Fachbereich Veterinärmedizin



    Charakterisierung und Modifikation eines dynamischen In-vitro-Leberzellenkulturmodells für den Ersatz von Tierversuchen in der biomedizinischen Forschung (1997)

    Unger, Juliane
    Berlin, 1997 — 149 Seiten
    Institut für Tierschutz und Tierverhalten

    Königsweg 67
    Gebäude 21, 1. OG
    14163 Berlin
    Tel.: +49 30 838 62901 (Sekretariat)
    email: tierschutz@vetmed.fu-berlin.de

    Abstract / Zusammenfassung

    International efforts to develop hybrid liver support systems have provided a variety of potentialin-vitro systems which pending on necessary modifications will be suitable to investigate hepaticfunctions. The present study analyses the "dynamic bioreactor culture system" first described byGerlach ( 1 994a) using primary pig hepatocytes as cellular template. The criteria derived from thisstudy established where modifications of the current design are mandatory. Furthermore, acomparison between the dynamic bioreactor culture and static culture models highlighted theinherent potential and limitations of both approaches.A substantial part of the project was focussed on material studies and technical improvements tothe bioreactor system since many results obtained over the course of this work suggested thatproperties of the materials used had a significant influence on the outcome of biotransformationexperiments. A systematic investigation of the interactions between PVC-, PU- and silastic tubingsthat are all commonly used in biomedical research and the xenobiotic compounds followed in thisstudy revealed strong and unpredictable interactions. The results of this analysis proved essentialfor an interpretation of the results obtained from biotransformation expenments and pointedtowards some essential improvement to the experimental design. For instance the replacement ofsilastic tubing by a recently developed membrane type (Mitsubishi Corporation) with low substratebinding properties would eliminate a major cause of uncontroled xenobiotica adsorption. Notably,another factor complicating the adsorption properties of the system was excluded by omitting theuse of a biomatrix. While cell embedding is helpful for some applications the histological resultsobtained from the analysis of bioreactor cores indicated that the naturally occuring aggregation ofthe hepatocytes inside the culture compartments achieved comparable cytoprotective effects.Additional modifications to he capillary configuration and the ratio between cell number andculture surface area represented another step towards a system allowing longterm cultivation offunctionally diverse and fully differentiated hepatocytes. Equally important, the advances madetowards a fully automated perfusion-monitor reached an unprecedented technical standardcompared to other up to date hepatocyte culture models.Besides technical improvements, parts of the work aimed at the determination of suitable criteria toassess cell viability througout the course of extended experiments and to establish possiblerelationships between apparent viability parameters and the capacity of the cells to adequatelymodify the test substrates lidocaine, testosterone and lormetazepame. Simple trypan blue exclusion, morphology and the CYP450-content were used for j udging cell viability at the stage of cellisolation. However, none of these parameters was suitable for monitoring extended bioreactorcultures. Accordingly, enzyme liberation (LDH, AST, GLDH, ALT, gGT), Na+, K+, Cl-, osmolality,urea synthesis. glucose- and phosphor utilisation, amino acid consumption, CO2-release and themorphology of cells in prallel monolayer control cultures were monitored during extendedbioreactor runs. The development of a new embedding technique allowed a final histologicalassessment of the reactor core which proved helpful in the interpretation of some parametersanalysed. Results obtained from these studies suggested that a number of commonly acceptedstandards may require reevaluation. For instance, the histological analysis of several bioreactorcultures after 1-2 weeks in-vitro culturing may not be caused by the stabilization of the hepatoctesbut is more likely to reflect a loss in their ability to newly synthesize the relevant enzymes.Furthermore, a more detailed analysis of the apparent alterations in the urea concentration clearlyshowed that this parameter was greatly influenced by the medium composition and therefore doesnot represent a reliable indicator for metabolic competence on its own. Similar observations weremade for some other parameters like glucose and amino acid utilization emphazising the fact thatconclusions drawn from single parameters are likey to be incorrect.Liver function in-vivo is at the heart of maintaining metabolic homeostasis. The requireddifferentiation and compartimentalization of hepatocytes is a severe problem in any kind ofdynamic culture model. For example, the homogenous pO2 used in other established culturesystems causes a loss of liver specific cell differentiation and spatial organization. While this is nota general problem in our culture model several! improvements had to be made to achieve a moreefficient oxygenation thus producing higher cell survival rates. Changes in theoxygenationmembrane material used, an increase of total oxygenation surface and the decreaseof the spacing between the capillaries where some of the parameters needed to be optimized. Asecond commonly observed complication is caused by medium induced malnutrubon (too muchglucose, low levels of fatty acids, ketone bodies and proteins, lack of regulations by hormones;Seifter and Englard, 1988). Under these conditions a malfunction is likely to develop. The resultingloss of cellular integrity causes earlier cell dash, abnormalities in basic metabolic activities and thecapacity to perform adequate biotransformation of xenobiotica. Therefore, the development of aprocedure that allows the controled operation of he biorector was a fundamental contributiontowards an experimental design that will allow a more systematic investigation of the parametersgoverning dynamic culture systems.ln summary, this study ha! established a number of criteriaand directions for further improvements in bioreactor design and operation. Optimization of reactorspecifications like the overall dimensions, the ratio of cells to culture surface, oxygenationconditions and medium formulations (hepatocyte specific energy supply by galactose, ketonebodies and fatty acids) will be essential to further decrease artificial metabolic activities, extendpotential culture duration and more efficiently mimic hepatic function and differentiation.Combiningthese efforts with additional improvements in the capacity to control the system A complexco-culture technique with several different cell populations should be well within reach. Thepossibility to investigate inflammatory and immunologicallymediated hepatotoxicity as well as tostudy models for infectious diseases of the liver by an in-vitro approach are challengingperspectives and greatly emphazise he need to reinforce efforts in this area of biomedicalresearch.