Expression Of Liver-specific Functions Research Articles

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.

Membrane Bioreactor for Expansion and Differentiation of Embryonic Liver Cells

There is a growing demand for the expansion and differentiation of stem cells for cell therapies, tissue engineering, and model systems for drug screening. Current methods for stem cell production are based on the use of batch tissue culture flasks, which have several drawbacks. In this paper we report on the use of a crossed hollow fiber membrane bioreactor for the expansion and differentiation of embryonic liver cells, which have been used as an alternative model of human liver progenitor cells. The bioreactor is based on two bundles of fiber (PEEK-WC HF and PES-HF) which are cross-assembled in an alternating manner. This bioreactor geometry ensures high mass exchange of nutrients and metabolites, which is important for cell proliferation and differentiation. The membrane bioreactor, thanks to its optimized fluid dynamics and mass transport and the adequate surface properties of hollow fibers, was able to guide the expansion and differentiation of liver progenitor cells in mature hepatocytes as demonstrated by their expression of liver specific functions in terms of urea synthesis, albumin production, and diazepam drug biotransformation.

Hepatocyte spheroid culture on a polydimethylsiloxane chip having microcavities

A two-dimensional microarray technique of spherical multicellular aggregates (spheroids) using a microfabricated polydimethylsiloxane (PDMS) chip and the expression of liver-specific functions of primary rat hepatocytes on the chip were investigated. The PDMS chip, which was fabricated by a photolithography-based technique, consisted of approximately 2500 cylindrical microcavities (approximately 1100 cavities/cm2) in a triangular arrangement of 330 μm pitch on a PDMS plate (20 × 20 mm); each cavity measured 300 μm in diameter and 100 μm in depth. Most hepatocytes on the PDMS chip gradually gathered and subsequently formed a single spheroid in each cavity until 3 days of culture. A part of the spheroid was attached to the bottom or wall surface of the microcavity, and the spheroid configuration was maintained for at least 14 days of culture. Albumin secretion, ammonia removal and ethoxyresorufin O-dealkylase (EROD) activity, which is a cytochrome P-450-dependent reaction, of hepatocytes on the PDMS chip were higher than those of a monolayer dish or a flat PDMS dish without microcavities, and were maintained for at least 10 days of culture. The spheroid microarray technique appears to be promising in the development of cell chips and microbioreactors.

Spheroid formation and expression of liver specific functions of primary rat hepatocytes co-cultured with bone marrow cells

We have developed a new co-culture system consisting of adhesive bone marrow cells (A-BMCs), non-adhesive bone marrow cells (NA-BMCs) and hepatocytes with improved hepatocyte immobilization efficiency and better maintenance of liver specific functions. The composition of the inoculated cells affected the morphology of hepatocytes. Spheroids formed spontaneously when rat hepatocytes were co-cultured with bone marrow cells (BMCs). However, the addition of NA-BMCs to existing hepatocyte monolayers did not change their morphologies to spheroids. On the other hand, NA-BMCs dramatically increased hepatocyte immobilization efficiency. Hepatocytes co-cultured with either NA-BMCs or A-BMCs maintained their albumin production activities significantly better than hepatocyte culture alone. Interestingly, the two fractions of BMCs appear to have combination effects on hepatocytes to maintain albumin production activity. We conclude that co-culture of hepatocytes and BMCs is an effective strategy to enhance hepatocyte immobilization efficiency and functions in vitro.

Expression of liver-specific functions by rat hepatocytes seeded in treated poly(lactic-co-glycolic) acid biodegradable foams.

Techniques of liver replacement would benefit patients awaiting donor livers and may be a substitute for transplantation in patients whose livers can regenerate. Poly(lactic-co-glycolic acid) (PLGA) copolymers are biodegradable and have been shown to be useful as scaffolds for seeding and culturing various types of cells. In this study, foam disks were prepared from PLGA (lactic-to-glycolic mole ratio of 85:15) by lyophilization of benzene (5% w/v) solutions. These disks were then used as scaffolds for rat hepatocyte culture. Foams were coated with either a type I collagen gel (0.1% w/v), coated with gelatin (5% w/v), or treated with oxygen plasma (25 W, 90 s) to modify their surface chemistry and wettability. The disks were then seeded with rat hepatocytes (10(6)/mL) and cultured for a period of 2 weeks. All surface treatments resulted in increased hydrophilicity, the greatest being obtained by collagen treatment (contact angle < 10 degrees ), and a minimal decrease in void fraction (5%). DNA content after a 2-week culture period increased proportionally with the wettability of the treated foam surface. Urea synthesis in untreated foams averaged 15.3 +/- 2.3 microg/h/microg DNA, which was significantly higher than that for controls, whereas gelatin and collagen treated foams exhibited urea synthetic rates below the control levels at all times. The DNA content decreased significantly by about 50% between days 1 and 12. PLGA foams, treated and untreated, represent a promising scaffold for scaling up hepatocyte cultures.

Formation of cylindrical multicellular aggregate (cylindroid) and expression of liver specific functions of primary rat hepatocytes.

In our studies of the development of a hybrid artificial liver, we investigated the formation of cylindrical multicellular aggregate (cylindroid) of primary rat hepatocytes on a pressed sheet of polyurethane foam (pressed-PUF) as a culture substratum. Hepatocytes formed cylindroids by attaching to a pressed-PUF surface, peeling off from the surface and aggregating. The diameter and length of most cylindroids were approximately 200-500 mum and 500 mum-2 mm, respectively. The activities of liver specific functions (albumin secretion and ammonia metabolism) of hepatocyte cylindroids were equivalent to or higher than those of hepatocyte spheroids. These results suggest that hepatocyte cylindroids can maintain highly differentiated functions longer than hepatocyte spheroids, and that a PUF/cylindroid culture may be effective to develop of a hybrid artificial liver.

Effect of Kurozu(Brewed Rice Vinegar) on Maintenance of Primary Culture of Rat Hepatocytes.

Kurozu is a brewed rice vinegar prepared by a traditional method in Kagoshima prefecture, Japan, and is regarded as a healthful food. The effects of the non-volatile components obtained from Kurozu (the residue of Kurozu) on the primary monolayer culture of rat hepatocytes were examined. In serum-free L-15 medium supplemented with the residue of Kurozu, about 60% of the hepatocytes inoculated to a dish survived for 6 days of culture. In contrast, without the residue, the number of hepatocytes decreased to less than one-third of the inoculated cells during the culture. The amount of albumin secreted in the medium was monitored as an expression of liver-specific function. In the medium with the residue of Kurozu, the rat hepatocytes secreted a considerable amount of albumin in 4-day culture (2.5 μg/ml medium/24 h), and even at 6 days (1.3 μg/ml medium/24 h), while albumin secretion by the hepatocytes cultured without the residue decreased rapidly with time. These results suggest that some components in Kurozu residue are effective to maintain the primary culture of rat hepatocytes and thus for albumin production by the hepatocytes.

The formation of a spheroid of primary hepatocytes and the expression of liver-specific functions depend on the characteristics of polyurethane foam

Rapid formation of spherical multicellular aggregates (spheroids) of hepatocytes and expression of liver-specific functions are important in developing a hybrid artificial liver. The relation between these points and the surface characteristics of the culture substratum are investigated in this article. Polyurethane foam (PUF) was used as a culture substratum for hepatocytes. Rat, dog, and porcine primary hepatocytes spontaneously formed a spheroid within 1.5 days in the pores of a hydrophilic PUF with a contact angle of 53.4±2.7°. Rat and dog primary hepatocytes formed a spheroid within 3 and 7 days of culture, respectively, in hydrophobic PUF with a contact angle of about 100°. The rates of albumin secretion and ammonia metabolism at 5 days of culture of porcine hepatocytes were 31 μg of albumin/106 nuclei/day and 0.018 μmol of NH3/106 nuclei/h, respectively, about 2 times higher than the values with hydrophobic PUF. It is very important to control the hydrophilicity of the PUF surface to develop an effective artificial liver module.

Primary culture of chicken hepatocytes in serum-free medium (pH 7.8) secreted albumin and transferrin for a long period in free gas exchange with the atmosphere

To study the liver functions of chicken, we examined the primary culture of chicken hepatocytes, and found an easy method of long-term culture with free atmosphere exchange. Chicken hepatocytes were obtained by collagenase perfusion and cultured at 37°C as a monolayer without substratum in serum-free L-15 medium (pH 7.8) with free atmosphere exchange. The amounts of albumin and transferrin in medium were assayed by ELISA. The culture of chicken hepatocytes was maintained in the serum-free L-15 medium (pH 7.8) at 37°C with free atmosphere exchange for 20 days. The amount of albumin secreted in the medium decreased to low levels early in culture; however, this was followed by marked increase from day 9 to day 17 of culture. The amount of transferrin was constant until day 6, then it too increased considerably with further culture. We reported an easy method for the simple monolayer culture of chicken hepatocytes in serum-free L-15 medium (pH 7.8) with free atmosphere exchange over an extended period. Expression of liver-specific functions, viz. albumin and transferrin synthesis, was observed after 1 week of culture.

Expression of liver-specific functions in rat hepatocytes following sublethal and lethal acetaminophen poisoning

Aim: In order to study the short-term effect of moderate and severe reduction of liver function by acetaminophen poisoning of different severity on gene expression for liver-specific functions, rats were given 3.75 and 7.5 g per kg body weight acetaminophen intragastrically. The lower dose is associated with low mortality; after the higher dose, most rats die at between 12 and 24 h. Methods: In the morning 1 1 2 ,3,6,9, and 12 h after the injection, the rats were killed and RNA was extracted from liver tissue. By slot-blot hybridization mRNA steady-state levels were determined for enzymes involved in metabolic liver functions, i.e. unreagenesis, gluconeogensis, and drug metabolism, for acute phase proteins, “house-keeping” proteins, and for proteins related to liver regeneration. Results were expressed as per cent of the level in similarly fasted, untreated rats of the same stock Results: After the smaller dose of acetaminophen, most of the examined mRNA levels were increasing during the experimental period, being two- to four-fold elevated in relation to control after 6 to 12 h. Rats receiving the lethal dose either showed no or a later and smaller increase, and in several cases a fall towards the end of the experiment. The greatest differences were seen for mRNA of arginase, β-fibrinogne, α1-acid glycoprotein, α-tubulin, histone 3, TGFβ, and cyclin d, i.e. proteins associated with acute phase response and liver cell replication and maintenance. Conclusions: It is concluded that reversible intoxication with acetaminophen induces an adaptive modulation of mRNA expression of liver functions and regeneration which is lacking after severe intoxication. This adaptation, with emphasis on acute phase response and regeneration, may be crucial for recovery after acetaminophen intoxication. If this also applies to the intoxication in man, estimates of the corresponding variables may be clues to the prognosis of acetaminophen-induced fulminant hepatic failure.

Effect of 350 kDa Glycoprotein in Royal Jelly on Primary Culture of Rat Hepatocytes.

The functions of the 350 kDa glycoprotein in royal jelly (RJ) on the primary monolayer culture of rat hepatocytes were examined. In the serum-free L-15 medium supplemented with the RJ protein, the monolayer culture of hepatocytes was maintained for 20 days. In contrast, without the RJ glycoprotein, the number of the hepatocytes which adhered to the dish decreased to less than one-half the number of inoculated cells during 20 days of culture. The amount of albumin secreted in the medium was monitored as an expression of liver-specific function. In the medium with the RJ glycoprotein, the hepatocytes maintained the secretion of albumin at a similar high level for 2 days compared to that in the starting culture, followed by diminishment to a low level. In contrast, the secretion of albumin by the hepatocytes cultured without RJ glycoprotein was decreased rapidly with time. As a control experiment, bovine serum albumin added to the medium did not show any effect both on the maintenance or albumin synthesis of rat hepatocytes.

Expression of liver-specific functions and secretion of a hepatocyte growth factor by a newly established rat hepatoma cell line growing in a chemically-defined serum-free medium

A rat hepatoma cell line 3'-mRLh-2 was established from 3'-methyl-4-dimethylaminoazobenzene-induced hepatoma. Cells proliferated well in 5Fs-DM-160, a chemically-defined serum-free medium; population doubling time was 68.5 h, and modal chromosome number was 81 (21%). The cells were transplantable, and the transplanted tumors were histologically diagnosed as hepatocellular carcinoma. They were cytochemically positive for gamma-glutamyl transpeptidase. The cells possessed about 30% of tyrosine aminotransferase activity level in the rat liver, and showed 5.5 to 7.4-fold induction of this enzyme activity in response to dexamethasone. Also, the cells secreted alpha-fetoprotein, albumin, transferrin, alpha 1-acid glycoprotein, C-reactive protein, fibrinogen, complement component C3, and other five-serum proteins. Furthermore, the conditioned medium stimulated DNA synthesis in primary cultures of adult rat hepatocytes in a dose-dependent, saturable manner and in the absence of epidermal growth factor. These properties of the cell line 3'-mRLh-2 were compared with those of the popular rat hepatoma cell lines, such as H4-II-E-C3 from Reuber hepatoma H35 and HTC from Morris hepatoma 7288C.

Presence of cytochrome P-450- and cytochrome P-448-dependent monooxygenase functions in hepatoma cell lines

Cell lines derived from Reuber H-4-II-E hepatoma cells and their hybrids that differ in the expression of liver-specific functions are shown to contain different forms of monooxygenases. According to 1) the specificity toward the substrates benzo(a)pyrene, aldrin and chenodexycholic acid, 2) the kinetics of the epoxidation of aldrin, 3) the response to inducers, such as benz(a)anthracene and dexamethasone, and 4) the in vitro modifier 7,8-benzoflavone, the monooxygenases predominating in differentiated cell lines belong to the cytochrome P-450-dependent enzyme(s), those in the less differentiated lines belong to the cytochrome P-448-dependent form(s).

Characterization of differentiated and dedifferentiated clones from a rat hepatoma

An analysis of clonal variability of derivatives of the rat hepatoma line H4IIEC3 has shown that the overwhelming majority of clones express in a stable fashion a number of liver specific functions, including secretion of serum albumin, activity of the liver specific isozymes of alcohol dehydrogenase (EC1.1.1.1) and aldolase (EC4.1.2.13), and high basal activity and hormone inducibility of tyrosine aminotransferase (EC2.6.1.5) and alanine aminotransferase (EC2.6.1.2). The differences in level of expression of these functions cover a range of five to ten-fold, and the variations do not appear coordinated within or between clones. Seven clones, which differ from the above ones both in morphology and in the expression of liver specific functions, have been isolated. In five of them, no expression of any of the functions is detectable, while two of them show diminished but significant expression of two or three of the functions. In addition, an unexplained negative correlation between activity of glucose-6-phosphate dehydrogenase (EC1.1.1.49) and the expression of liver specific functions is described. L'analyse de la variabilité des clones dérivés de la souche d'hépatome de rat H4IIEC3 a montré que la très grande majorité de ceux-ci expriment de façon stable un certain nombre de fonctions spécifiques de la différenciation hépatique, notamment la sécrétion de sérum albumine, l'activité des iso-enzymes spécifiques du foie de l'alcool déshydrogénase (EC.1.1.1.1) et de l'aldolase (EC.4.1.2.13), le haut niveau de base et l'inductibilité par les hormones stéroïdes de la tyrosine aminotransférase (EC.2.6.1.5) et de l'alanine aminotransférase (EC.2.6.1.2). Les différences entre les clones des niveaux d'expression de ces fonctions sont d'un facteur cinq à dix et ces variations ne paraissent pas être coordonnées. Sept clones distincts des précédents par leurs morphologies et l'expression des fonctions différenciées ont été isolés. Cinq d'entre eux n'expriment aucune de ces fonctions, et deux autres en expriment deux ou trois à un taux réduit mais significatif. De plus, une corrélation inverse, qui reste inexpliquée, entre l'activité glucose-6-phosphate déshydrogénase (EC.1.1.1.49) et l'expression des fonctions différenciées est décrite.