffer, stellate, and sinusoidal endothelium, respectively), encapsulated in liver derived ECM-Alginate composite, was bioengineered. When the microtissues were treated with free fatty acids (FFAs) including Oleic acid (6.6×10-4M) and Palmitic acid (3.3×10-4M), they displayed the key features of NAFLD, including similar pattern of transcripts for genes involved in lipid metabolism, inflammation, insulin-resistance, and fibrosis, as well as pro-inflammatory and pro-fibrotic cytokines' secretions and intracellular lipid accumulation. Continuing FFAs supplementation, we demonstrated that the NAFLD phenomenon was established on day 3 and progressed to the initial fibrosis stage by day 8. Furthermore, this model was stable until day 12 post FFAs withdrawal on day 3. Moreover, administration of an anti-steatotic drug candidate, Liraglutide (15 µM), on the NAFLD microtissues significantly ameliorated the NAFLD phenomenon. Overall, we bioengineered a drug-responsive, cost-benefit liver microtissues which can simulate the initiation and progression of NAFLD. It is expected that this platform could potentially be used for studying molecular pathogenesis of NAFLD and high-throughput drug screening. See also the graphical abstract(Fig. 1).

A portable micro-nanochannel bio-3D printed liver microtissue biosensor for DON detection.

We investigated a portable micro-nanochannel biosensor 3D-printed liver microtissues for rapid and sensitive deoxynivalenol (DON) detection. The screen-printed carbon electrode (SPCE) was modified with nanoporous anodic aluminum oxide (AAO), gold nanoparticles (AuNPs), and cytochrome C oxidase (COx) to enhance sensor performance. Gelatin methacrylate hydrogel, combined with hepatocellular carcinoma cells, formed the bioink for 3D printing. Liver microtissues were prepared through standardized and high-throughput techniques via bio-3D printing technology. These microtissues were immobilized onto modified electrodes to fabricate liver microtissue sensors. The peak current of this biosensor was positively correlated with DON concentration, as determined by cyclic voltammetry (CV), within a linear detection range of 2∼40 µg/mL. The standard curve equation is denoted by ICV(µA) = = 18.76956 + 0.03107CDON(µg/mL), with a correlation coefficient R2 was 0.99471(n＝3). A minimum detection limit of 1.229 µg/mL was calculated from the formula, indicating the successful construction of a portable micro-nanochannel bio-3D printed liver microtissue biosensor. It provides innovative ideas for developing rapid and convenient instrumentation to detect mycotoxin hazards after grain production. It also holds significant potential for application in the prediction and assessment of post-production quality changes in grain.

Bioengineering liver microtissues for modeling non-alcoholic fatty liver disease.

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.

Evaluation of Human Liver Microtissues for Drug Screening on Schistosoma mansoni Schistosomula.

Schistosomiasis is a major neglected tropical disease with more than 200 million infections annually. Despite only one drug, praziquantel, being available, the drug pipeline against schistosomiasis is empty, and drug screening tools have limitations. We evaluated the potential of human liver microtissues (hLiMTs) in antischistosomal drug discovery. Because hLiMTs express all human P450 enzymes, they are an excellent tool to evaluate compounds' bioinactivation, bioactivation, and toxicity. To validate the metabolic conversion capacity of hLiMTs, we first quantified (R)- and (S)-praziquantel and the main metabolite trans-OH-praziquantel following incubation with 0.032-50 µM (0.01-15.62 µg/mL) praziquantel for up to 72 h by a validated LC-MS/MS method. We cocultured hLiMTs with newly transformed schistosomula (NTS) and evaluated the antischistosomal activity and cytotoxicity of three prodrugs terfenadine, tamoxifen citrate, and flutamide. HLiMTs converted 300-350 ng (R)-praziquantel within 24 h into trans-OH-praziquantel. We observed changes in the IC50 values for terfenadine, flutamide, and tamoxifen citrate in comparison to the standard NTS assay in vitro. Cytotoxicity was observed at high concentrations of flutamide and tamoxifen citrate. An in vitro platform containing hLiMTs could serve as an advanced drug screening tool for Schistosoma mansoni, providing information on reduced or increased activity and toxicity.

Quantification of non-alcoholic fatty liver disease progression in 3D liver microtissues using impedance spectroscopy.

Non-alcoholic fatty liver disease (NAFLD) has become a global pandemic. However, a pharmacological cure has not been approved for NAFLD treatment. The greatest barriers to the development of new treatments are the ambiguous criteria among the NAFLD stages and the lack of quantitative methodologies for its disease assessment in a translatable preclinical model. In this study, we developed impedance assessment systems to quantify NAFLD progression in three-dimensional (3D) liver microtissue (hMT). The hMT model undergoing NAFLD represents clinical-like characteristics for a range of stages, such as lipid accumulation, cell ballooning, and stiffening. Each stage can be quantitatively assessed by an impedance system with microchannels under constant or dynamic pressure, depending on the relevant mechanical and morphological changes used in the clinical assessment of NAFLD. We determined a correlation between the impedance parameters and pathophysiological characteristics, such as gap widening and cytoplasmic deformation associated with NAFLD progression using bioimpedance simulation, showing hMTs struggling to return to normal states. In addition, we identified the relative stiffness to assess fibrogenesis from the correlation of resistance change and elongation length into the smaller channel of hMTs. We hope this methodology will have a significant impact on drug development by facilitating improved NAFLD assessment.

Fabrication of liver microtissue with liver decellularized extracellular matrix (dECM) bioink by digital light processing (DLP) bioprinting.

As one of the most effective treatments of end-stage liver disease, liver transplantation still suffers from a shortage of donor organs or a low degree of engraftment. Thus, alternatives to liver transplantation, such as liver support systems, have to be extensively explored. In this study, a novel liver microtissue with an inner gear-like structure, which achieved a larger body surface area, was designed and manufactured to improve hepatic functional restoration. The liver-specific bioinks were developed by combining photocurable methacrylated gelatin (GelMA) with liver decellularized extracellular matrix (dECM), and human-induced hepatocytes (hiHep cells) were encapsulated to form cell-laden bioinks. The mechanical properties, swelling, and cytocompatibility of GelMA/dECM bioinks were carefully characterized before 3D printing. Then, the digital light process (DLP)-based bioprinting was used to fabricate the liver microtissue, and liver dECM was found to improve both the printability and cell viability of GelMA bioinks. hiHep cells were also found to spread farther and have better hepatocyte-specific functions (albumin secretion and urea) in the liver microtissue when liver dECM was added to the GelMA bioinks. Our results provide a promising liver dECM-based cell-laden bioink for liver microtissue fabrication, which would be a potential liver tissue engineering product to help restore hepatic functions.