Evaluation of a novel bioartificial liver in rats with complete liver ischemia: treatment efficacy and species-specific alpha-GST detection to monitor hepatocyte viability.

BACKGROUND/AIMS: There is an urgent need for an effective bioartificial liver system to bridge patients with fulminant hepatic failure to liver transplantation or to regeneration of their own liver. Recently, we proposed a bioreactor with a novel design for use as a bioartificial liver (BAL). The reactor comprises a spirally wound nonwoven polyester fabric in which hepatocytes are cultured (40 x 10(6) cells/ml) as small aggregates and homogeneously distributed oxygenation tubing for decentralized oxygen supply and CO2 removal. The aims of this study were to evaluate the treatment efficacy of our original porcine hepatocyte-based BAL in rats with fulminant hepatic failure due to liver ischemia (LIS) and to monitor the viability of the porcine hepatocytes in the bioreactor during treatment. The latter aim is novel and was accomplished by applying a new species-specific enzyme immunoassay (EIA) for the determination of porcine alpha-glutathione S-transferase (alpha-GST), a marker for hepatocellular damage. METHODS: Three experimental groups were studied: the first control group (LIS Control, n = 13) received a glucose infusion only; a second control group (LIS No-Cell-BAL, n = 8) received BAL treatment without cells; and the treated group (LIS Cell-BAL, n = 8) was connected to our BAL which had been seeded with 4.4 x 10(8) viable primary porcine hepatocytes. RESULTS/CONCLUSIONS: In contrast to previous comparable studies, BAL treatment significantly improved survival time in recipients with LIS. In addition, the onset of hepatic encephalopathy was significantly delayed and the mean arterial blood pressure significantly improved. Significantly lower levels of ammonia and lactate in the LIS Cell-BAL group indicated that the porcine hepatocytes in the bioreactor were metabolically activity. Low pig alpha-GST levels suggested that our bioreactor was capable of maintaining hepatocyte viability during treatment. These results provide a rationale for a comparable study in LIS-pigs as a next step towards potential clinical application.

Does the extend of the culture time of primary hepatocytes in a bioreactor affect the treatment efficacy of a bioartificial liver?

The purpose of this study was to investigate whether the efficacy of our novel extracorporeal bioartificial liver (BAL) to support rats with complete liver ischemia (LIS) could be improved by extending the culture time of freshly isolated porcine hepatocytes from 14 hours to 38 hours. The results showed that survival as well as porcine hepatocyte integrity improved, the onset of coma delayed, and the ammonia levels decreased in LIS rats of the 38 hour group compared to the 14 hour group, but no statistically significant differences were observed. In the 38 hour group, but not the 14 hour group, the onset of hepatic encephalopathy was significantly delayed and ammonia metabolism significantly improved compared to the LIS rats in control groups that only received a glucose infusion or were connected to a BAL without cells. In conclusion, prolonged hepatocyte recovery favoured all investigated parameters, although not all observed effects were statistically significant. More research is required to find out how long primary hepatocytes should be cultured in a bioreactor for optimal BAL support.

Commercially available media for flushing extracorporeal bioartificial liver systems prior to connection to the patient's circulation: an in vitro comparative study in two and three dimensional porcine hepatocyte cultures.

Extracorporeal bioartificial liver (BAL) systems based on hepatocytes need to be flushed before clinical application, as hepatocyte culture media are not approved for medical use. Commercially available 0.9% NaCl solution and hemofiltration solution (both supplemented with 10% human albumin) were investigated in vitro to test their potential to wash BAL systems with minimal stress for the cultured hepatocytes. After a 2 hour incubation, the lidocaine metabolising capacity and release of liver enzymes were assessed. As hepatocytes have been cultured in bioreactors in either two or three dimensional cell configurations, we tested the media in respectively hepatocyte monolayers cultures and in our newly developed bioreactor in which hepatocytes reorganise as small hepatocyte aggregates. The three dimensional hepatocyte cultures tolerated both media well, and no significant differences were seen compared with hepatocytes cultured in Williams' E (reference hepatocyte culture medium). The two dimensional hepatocyte cultures tolerated the supplemented hemofiltration solution and the reference medium equally well, but the condition of the porcine hepatocytes monolayer cultures was significantly impaired when incubated with the supplemented physiological saline solution. In conclusion, as a supplemented physiological saline solution may have detrimental effects on the condition of the hepatocytes, the more complex hemofiltration solution (bicarbonate buffered, glucose, essential minerals) was considered the better alternative for flushing bioartificial liver systems.

In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates.

BACKGROUND/AIMS: The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy. We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli. METHODS: The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e. a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO2 removal. Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact. Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion. RESULTS: Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo. The biochemical performance of the bioreactor remained stable over the investigated period. The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures. Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size. CONCLUSIONS: The novel bioreactor showed encouraging efficiency. The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failure.

Immunological consequences of the use of xenogeneic hepatocytes in a bioartificial liver for acute liver failure.

The use of cells from xenogeneic origin in a bioartificial liver can have a number of immunological consequences, not only for the cells in the bioartificial liver but also for the patient receiving the bioartificial liver treatment. The impact of these consequences will depend on the immune status of the patient receiving bioartificial liver treatment, the duration and frequency of the treatment and on the extent of interaction between the patients blood (or plasma) and the xenogeneic liver cells. In an experimental model we infused rats with a culture supernatant of pig hepatocytes and demonstrated using Western blots and immunohistological techniques that antibodies are raised against the very small amounts of the pig hepatocyte-derived proteins present in the culture medium. Potential problems of bioartificial liver destruction and the possibility of hypersensitivity reactions due to the secretion of xenogeneic proteins into the circulation of the patient are discussed. Because the liver has an important role in the clearance of immune complexes it is concluded that precautions should be taken when (repeated) application of a xenogeneic bioartificial liver in patients with liver failure is considered.

Functional activity of isolated pig hepatocytes attached to different extracellular matrix substrates. Implication for application of pig hepatocytes in a bioartificial liver.

For the manufacture of a bioartificial liver for human application, large amounts of viable and active hepatocytes are needed. Pig hepatocytes are considered to be the best alternative to scarce human hepatocytes. In vitro hepatocyte functions have so far been tested under different circumstances, mainly with rat hepatocytes. Pig hepatocytes were isolated with a single two-step isolation procedure, resulting in a high yield of viable hepatocytes. The hepatocytes were tested for their ability to synthesise urea, to metabolise 7-ethoxycoumarin (cytochrome P450 activity), and to synthesise and secrete proteins. These activities of hepatocytes while attached to tissue culture plastic were compared to the activity of the cells attached to several extracellular matrix constituents: collagen I and IV, laminin, fibronectin, Engelbreth-Holm-Swarm Natrix and in the presence of Matrigel. With the exception of Matrigel, neither of the extracellular matrix substrates enhanced pig hepatocyte function compared to tissue culture plastic. However, relatively large amounts of murine proteins leak out of the Matrigel. The advisability of using Matrigel or other extracellular matrix proteins in a bioartificial liver loaded with pig hepatocytes is discussed.

Glucocorticoids and catecholamines as mediators of acute-phase proteins, especially rat alpha-macrofoetoprotein.

Adrenal hormones were studied as possible triggering substances of the synthesis of acute-phase reactants in rats. alpha-Macrofoetoprotein, which rises sharply in concentration during inflammation, was used to monitor the acute-phase reaction. In normal rats glucocorticoids and catecholamines induce alpha-macrofoetoprotein synthesis; glucocorticoids only increase alpha-macrofoetoprotein to moderate levels in plasma, but catecholamines enhance alpha-macrofoetoprotein synthesis to very high levels, comparable with those observed in the post-injury phase. However, catecholamines in vivo also activate the adrenal cortex, suggesting a synergistic effect of both kinds of adrenal hormones. Our study showed that in adrenalectomized rats, the effect of catecholamines on alpha-macrofoetoprotein synthesis is greatly diminished, whereas the moderate effect of glucocorticoids remains. Combination of glucocorticoids and catecholamines induces extremely high alpha-macrofoetoprotein levels in both adrenalectomized and normal rats. With crossed immunoelectrophoresis it was shown that other acute-phase reactants, such as haptoglobin and alpha 1-major acute-phase protein, are affected differently by the hormones. Contrary to glucocorticoids, catecholamines give a pattern comparable with that found after surgical injury.

Influence of liver isoferritin profile on quantitative measurement of total ferritin protein in human liver, with emphasis on iron overload.

Ferritin was isolated from normal human liver and from iron-loaded human liver by gel chromatography and by ultracentrifugation. From each of these ferritin batches several isoferritin fractions were isolated by preparative isoelectric focusing. It was our aim to have at our disposal isoferritin fractions with relatively large pI ranges but distinctly different isoferritin profiles on analytical isoelectric focusing. These fractions were compared for their immunoreactivity. The total protein in the isoferritin fractions was measured by the nitrogen content. The immunoreactivity of the isoferritin fractions was measured as the ratio of the reaction in immunoassays to the nitrogen content of these fractions. We used radial immunodiffusion and enzyme-linked immunoassay to measure immunoreactivity. The immunoreactivity did not change obviously with the isoferritin composition of the isolated fractions. It is concluded that pathological changes in the isoferritin composition that might occur in liver ferritin during iron overload does not significantly influence the quantitative measurement of liver ferritin protein by immunological methods.

Inhibition of polymorphonuclear leukocyte chemotaxis by alpha-macrofetoprotein, an acute-phase reactant of the rat.

alpha-Macrofetoprotein (alpha M FP) is a normal fetal plasma constituent in the rat, with very low plasma levels in the adult phase but rising sharply after injury. This fetal acute-phase protein is a strong inhibitor of inflammatory edema. Fetal inflammatory reactions show diminished exudation, but also impaired emigration of polymorph nuclear cells (PMNs). Therefore we studied the effect of alpha M FP on chemotaxis of PMN in vitro and in vivo. In vitro experiments showed a strong inhibitory effect on casein-induced leukotaxis (Boyden technique) with a clear dose-effect relationship. In vivo with glycogen-induced pleurisy and peritonitis, high alpha M FP levels are accompanied by diminished PMN emigration and vice versa. The significance of these findings is discussed in relation to fetal pathology and also as a model showing the modulating effects of acute-phase proteins on the inflammatory reaction induced by tissue injury.

Inflammation inhibiting properties of rat alphaM foetoprotein (rat-alpha2 macroglobulin), an acute phase reactant.

alphaMFoetoprotein of the rat (rat alpha2macroglobulin) is present in serum during foetal development. After birth, alphaMFP declines rapidly, but the protein returns after injury. The injury we used consisted of skin and muscle incision, laparotomy, BaSO4 i.p. The protein occurs also during liverregeneration, livercardinogenesis en during infections. Therefore, alphaMFP can be considered as an acute phase reactant. We found that this protein strongly inhibits inflammatory exudation caused by carrageenin, histamin, prostaglandin E2, 5HT and bradykinin. Furthermore, we found that alphaMFP suppresses completely inflammatory reactions during Ga1.N-hepatitis. In this condition, the primary biochemical lesion, consisting of liver UDPG depletion, does occur in spite of the protective effect of alphaMFP. Thus, the inflammatory inhibiting effects of alphaMFP seem to be an important mechanism reducing inflammatory reaction patterns.

Lacoferrin in rabbit bile: its relation to iron metabolism.

The relation of biliary lactoferrin concentration and the iron status of the body was studied in normal, anaemic and iron-loaded rabbits. In anaemic rabbits lactoferrin concentrations rose to two to three times the original values. Loading with iron resulted in a return to normal values. Mobilization of iron with desferrioxamine also gave a rise in lactoferrin concentration in bile. Lactoferrin may have a regulatory function in situations of enhanced iron absorption or mobilization of iron from depots.