Increase In Albumin Secretion Research Articles

Cultivation of Fetal Liver Cells in a Three-Dimensional Poly-L-Lactic Acid Scaffold in the Presence of Oncostatin M

To investigate the feasibility of fetal liver cells for liver tissue engineering, the supporting function of poly-L-lactic acid (PLLA) for fetal liver cells and the effects of oncostatin M (OSM) on hepatic differentiation were studied. After preparing three-dimensional biodegradable PLLA scaffold having a well-developed open-pore structure by a gas-forming method with ammonium chloride particles as a porogen and a gas-forming reagent, fetal liver cells separated from E14.5-C57BL/6CrSlc murine embryos were inoculated in the PLLA scaffolds. Cells were cultured in Williams' E medium with or without OSM (10 ng/ml) for 30 days with a medium change every 2 days. Results showed that there were significant increases in the number of cells and in albumin secretion in PLLA culture compared with in monolayer culture on day 15. In addition, a significant increase in albumin secretion was observed in OSM-added PLLA culture compared with OSM-free culture, and there was only a slightly enhanced albumin secretion in monolayer cultures with OSM. These results suggest that PLLA may enhance the biological activity of OSM for inducing maturation of fetal liver cells. Interestingly, the number of cells in PLLA culture with OSM decreased compared with OSM-free PLLA culture at day 15. This may be because promotion of hepatic development by OSM simultaneously suppressed in vitro hematopoiesis (i.e., blood cell production). In summary, our results indicate that the three-dimensional PLLA scaffold is a good support material for the cultivation of fetal liver cells and that OSM is capable of not only terminating hematopoiesis of the fetal liver but also stimulating the maturation of hepatic parenchymal cells in vitro.

The Effect of Coculture with Nonparenchymal Cells on Porcine Hepatocyte Function

Porcine hepatocytes are currently being investigated as a therapy for patients suffering from acute liver failure. Incorporating hepatocytes in an extracorporeal device that would stabilize a patient until transplantation or recovery could dramatically decrease the mortality rate associated with this disease. The ability to maximize hepatocyte function would contribute significantly to being able to provide the required cell mass in a device of reasonable size. Several approaches have been effective at increasing rat hepatocyte function in vitro, including coculture with nonparenchymal cells. In this study, we investigated the effect of the addition of 3T3 cells to porcine hepatocyte culture and found that while there was an increase in albumin secretion, there was little or no effect on urea synthesis or cytochrome P450 activity.

Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures.

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.

Long-Term Culture of Fetal Liver Cells Using a Three-Dimensional Porous Polymer Substrate

To develop a bioartificial liver, long-term culture of fetal liver cells over a month's time was performed under three different culture conditions, i.e., stationary cultures and shaken-flask cultures, both by using a substratum made of porous polyvinyl formal (PVF) resin and conventional monolayer dish cultures as controls. Time course changes in cell numbers and albumin secretion were evaluated in cultures using Williams' E medium (WE) or minimum essential medium alpha (aMEM) supplemented with serum and hormones. In the WE medium, the numbers of fetal liver cells in all culture conditions gradually decreased with time, and albumin secretion rates rapidly decreased. In the stationary cultures using PVF, however, a significant increase in albumin secretion was observed after two weeks of culture. When cells were cultured in aMEM, the fetal liver cells exhibited sufficient proliferation in stationary and monolayer cultures, although albumin secretion rates per single cell were lower than those in WE. On the basis of these results, another series of culture experiments were performed, in which aMEM was used for the first 10 days to encourage cell proliferation, and the medium was changed to WE afterward. In these cultures, albumin secretion rates in the stationary cultures dramatically increased after the medium exchanges and were maintained at these high levels throughout the remaining culture period.

Enhanced Secretion of ApoB by Transfected HepG2 Cells Overexpressing Fibrinogen

HepG2 cells stably transfected with cDNA-encoding single fibrinogen chains overexpress fibrinogen and have increased (4-fold) secretion of apolipoprotein B. Overexpression of fibrinogen does not affect the secretion of three representative acute-phase proteins but causes a small increase in albumin secretion. Enhanced apolipoprotein B secretion is due to less intracellular degradation and not to increased expression. The increased secretion of apolipoprotein B is independent of the acute-phase response, since stimulation of fibrinogen gene expression by interleukin 6 did not affect secretion. HepG2 cells overexpressing fibrinogen chains had increased 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels, enhanced cholesterol production but normal levels of triglyceride and phospholipid synthesis and of sterol response binding proteins. These results, that associate overexpression of fibrinogen with enhance apolipoprotein B secretion, may be significant since epidemiological studies indicate that elevated levels of fibrinogen and lipids are independent risk factors in coronary artery disease.

Inhibition of Lipopolysaccharide and Cytokine Mixture-Mediated Hepatocyte Nitric Oxide Synthesis by Dimethyl Sulfoxide

Treatment and pretreatment of hepatocytes with 2% dimethyl sulfoxide (DMSO) inhibited lipopolysaccharide and cytokine mixture (LPS/CM)-mediated NO synthesis in hepatocytes without any obvious effects on cell viability. DMSO at concentrations of 0.5–4% stimulated DNA replication and increased albumin secretion in LPS/CM-treated hepatocytes. Genisein, a inhibitor of protein tyrosine kinase (PTK), inhibited LPS/CM-mediated NO synthesis in hepatocytes. These results suggest that PTK is critical for hepatocyte NO synthesis, and DMSO-inhibited NO synthesis may be associated with prevention of LPS/CM-induced PTK activation in hepatocytes.

Engineering of a sugar-derivatized porous network for hepatocyte culture

Many tissue engineering applications require a scaffold or template conducive to cell attachment and maintenance of functions. It may also be advantageous in some cases for these scaffolds to have a controlled porous architecture to facilitate cellular or tissue ingrowth. In this study, we have engineered a porous carbohydrate-derivatized substrate for hepatocyte culture. Polystyrene foams, with pore sizes up to 100 μm, fabricated by phase separation from a homogeneous naphthalene solution, were derivatized with lactose and heparin, both of which are known to promote rat hepatocyte attachment and maintenance of its differentiated functions. Rat hepatocytes cultured on these derivatized foams exhibited a rounded cellular morphology with many microvilli evident on the surface of the cells. The hepatocytes showed an increase in albumin secretion for the first 3 days of culture in a defined, serum-free medium, and dropped back to initial levels by the end of 7 days. The production of cytochrome P 450-dependent hydroxytestosterone metabolites were also measured. Two testosterone metabolites were maintained and five others were present but decreased over a culture period of 1 week. These carbohydrate-derivatized porous substrates may be useful for largescale culture of hepatocytes, toxicology screening and for use in a liver assist device.

Long-term co-cultures of adult human hepatocytes with rat liver epithelial cells: modulation of albumin secretion and accumulation of extracellular material.

High yields of viable human hepatocytes were obtained by enzymatic perfusion of the left hepatic lobe of kidney donors and cultured alone or with an epithelial cell line derived from rat liver. In conventional cultures, human hepatocytes did not survive more than 2 to 3 weeks and by Day 8 decreased their ability to secrete albumin. When co-cultured, they survived for more than 2 months and secreted high levels of albumin even in a serum-free medium. This long-term survival appeared to correlate with production of an extracellular material which is rich in Type III collagen. In vitro phenotypic alterations of parenchymal cells were reversed by addition of rat liver cells and were characterized by recovery of cuboidal morphology, increased albumin secretion and a shift from Type I to Type III collagen deposition. Rat liver epithelial cells could not be replaced by nonhepatic epithelial cells. These observations suggest that when adult human hepatocytes are maintained in a culture which closely resembles their in vivo environment, they are capable of continuing to actively express specific cell functions.

Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells

Exposure of BW77-1 and BW77-2 mouse hepatic tumour cells to the polar solvent dimethylsulphoxide (DMSO) altered extracellular accumulation of albumin and alpha-foetoprotein (AFP) and perturbed their cell cycle kinetics. The amount of albumin secreted into the culture growth medium was dependent on the concentration of DMSO used. Hepatic tumour cells cultured in 1 and 2% DMSO accumulated 50% and 111% more albumin, respectively, than non-DMSO-stimulated cells during the final 24 h of a 4-day exposure to the polar solvent. Commitment of mouse hepatoma cells to increased albumin secretion was temporally dependent, requiring a minimum of 48 h in the presence of DMSO. The AFP level in 1% DMSO-treated cultures was also significantly increased, compared with control cells. Unlike albumin secretion, however, exposure of hepatic tumour cells to 2% DMSO did not further increase (but slightly decreased) extracellular AFP accumulation. Treatment of BW77-1 cells with DMSO resulted in a gradual decline in the percentage of 2C DNA content cells (diploid G1 population) and in a corresponding increase in the proportion of cells with a 4C DNA content (generation of either a G2 or tetraploid G1 population). The extent of this shift directly reflected the concentration of polar solvent in the medium and paralleled the DMSO-induced stimulation in albumin secretion. DMSO-stimulated hepatic tumour cells, therefore, may prove useful in the elucidation of specific regulatory events underlying control of gene expression during the hepatocyte cell cycle.