Glial Cell Line-Derived Neurotrophic Factor Enhances Autophagic Flux in Mouse and Rat Hepatocytes and Protects Against Palmitate Lipotoxicity.

Glial cell line-derived neurotrophic factor (GDNF) is a protein that is required for the development and survival of enteric, sympathetic, and catecholaminergic neurons. We previously reported that GDNF is protective against high fat diet (HFD)-induced hepatic steatosis in mice through suppression of hepatic expression of peroxisome proliferator activated receptor-γ and genes encoding enzymes involved in de novo lipogenesis. We also reported that transgenic overexpression of GDNF in mice prevented the HFD-induced liver accumulation of the autophagy cargo-associated protein p62/sequestosome 1 characteristic of impaired autophagy. Here we investigated the effects of GDNF on hepatic autophagy in response to increased fat load, and on hepatocyte mitochondrial fatty acid ß-oxidation and cell survival. GDNF not only prevented the reductions in the liver levels of some key autophagy-related proteins, including Atg5, Atg7, Beclin-1 and LC3A/B-II, seen in HFD-fed control mice, but enhanced their levels after 12 weeks of HFD feeding. In vitro, GDNF accelerated autophagic cargo clearance in primary mouse hepatocytes and a rat hepatocyte cell line, and reduced the phosphorylation of the mechanistic target of rapamycin complex downstream-target p70S6 kinase similar to the autophagy activator rapamycin. GDNF also enhanced mitochondrial fatty acid ß-oxidation in primary mouse and rat hepatocytes, and protected against palmitate-induced lipotoxicity. Conclusion: We demonstrate a role for GDNF in enhancing hepatic autophagy and in potentiating mitochondrial function and fatty acid oxidation. Our studies show that GDNF and its receptor agonists could be useful for enhancing hepatocyte survival and protecting against fatty acid-induced hepatic lipotoxicity.

PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. CONCLUSION: PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host-HBV interaction, thus providing new insights into targeted therapeutic intervention. (Hepatology 2017;66:398-415).

Functional lipidomics: Palmitic acid impairs hepatocellular carcinoma development by modulating membrane fluidity and glucose metabolism.

Lipids are essential cellular components and energy sources of living organisms, and altered lipid composition is increasingly recognized as a signature of cancer. We performed lipidomic analysis in a series of hepatocellular carcinoma (HCC) cells and identified over 1,700 intact lipids originating from three major lipid categories. Comparative lipidomic screening revealed that 93 significantly changed lipids and decreased palmitic acyl (C16:0)-containing glycerophospholipids were positively associated with metastatic abilities of HCC cells. Furthermore, both in vitro and in vivo experiments demonstrated that C16:0 incubation specifically reduced malignant cell proliferation, impaired cell invasiveness, and suppressed tumor growth in mouse xenograft models. Biochemical experiments demonstrated that C16:0 treatment decreased cell membrane fluidity and limited glucose metabolism. A phosphoproteomics approach further revealed such C16:0 incubation attenuated phosphorylation levels of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) pathway proteins. Multiple reaction monitoring analysis of 443 lipid molecules showed 8 reduced C16:0-containing lipids out of total 10 altered lipids when cancer tissues were compared with adjacent nontumor tissues in a cohort of clinical HCC specimens (P < 0.05). CONCLUSION: These data collectively demonstrate the biomedical potential of using altered lipid metabolism as a diagnostic marker for cancerous cells and open an opportunity for treating aggressive HCCs by targeting altered C16:0 metabolism. (Hepatology 2017;66:432-448).

Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities.

Cancer cell lines of human tissue origin have been extensively used to investigate antiproliferative activity and toxicity of herbal extracts, isolated compounds, and anticancer drugs. These cell lines are genetically and/or epigenetically well characterized to determine the altered expression of proteins within given cellular pathways and critical genes in cancer. Human derived hepatoma (HepG2) cell line has been extensively exploited to examine cytoprotective, antioxidative, hepatoprotective, anti-hepatoma, hypocholesterolemic, anti-steatosis, bioenergetic homeostatic and anti-insulin resistant properties. Moreover, mechanism of action of various botanicals and bioactive constituents has been reported using these cells. HepG2 cells have significant differences as compared to primary hepatocytes with respect to expression of cytochrome P450 enzymes and xenobiotic receptors in conventional in vitro culture conditions. Therefore, strategies have been employed to overcome limitations of two dimensional (2D) in vitro HepG2 cell culture in order to recognize functional biomarkers more accurately and to boost its predictive value in clinical research. In consequence, three dimensional (3D) human hepatoma cell culture models are being developed as a resource to achieve these goals of simulating the in vivo tumor microenvironment. It is assumed that bioengineered 3D hepatoma cell culture models can provide significant assistance in scrutinizing the molecular response of herbal natural products to recognize novel prognostic targets and crucial biomarkers in treatment strategies for cancer patients in near future.

[Effect of shift rotation culture on formation and activity of encapsulated hepatocytes aggregates].

Objective: To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates. Methods: AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC). Results: On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all P<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all P<0.01). Conclusion: Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.

Metabolism and cytotoxic effects of phosphatidylcholine hydroperoxide in human hepatoma HepG2 cells.

In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis.

Regulation of HepG2 cell apoptosis by hepatitis C virus (HCV) core protein via the sirt1-p53-bax pathway.

Hepatitis C virus (HCV) core protein stimulates many signaling pathways related to apoptosis inhibition resulting in hepatocellular carcinoma (HCC). It has been reported that sirt1 is involved in regulating apoptosis; therefore, we investigated the influence of HCV core protein on sirt1 expression and apoptosis in human HepG2 cells. Our study showed that HCV core protein inhibited apoptosis of HepG2 cells as well as caspase-3 expression and activity (P < 0.05). At the same time, sirt1 expression was increased at both the mRNA (P < 0.05) and protein (P < 0.05) levels. Furthermore, apoptosis inhibition was reversed when sirt1 was knocked down (P < 0.05). Our study provides further evidence that the sirt1-p53-Bax signaling pathway plays an important role in regulating the suppression of cell apoptosis induced by HCV core protein.

Anti-proliferative effect of Fe(III) complexed with 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone in HepG2 cells.

We previously developed a chelating ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB-ASH), which can chelate Fe(III) to form a complex. The HMB-ASH-Fe(III) complex exhibits a dose-dependent anti-proliferative effect in HepG2 cells, whereas the ligand, HMB-ASH, and Fe(III) alone had no considerable effect. The HMB-ASH-Fe(III) complex was composed of Fe(III):HMB-ASH (1:2), as determined by high-performance liquid chromatography with high-resolution mass spectrometry. The IC50 value was approximately 20 µM, which was comparable to those of the anti-cancer drugs oxaliplatin (OXP) and etoposide (ETP) under the same conditions. Similar to OXP and ETP, HMB-ASH-Fe(III) induced apoptosis in HepG2 cells, as revealed by terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling assay.

Transient knockdown of presenilin-1 provokes endoplasmic reticulum stress related formation of autophagosomes in HepG2 cells.

The involvement of presenilins in the endoplasmic reticulum (ER) related autophagy was investigated by their transient knockdown in HepG2 cells. The silencing of PSEN1 but not of PSEN2 led to cell growth impairment and decreased viability. PSEN1 silencing resulted in ER stress response as evidenced by the elevated levels of glucose regulated protein 78 (Grp78), protein disulfide isomerase (PDI), and CCAAT/enhancer-binding protein homologous protein (CHOP) and by the activation of activating transcription factor 6 (ATF6). The activation of autophagy was indicated by the increased procession of microtubule-associated light chain 3 protein isoform B (LC3B) and by decreased phosphorylation of mammalian target of rapamycin (mTOR) and 70kDa ribosomal protein S6 kinase (p70S6K). Formation of ER-related cytoplasmic vacuolization colocalizing with the autophagic marker LC3B was also observed. The morphological effects and LC3B activation in presenilin-1 knockdown cells could be prevented by using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin or by calcium chelation. The results show that presenilin-1 hampers the ER stress dependent initiation of macroautophagy.

miR-203 inhibits proliferation of HCC cells by targeting survivin.

To validate whether down-regulation of microRNA-203 (miR-203) in hepatocellular carcinoma (HCC) is involved in HCC progression by targeting survivin. MiR-203 mimics was transfected into HepG2 cells to enhance miR-203 expression, and miR-203 inhibitor was transfected into HepG2 cells to inhibit miR-203 expression. The effect of up-regulation and down-regulation of miR-203 on survivin expression of HepG2 cells was evaluated using Western blot assay. The effect of miR-203 or survivin expression on the proliferation of HepG2 cells was detected using the CKK-8 assay. Over-expression of miR-203 significantly inhibited the expression of survivin in HepG2 cells (p < 0·05), and down-expression of miR-203 significantly promoted the expression of survivin in HepG2 cells (p < 0·05). Both over-expression of miR-203 and down-regulation of survivin suppressed proliferation of HepG2 cells significantly compared with negative control. Low expression of miR-203 contributes to the progression of HCC via targeting survivin.

Ultrastructural and molecular analyzes of insulin-producing cells induced from human hepatoma cells.

BACKGROUND AIMS: Diabetes type I is an autoimmune disease characterized by the destruction of pancreatic insulin-producing (beta-) cells and resulting in external insulin dependence for life. Islet transplantation represents a potential treatment for diabetes but there is currently a shortage of suitable organs donors. To augment the supply of donors, different strategies are required to provide a potential source of beta-cells. These sources include embryonic and adult stem cells as well as differentiated cell types. The main goal of this study was to induce the transdifferentiation (or conversion of one type cell to another) of human hepatoma cells (HepG2 cells) to insulin-expressing cells based on the exposure of HepG2 cells to an extract of rat insulinoma cells (RIN). METHODS: HepG2 cells were first transiently permeabilized with Streptolysin O and then exposed to a cell extract obtained from RIN cells. Following transient exposure to the RIN extract, the HepG2 cells were cultured for 3 weeks. RESULTS: Acquisition of the insulin-producing cell phenotype was determined on the basis of (i) morphologic and (ii) ultrastructural observations, (iii) immunologic detection and (iv) reverse transcription (RT)-polymerase chain reaction (PCR) analysis. CONCLUSIONS: This study supports the use of cell extract as a feasible method for achieve transdifferentiation of hepatic cells to insulin-producing cells.

In vitro and QSAR studies of cucurbitacins on HepG2 and HSC-T6 liver cell lines.

The aim of this study was to evaluate cucurbitacins (Cucs) liver protective activity in vitro and conduct QSAR studies against lipophilicity and ab initio descriptors. Nine Cucs were isolated from Cucurbitaceae plants and eight prepared by C2-alkylation or C16-acylation. Ten Cucs demonstrated protective activity on human hepatocyte-derived HepG2 cells exposed to CCl(4) (EC(50)=2.4-45.3 µM) with good margin to toxicity (T/A). All Cucs exhibited anti-proliferative effect on serum-activated rat stellate cells, HSC-T6 (EC(50)=0.02-4.12 µM) with high T/A. While silybin is nontoxic, its protection is lower compared to Cuc D (3), iso-D (4), I (5), B (11), E (12), I-Me (6), L-Me (7), and E-Me (13) on both cell lines. Strong correlations were found for lipophilicity with both protection and toxicity on HepG2. Lipophilicity correlated only with toxicity on HSC-T6. Consequently, we suggest that Cucs are potential hepatoprotective agents against fibrosis that deserve further examination.

Cryopreservation of encapsulated liver spheroids using a cryogen-free cooler: high functional recovery using a multi-step cooling profile.

Acute liver failure has high mortality with unpredictable onset. A bioartificial liver, comprising alginate-encapsulated HepG2 spheroids, could temporarily replace liver function but must be cryopreservable. For clinical use, contamination risks from liquid coolants for cryopreservation and storage should be minimized. A cryogen-free cooler was compared to nitrogen vapour-controlled cryopreservation of alginate-encapsulated liver cell spheroids (AELS). AELS were cooled using a multi-step, slow-cooling profile in 12 percent v/v Me2SO Celsior and stored in liquid nitrogen; temperatures were recorded throughout, and the AELS were assayed at 24, 48 and 72 hours post-warming and results compared to unfrozen control values. Viability was assessed by fluorescent staining and quantified using image analysis; cell numbers were quantified using nuclear counts, and cell function using albumin synthesis. The cryogen-free cooler performed the cooling profile as desired, apart from one step requiring a rapid cool ramp. Viability, cell numbers and function were similarly decreased in both cryopreserved groups to about 90 percent, 70 percent and 65 percent of the controls respectively. This technology offers a clinic alternative to liquid nitrogen-coolant cryopreservation.

[The CD133 polyclonal antibody generation and cancer stem cells identification].

OBJECTIVE: To generate the cancer stem cells (CSCs) specific protein CD133 polyclonal antibody for the study of the biological characteristics of CSCs in tumor tissues and CSCs screening for the mouse model. METHODS: The extracellular peptide of the human CD133 was injected into rabbits to generate polyclonal antibody which was used for glioblastoma(GBM) Western blot and immunohistochemistry. RESULTS: The CD133 antiserum we made could detect both overexpressed myc-CD133 and endogenous CD133 efficiently by Western blot. Immunohistochemistry indicated that the CD133 polyclonal antibody can label CSCs in GBM sections. CONCLUSION: High efficient and specific CD133 antibody was generated successfully and could be used to label CSCs in tumor sections and screen CSCs for the mouse model.

Assessing the toxicity in vitro of degradation products from deoxynivalenol photocatalytic degradation by using upconversion nanoparticles@TiO2 composite.

Deoxynivalenol (DON) is one of the most globally prevalent mycotoxins mainly produced by Fusarium species. It can cause pollution to water environmental quality due to its water solubility. Therefore, it is necessary to develop a green and efficient detoxification technology for DON. More importantly, the toxicity of the degradation products should be assessed. Photocatalytic degradation technology has attracted increasing attention in the field of pollutants treatment, especially for wastewater treatment. Herein, the as-prepared NaYF4:Yb,Tm@TiO2 composite (UCNP@TiO2) was employed as a novel photocatalyst for the NIR-enhanced photocatalytic degradation of DON. Three intermediate products were identified by using the ESI/MS analysis and secondary mass spectrogram, with the m/z values of 329.399, 311.243 and 280.913, respectively. Furthermore, the in vitro safety of the product mixtures with various degradation time (30 min, 60 min, 90 min and 120 min) were evaluated through the influences on cell viability, cell morphology, cell cycle, intracellular reactive oxygen species (ROS) level, cell apoptosis and antioxidant capacity of HepG2 cells. There were no significant differences in these investigated indicators between the control (free of DON) and 120 min products treatment. Overall, the results indicated that the toxicity of degradation products after 120 min irradiation was much lower and even nontoxic than that of DON.

Alginate-encapsulated HepG2 cells in a fluidized bed bioreactor maintain function in human liver failure plasma.

Alginate-encapsulated HepG2 cells cultured in microgravity have the potential to serve as the cellular component of a bioartificial liver. This study investigates their performance in normal and liver failure (LF) human plasma over 6-8 h in a fluidized bed bioreactor. After 8 days of microgravity culture, beads containing 1.5 x 10(9) cells were perfused for up to 8 h at 48 mL/min with 300 mL of plasma. After exposure to 90% LF plasma, vital dye staining showed maintained cell viability, while a 7% increase in lactate dehydrogenase activity indicated minimal cell damage. Glucose consumption, lactate production, and a 4.3-fold linear increase in alpha-fetoprotein levels were observed. Detoxificatory function was demonstrated by quantification of bilirubin conjugation, urea synthesis, and Cyp450 1A activity. These data show that in LF plasma, alginate-encapsulated HepG2 cells can maintain viability, and metabolic, synthetic, and detoxificatory activities, indicating that the system can be scaled-up to form the biological component of a bioartificial liver.

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies.

BACKGROUND: Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes. OBJECTIVE: For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes. METHODS: POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method. RESULTS: The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3(p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models. CONCLUSION: In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.

Comparative analysis of toxicity reduction of wastewater in twelve industrial park wastewater treatment plants based on battery of toxicity assays.

Wastewater treatment plants (WWTPs) in industrial parks provide centralized treatment for industrial and domestic wastewater. However, the information on toxicity reduction of wastewater and its correlation with treatment process in industrial park is limited. This study compared the toxicity reduction of wastewater in 12 industrial park WWTPs based on battery of toxicity assays. Nine toxic endpoints involving microorganism, phytoplankton, zooplankton, plant and human cell lines were applied. All the influents of WWTPs induced high toxicities, which were significantly reduced after the treatments from 7 of the studied WWTPs. However, the effluents of five WWTPs induced higher toxicity in one or more toxic endpoints compared to the influents. This study also found that most of anaerobic-anoxic-oxic (A2/O)-based processes had good removal efficiency of wastewater toxicity, while the sequencing batch reactor (SBR)-based processes had the lowest removal efficiency. Moreover, low correlation coefficients were obtained among all toxic endpoints, indicating that battery of toxicity assays was necessary to completely characterize the toxicity and risk of wastewater in industrial parks. This study shed new lights to the toxicity reduction of wastewater and its correlation with treatment process, which is very useful for the design, management and operation of WWTPs in industrial parks.

Generation of Multilayered 3D Structures of HepG2 Cells Using a Bio-printing Technique.

BACKGROUND/AIMS: Chronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare. METHODS: A 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liverspecific markers was quantified on days 1, 7, 14, and 21. RESULTS: The cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture. CONCLUSIONS: The 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver.

Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells.

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.