Maintenance of hepatocyte functions by coculture with bone marrow stromal cells

Rat hepatocytes and bone marrow stromal cells (BMSCs) were cocultured with the aim of maintaining differentiated hepatocyte functions. After BMSCs were expanded to a confluent monolayer, freshly isolated hepatocytes were cultured with them, separated by a semipermeable membrane. The BMSCs significantly increased the urea synthesis and albumin secretion activities of the hepatocytes. Conditioned medium prepared from a BMSC monoculture had the same effect. Further study showed that interleukin-6 was involved in the maintenance of urea synthesis and another factor in the maintenance of albumin secretion.     

Micropatterned organoid culture of rat hepatocytes and HepG2 cells

The culture of liver cell organoids (multicellular aggregates) such as spheroids or cylindroids, which can strongly express liver functions, has been advocated as a useful technique that has advantages over monolayer culture. This paper describes a micropatterning technique for obtaining spheroids and cylindroids by using rat hepatocytes or HepG2 cells. We developed culture chips that comprised multiple, circular or rectangular microwells; the bottom surface of each microwell was modified with collagen to create a cell adhesion area, and the entire microwell, excluding the collagen-coated spots, was modified with polyethylene glycol (PEG) to create a nonadhesive area. Rat hepatocytes and HepG2 cells formed uniform spheroids and cylindroids on the circular and rectangular chips, respectively. Consequently, two-dimensional micropatterned chips containing homogeneous spheroids or cylindroids were generated. The expression of liver functions (protein secretion and ammonia removal) was greater in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture. Thus, this chip technology has potential for use in various applications that involve organoid culture.     

Effects of the mycotoxin deoxynivalenol on human primary hepatocytes

Toxic effects of the mycotoxin deoxynivalenol (DON) observed in animals range from diarrhea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. In the last years, DON has been tested in hepatocytes of several animal species for its cytotoxicity. However, these tests are limited to the use of animal cells. No studies using human hepatocytes are available. Further investigations with the human hepatocellular liver carcinoma cell line HepG2 might be limited due to the disadvantages of cell lines (e. g. immortalization, tumor derivation, longtime cultivation) and do not necessarily reflect the response of normal human cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human primary hepatocytes and compared these data to the effects in the HepG2 cell line. Cell viability, apoptotic and necrotic cell death, albumin secretion and metabolic activity were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary hepatocytes. Viability, protein content and albumin secretion were reduced in a dose-dependent manner. The apoptotic key enzyme caspase-3 was activated, while LDH release occurred only after long incubation time due to a secondary necrosis. Furthermore, we studied the metabolism of DON using LC-MS/MS. DON was neither metabolized by primary hepatocytes cells nor by the HepG2 cell line.     

Protective effects of protein hydrolysate from marine microalgae Navicula incerta on ethanol-induced toxicity in HepG2/CYP2E1 cells

Ethanol is independently known to cause tissue damage through various mechanisms. This study was designed to evaluate the protective effect of marine microalgae, Navicula incerta protein enzymatic hydrolysates (NEHs) against ethanol-induced hepatotoxicity in HepG2 cells transfected with human CYP2E1. Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. When the alcohol-induced cells were treated with NEHs at various concentrations, there was a dose-dependent decrease of gamma-glutamyl transpeptidase (GGT) activity in the culture media and loss of cell viability. Among the NEHs constituents the hydrolysates which were obtained by papain (P-NEH), pronase-E (PR-NEH) and α-chymotrypsin (A-NEH) activity attenuated the ethanol cytotoxicity effectively, respectively. The activity appeared to be a GGT inhibitor. Therefore, the cytoprotective effects at alcohol-induced HepG2/CYP2E1 cells could be attributed to the inhibition of GGT activity by NEHs. This study suggests that NEHs have enough potential to be considered as highly active compounds against ethanol toxicity which leads NEHs to be significant nutraceuticals.     

琐琐葡萄总黄酮在体外对乙型肝炎病毒的影响

目的：考察琐琐葡萄总黄酮体外抗乙型肝炎病毒(HBV)的效果。方法：用HBV DNA转染人肝癌细胞所得的HepG 2.2.15细胞株为模型,ELISA法测定用药后8d细胞培养上清液中HBsAg和HBeAg含量,计算琐琐葡萄总黄酮对HBsAg和HBeAg分泌的抑制率,荧光定量PCR法测定HBV DNA的变化,MTT比色法检测细胞毒性。结果：琐琐葡萄总黄酮在12.5～100μg/mL范围内,均能不同程度地减少细胞培养上清液中HBsAg和HBeAg的含量,且细胞毒性小,半数中毒剂量(TC50)为284.91μg/mL,对HBsAg半数有效剂量(IC50)为327.56μg/mL、对HBeAg为215.34μg/mL,对HBsAg治疗指数(TI)为0.87、对HBeAg治疗指数为1.32;总黄酮质量浓度100μg/mL时对HBV DNA的抑制率为47.63%。结论：琐琐葡萄总黄酮具有一定的体外抗HBV作用。     

Construction of heterotypic cell sheets by magnetic force-based 3-D coculture of HepG2 and NIH3T3 cells

Heterotypic 3-D coculture is essential to mimic tissues and organs, because cell-cell interaction between various types of cells is believed to be important for the activation of cellular functions. In this study, magnetic force was applied to construct a 3-D coculture system of HepG2 and NIH3T3 cells as a model of hepatocytes and mesenchymal cells. Magnetite cationic liposomes (MCLs) were used to label target cells. NIH3T3 cells labeled with MCLs were seeded onto ultralow-attachment plates, whose surface is composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. When a magnet was placed under the plate, cells accumulated on the bottom of the well. After a 24-h incubation period, the cells formed a multilayered cell sheet, which contained the major mesenchymal extracellular matrix (ECM) components (fibronectin and type I collagen), suggesting that the use of stromal NIH3T3 cells gave sufficient strength to cell sheets. Both NIH3T3 and HepG2 cells were labeled with MCLs, and cocultured by two methods: NIH3T3 cell sheets were constructed and HepG2 cells were subsequently seeded onto NIH3T3 cell sheets, and then allowed to form layered cell sheets by applying magnetic force; or NIH3T3 and HepG2 cells were mixed and then allowed to form mixed cell sheets by applying magnetic force. These heterotypic multilayered cell sheets were successfully constructed and an enhanced albumin secretion by HepG2 cells was observed. These results suggest that the new tissue engineering technique using magnetite nanoparticles and magnetic force, to which we refer to as magnetic force-based tissue engineering (Mag-TE), is a promising approach to construct multilayered cell sheets consisting of heterotypic cocultured cells.     

Organotypic co-culture system to study plant extract bioactivity on hepatocytes

Natural plant extracts are complex mixtures of molecules that have to be uptaken and metabolized, at least by the intestinal barrier, before they reach their body targets. This paper evaluates the feasibility of using a co-culture system to assay the bioactivity of plant extract on hepatic cells closest to the in vivo situation. Caco-2 cells, mimicking intestinal barrier roles, were grown on inserts on a monolayer of HepG2 hepatocytes for 24 h. The co-culture induced some adaptations, but the Caco-2 cells showed no differences in their abilities to filter and metabolize grape-seed derived extract (GSPE) components. The co-culture clearly reproduced the ability of a procyanidin extract to decrease the triglyceride secretion of hepatocytes, a proven in vivo effect. This system mimics a human physiological system that is useful for assaying the bioactivity of extracts and may be able to be used for developing new functional foods.     

Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes

A hepatocyte growth factor (HGF)/heparin-immobilized collagen system was used as a synthetic extracellular matrix for hepatocyte culture. The albumin synthesis, nucleus numbers and morphology of the hepatocytes were determined separately to evaluate the hepatocyte number and hepatocyte-specific function under this system. The benefits of the HGF/heparin-immobilized collagen system for hepatocyte culture were confirmed by three types of culture methods in vitro, namely 2D film cultures, 2D gel cultures and 3D gel cultures. In 2D collagen film cultures, hepatocytes exhibited the highest albumin synthesis (1.42 μg/well/day) in HGF/heparin-immobilized collagen films at 7 days of culture. Heparin inhibited hepatocyte adhesion while HGF promoted hepatocyte migration, and spheroid formation was easily detected in HGF/heparin-immobilized collagen films. In 2D collagen gel cultures, albumin synthesis of around 15 μg/well/day was detected and maintained for more than 18 days on HGF/heparin-immobilized collagen gels. Similar findings were obtained in 3D HGF/heparin-immobilized collagen gel cultures, which exhibited albumin synthesis of up to 30 μg/well/day. The albumin synthesis by hepatocytes was two-fold higher in 3D gel cultures compared with 2D gel cultures, and was maintained for over 2 weeks compared with 2D film cultures using the HGF/heparin-immobilized collagen system. Taken together, the HGF/heparin-immobilized collagen system was effective for albumin synthesis by hepatocytes in both 2D film cultures and 3D gel cultures, and therefore shows good potential for tissue engineering use.     

Cultivation of rat hepatocytes in a medium based on commercially available infusate solutions

The possibility of using commercially available infusate solutions as a culture medium for hepatocytes was investigated in primary monolayer cultures of rat hepatocytes. The addition of Ca2+ to the infusate medium was necessary for hepatocytes to express their albumin secreting ability. The infusate medium supplemented with hormones (10(-7) M insulin and 10(-7) M dexamethasone) and Ca2+ (72.5 mg/l) allowed hepatocytes to produce albumin of an amount comparable to that produced in Williams' E medium. The activity of released lactate dehydrogenase (LDH) was kept at a low level throughout the cultivation in the infusate medium.     

Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells

Human hepatic cancer cells, HepG2, formed spheroids on a poly-L-glutamic acid-coated dish. Doxorubicin (DOX) efflux activity of the cells in spheroid culture was higher than that in monolayer culture due to the higher expression of MDR1 protein of the cells in spheroids compared with those in monolayer. The amount of MDR1 per cell in spheroids was similar to that of hepatic tumor tissue in vivo. Consequently, it was suggested that the drug efflux activity of cells in spheroid culture reflected the activity of hepatic cancer cells. Furthermore, the IC(50) of DOX and epirubicin (EPI) in HepG2 cells, both of which are known to be exported by MDR1, were higher in spheroid compared with monolayer cells, while IC(50) of 5-fluorouracil (5-FU), which is not exported by MDR1 protein, was almost the same in both types of culture. The higher IC(50) of DOX and EPI in HepG2 cells in spheroid culture was associated with a higher efflux activity of the drugs in the spheroid-cultured cells, which appeared to reflect the IC(50) of DOX and EPI in cancer cells in vivo. Therefore, a spheroid culture of hepatic cancer cells seems to provide a promising cell-based in vitro assay system for examining the proper IC(50) values of anticancer agents that would reflect the drug resistance of cancer cells in vivo. In addition, the system would be useful in screening for inhibitors of MDR1 activity, which will help to overcome the multidrug resistance of cancer cells.     

普洱茶茶色素对HepG2细胞脂质代谢的影响及作用机理

以正常培养和油酸诱导培养的人肝癌细胞系(human hepatocellular liver carcinoma cell line,Hep G2)细胞为模型,通过测定普洱茶茶色素对Hep G2细胞内甘油三酯(triglyceride,TG)和总胆固醇(total cholesterol,TC)含量,细胞中脂肪酸合成酶(fatty acid synthase,FAS)、固醇调节元件结合蛋白1c(sterol regulatory element binding protein 1c,SREBP-1c)、三磷酸腺苷结合转运子A1(ATP binding cassette transporter A1,ABCA1)、胆固醇7α-羟化酶(cholesterol 7α-hydroxylase,CYP7A1)的转录水平,磷酸化腺苷酸活化蛋白激酶(phospho-AMPactivated protein kinase,p-AMPK)的蛋白表达水平研究普洱茶茶色素的减肥降脂作用机制。结果显示,普洱茶茶色素能明显降低油酸诱导的Hep G2细胞模型中TG和TC含量,作用程度依赖于普洱茶的作用质量浓度。但对正常培养的Hep G2的TG、TC作用影响不显著。经过普洱茶茶色素作用油酸诱导Hep G2细胞24 h后,能显著下调细胞的FAS和SREBP-1c的m RNA表达水平(P0.05),显著上调ABCA1的转录水平(P0.05),且使CYP7A1的转录水平呈上升趋势,并显著上调p-AMPK蛋白的表达量(P0.05)。因此,普洱茶茶色素可通过调控上述调控因子和酶的表达而改善油酸诱导下Hep G2细胞的脂质代谢水平。     

Morphology of spheroidal hepatocytes within injectable, biodegradable, and thermosensitive poly(organophosphazene) hydrogel as cell delivery vehicle

An aggregate of specific cells is often regarded as a better form of single-cell in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, cultivated as spheroids and entrapped in a synthetic thermoreversible extracellular matrix, were examined for differentiation morphology and enhanced liver-specific functions and compared with a control set (single-cell hepatocytes). A copolymer of poly(organophsophazene) hydrogel, an injectable, biodegradable, and thermosensitive matrix, was used to entrap hepatocytes as spheroids or single cells. In a 28-d culture period, the spheroidal hepatocytes maintained a higher viability and produced albumin and urea at constant rates, whereas the single hepatocytes showed a reduction in the level of albumin secretion in the hydrogel. The culturing of spheroidal hepatocytes is a potentially useful three-dimensional cell system for application in bioartificial liver devices and bioreactors.     

Effects of ready to drink teas on AhR- and PXR-mediated expression of cytochromes P450 CYP1A1 and CYP3A4 in human cancer cell lines and primary human hepatocytes

A variety of xenobiotics are taken in the diet and they can interfere with regulatory pathways of drug metabolizing enzymes in humans. This can result in food-drug interactions, which is undesirable clinical situation where drug pharmacokinetics are influenced by dietary compounds. Xenobiotics-mediated food-drug interactions include the induction of drug metabolizing cytochromes P450. The expression of the most important inducible cytochromes CYP1A and CYP3A4 are regulated by xenoreceptors PXR and AhR.We examined extracts from 17 different flavoured ready to drink teas (RDTs) for their capabilities to activate PXR and AhR receptors and to induce CYP3A4 and CYP1A genes. Primary cultures of human hepatocytes and cancer cell lines HepG2 and LS174T were used as in vitro models. Gene reporter assays, RT-PCR and Western blots were performed.We identified three RDTs that induced CYP3A4 mRNA and protein, implying a potential for food-drug interactions. Several RDTs slightly elevated CYP1A1 expression or activated AhR.     

酒精对人原代培养肝细胞的氧化损伤与CYP 2E1关系的研究

目的 研究急性酒精暴露下对人原代培养肝细胞中CYP 2E1依赖的毒性作用和氧化损伤。方法 分离培养人原代肝细胞，以25-100mmol/L乙醇作用于人原代肝细胞9h及100mmol／L乙醇作用于人原代肝细胞0～24h后，检测人原代肝细胞中CYP 2E1的含量，并研100mmol/L乙醇作用于人原代肝细胞0～24h后，天冬氨酸转胺酶(aspartate transaminase，AST)的释放量及肝细胞中谷胱甘肽(Glutathione，GSH)、丙二醛(Malonclialdehyde，MDA)的含量。结果 急性酒精暴露导致人原代肝细胞中CYP 2E1的释放增加，并呈明显的剂量效应和时间效应关系；在100mmol/L乙醇作用下，AST和MDA明显升高，在0～24h内呈明显的时间效应关系，而GSH含量在6h后明显降低。结论 100mmol／L乙醇急性暴露可导致人原代培养肝细胞明显的氧化损伤，这种损伤与CYP 2E1活性的变化直接相关。     

Structure-dependent relative toxic potencies of selected pyrrolizidine alkaloids

Pyrrolizidine alkaloids are naturally occurring secondary plant metabolites mainly found in plant families of Asteraceae, Boraginaceae, and Fabaceae. Chemically, Pas consist of a pyrrolizidine core bearing hydroxyl groups, the so-called necine base, and mono- or dicarboxylic necine acids bound to the pyrrolizidine core via ester linkages. 1,2-unsaturated PAs are hepatotoxic, genotoxic, and carcinogenic due to the highly reactive pyrrolic metabolites formed by cytochrome P450 monooxygenases (CYPs) primarily in the liver. The presence of PAs as frequent contaminants in the wide variety of food and feed products has to be considered a relevant safety issue. Based on the currently available data, the risk assessment of PAs was mainly approached using the two most toxic potent congeners, i.e., lasiocarpine and riddelliine. However, it is well recognized that toxicity is differing significantly between the congeners related to their structural features. The risk of PA-containing products is indeed overestimated, and a comprehensive risk assessment should take these differences into account. After analyzing the data of many PAs, Merz and Schrenk derived interim Relative Potency (iREP) factors to present the differences in their toxicity between the sub-groups of PA congeners concerning their structural features. The use of such iREP factors could probably provide a more scientific basis for PA risk assessment until sufficient experimental analysis of the toxicities of individual congeners is applied. To obtain a better understanding of the relationship between structure and toxicity of PA congeners and provide more evidence for further refinement of relative (toxic) potency factors, data of the in vitro cytotoxicity, genotoxicity, and mutagenicity of diverse individual PA congeners (lasiocarpine, monocrotalineφ, retrorsine, senecionine, seneciphyllineφ, echimidineφ, europineφ, heliotrineφ, indicine, and lycopsamine) has been generated in our project supported by Kooperation Phytopharmaka. Among them, lasiocarpine, retrorsine, senecionine, indicine and lycopsamine have been investigated on my part. Cytotoxicity was assessed using the Alamar blue assay in primary rat hepatocytes, HepG2 cells, and the HepG2 (CYP3A4) cell line. In HepG2 cells, none of the selected PAs exhibited cytotoxic effects, probably due to the lack of CYPs. In primary rat hepatocytes as well as in HepG2(CYP3A4) cells, a clear structure dependent cytotoxicity could be demonstrated. The role of CYP450 enzymes in metabolic activation was further confirmed using an inhibition assay. A kinetic assay analyzing 7-benzyloxyresorufin-O- dealkylation (BROD) was used for measuring the activity of CYP450 enzymes. Furthermore, the utilization of a glutathione-reductase-DTNB recycling assay indicated that glutathione might not play a critical role in PA-induced cytotoxicity. A micronucleus test was used for determining the PA-induced clastogenic genotoxicity. All selected PA congeners exhibited concentration-dependent toxicity in the HepG2 (CYP3A4) cells. The relative potencies of PA congeners estimated from Alamar blue assay and micronucleus assay are generally consistent with the following ranking: lasiocarpine > senecionine > seneciphylline > retrorsine > heliotrine (?) echimidine > europine = indicine = lycopsamine = monocrotaline. The relative toxic potencies evaluated based on our findings were not completely consistent with the iREP classification previously reported by Merz and Schrenk. Monocrotaline in both assays exhibited considerably lower toxic potency. Echimidine, however, was more toxic than expected. On the other hand, mutagenicity was measured in Ames fluctuation assay with Salmonella typhimurium strains TA98 and TA100. None of the selected PA congeners up to 300 μM showed mutagenic effects despite metabolic activation with S9-mix.     

Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture

Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.     

Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH)

We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.     

Anti‐proliferative activity of bovine blood hydrolysates towards cancer cells in culture

Four proteins, α/β globulin, serum albumin, γ‐globulin and fibrinogen, were isolated from bovine blood and hydrolysed using papain. Hydrolysates were assessed for non‐cellular and cellular antioxidant activity. The anti‐proliferative activity of hydrolysed fractions was assessed in a number of cancer cell lines including U937 lymphoma cells, MCF‐7 breast cancer cells, HepG2 hepatocytes and Caco‐2 epithelial colorectal adenocarcinoma cells. Anti‐inflammatory activity of the hydrolysates was also assessed. Hydrolysates generated from γ‐globulin or fibrinogen had significant antioxidant activity in non‐cellular assays. Hydrolysates were also found to be highly toxic to different cancer cell lines, in particular U937 lymphoma cells when assessed using the MTT assay. The fibrinogen hydrolysate was the most toxic sample and toxicity appeared to correlate with its non‐cellular antioxidant activity. None of the hydrolysates had significant anti‐inflammatory activity. The high cytotoxicity of the γ‐globulin and the fibrinogen hydrolysates towards cancer cells may indicate a potential use as anti‐proliferative agents.     

Availability of bone marrow stromal cells in three-dimensional coculture with hepatocytes and transplantation into liver-damaged mice

Rat bone marrow stromal cells (BMSCs) were cultured in porous hydroxyapatite (HA) disks for 2 weeks to form a cell layer on the surface. Freshly isolated hepatocytes were then inoculated into both BMSC-cultured and non-treated HA disks. Hepatocytes cocultured with BMSCs secreted significantly more albumin than those in monoculture in vitro. The cell-packed HA disks were implanted into the peritoneal cavity of Nagase analbuminemia rats (NARs), and 4 weeks later, blood samples were collected to measure the albumin concentration. The cotransplantation of BMSCs with hepatocytes significantly increased the serum albumin concentration in NARs. The HA disks coculturing mice hepatocytes and BMSCs were also implanted into mice, in which liver damage had been induced using carbon tetrachloride and phenobarbital. The decreased serum albumin level in liver-damaged mice was completely recovered by the transplantation of hepatocytes and BMSCs. The serum level of IL-6 in liver-damaged mice was also increased by the cotransplantation of BMSCs and hepatocytes. Thus, the transplantation of BMSCs appears to have a systemic effect on recipients through the increase in the serum cytokine level as well as a local effect on cotransplanted hepatocytes.