Native Liver Tissue Research Articles

Perfusion of hepatocellular carcinomas measured by diffusion-derived vessel density biomarker: Higher hepatocellular carcinoma perfusion than earlier intravoxel incoherent motion reports.

Diffusion-derived vessel density (DVDD) is a physiological surrogate of the area of microvessels per unit tissue area. DDVD is calculated according to DDVD(b0b2) = Sb0/ROIarea0 - Sb2/ROIarea2, where Sb0 and Sb2 refer to the liver signal when b is 0 or 2 s/mm2. Pathohistological studies and contrast-enhanced CT/MRI data showed higher blood volume in hepatocellular carcinoma (HCC) relative to native liver tissue. With intravoxel incoherent motion (IVIM) imaging, most authors paradoxically reported a decreased perfusion fraction of HCC relative to the adjacent liver. This study applied DDVD to assess the perfusion of HCC. MRI was performed with a 3.0-T magnet. Diffusion-weighted images with b-values of 0 and 2 s/mm2 were acquired in 72 HCC patients. Thirty-two patients had microvascular invasion (MVI(+)) and 40 patients did not have microvascular invasion (MVI(-)). Fifty-eight patients had Edmondson-Steiner grade I or II HCC, and 14 patients had Edmondson-Steiner grade III or IV HCC. DDVD measurement was conducted on the axial slice that showed the largest HCC size. DDVD(b0b2) T/L = HCC DDVD(b0b2)/liver DDVD(b0b2). DDVD(b0b2) T/L median (95% confidence interval) of all HCCs was 2.942 (2.419-3.522), of MVI(-) HCCs was 2.699 (2.030-3.522), of MVI(+) HCCs was 2.988 (2.423-3.990), of Edmondson-Steiner grade I/II HCCs was 2.873 (2.277-3.465), and of Edmondson-Steiner grade III/IV HCCs was 3.403 (2.008-4.485). DDVD(b0b2) T/L approximately agrees with contrast agent dynamically enhanced CT/MRI literature data, whereas it differs from earlier IVIM study results, where HCC perfusion fraction was paradoxically lower relative to native liver tissue. A weak trend was noted with MIV(+) HCCs had a higher DDVD(b0b2) T/L than that of MVI(-) HCCs, and a weak trend was noted with the poorly differentiated group of HCCs (Edmondson-Steiner grade III and IV) had a higher DDVD(b0b2) T/L than that of the better differentiated group of HCCs (Edmondson-Steiner grade I and II).

Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

T2 relaxation time elongation of hepatocellular carcinoma relative to native liver tissue leads to an underestimation of perfusion fraction measured by standard intravoxel incoherent motion magnetic resonance imaging.

T2 relaxation time elongation of hepatocellular carcinoma relative to native liver tissue leads to an underestimation of perfusion fraction measured by standard intravoxel incoherent motion magnetic resonance imaging.

Relationship of the expression of the NF1, p53, bcl-2, pRB proteins with DNA/RNA of viruses and mutations of the BRCA 1/2 and hMSH2 genes in liver cancer

Among tumors of the gastrointestinal tract, the incidence and mortality from primary liver cancer remains high. Over the past 20 years, the incidence of liver cancer in Belarus has increased by 45.7 %. The aim of the study was to establish the relationship between the expression of the NF1, p53, bcl-2 proteins and the pRB genes with DNA/RNA of viruses and mutations of the BRCA 1/2 and hMSH2 genes in liver cancer. Patients (n = 303) with morphologically established primary liver cancer were examined. A molecular biological and immunological study was performed: antibodies to cell-cycle NF1, p53, bcl-2, pRB proteins, DNA/RNA of viruses, the BRCA 1/2, hMSH2 gene. The levels of antibodies to p53, bcl-2, pRB, and NF1 in the serum of healthy individuals differed from those in blood samples and tissue extracts from patients with hepatitis C, liver cancer and in native liver tissue samples. Mutations in liver cancer were (p = 0.004): in the tumor tissue – 6.13 %, in the blood – 0.85 %. Changes in the hMSH2 gene in the tissue were set at 15.48 %. DNA/RNA was isolated in the tumor tissue: HSV 1/2 – 56.1 %, HHV6 – 24.4, CMV – 17.1, HCV – 17.1, HBV – 4.9, HPV – 4.1, VEB – 2.4 %. Including mixed persistence – 26.8 %. In hepatitis (C, B) and liver cancer with CMV carriers, the expression of bcl-2, NF1 and pRb1 (p = 0.001) increases. The Epstein–Barr virus leads to the activity of the p53 protein in liver cancer (p = 0.003). The p53, bcl-2, pRB, and NF1 proteins in the liver tissue do not depend on the age and sex of patients. The degree of their influence on the function of genes with regard to DNA/RNA of viruses and mutations present in the genome (BRCA 1/2, hMSH2) suggests the degree of oncogenicity of viruses. The concentrations of p53, bcl-2, pRB, and NF1 in the blood, exceeding the predicted ones, for 50.8 year old persons, indicate the risk of development/presence of a tumor process in the liver.

Efficient hepatic differentiation of hydrogel microsphere-encapsulated human pluripotent stem cells for engineering prevascularized liver tissue

Liver tissue engineering is promising as an alternative strategy to treat liver failure. However, generating functional hepatocytes from stem cells is conventionally restricted by the immature status of differentiated cells. Besides, embedding hepatocytes in bulk scaffold is limited by a lack of vascularity and low cell-packing density. Here, we fabricate collagen type I (COL1) microspheres for efficient hepatic differentiation of pluripotent stem cells and subsequent assembly of prevascularized liver tissue (PLT). Using a microfluidic platform, we demonstrate that hydrogel COL1 microspheres (mCOL1) encapsulating human embryonic stem cells (hESCs) can be reproducibly generated and efficiently differentiated into hepatocyte-like cells (HLCs) microspheres for the first time. Compared with other culture configurations such as encapsulation of hESC in a bulk COL1 hydrogel and 2D monolayer culture, mCOL1 with high uniformity produce HLC microspheres of improved maturity based on comprehensive analyses of cell morphology, transcriptome profile, hepatic marker expression and hepatic functions. In addition, these HLC microspheres can be applied as building blocks to self-assemble with endothelial cells to construct a dense PLT. The PLT resembles native liver tissue with high cell-packing density, shows successful engraftment in mice liver following implantation, and exhibits improved hepatic function in vivo. Overall, it is believed that this multiscale technology will advance the fabrication of stem cell-based liver tissue for regenerative medicine, drug screening, and in vitro liver modeling.

OR22-1 Temporal And Spatial Cellular Heterogeneity Of Hepatocyte Metabolism

Abstract Gene expression plays a pivotal role in the long-term regulation of gluconeogenesis and lipogenesis in the liver. Although most of the previous studies were analyzed as a whole liver, it is known that hepatocytes across the liver display specific aspects of liver function in a zonation dependent manner. Using a combination of single-cell RNA sequencing (scRNA-seq) and single molecule Fluorescence In Situ Hybridization (smFISH) technologies, we examined several novel molecular and cellular aspects of the normal regulation of hepatocyte metabolic gene expression during the transitions from the fed to fasted, and subsequent starvation states in male C57BL/6J mice. Targeted scRNA-seq was used to quantitively analyze the temporal and spatial differences of gluconeogenic and lipogenic genes following a post-prandial time course. Initially, in the fed state lipogenic genes (such as Fasn) were highly expressed in both the periportal (PP) and pericentral (PC) hepatocytes that were rapidly turned off as the mouse entered the fasted state. In contrast, gluconeogenic genes (such as Pck1) were expressed in a subset of PP hepatocytes in the fully fed state. As the mouse entered the fasted state gluconeogenic genes became activated primarily in the PP hepatocytes. However, as the mouse enters a starvation state the PC hepatocytes also increase their expression of gluconeogenic genes. Analyses by smFISH not only confirmed the scRNA-seq data but is also applicable to determine the spatial context in the native liver tissue as well as analyzing the transcriptional regulation of gluconeogenic and lipogenic genes at the single molecule resolution. In the fed state, few PP hepatocytes expressed gluconeogenic transcription sites (TS), and during a fasted time-course, over time the TS expanded outwards to the PC hepatocytes. On the other hand, lipogenic TS were expressed in both PC and PP hepatocytes in the fed state, but during a fasted time-course, TS were not detected. In summary, these data suggest that hepatocyte zonation of gluconeogenic genes is dynamic and can vary substantially depending upon the fasted and fed states. Understanding the regulation of the liver hepatocyte normal physiology will provide the molecular basis for which we can more precisely determine the dysregulation that occurs in states of insulin resistance. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.

Hepatogenic differentiation of adipose-derived mesenchymal stem cells directed by topographical cues: a proof of concept study

Topography and roughness are key features that can direct the behavior of cells. Here, we have developed a surface pattern comparable to the features and roughness present in the native liver tissue by virtue of the liver extracellular matrix. This study used polydimethylsiloxane to get an imprint of the topographical pattern of the native liver and used this imprint as a template to guide the differentiation of adipose-derived mesenchymal stem cells. The results showed a similar gene and protein expression profile to that of hepatocytes, showing potential in the field of scaffold development for tissue engineering applications.

Gelatin-Based Hybrid Hydrogel Scaffolds: Toward Physicochemical Liver Mimicry.

There exists a clear need to develop novel materials that could serve liver tissue engineering purposes. Those materials need to be researched for the development of bioengineered liver tissue as an alternative to donor livers, as well as for materials that could be applied for scaffolds to develop an in vitro model for drug-induced liver injury (DILI) detection . In this paper, the hydrogels oxidized dextran-gelatin (Dexox-Gel) and norbornene-modified dextran-thiolated gelatin (DexNB-GelSH) were developed, and their feasibility toward processing via indirect 3D-printing was investigated with the aim to develop hydrogel scaffolds that physicochemically mimic native liver tissue. Furthermore, their in vitro biocompatibility was assessed using preliminary biological tests using HepG2 cells. Both materials were thoroughly physicochemically characterized and benchmarked to the methacrylated gelatin (GelMA) reference material. Due to inferior properties, Dexox-gel was not further processed into 3D-hydrogel scaffolds. This research revealed that DexNB-GelSH exhibited physicochemical properties that were in excellent agreement with the properties of natural liver tissue in contrast to GelMA. In combination with an equally good biological evaluation of DexNB-GelSH in comparison with GelMA based on an MTS proliferation assay and an albumin quantification assay, DexNB-GelSH can be considered promising in the field of liver tissue engineering.

Surface wave elastography using high speed full-field optical interferometry

The assessment of mechanical stiffness is an essential diagnostic tool for investigating the biomechanical properties of biological tissues. Surface wave elastography (SWE) is an emerging technique to quantify elastic properties of tissues in clinical diagnosis. High-speed optical imaging combined with SWE has enormous potential in quantifying the elastic properties of tissues at microscale resolutions. In this study, we implement surface wave elastography using high-speed optical interferometry to characterize the elastic properties of tissue-mimicking phantoms and ex-vivo native caprine liver tissue by imaging the surface wave induced by an electromechanical actuator. The sinusoidal mechanical excitations ranging from 120 Hz to 1.2 kHz on the surface of tissues are captured using a high-speed camera with a frame rate of 4 kHz at micrometer resolutions. The surface wavefront reconstruction is performed using a phase-shifting algorithm and linear regression is used to calculate the surface wave velocity. The mechanical stiffness estimated from the optical system is compared with the results of mechanical compression testing measurements. The results from this multimodal platform combining optical interferometry and vibrational spectroscopy using SWE are highly promising towards a non-invasive or minimally invasive imaging for in-vivo and ex-vivo mechanical characterization of tissues with future clinical applications.

Radially Aligned Porous Silk Fibroin Scaffolds as Functional Templates for Engineering Human Biomimetic Hepatic Lobules.

Bioengineering functional hepatic tissue constructs that physiologically replicate the human native liver tissue in vitro is sought for clinical research and drug discovery. However, the intricate architecture and specific biofunctionality possessed by the native liver tissue remain challenging to mimic in vitro. In the present study, a versatile strategy to fabricate lobular-like silk protein scaffolds with radially aligned lamellar sheets, interconnected channels, and a converging central cavity was designed and implemented. A proof-of-concept study to bioengineer biomimetic hepatic lobules was conducted through coculturing human hepatocytes and primary endothelial cells on these lobular-like scaffolds. Relatively long-term viability of hepatocyte/endothelial cells was found along with cell alignment and organization in vitro. The hepatocytes showed special epithelial polarity and bile duct formation, similar to the liver plate, while the aligned endothelial cells generated endothelial networks, similar to natural hepatic sinuses. This endowed the three-dimensional (3D) tissue constructs with the capability to recapitulate hepatic-like parenchymal-mesenchymal growth patterns in vitro. More importantly, the cocultured hepatocytes outperformed monocultures or monolayer cultures, displaying significantly enhanced hepatocyte functions, including functional gene expression, albumin (ALB) secretion, urea synthesis, and metabolic activity. Thus, this functional unit with a biomimetic phenotype provides a novel technology for bioengineering biomimetic hepatic lobules in vitro, with potential utility as a building block for bioartificial liver (BAL) engineering or as a robust tool for drug metabolism investigation.

Characterization of Indonesia Decellularized Liver Cubes Scaffold using Scanning Electron Microscopy

Liver biological scaffold was developed in order to resemble native liver tissue environment. It can be achieved by decellularizing native liver tissue that will remove cells and preserve extracellular matrix (ECM). Furthermore, ECM fibers are arranged in a special pattern, which affect liver cell polarity and topography that are important for cells’ implantation, proliferation and differentiation. Therefore, the aim of this study was to evaluate liver cube scaffold topography that was decellularized with fixed multiple sites syringe injection (Indonesia patent number: S00201907930).Rat liver cubes (n=3) underwent decellularization with Ethylene Glycol Tetraacetic Acid (EGTA) immersion and increased Sodium Dodecyl Sulfate (SDS) concentrations using previous multiple sites syringe injection protocol study. Deoxyribonucleic Acid (DNA) concentrations were measured to confirm less DNA materials remaining in scaffolds. Scanning Electron Microscope (SEM) analysis of scaffolds were conducted for topographic characterization compared to undecellularized liver control. Molecular analysis of DNA concentration showed complete removal of DNA material. SEM analysis gave appearance of intact liver cube scaffold microarchitecture. Liver cubes decellularization using multiple sites syringe injection showed good topographic liver scaffold characterization.

Decellularized caprine liver-derived biomimetic and pro-angiogenic scaffolds for liver tissue engineering

The development of a pre-vascularized liver tissue construct with native-like 3-dimensional (3D) microarchitecture and extracellular matrix (ECM) composition is essential to meet the current demand for liver transplantation. Here, we report the methodology and in-depth characterization of the decellularized caprine liver scaffold (CLECM-S) for tissue engineering application. CLECM-S retained crucial ECM components and structural features similar to the native liver tissue. For comparative evaluation, conventional glutaraldehyde crosslinked collagen scaffold (Col-S) was taken as a control. CLECM-S underwent a slow but sustained swelling and collagenase mediated degradation and had the mechanical stiffness closer to that of the native human liver. HepG2 cells cultured on CLECM-S exhibited an enhanced expression of mature and functional hepatocyte markers. In addition, CLECM-S also showed pro-angiogenic properties as confirmed by Chick Chorioallantoic Membrane (CAM) assay. Upon implantation in a mouse model, the scaffolds did not elicit any significant immunogenic response. These results, together, provide a solid evidence depicting superiority of CLECM-S over conventional Col-S for liver tissue engineering applications.

Fabrication of perfusable 3D hepatic lobule-like constructs through assembly of multiple cell type laden hydrogel microstructures

The in vitro reproduction of three-dimensional (3D) cellular constructs to physiologically mimic human liver is highly desired for drug screening and clinical research. However, the fabrication of a liver-mimetic 3D model using traditional bottom-up technologies is challenging owing to the complex architecture and specific functions of real liver tissue. This work proposes a versatile strategy for spatially assembling gear-like microstructures encapsulating multiple cell types, and reorganizing them into 3D lobule-like micro-architecture with physiological relevance to native liver tissue. Gear-like microstructures were fabricated by photo-crosslinking poly(ethylene glycol) diacrylate (PEGDA) hydrogel mixed with hepatocytes and fibroblasts, in a digital micromirror device (DMD)-based microfluidic channel. The microstructures were assembled through coordinated micromanipulation based on local fluid force, and spatially self-aligned through hydrophilic–hydrophobic interactions into a 3D integrated construct with lobule-like morphology and a perfusable central lumen. The resulting 3D lobule-like constructs allowed long-term co-culture of hepatocytes and fibroblasts with high cell viability. The co-cultured constructs enhanced hepatocyte proliferation and spreading, as well as liver functions including a 50% increase in albumin secretion and urea synthesis. For hepatotoxicity assessment, the 3D lobule-like construct enabled drug perfusion through its built-in lumen for simulation of drug diffusion in the liver, which could improve the response sensitivity and efficiency to hepatotoxic drug. These results demonstrated that this method provides a valuable 3D co-culture model with perfusable lobule-like architecture and physiological functions, which has potential applications in drug discovery and tissue engineering applications.

Preparation and Culture of Human Liver Resident Immune Cells

Co-cultivation of tumor cells and liver resident immune cells or other non-parenchymal cells (NPCs) from the same donor is important for the study of cancer metastasis. So far, little is known about the mechanism of tumor cell or pathogen clearance, leukocyte infiltration, and immune cell recruitment in the human liver. To investigate these processes in vitro, the use of primary human hepatocytes and non-parenchymal cell, especially immune cell, co-culture systems play essential roles in the establishment of cell-cell and cell-extracellular matrix communications similar to native liver tissues. Hepatic non-parenchymal cells mainly comprise liver sinusoid endothelial cells (LSECs), microvascular endothelial cells, hepatic stellate cells, Kupffer cells (KCs), natural killer T (iNKT) cells, and dendritic cells (DCs). Here we describe procedures for preparation, isolation, and culture of human liver resident immune cells and other non-parenchymal cells. © 2018 by John Wiley & Sons, Inc.

Regulatory Dendritic Cell (DCreg) Cell Infusion in Living Donor Liver Transplantation

Background Based on strong preclinical data showing that regulatory dendritic cells (DCreg) of donor origin can promote long-term graft survival in rodents and suppress allograft rejection in non-human primates, we have embarked on a first-in-human phase I/II safety and preliminary efficacy trial of donor-derived DCreg in living donor liver transplantation (LTx). Methods In the study protocol, DCreg are generated from elutriated monocytes isolated from the leukapheresed donor,14-28 days (d) before transplant. The donor-derived DCreg are infused iv (target range 2.5-10.106/kg) 7 d before transplant (d −7), while a half dose of MPA is administered from d −7 to d 0. Standard-of-care immunosuppression (steroid, MPA, tacrolimus) is administered post-LTx, and in those patients that meet eligibility criteria for weaning, immunosuppressive therapy is gradually withdrawn, starting with MPA reduction 6 mths post-LTx. Sequential immunological analyses are conducted on recipient blood and tissue samples to track the donor-derived DCreg, evaluate anti-donor reactivity and examine potential mechanisms of immune hyporesponsiveness. In the first LTx recipient (HLA-B12+) to receive donor-derived (HLA-A3+) DCreg, peripheral blood samples were obtained before and after cell infusion; native liver tissue was obtained at the time of graft implantation. Results Donor-derived DCreg generated for infusion were HLA-DR+, CD1c−, CD11c+, CD83−, IRF4lo and expressed a high programed death ligand-1 (PD-L1):CD86 ratio. In parallel small scale in vitro functional analyses, the DCreg failed to respond to LPS, and induced donor-specific hyporesponsiveness of recipient CD4 and CD8 T cells. The donor-derived DCreg (HLA-A3+) displaying a similar phenotype to that exhibited pre-infusion, were detected in whole blood immediately after completion of the cell infusion that comprised 5.106 DCreg/kg (450.106 total DCreg). No infusion reaction or cytokine release response was observed. Three days post-infusion (d −4), HLA-A3+ donor-derived DC could no longer be detected in blood, although a small population of DC co-expressing both recipient (HLA-B12) and donor MHC (HLA-A3), ie cross-dressed DC, were evident in the circulation. At the time of transplant, before graft implantation, cross-dressed (HLA-A3+B12+) DC could also be detected in non-parenchymal cell populations isolated from the diseased native liver. Using a 14-color panel of mAbs, these tissue-resident cross-dressed DC were PD-L1hi, IRF4lo and Foxp3hi Conclusions After safe infusion of donor-derived DCreg into a prospective living donor LTx recipient, cross-dressed DC were detected in the peripheral blood within a few days and in the native diseased liver at the time of graft implantation. These cross-dressed DC, that appear to have acquired donor MHC from the infused DCreg, exhibit low levels of the DC transcription/maturation factor IRF4 and high levels of PD-L1 and Foxp3, suggesting possible in vivo regulatory function.

Intermittent Pringle maneuver may be beneficial for radiofrequency ablations in situations with tumor-vessel proximity.

BackgroundRadiofrequency ablation (RFA) represents a treatment option for non-resectable liver malignancies. Larger ablations can be achieved with a temporary hepatic inflow occlusion (Pringle maneuver – PM). However, a PM can induce dehydration and carbonization of the target tissue. The objective of this study was to evaluate the impact of an intermittent PM on the ablation size.MethodsTwenty-five multipolar RFAs were performed in porcine livers ex vivo. A perfused glass tube was used to simulate a natural vessel. The following five test series (each n=5) were conducted: (1) continuous PM, (2–4) intermittent PM, and (5) no PM. Ablations were cut into half. Ablation area, minimal radius, and maximal radius were compared.ResultsNo change in complete ablation size could be measured between the test series (p>0.05). A small rim of native liver tissue was observed around the glass tube in the test series without PM. A significant increase of ablation area could be measured on the margin of the ablations with an intermittent PM, starting without hepatic inflow occlusion (p<0.05).ConclusionAn intermittent PM did not lead to smaller ablations compared to a continuous or no PM ex vivo. Furthermore, an intermittent PM can increase the ablation area when initial hepatic inflow is succeeded by a PM.

Can point shear wave elastography differentiate focal nodular hyperplasia from hepatocellular adenoma.

PurposeFocal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are liver tumors that require different management. We assessed the potential of point shear wave elastography (pSWE) to differentiate FNH from HCA and the interobserver and intraobserver reliability of pSWE in the examination of these lesions and of native liver tissue (NLT).MethodsThe study included 88 patients (65 FNH, 23 HCA). pSWE was performed by two experienced liver sonographers (observers 1 [O1] and 2 [O2]) and acquired within the lesion of interest and NLT. Group differences, optimal cutoff for characterization and interobserver reliability was assessed with Mann‐Whitney‐U, area under the ROC curce (AUROC) and intraclass correlation coefficient (ICC). Intraobserver reliability in NLT was assessed in 20 healthy subjects using ICC.ResultsMedian stiffness was significantly higher in FNH than in HCA (7.01 kPa vs 4.98 kPa for O1 (P = 0.017) and 7.68 kPa vs 6.00 kPa for O2 (P = 0.031)). A cutoff point for differentiation between the two entities could not be determined with an AUROC of 0.67 (O1) and 0.69 (O2). Interobserver reliability was good for lesion‐ stiffness (ICC = 0.86) and poor for NLT stiffness (ICC = 0.09). In healthy subjects, intraobserver reliability for NLT‐stiffness was poor for O1 (ICC = 0.23) and moderate for O2 (ICC = 0.62).ConclusionThis study shows that pSWE cannot reliably differentiate FNH from HCA. Interobserver and intraobserver reliability for pSWE in NLT were insufficient. Interpretation of results gained with this method should be done with great caution.

Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction.

There is a significant need for in vitro methods to study drug-induced liver injury that are rapid, reproducible, and scalable for existing high-throughput systems. However, traditional monolayer and suspension cultures of hepatocytes are difficult to handle and risk the loss of phenotype. Generally, three-dimensional (3D) cell culture platforms help recapitulate native liver tissue phenotype, but suffer from technical limitations for high-throughput screening, including scalability, speed, and handling. Here, we developed a novel assay for cytochrome P450 (CYP450) induction/inhibition using magnetic 3D cell culture that overcomes the limitations of other platforms by aggregating magnetized cells with magnetic forces. With this platform, spheroids can be rapidly assembled and easily handled, while replicating native liver function. We assembled spheroids of primary human hepatocytes in a 384-well format and maintained this culture over five days, including a 72 h induction period with known CYP450 inducers/inhibitors. CYP450 activity and viability in the spheroids were assessed and compared in parallel with monolayers. CYP450 activity was induced/inhibited in spheroids as expected, separate from any toxic response. Spheroids showed a significantly higher baseline level of CYP450 activity and induction over monolayers. Positive staining in spheroids for albumin and multidrug resistance-associated protein (MRP2) indicates the preservation of hepatocyte function within spheroids. The study presents a proof-of-concept for the use of magnetic 3D cell culture for the assembly and handling of novel hepatic tissue models.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor.

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures.

Occult hepatitis B virus infection predicts de novo hepatitis B infection in patients with alcoholic cirrhosis after liver transplantation.

Occult hepatitis B virus infection (OBI) in patients undergoing liver transplantation (LT) is a suspected source of de novo hepatitis B virus (HBV) infection after LT. This study aimed to investigate the prevalence of OBI in liver transplant recipients with alcoholic cirrhosis and demonstrate the association between OBI and de novo HBV infection after LT in these patients. Forty-three patients with alcoholic cirrhosis who were negative for HBsAg before LT were recruited in this retrospective study. DNA was extracted from paraffin-embedded native liver tissues and quantified for HBV DNA by real-time PCR. Correlation between de novo HBV infection after LT (positive HBsAg and/or detectable HBV DNA in serum) and detection of intrahepatic HBV DNA before LT was analysed. Detectable HBV DNA in the explanted liver was found in 41.9% (18/43) of the patients and was thus defined as OBI, which was correlated with the presence of serum hepatitis B core antibody (P = 0.008). De novo HBV infection occurred in 18.6% (8/43) of the recipients at a median of 10 months after LT. The rate of de novo HBV infection was 38.9% (7/18) in patients with OBI, compared with 4% (1/25) in patients without OBI (P = 0.004). Furthermore, de novo HBV infection was inversely correlated with the presence of hepatitis B surface antibody in recipients with OBI (P = 0.026). With a prevalence of 41.9% in liver transplant recipients with alcoholic cirrhosis, OBI in the native liver can predict de novo HBV infection after LT.