Three-dimensional high-density culture of HepG2 cells in a 5-ml radial-flow bioreactor for construction of artificial liver

A three-dimensional high-density cell culture is essential for the construction of an artificial tissue. Many researchers have reported that three-dimensional cell culture enhances cell function. The use of a radial-flow bioreactor (RFB) has enabled the cultivation of cells at high density for constructing a three-dimensional tissue. In this study, we have developed a novel, small RFB, which has a bed volume of 5 ml and is equipped with a porous support as an immobilized scaffold; its performance was tested using the hepatoblastoma cell line, HepG2. Among the other supports tested here, hydroxyl apatite was selected from the viewpoint of its ability to support good cell growth at high density with uniform distribution in a bioreactor. The HepG2 cells grew well in the scaffold under a sufficient supply of nutrients by radial flow and were used to construct a three-dimensional tissue in the scaffold. The concentration of the cells cultivated in this 5-ml RFB reached 10(8) cells/ml and the glucose consumption rate was almost similar to that obtained when using a 30-ml RFB, which has already been reported previously. This high glucose consumption continued over 7 d after the growth phase. Furthermore, albumin production was maintained in the stable phase. Gene expression profiles of cells obtained from long-term cultures in the 5-ml RFB were analyzed. It was found that the expressions of genes encoding the cell cycle-related proteins, cyclins, and cell cycle division 2 (cdc2) were suppressed in the stable phase. In addition, the number of cells incorporating 5'-bromo-2'-deoxyuridine (BrdU) in the stable phase markedly decreased compared with that in the growth phase. These results indicated that the majority of cells in the stable phase remain in the G0/G1 phase. Furthermore, this implies that the three-dimensional tissue constructed in the 5-ml RFB showed the high function similar to a normal liver in the human body. Therefore, the 5-ml RFB was considered as a useful tool and a substitute method for animal experiments.     

Vanillin rich fraction regulates LDLR and HMGCR gene expression in HepG2 cells

Vanillin and its analogs have been exploited for their various health benefits. This work aimed to investigate the antioxidant properties and regulatory effects of vanillin rich fraction (VRF) extracted from vanilla pods using the supercritical fluid extraction (SFE) and commercial vanillin on low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) gene expression in HepG2 cells. The vanillin content in the VRF was 2.6% (w/w) obtained at a temperature of 80 °C and a pressure of 600 bar. The VRF exhibited better antioxidant activity compared to the vanillin in DPPH and BCB tests. LDLR mRNA level was increased significantly by 2, 3 and 1.3 fold in the VRF treated cells at 100, 200 and vanillin treated cells at 100, respectively, compared with untreated cells. On the other hand, the HMGCR mRNA level was decreased significantly by 14, 58 and 13% respectively, in the VRF treated cells at 100, 200 and V treated cells at 100, respectively, compared with untreated cells. The VRF showed potential antioxidant activity and regulated genes involved in cholesterol metabolism including LDLR and HMGCR in dose-dependent manner.     

Effect of bottom clearance on performance of airlift bioreactor in high-density culture of Panax notoginseng cells

A fed-batch cultivation of Panax notoginseng cells in a concentric-tube airlift reactor was performed to study the effects of bottom clearance on cell growth and the production of ginseng saponin and polysaccharide. At a bottom clearance of 4.0 cm, the highest cell density of 29.1+/-1.6 g/l by dry weight was obtained, and the accumulation of saponin and polysaccharide also reached a maximum, i.e., 2.39+/-0.43 and 2.73+/-0.40 g/l, respectively. Cell growth and metabolite production were limited at a small (2.5 cm) or large (5.0 cm) bottom clearance. By analyzing the time constants of mixing, mass transfer and oxygen consumption, bulk gas-liquid oxygen transfer was found to be responsible for the growth limitation at a small bottom clearance (2.5 cm). The decrease in cell density at a large bottom clearance (5.0 cm) was related to cell sedimentation at the reactor bottom. This work is beneficial for the scale-up and efficient operation of the airlift reactor in cell cultures.     

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.     

Antioxidant behaviour of carotenoids highly accumulated in HepG2 cells

The antioxidant behaviour of major dietary carotenoids accumulated at high concentrations in human hepatoma HepG2 cells was evaluated, in comparison with α-tocopherol. The cells that accumulated carotenoids and α-tocopherol at levels higher than the values reported in the human liver were exposed to mild oxidative stress with tert-butylhydroperoxide. β-Carotene (>2.6 nmol/mg protein) and astaxanthin (>1.8 nmol/mg protein) significantly suppressed lipid peroxidation, while β-cryptoxanthin and lutein did not. α-Tocopherol remarkably suppressed lipid peroxidation with an IC₅₀ value of 0.16 nmol/mg protein. Neither α-tocopherol nor any of the carotenoids except for lycopene showed pro-oxidant action even at high cellular concentrations. The antioxidant behaviours of carotenoids in a cellular milieu were quite different from those previously found in liposomes and homogeneous solutions. Further studies are required to assess the implications of the antioxidant behaviours found in the cultured cells on human health.     

小麦低聚肽对HepG2细胞胆固醇代谢的影响

目的:在对小麦低聚肽的基础理化成分和分子量分布进行分析的基础上,观察不同浓度的小麦低聚肽对人肝癌细胞株HepG2胆固醇代谢的影响。方法:将培养的HepG2细胞随机分为5组:对照组、2、4、6、8g/L小麦低聚肽实验组。经小麦低聚肽作用24h后,进行反转录-聚合酶链反应（RT-PCR）,定量分析低密度脂蛋白受体（LDLR）和3-羟基-3-甲基戊二酸单酰辅酶A还原酶（HMGCR）mRNA的表达。结果:与对照组相比,经小麦低聚肽作用后,各实验组HepG2细胞LDLR mRNA表达明显增加（p<0.05或p<0.01）,其中6g/L小麦低聚肽组的LDLR mRNA表达增加最多;HMGCR mRNA表达均降低,2、4、6g/L小麦低聚肽组的HMGCR mRNA表达极显著降低（均为p<0.01）,其中2g/L小麦低聚肽组的HMGCR mRNA表达降低最多。结论:不同浓度的小麦低聚肽均能升高HepG2细胞内LDLR mRNA水平和降低HMGCR mRNA水平,从而起到降低胆固醇的作用,为其开发利用提供了一定的实验依据和理论基础。      

根皮苷对油酸诱导HepG2细胞脂肪及胆固醇代谢基因表达的影响

以噻唑兰(MTT)比色法检测细胞活力,筛选出合适的根皮苷药物浓度及油酸浓度,建立油酸诱导肝癌细胞(Hep G2)产生脂肪堆积体外模型。分别以MTT值、Hep G2细胞形态、Oil Red O脂滴染色、甘油三酯(TG值)、胆固醇(TC值)、脂肪代谢及胆固醇代谢基因SREBP-1C、FAS、CPT-1、PPARα、HMGCR mRNA表达水平为指标,考察根皮苷对油酸高脂模型细胞的影响。根皮苷能够显著抑制油酸诱导的Hep G2细胞脂肪堆积作用,90μmol/L根皮苷能够降低脂肪合成相关基因的表达,提高脂肪氧化代谢基因的表达,降低胆固醇合成相关基因的表达。     

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.     

金松酸对HepG2细胞中甘油三酯蓄积的影响

为探讨香榧金松酸(SA)对甘油三酯(TG)蓄积的影响及其作用机制,本研究以HepG2为模型细胞,首先采用MTT法确定油酸(OA)与SA的临界安全浓度,然后通过油红染色法观察细胞内脂质积累情况并定量测定了细胞内TG含量,最后利用RT-qPCR分析SA对细胞脂质代谢关键基因的影响。结果显示：OA和SA总添加量为500 mol/L,随着SA替换量从0%上升到100%,细胞内TG蓄积明显降低,从0.033 mmol/g减低到0.017 mmol/g;脂肪酸合成关键基因FAS、ACC1、SCD1、SREBP1的表达量分别降低了40.92%、62.01%、25.56%、43.79%,脂肪酸氧化关键基因SCD1、PPARα的表达量分别提高了143.94%、445.11%,TG分解基因ATGL的表达量增加了123.71%。因此,SA显著抑制了细胞TG蓄积,其可能机制是SA通过上调TG分解代谢关键酶同时下调TG合成代谢关键酶。作为天然食物成分,SA在减肥食品开发方面具有良好的应用前景。     

Antiproliferative and cytoprotective activities of a phenolic-rich juice in HepG2 cells

Antioxidant phytochemicals, especially the phenolics found in fruits and vegetables, have been proposed as the major bioactive compounds providing the health benefits associated with diets rich in plant-foods. In the present study, we aimed to evaluate the modulatory effect of a functional juice rich in phenolic compounds on cell proliferation and on the protection from oxidative stress caused by tert-butylhydroperoxide (t-BOOH) and hydrogen peroxide (H₂O₂) in HepG2 cells. Cell growth and cell viability were evaluated by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Lipid peroxidation was measured as the thiobarbituric acid reacting substances (TBARS) assay. Glutathione peroxidase (GPx) and glutathione-s-transferase (GST) activities were assayed by colorimetric assays. In order to elucidate the action mechanisms of juice phenolics, the protective effects of the juice were compared to those of the radical scavenger N,N′-diphenyl-1,4-phenylene-diamine (DPPD) and the iron chelator o-phenanthroline (o-phe). Cell proliferation was inhibited in a dose-dependent manner by juice extracts. Sub-toxic concentrations of phenolics (2–30 μg/mL of medium) increased cell survival and a decreased lipid peroxidation after exposure to both H₂O₂ and t-BOOH. Both juice and o-phe decreased the t-BOOH-induced GPx activation, but not that of GST. DPPD suppressed the t-BOOH-induced GST activation but not that of GPx. Our results support the efficacy of natural phenolics from grapes and berries in offering protection against oxidative stress. The possible role of an iron chelating mechanism, potentially of biological relevance, is proposed as being partially responsible for the health benefits provided by phenolic-rich processed foods.     

6-shogaol attenuates H2O2-induced oxidative stress via upregulation of Nrf2-mediated γ-glutamylcysteine synthetase and heme oxygenase expression in HepG2 cells

Food Science and Biotechnology - The signaling pathway by which 6-shogaol protects HepG2 cells against H2O2-induced oxidative stress was investigated. Cellular anti-oxidant activities, the GSH...     

D-手性肌醇及D-松醇对缓解HepG2细胞胰岛素抵抗的作用

对D-手性肌醇及D-松醇缓解胰岛素抵抗作用进行评价,选用HepG2人肝癌细胞建立胰岛素抵抗细胞模型。将实验分为正常对照组和D-手性肌醇及D-松醇实验组,分别设立50、100、200、400mg/L4个剂量。采用四甲基偶氮唑盐法(MTT法)检测受试样品对细胞增殖的影响。选用1×10-6mol/L浓度的胰岛素诱导细胞建立胰岛素抵抗模型,给予不同浓度D-手性肌醇及D-松醇,考察二者对胰岛素抵抗HepG2细胞葡萄糖消耗量的影响。实验结果表明,在200~1000 mg/L的浓度范围内D-手性肌醇和D-松醇对HepG2细胞的正常增殖无显著影响。D-手性肌醇浓度为50、100和200 mg/L时,对胰岛素抵抗HepG2细胞的葡萄糖消耗量与模型对照组相比有显著促进作用(p0.05),浓度为400 mg/L时,有极显著性差异(p0.01);D-松醇浓度为200 mg/L时,对胰岛素抵抗HepG2细胞的葡萄糖消耗量与模型对照组相比有显著促进作用(p0.05),浓度为400 mg/L时,有极显著性差异(p0.01)。因此,D-手性肌醇和D-松醇均能够缓解HepG2细胞胰岛素抵抗现象,且在1000 mg/L浓度范围内对HepG2细胞正常增殖无显著影响。     

二氢杨梅素和杨梅苷抑制HepG2细胞的协同作用

目的:探讨藤茶中的主要活性成分二氢杨梅素（DMY）和杨梅苷（MYT）抑制HepG2细胞增殖的协同作用。方法:通过对体外培养的细胞分别给予DMY、MYT和DMY:MYT为1:4、1:2、1:1、2:1、4:1、8:1的混合物,采用MTT法和荧光染色法测定其对HepG2细胞活力的影响,并用合用指数（CI）和等效曲线法分析二者的协同作用。结果:DMY和MYT能够抑制HepG2肿瘤细胞的增殖,且IC₅₀分别为56.46和95.01 μmol/L。当二者比例为1:4～8:1时,对HepG2肿瘤细胞增殖的IC₅₀为40.90～73.00 μmol/L,CI值为0.60～0.86,其中1:1时协同作用最强,等效线法也表明二者存在协同作用。结论:DMY和MYT联用能够有效提升单体化合物对HepG2细胞的抑制作用,呈现出协同作用,这为藤茶进一步的功能研究和应用提供了依据。     

Investigation of the cytotoxicity of food-grade nanoemulsions in Caco-2 cell monolayers and HepG2 cells

Nanoemulsions represent one of the emerging formulations for nutraceutical delivery. However, the possible toxicity associated with the small droplet size (diameter <200 nm) is still unknown. In this study, three nanoemulsions emulsified by modified starch, Tween 20 and whey protein isolate, respectively, were prepared and their cytotoxicity was examined by comparing with the corresponding micron-sized emulsions. Caco-2 cell monolayers were used to mimic the small intestine epithelium. Integrity of the cell membrane and tight junctions was tested by measuring the lactate dehydrogenase leakage and transepithelial electrical resistance, respectively. All three nanoemulsions did not reveal significant difference from their micron-sized counterparts, suggesting no apparent toxicity of the nanoemulsions on the small intestine. Meanwhile, the possible hepatic toxicity was investigated using MTT assay on HepG2 cells. It was found that nanoemulsions made with modified starch and whey protein isolate, but not Tween 20, affected the cell viability/proliferation more than did the micron-sized emulsions. Further in vivo investigation is required to examine the possible hepatic toxicity of nanoemulsions.     

过氧化氢诱导人肝癌细胞产生氧化应激最佳作用浓度的研究

不同浓度过氧化氢作用人肝癌细胞(HepG2)4 h后,用单细胞凝胶电泳技术测定DNA损伤状况,分光光度法测定MDA含量、SOD及GSH-Px活性,研究过氧化氢的最佳作用浓度,构建体外氧化应激细胞模型。实验结果表明,过氧化氢的最佳作用浓度为100μmol/L,作用细胞的时间选择4 h,可以成功构建以过氧化氢为氧化应激诱导剂的HepG2细胞体外氧化应激模型。