Induction of uncoupling protein (UCP) 2 in primary cultured hepatocytes.

Uncoupling protein 2 (UCP2) mRNA expression and function was examined in rat primary cultured hepatocytes. UCP2 mRNA was not expressed in freshly isolated hepatocytes, but appeared during a 24-144 h primary culture period. Isolated mitochondria from 144 h cultured hepatocytes showed a lower oxygen consumption rate in the presence of succinate and ADP. However, the ratio of the oxygen consumption rate when media contained succinate alone to that with succinate and ADP was increased by 166% versus control mitochondria. Moreover, the mitochondrial potential in the presence of succinate was decreased by 60%, indicating the potential role of UCP2 in hepatocyte mitochondria as an active uncoupler.     

Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E

Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic conditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of antioxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the protective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated.Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vitamin E at 18 μg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic conditions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients.     

Physiological oxygen tension modulates the chemically induced mitogenic response of cultured rat hepatocytes

Freshly isolated rat hepatocytes were cultured at periportal- (13% O₂) or perivenous-like (4% O₂) oxygen tension and exposed to subtoxic exposure levels of cyproterone acetate (CPA: 10–330 μM), phenobarbital (PB: 0.75-6 mM), and dimethylsulfoxide (DMSO: 0.1–3.3%) from 24–72 h after seeding. Induced alterations in ploidy, in the number of S-phase cells, the degree of binuclearity, and cellular protein content were determined by twin parameter protein/DNA flow cytometry analysis of intact cells and isolated nuclei. CPA and PB increased whereas DMSO decreased dose dependently the total number of S-phase cells. The changes differed within individual ploidy classes and were modulated by the oxygen tension. CPA increased and DMSO decreased the number of S-phase cells preferentially among the diploid hepatocytes at periportal-like oxygen tension. In contrast, PB increased binuclearity and S-phase cells mainly among the tetraploid hepatocytes at perivenous-like oxygen tension. Cellular protein content increased dose dependently after exposure to the hepatomitogens (CPA, PB) and decreased after exposure to DMSO at both oxygen tensions. Comparison with in vitro data proves that chemicals which interact with cells from the progenitor liver compartment (CPA, DMSO) exert their mitogenic activity best in cultures at periportal-like oxygen tension preferentially in diploid hepatocytes, whereas chemicals which affect cells from the functional compartment show a higher activity at perivenous-like oxygen tension. Physiological oxygen tension seems to be an effective modulator of the proliferative response of cultured rat hepatocytes similar to that expected for periportally or perivenously derived hepatocytes. © 1993 Wiley-Liss, Inc.     

Enhanced maintenance and functions of rat hepatocytes induced by combination of on-site oxygenation and coculture with fibroblasts

In in vivo liver tissue, each hepatocyte has intimate interactions not only with adjacent hepatocytes but also with nonparenchymal cells in a three-dimensional (3D) manner. We recently reported that hepatic function is highly maintained on collagen covalently immobilized poly-dimethylsiloxane (PDMS) membranes through which oxygen is supplied directly to the cells. In this study, to further enhance performances of hepatocytes culture, we investigated cocultivation of rat hepatocytes with a mouse fibroblast, NIH/3T3 (3T3) in the same PDMS membranes. Various functions of hepatocytes were better maintained on the membrane at remarkably higher levels, particularly albumin secretion on such coculture was about 20 times higher than that in conventional coculture on tissue-culture-treated polystyrene (TCPS) surfaces. The remarkable functional enhancements are likely to be explained by the net growth of hepatocytes (from 1.2- to 1.4-fold inoculated number) and very intimate contact between hepatocytes and 3T3 cells in almost continuous double-layered structures under the adequate oxygen supply. The results demonstrate that simultaneous realization of different requirements toward mimicking in vivo liver tissue microstructure is effective in improving performance of hepatocytes culture system.     

Rapid and enhanced repolarization in sandwich-cultured hepatocytes on an oxygen-permeable membrane

In this study, we established rat primary hepatocyte sandwich cultures on oxygen-permeable membranes and investigated the change in their repolarization. Functional bile canaliculi in sandwich-cultured hepatocytes on oxygen-permeable polydimethylsiloxane (PDMS) membranes were re-established more quickly than those in a conventional sandwich culture on polystyrene (PS). This enhanced biliary excretory activity was also observed in hepatocytes on another oxygen-permeable membrane plate but not on a PDMS surface whose oxygen permeability is blocked. An apical efflux transporter protein, Mrp2, was more rapidly distributed in hepatocytes cultured on PDMS membranes than in hepatocytes cultured on conventional PS plates. Moreover, the area of distribution of the Mrp2 in polarized hepatocytes cultured on PDMS membranes was more widespread than that for the hepatocytes grown on sandwich-cultured PS plates. The observation of ultrastructure in transmission electron microscopy clearly confirmed the presence of bile canalicular lumens possessing microvilli and tight junctions. Additionally, we demonstrated that the 7-ethoxyresorufin-O-deethylation activity of hepatocytes on PDMS membranes was also improved as compared to those on a PS surface. Therefore, sandwich-cultured hepatocytes on oxygen-permeable substrates can provide a simple tool for predicting the hepatic metabolism and toxicity of xenobiotics in vivo with short span and low cost in the course of drug discovery and evaluation.     

Gas‐permeable membranes and co‐culture with fibroblasts enable high‐density hepatocyte culture as multilayered liver tissues

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three‐dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell–cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate‐based format, enabling high density hepatocyte culture as a stable 3D‐multilayer. Multilayered co‐cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen‐conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co‐cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate‐based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co‐cultures were maintained for at least 1 week; the so‐cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate‐based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

The importance of proline on long-term hepatocyte function in a collagen gel sandwich configuration: regulation of protein secretion

A serum-free culture system for primary hepatocytes which maintains stabel high-level hepatocyte function for prolonged periods in culture has been developed. Isolated rat primary hepatocytes were cultured in serum-free media between two layer of gelled collagen in a sandwich configuration which reinstates the cellular polarity necessary for long-term function in vitro. Thsee serum-free hepatocyte cultures maintained near physiological rates of albumin and transferrin secretion for a minimum of 26 days in culture. L-Proline was shown to be critical for both the approach to steady state and maximal level of protein secretion. Analysis of does-response data gave K(m) values of 2.9 and 1.7 mug/mL for albumin and transferrin secretion, respectively.     

Structural reaction pattern of hepatocytes following exposure to hypotonicity

Isolated rat hepatocytes were exposed to hypotonic media (225 mosmol/l) for 5 and 15 min and processed for a quantitative electron microscopic stereologic analysis. Within 5 min of hypotonicity, the hepatocyte volume increased by 25% and thereatter displayed a volume regulatory decrease leading to mean cellular volume, which was 16% above that of controls. Stereologic analysis of the major subcellular compartment, the cytosol, showed an identical change as the whole cell. In contrast to that, the mitochondrial compartment increased in volume by 30% within the first 5 min of exposure and returned by regulatory volume decrease back to values of the isotonic controls after 15 min of hypotonicity. In contrast, hypotonicity (220 mosmol/l)-stimulation of flux through mitochondrial glutaminase and the glycine cleavage enzyme complex, as assessed by ¹⁴CO₂ production from [1-¹⁴C]glutamine or [1-¹⁴C]glycine in isolated perfused rat liver persisted throughout a 15-min period of hypotonic exposure. Thus hypotonicity-induced alterations of mitochondrial metabolism apparently do not parallel the time course of mitochondrial volume changes. This suggests that persistent mitochondrial swelling is not required for functional alterations, but that the latter may be triggered by the initial swelling of mitochondria. Hypotonic exposure did not alter the nuclear volume of isolated hepatocytes. Cell membrane surface nearly doubled after 5 min of hypotonic exposure, but returned within 15 min of exposure to values observed in normotonic media. This may reflect the participation of exocytosis in hepatocyte volume regulation. © 1993 Wiley-Liss, Inc.     

Lipid peroxidation is not the cause of lysis of human erythrocytes exposed to inorganic or methylmercury

Exposure of human erythrocytes in a 50% hematocrit to 0.5-1 mM Hg2+ initiated immediate hemolysis which proceeded at a constant rate without any formation of lipid hydroperoxides. Treatment of 0.03% hematocrits with 0.4 ppm of Hg2+ or 40 ppm of methylmercury caused rapid hemolysis after a short lag period. The kinetics of the process were unaltered by saturation of the cell suspensions with oxygen, by its replacement with He or CO, or by variation in the level of vitamin E in the membranes. The results show that peroxidation of erythrocyte membrane lipids is not the cause of hemolysis induced by either Hg2+ or methylmercury.     

Autophagy

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.     

Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.     

Influence of whole-body gamma-irradiation upon rat erythrocyte: lipid peroxidation and osmotic fragility

The effects of whole-body gamma-irradiation of rats (8 Gy) on erythrocyte enzymes and biochemical components involved in lipid peroxidation were studied. Decreased superoxide dismutase and glutathione reductase activities, and lowered concentrations of reduced glutathione, were found to be the main factors responsible for the observed increase in lipid peroxidation in the erythrocytes of irradiated rats. This increased lipid peroxidation did not result in a greater tendency to hemolysis in hypotonic media; on the contrary, the mean osmotic fragility was decreased at days D + 1 and D + 3 after irradiation. The behavior of the erythrocyte populations towards hemolysis in hypotonic media appeared to be most homogeneous at days D + 4 and D + 8 after irradiation, which correspond to maxima of malonic dialdehyde concentrations in erythrocytes. Such a synchrony of variations suggests that crosslinking of primary amino groups of proteins or phospholipids by malonic dialdehyde might produce a rigidification in erythrocyte membranes, possibly leading to a more homogeneous behavior of the erythrocyte populations towards hemolysis in hypotonic media.     

Stabilization of erythrocytes against oxidative and hypotonic stress by tannins isolated from sumac leaves (Rhus typhina L.) and grape seeds (Vitis vinifera L.)

Erythrocytes are constantly exposed to ROS due to their function in the organism. High tension of oxygen, presence of hemoglobin iron and high concentration of polyunsaturated fatty acids in membrane make erythrocytes especially susceptible to oxidative stress. A comparison of the antioxidant activities of polyphenol-rich plant extracts containing hydrolysable tannins from sumac leaves (Rhus typhina L.) and condensed tannins from grape seeds (Vitis vinifera L.) showed that at the 5-50 μg/ml concentration range they reduced to the same extent hemolysis and glutathione, lipid and hemoglobin oxidation induced by erythrocyte treatment with 400 μM ONOO(-) or 1 mM HClO. However, extract (condensed tannins) from grape seeds in comparison with extract (hydrolysable tannins) from sumac leaves stabilized erythrocytes in hypotonic NaCl solutions weakly. Our data indicate that both hydrolysable and condensed tannins significantly decrease the fluidity of the surface of erythrocyte membranes but the effect of hydrolysable ones was more profound. In conclusion, our results indicate that extracts from sumac leaves (hydrolysable tannins) and grape seeds (condensed tannins) are very effective protectors against oxidative damage in erythrocytes.     

Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.     

Effect of 2,5-anhydro-D-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential (V(m)) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V(m) of hepatocyte couplets. Increasing intracellular Ca(2+) by addition of thapsigargin or ionomycin also did not evoke a change of V(m). This is most likely due to a lack of Ca(2+)-dependent K(+) channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na(+)/K(+) ATPase by ouabain. Ouabain was also able to block 2, 5-AM's effect on monolayer V(m). Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.     

Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: influence on cellular DNA and protein content monitored by flow cytometry

Primary hepatocytes were cultured at oxygen tensions similar to those reported to be present in periportal (13% O2) and pericentral (4% O2) regions of the liver lobules. Cellular DNA and protein content of individual hepatocytes were determined simultaneously by two-parameter (DNA/protein) flow cytometry after 1, 4, and 7 days in culture. pO2 tensions monitored on line in conventional plastic culture dishes revealed that the depletion of the pO2 in the culture medium depended on the number of hepatocytes plated. When cultured as monolayer after 4-7 days at periportal (13% O2) and more pronounced at pericentral oxygen concentration (4% O2), up to 90% of the hepatocytes showed degenerated nuclei but normal protein content. By using culture dishes with teflon membrane bottoms the oxygen tension in the culture medium was accurately maintained by the incubator atmosphere. At pericentral oxygen tension the fraction of 2N cells increased by about 20%. That of the 4N cell was not affected, and the contribution of 8N hepatocytes dropped to 70% compared to cultures at periportal oxygen tension. Concomitantly, in the 4% O2 hepatocyte cultures the protein content of the 2N and the 4N cells was better preserved and increased by up to 10%. These results suggest that in vitro at pericentral oxygen conditions (4% O2) ageing of hepatocytes is delayed, regenerating processes are better maintained, and, furthermore, freshly isolated 4N hepatocytes have the potency to adapt their metabolism in vitro to periportal as well as to perivenous oxygen tensions.     

Protective effect of S-adenosylmethionine on cellular and mitochondrial membranes of rat hepatocytes against tert-butylhydroperoxide-induced injury in primary culture

Accumulating evidence that administration of S-adenosylmethionine (SAMe) protects hepatocytes against oxidative stress-mediated injury led us to evaluate the effect of SAMe on hepatocyte injury induced in culture by oxidant substance tert-butylhydroperoxide (1.5 mM tBHP) with regard to prevent mitochondrial injury. The pretreatment of hepatocyte culture with SAMe in doses of 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 mg/l for 30 min prevented the release of LDH from cells incubated for 30 min with tBHP in a dose dependent manner. The inhibitory effect of SAMe on lipid peroxidation paralleled the effect on cell viability. SAMe also moderated the decrease of the mitochondrial membrane potential induced by tBHP. Our results indicate that the inhibition of lipid peroxidation by SAMe can contribute to the prevention of disruption of both cellular and mitochondrial membranes. While the protective effect of SAMe against tBHP-induced GSH depletion was not confirmed, probably the most potent effect of SAMe on membranes by phospholipid methylation should be verified.     

Interaction between plant polyphenols and the erythrocyte membrane

The purpose of these studies was to determine the effect of polyphenols contained in extracts from apple, strawberry and blackcurrant on the properties of the erythrocyte membrane, treated as a model of the biological membrane. To this end, the effect of the substances used on hemolysis, osmotic resistance and shape of erythrocytes, and on packing order in the hydrophilic region of the erythrocyte membrane was studied. The investigation was performed with spectrophotometric and fluorimetric methods, and using the optical microscope. The hemolytic studies have shown that the extracts do not induce hemolysis at the concentrations used. The results obtained from the spectrophotometric measurements of osmotic resistance of erythrocytes showed that the polyphenols contained in the extracts cause an increase in the resistance, rendering them less prone to hemolysis in hypotonic solutions of sodium chloride. The fluorimetric studies indicate that the used substances cause a decrease of packing order in the hydrophilic area of membrane lipids. The observations of erythrocyte shapes in a biological optical microscope have shown that, as a result of the substances’ action, the erythrocytes become mostly echinocytes, which means that the polyphenols of the extracts localize in the outer lipid monolayer of the erythrocyte membrane. The results obtained indicate that, in the concentration range used, the plant extracts are incorporated into the hydrophilic area of the membrane, modifying its properties.     

Mechanical lysis of human erythrocytes. Membrane stabilization by plasma proteins]

Mechanical properties of erythrocyte membranes play an important role in red cell functions. Stability of human erythrocytes under deforming mechanical tensions which occur in the rapidly moving fluid is studied. The activation energy of the mechanical hemolysis determined by the temperature dependence of the hemolysis rate is 55 + 7 kJ/mol. The fragility of erythrocytes rises sharply as the salt concentrations increase. Glutaric dialdehyde forms a certain number of interprotein bonds which increase the fragility of erythrocytes. The mechanical stability of the erythrocyte membrane falls at high (0.5 M) ethanol concentrations. Blood plasma proteins, particularly human serum albumin, have a pronounced stabilizing effect. The hemolysis occurring during the rapid mixing is not probably associated with an osmotic mechanism since high sucrose concentrations do not prevent this process. The mechanical hemolysis depends both on the deforming tension arising in the membrane and on the state of the erythrocyte membrane.     

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O₂ (+)] or physiological oxygen concentrations [10%‐O₂ (+), 5%‐O₂ (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O₂ (?)]. The results indicated that the hepatocytes under 10%‐O₂ (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O₂ (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014