UvA-DARE (Digital Academic Repository) Development of an immortalised human hepatocyte cell line for the AMC Bio- Artificial Liver Deurholt, T. Publication date 2007 Document Version Final published version Link to publication Citation for published version (APA): Deurholt, T. (2007). Development of an immortalised human hepatocyte cell line for the AMC Bio-Artificial Liver. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. 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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:06 Oct 2021 Development of an immortalised human hepatocyte cell line for the AMC Bio-Artificial Liver Tanja Deurholt Colofon The work described in this thesis was financially supported by: - the European Union - Hep-Art Medical Devices B.V., the Netherlands - Technology Foundation (STW) of NWO, the Netherlands The publication of this thesis was financially supported by: - Hep-Art Medical Devices B.V., the Netherlands - Nederlandse Vereniging voor Hepatologie, Haarlem, the Netherlands - Van Gool Stichting, Amsterdam, the Netherlands - Academic Medical Center, Amsterdam, the Netherlands - AMC Liver Center, Amsterdam, the Netherlands Printed by PrintPartners Ipskamp ISBN 978-90-9022509-8 DEVELOPMENT OF AN IMMORTALISED HUMAN HEPATOCYTE CELL LINE FOR THE AMC BIO-ARTIFICIAL LIVER ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof.dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op donderdag 6 december 2007, te 10.00 uur door Tanja Deurholt geboren te Zaandam Promotiecommissie: Promotor: Prof.dr. R.P.J. Oude Elferink Co-promotores: Dr. R.A.F.M. Chamuleau Dr. R. Hoekstra Overige leden: Prof.dr. W.H. Lamers Prof.dr. T.M. van Gulik Prof.dr. P.L.M. Jansen Prof.dr. G.M.M. Groothuis Prof.dr. B. Clement Dr. A.J. Meijer Faculteit der Geneeskunde Aan mijn ouders T a b l e o f C o n t e n t s Chapter 1 General introduction and outline of the thesis page 9 Chapter 2 Assessment of in vitro applicability of reversibly page 33 immortalised NKNT-3 cells and clonal derivatives Chapter 3 In vitro functionality of human fetal liver cells and page 53 clonal derivatives under proliferative conditions Chapter 4 Novel immortalised human fetal liver cell line, page 75 cBAL111, has the potential to fully differentiate into functional hepatocytes Chapter 5 The effect of culture time on the in vitro hepatic page 95 functionality of the immortalised human fetal liver cell line cBAL111 Chapter 6 Increased reproducibility of quantitative reverse page 117 transcriptase-PCR Chapter 7 Summary and general discussion page 127 Nederlandse samenvatting page 133 Dankwoord page 139 C h a p t e r 1 General introduction and outline of the thesis Robert Chamuleau1 Tanja Deurholt2 and Ruurdtje Hoekstra2,3 1Department of Hepatology, 2AMC Liver Center and 3Surgical Laboratory, Academic Medical Center, University of Amsterdam, the Netherlands Modified from: Metabolic Brain Disease, volume 20, number 5, page 327-335, December 2005 General introduction and outline of the thesis General introduction Summary Acute liver failure (ALF) is a serious disease that is only effectively treated by orthotopic liver transplantation. However, the shortage of liver donors and the mortality, morbidity and life- long immunosuppressive therapy related with liver transplantation have urged the development of complementary or alternative therapies. The search for these therapies is impaired by the high variability in etiology and pathogenesis of ALF. Nevertheless, bioartificial livers (BALs) have shown to be effective in supporting the liver function in animals with ALF. Freshly isolated or cryopreserved porcine or human hepatocytes have been most frequently used as biocomponent in clinically applied BALs. Phase 1 studies of all biocomponent modalities showed safety, feasibility, and improvement of patient’s biochemical, neurological, and hemodynamic parameters. However, the xenotransplantation- related disadvantages of porcine cells and the shortage of primary human hepatocytes have urged the exploration of alternative biocomponents. The future lies in the development of one or more human hepatocyte cell lines, which will have minimal immunogenicity, no risk of xeno-zoonosis, and the requested functionality and availability. Primary sources for the development of a human hepatocyte cell line are liver-tumour-derived cell lines, immortalised mature or fetal hepatocytes and stem cells. However, in all cell types tested so far, the in vitro differentiation cannot be stimulated to such an extent that their functionality reaches that of primary human hepatocytes and a pilot- controlled clinical trial with C3A cells in ALF patients did not show significant improvement of survival. More insight in differentiation-promoting factors and the influence of matrix and co- culture conditions to optimize the functionality of the human hepatocyte cell lines is necessary. 10 General introduction and outline of the thesis 1. Liver support systems for Acute Liver Failure 1.1 Acute Liver Failure The liver performs many different functions to ensure blood homeostasis: hepatocytes synthesize various proteins, including clotting factors; they produce bile and regulate carbohydrate, fat and protein metabolism; they detoxify ammonia and many other substances, including drugs (1). When liver function fails in acute liver failure (ALF), all these processes are disturbed. ALF is therefore a severe liver disease with mortality between 60 and 90%, depending on the cause. ALF can develop as a consequence of many different etiologies, like viral hepatitis, acetaminophen or other drug intoxication, metabolic diseases like Wilson’s disease, or primary non-functioning graft after liver transplantation. ALF patients most often present with jaundice, which is caused by the accumulation of serum bilirubin, combined with anorexia, nausea, vomiting and abdominal pain. At the time of presentation, most of the liver damage has already taken place. ALF progresses with the development of coagulopathy, cerebral oedema resulting in drowsiness and progressing to coma, renal impairment, hydrodynamic instability, increased susceptibility to infection and multi-organ failure (1). Each subgroup of ALF patients shows specific complications. For example, empirical evidence shows that 30% of the patients with acetaminophen induced ALF develop acidosis, where in other etiologies only 5% of the patients show pH disturbance; the underlying mechanism is unclear (2). The pathogenesis of cerebral oedema, which accounts for most deaths, is still heavily debated. A combination of vasogenic and cytotoxic mechanisms is probably involved and increased permeability of the blood-brain barrier, accumulation of astrocytic glutamine, disturbance of the neuronal neurotransmitter balance as well as the accumulation of inflammatory cytokines may play a role (2). The only life-saving therapy, orthotopic liver transplantation (OLT), is limited by the scarcity of donor livers and is associated with a relatively high mortality. Adam et al. reported a survival of 66% and 61% at 5 and 8 years, respectively, of 22,089 patients who had undergone OLT procedures during 1988–1997 in 103 active centers (3). The frequency of death declined from the first week onwards and 65% of deaths occurred in the first 6 months. In addition to mortality, OLT has considerable morbidity and transplanted patients need lifelong immunosuppressive therapy. Further medical treatment of ALF patients is mainly supportive, including mechanical ventilation and aggressive daily microbiological surveillance until recovery or until OLT is available. To bridge patients with ALF more successfully to OLT or even to regeneration of the native liver, a diversity of liver support systems has been developed. 11 General introduction and outline of the thesis 1.2 Liver support systems The first liver support therapies, already in the 1950s, were artificial devices that mainly focused on the detoxifying function of the liver (4-6). However, the limited function of these systems could not elongate life span, although neurological status improved. Currently, the molecular adsorbent recirculating system (MARS) is the only artificial system that did show improved survival in a controlled trial in a subgroup of patients with hepatorenal syndrome (7). One of the main reasons for the limited success of artificial devices, is most likely the non-specific absorption, e.g. the MARS cannot distinguish harmful molecules from substances that might be beneficial to recovery of the liver, like hepatocyte growth factor (HGF) (8). In addition, systems based on absorption