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Zusammenfassung
Abstract
In vitro models for investigating cellular mechanism have been shown to be a valuable tool, since the use of animal models re-vealed some significant differences in cell metabolism in particular for the human liver. The development of an in vitro model based on primary human hepatocytes were sought to overcome limitations of hepato-carcinogenic cell lines. Due to ethical aspects human ...
Zusammenfassung
Abstract
In vitro models for investigating cellular mechanism have been shown to be a valuable tool, since the use of animal models re-vealed some significant differences in cell metabolism in particular for the human liver. The development of an in vitro model based on primary human hepatocytes were sought to overcome limitations of hepato-carcinogenic cell lines. Due to ethical aspects human tissue source is limited to surgical liver specimen. Therefore isolation and culture of primary liver cells from human tissues represents a prerequisite not only for in vitro testing but also for therapeutic concepts in liver cell transplantation. Protocols for isolation and cultivation needs to be validated regarding donor variability and methodological procedures as well as liver specific function. Primary human liver cells were isolated using a high pressure perfusion with EGTA/collagenase digestion. Purified cell fractions were characterised by their morphology, immunohistochemistry and by vitamin A autofluorescence. Isolated hepatocytes were stored in a modified UW solution at 4°C up to 72 hours and cultivated in a collagen gel sandwich system up to 4 weeks. For activation of hepatic stellate cells, cells were seeded on petri dishes for at least 24 days. Cell viability, yield, attachment, induction of CYP-450 isoenzymes and gene expression were correlated to patients and method specific parameters.Isolation of human liver cells (w=143, 41±34g) resulting in an average cell yield of 2xl07 cells/g with 82% viability. During 4°C storage (48 h) attachment rate and viability decreased to 71% and 57%, respectively. Additionally, cell viability (75%) and attachment rate (72%) was significantly reduced by pringle manoeuvre (<30min). In all cases cell function recovered during a culture period of 4 days. Neoadjuvant chemotherapy as well as ischemia (<20 min) of donors resulted in altered CYP 450 activity (2B6, 2C19 3A4, ECOD). Collagenase digestion were responsible for not detecting distinct surface resident proteins, which partly recovered during cultivation. Long-term culture experiments comparing rat with human hepatocytes demonstrated, that induction of porphyrin metabolism resulted in a liver specific cell damage (LDH increase) only with human hepatocytes reversible by adding superoxid-dismutase (SOD). Further, human hepatocytes were infected in vitro by hepatitis B virus and showed a significant virus production. Using an in vitro model for human hepatic stellate cells it has been demonstrated that activated stellate cells, as well as hepatocytes, express CD-14 and toll-like receptor 2 (TR2).The human liver in vitro model using primary liver cells from surgical specimen enables researchers to investigate specific questions of human liver metabolism, that were impossible to be undertaken by animal models. Furthermore, in vitro models are a requirement developing strategies for cellular therapy in liver cell transplant.
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