Primary Culture of Chicken Hepatocytes as an in Vitro Model for Determining the Influence of Dioxin

An easy method for primary culture of chicken hepatocytes was developed to study the influence of dioxin on birds. Chicken hepatocytes could maintain gene expression and protein secretion of albumin for a long period in serum-free medium with free atmosphere exchange at 37°C. Moreover, the cells showed a sensitive response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by monitoring the expression of P450 1A, theta GST (θ-GST) and albumin genes.     

Primary culture of chicken hepatocytes as an in vitro model for determining the influence of dioxin

An easy method for primary culture of chicken hepatocytes was developed to study the influence of dioxin on birds. Chicken hepatocytes could maintain gene expression and protein secretion of albumin for a long period in serum-free medium with free atmosphere exchange at 37 degrees C. Moreover, the cells showed a sensitive response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) by monitoring the expression of P450 1A, theta GST (theta-GST) and albumin genes.     

Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture.

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.     

Biosynthesis of plasma proteins in serum-free medium by primary monolayer culture of rat hepatocytes

Morphologically intact rat hepatocytes separated by collagenase perfusion were cultured in L-15/fetal calf serum medium to form a monolayer. Thereafter the hepatocytes were grown in serum-free L-15 medium in which they produced and continuously released plasma proteins. The secreted plasma proteins were collected, separated and characterized by crossed immunoelectrophoresis. Most of the newly biosynthesized plasma proteins secreted into the medium during incubation for thirty hours had the same electrophoretic mobility, antigenicity and staining characteristics as their counterparts in rat serum. The addition of tritium labelled amino acid mixture to the culture medium revealed that the release of radioactively labelled plasma proteins into the culture medium was essentially linear during the thirty hour incubation period. However, saturation of the intracellular pool took place after ca. ten hours of incubation. Addition of leukocytic endogenous mediator, LEM, to cultures of rat hepatocytes caused a profound increase in the relative concentration of acute-phase proteins secreted into the culture medium.     

Culture conditions affect induction of vitellogenin synthesis by estradiol-17 beta in primary cultures of tilapia hepatocytes

In vitro synthesis of vitellogenin (VTG), a female-specific protein, after estradiol-17 beta (E(2)) treatment was compared among different culture conditions using the hepatocytes of tilapia, Oreochromis mossambicus. VTG was measured by enzyme-linked immunosorbent assay. Comparison of Leibovitz's L-15 medium (L-15), Williams' medium E (WE) and Medium 199 (M199), which have been used for hepatocyte cultures in certain teleost fishes, showed that monolayer formation of the hepatocytes on the plate in WE and M199 was faster than in L-15 at the beginning of the culture. Morphological differences in the hepatocytes among the culture media were not evident by 96 h after culture. VTG synthesis in L-15 after E(2) treatment was higher than in WE and M199. A concentration of NaHCO(3) at 5 mM in L-15 resulted in faster monolayer formation of the cells and higher VTG synthesis than at 0 and 23 mM. Primary culture of the tilapia hepatocytes at 28 degrees C showed higher synthesis of VTG than at 23 and 33 degrees C. These results suggest that nutritional requirements are vitally different among species, and there are optimal ranges in the pH and the temperature in cultured hepatocytes.     

Anchorage-independent colony formation of SV40 transformed BALB/c-3T3 cells in serum-free medium: role of cell- and serum-derived factors

The colony-forming response of SV40 transformed BALB/c-3T3 cells in agarose suspension culture was studied in a serum-free medium (with insulin, transferrin and serum albumin as the only macromolecular supplements) that was optimized for colony formation of fibronectin-attached monolayer cultures. In this serum-free medium, the SV3T3 cells fail to form colonies in agarose suspension. However, they can be induced to anchorage-independent colony formation by the growth factors that are additionally required by their untransformed counterparts for proliferation in monolayer culture. The SV3T3 cells are also rendered anchorage-independent for colony formation in serum-free medium by conditioned medium from dense monolayer serum-free SV3T3 cultures. These experiments suggest that it is the cell-substrate interaction that is responsible for the growth factor autonomy of fibronectin-attached transformed cells.     

The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes

The role of high-affinity specific transferrin receptors and low-affinity, non-saturable processes in the uptake of transferrin and iron by hepatocytes was investigated using fetal and adult rat hepatocytes in primary monolayer culture, rat transferrin, rat serum albumin and a rabbit anti-rat transferrin receptor antibody. The intracellular uptake of transferrin and iron occurred by saturable and non-saturable mechanisms. Treatment of the cells with the antibody almost completely eliminated the saturable uptake of iron but had little effect on the non-saturable process. Addition of albumin to the incubation medium reduced the endocytosis of transferrin by the cells but had no significant effect on the intracellular accumulation of iron. The maximum effect of rat serum albumin was observed at concentrations of 3 mg/ml and above. At a low incubation concentration of transferrin (0.5 microM), the presence of both rat albumin and the antibody decreased the rate of iron uptake by the cells to about 15% of the value found in their absence, but to only 40% when the diferric transferrin concentration was 5 microM. These results confirm that the uptake of transferrin-bound iron by both fetal and adult rat hepatocytes in culture occurs by a specific, receptor-mediated process and a low-affinity, non-saturable process. The low-affinity process increases in relative importance as the iron-transferrin concentration is raised.     

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.     

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.     

Ascorbic acid supplementation to primary culture of chicken hepatocytes with non-serum medium

Chicken liver is lack of ascorbic acid biosynthesis system, different from mammals and highly evoluted birds. Chicken hepatocytes cultured without ascorbate was expected to have lower ascorbate amounts than physiological levels. Intracellular was decreased as compared with intact liver by cell preparation performed with in situ collagenase perfusion. We added ascorbate to a primary culture of chicken hepatocytes in order to restore the amount of ascorbate. Serum-free Leivobitz's L-15 medium which do not contain ascorbate was used for control medium. Cells were cultured with several concentrations of ascorbate for 24 or 48 h. After ascorbate supplementation for 24 to 48 h, cellular ascorbate concentration increased depending on the dose of medium ascorbate. Medium lactate dehydrogenase activity derived from hepatocytes, an index of cell injury, decreased upon 5-100 mg/l of ascorbate supplementation for 48 h. Tyrosine aminotransferase activity, an index of liver function, increased following culture with 50 and 100 mg/l ascorbate for 48 h. The activities, however, decreased by supplementation with 1000 mg/l of ascorbate. In conclusion hepatocytes lost intracellular ascorbate during preparation by in situ collagenase perfusion. Supplementation of ascorbate restored cellular ascorbate concentration, lowered cell injury and raised tyrosine aminotransferase activitv in primary cultured chicken hepatocytes. Ascorbate treatment for 48 h at 50 mg/l was the best combination in this study for primary culture of chicken hepatpcyte with non-serum L-15 medium     

Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells.

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.     

Influence of branched-chain amino acid composition of culture media on the synthesis of plasma proteins by serum-free cultured rat hepatocytes

Summary Supplementation of Ham's F12 culture medium with essential amino acids (EAA) up to the rat plasma levels increased the rates of synthesis of albumin and transferrin by cultured rat hepatocytes by 1.3 and 1.7, respectively. Fifty percent of this increase could be attributed to three of the EAA: the branched-chain amino acids (BCAA: Leu Ile and Val). Non-branched-chain essential amino acids (non-BC-EAA) stimulated only 25% of the increase produced by the whole EAA mixture. When each EAA was tested individually, none of them caused an appreciable increase in albumin and transferrin in culture medium. When the concentrations of all EAA were raised to rat postprandial portal levels, albumin and transferrin synthesis rates reached a maximum, increasing by 3.2 and 3.5, respectively. Supplementation with BCAA at postprandial portal concentrations increased albumin and transferrin synthesis rates by 2.2 and 2.0, respectively, and had no noteworthy effect on the synthesis of cellular proteins. Non-BC-EAA at their postprandial portal concentrations increased albumin and transferrin synthesis rates by 1.7 and 1.9, respectively. Supplementation with alanine to reach a nitrogen content equal to that of the modified EAA-enriched medium had no stimulatory effect. Our results show that EAA have a specific effect on the synthesis of plasma proteins by cultured hepatocytes, and that BCAA at physiologic concentrations account for the major part of this stimulatory effect. Consequently, EAA and particularly BCAA concentration should be elevated in serum-free nutrient media to sustain maximum plasma protein synthesis.     

Long-Term continuous culture of hepatocytes in a packed-bed reactor utilizing porous resin

As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.     

High-Density culture of mouse-human hybridoma in serum-free defined medium

Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 x 10(7) cells/mL (viable cell density was 7.6 x 10(6) cells/mL), and the IgA productivity was around 20 mug/10(6) cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium.     

Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities

The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.     

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.     

A comparison of bovine serum albumin and chicken ovalbumin as supplements for the serum-free growth of chinook salmon embryo cells,CHSE-214.

Chicken ovalbumin and bovine serum albumin (BSA) were compared as supplements to the basal medium, L-15, for the serum-free cultivation of the Chinook Salmon Embryo cell line, CHSE-214. Unlike L-15 alone, ovalbumin and some commercial BSA preparations allowed cell proliferation and development of confluent monolayer cultures. However, only a fatty acid-free BSA (2%) supported continuous proliferation for two years through approximately 15 subcultivations. For this, subcultivation was achieved with non enzymatic cell dissociation solutions. Also, new serum-free subcultivation techniques were developed that utilized avian egg white trypsin inhibitors to terminate the action of either bovine or cod trypsin. Finally, CHSE-214 were successfully cryopreserved in 2% BSA, allowing all cell cultivation steps to be performed in the absence of FBS.     

Growth of human malignant lymphoid cell lines in serum-free medium

Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products.