Leonard M. Flendrig

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.

Significantly improved survival time in pigs with complete liver ischemia treated with a novel bioartificial liver.

Aim of the study was to evaluate treatment efficacy and safety of a scaled-up version of our porcine hepatocytes based BAL system in pigs with complete liver ischemia (LIS). Thirty-one pigs underwent total devascularization of the liver (LIS) by termino-lateral porta-caval shunts and sutures around the bile duct, the common hepatic and gastroduodenal arteries and their accessory branches. The hepato-duodenal ligament was completely transected. Four experimental groups were studied: the first control group (LIS Control, n = 10) received glucose infusion only, the second control group (LIS Plasmapheresis, n = 8) was connected to a centrifugal plasma-separator with a bottle representing the bioreactor volume, the third control group (LIS Empty-BAL, n = 5) received BAL treatment without cells, and the treated group (LIS Cell-BAL, n = 8) was connected for a maximum period of 24 hours to our scaled-up BAL seeded with around 14 billion viable primary porcine hepatocytes. BAL treatment significantly prolonged life in large animals (-35 kg) with complete LIS (Controls, mean ± SEM: 33.1 ± 3 h, Cell-BAL: 51.1 ± 3.4 h; p = 0.001; longest survivor 63 h). In addition, blood ammonia and total bilirubin levels decreased significantly, indicating metabolic activity of porcine hepatocytes in the bioreactor. No significant differences were noticed among the three control groups, indicating that there was no device effect and that the plasmapheresis procedure was well tolerated. No important adverse effectes were observed.

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.

Evaluation of a novel bioartificial liver in rats with complete liver ischemia: treatment efficacy and species-specific α-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.

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.

Possible immunological problems of bioartificial liver support

In the development of a bioartificial liver for clinical application the use of hepatocytes from xenogeneic origin is the most promising alternative for human hepatocytes due to the shortage of human donor material and the lack of a well functioning non-tumorigenous human hepatocyte cell line. The best candidate for the supply of hepatocytes is the pig since pig livers are easily obtained and large numbers of cells can be isolated. However, biosafety and immunological consequences should be considered when humans are to be treated with bioartificial livers loaded with pig hepatocytes. Biosafety problems, such as transmission of porcine pathogens will not be addressed in this paper. Immunological problems may arise if the patient develops an immune response to the xenogeneic cells or to the xenogeneic proteins derived from these cells. An immunological reaction could result in either destruction of the bioartificial liver or in hypersensitivity reactions in the patient receiving bioartificial liver treatment.

The bioartificial liver of the Academic Medical Center at Amsterdam

There is an urgent need for an effective liver support system to bridge patients with hepatic failure to liver transplantation or to a stage where their own liver is able to regenerate. In the past most attempts to reach this goal were focused on blood detoxification, based on the assumption that liver failure could be reversed if the associated toxins were removed from the circulation of the patient. Although improvement of the neurological status of patients has been reported, none achieved long-term survival [1]. It was therefore concluded that an effective liver support system should be able to perform the liver’s multiple synthetic and metabolic functions, including detoxification and excretion. The most logical approach to this problem is the introduction of active functioning hepatocytes. At present, the bioartificial liver is thought to hold the greatest promise for successful temporary liver support. The heart of such an extracorporeal system consists of a bioreactor: a cell culture device which comprises well nourished and oxygenated, viable, hepatocytes immobilized on a mechanical support and separated from the patient’s blood circulation by semi-permeable membranes. This paper reports on the principle and the in vitro and in vivo results of a highly original new design of bioartificial liver support developed at the Academic Medical Centre of the University of Amsterdam, the so called Academic Medical Centre Bioartificial Liver (AMC-BAL).