1. HYBRID LIVER SUPPORT SYSTEMS: ENZYME LIBERATION DURING IN-VITRO STUDIES AND FIRST CLINICAL TRIALS D. Kardassis*, B. Busse, G. Holland&, C. Mueller#, P. Neuhaus, J. Gerlach Department of Surgery, Institutes of Pathology& and Clinical Biochemistry#, Campus Virchow Clinic, Charité, Humboldt University, Berlin, Germany
Introduction. The development of hybrid liver support systems (LSS) as a bridge to liver transplantation for patients suffering from acute liver failure (ALF) has already enabled clinical trials. In order to evaluate the quality of hepatocyte cultures, cellular integrity and metabolic performance should be quantified. Measurement of specific release of hepatic enzymes before clinical application may act as a quality criterion. Furthermore, monitoring enzyme leakage during therapy could function as an indicator of both the status of the culture and the interaction between patients and LSS.
Methods. The BELS (Berlin Extracorporeal Liver Support system) (Figures 1 and 2) contains a bioreactor, made of different, three-dimensionally interwoven hollow fibre capillary systems which provide distributed perfusion and oxygenation (Figure 3). A monitor containing all facilities for maintenance and transport of the cell culture and a conventional plasma separation unit (Diapact CRRTTM, Braun Melsungen, Melsungen, Germany) complete the device. We determined the specific release of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamic acid dehydrogenase (GLDH) and lactate dehydrogenase (LDH) during 21 days in the BELS containing g primary porcine liver cells with an initial viability of 92-97% (n=16). Parenchymal and non-parenchymal cells formed aggregates along the capillaries (Figures 4 and 5). Additionally we measured concentrations of the aforementioned enzymes as well as porcine a-glutathione S-transferase (a-GST) during treatment of patients suffering from ALF (n=6) with the BELS.
Figure 1: Scheme of the BELS.
Figure 2: The Berlin Extracorporeal Liver Support system (BELS).
Results.
Poor quality culture
Figure 6: Progression of specific release (SER) of ALT, AST, GLDH and LDH during 21 days of liver cell co-culture (mean and standard error of the mean). Results from one poor quality culture with an initial viability of 70% (denoted J 33) are depicted for comparison with high quality cultures (n=16).
Figure 7: Levels of porcine a-GST during clinical application measured after contact of the patients’ plasma with the BELS. The dotted lines indicate pre-treatment and treatment interruption phases. The abrupt rise (*) in concentration during therapy of patient 6 was due to treatment interruption and plasma recirculation in the bioreactor for 6 hours.
Figure 3: Schematic view of the opened bioreactor housing and illustration of the independent capillary bundles.
Figure 8: Differences between AST levels in patients’ plasma during therapy before and after it has contacted the BELS.
Figure 9: Time course of total bilirubin (tBili) in patients’ plasma before and during therapy. Concentration was stabilised at pre-treatment levels or even decreased. Patient 2 suffered from haemolytic crisis and acute renal failure.
Figure 4: Scanning electron microscopy image of adherent liver cell co-culture between bioreactor capillaries.
Discussion. A three-dimensional bioreactor with sufficient supply of culture medium, nutrients and oxygenation can provide the environment for hepatic tissue components to re-organise after the traumatic procedure of isolation. Cell cultures with good viability distinguish themselves as soon as one day after isolation by low specific release of hepatic enzymes. Therefore this is a reliable quality criterion with regard to clinical applications (Figure 6).
During therapy no linear relationship between levels of common (non-specific) enzymes in the patient and the bioreactor could be established (Figure 8) in spite of its apparent biochemical activity (Figure 9). Nevertheless, measurement of species specific a-GST by ELISA-technique proved to be a valuable tool for biochemical detection of cellular damage in bioreactors during clinical application (Figure 7). a c b
Figures 5a, 5b, 5c: Electron microscopy images of hepatic cells cultivated in the bioreactor for 2-4 weeks: Extracellular matrix components (a), bile canaliculi and tight junctions between hepatocytes (b) and sinusoid-like structures performed by hepatocytes and non-parenchymal cells (c).