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Liver transplantation is currently the established treatment for patients with liver failure. Unfortunately there is a shortage of donor livers and therefore a number of patients die while on the waiting list for liver transplantation. Due to the high mortality rates and the increased waiting times for transplantation, there has been great interest in culturing hepatocytes in extracorporeal liver devices (known as bioartificial liver systems (BAL)). BAL systems may provide temporary liver support to bridge the patient prior to transplantation or to allow sufficient time for liver regeneration to occur. Many BAL systems have previously been developed and clinically tested on human patients. Apart from some minor improvement in the clinical status of patients all of the BAL systems failed to promote long-term survival of the patient. One critical point in BAL systems is the origin and functionality of hepatocytes used in the system. Hepatocytes derived from a non-human origin can be immunogenic and therefore not suitable for inclusion in BAL systems. On the other hand, human hepatocytes are in limited supply, as are the whole organs. One alternative to using freshly isolated hepatocytes or transformed cell lines is to generate hepatocyte-like cells from alternative sources. To address this issue we therefore focused on using hepatocytes derived from pancreatic cells, via a process termed transdifferentiation (or sometimes it is referred to as reprogramming) and in general means the conversion of one cell type to another. The model is based on culturing the rat pancreatic cell line AR42J-B13 (B13) with the synthetic glucocorticoid, dexamethasone (Dex). To develop proof-of-concept data for our BAL system we used a biocompatible matrix poly (lactic-co-glycolic acid) (PLGA) as a scaffold for culturing hepatocytes. This approach offers the option of transplanting the BAL system into the patient for the first time. I now provide evidence that the pancreatic B13 cells can be successfully cultured on biodegradable scaffolds of PLGA and transdifferentiated to hepatocyte-like cells with Dex (based on the morphological appearance). We were also interested in identifying novel glucocorticoid receptor interacting partners in the transdifferentiation of pancreatic cells to hepatocyte-like cells. To address this issue I developed B13 cell clones stably expressing functionally N- or C-terminal- Abstract V tagged glucocorticoid receptor. Stable expression of N- or C-terminal tagged glucocorticoid receptor enhanced the sensitivity to Dex. This resulted in increased cell death at higher concentrations of Dex but more efficient conversion of pancreatic cells to hepatocyte-like cells at lower concentrations of Dex compared to normal B13 cells. The B13 cell clones were used to develop a protocol for determination of the early signalling events (interacting partners) in the conversion of pancreatic cells to hepatocytes. The protocol includes gentle protein extraction from the B13 cells followed by purification of the EGFP tagged GR and analysis of co-purified proteins by mass spectrometry. I have now identified 11 potential GR interacting partners, 7 of which are already published and 4 are completely novel. These results suggest that the interacting partners may be involved in the reprogramming of one cell type to another.

The aim of this thesis was to assess feasibility of using chronically injured liver as a source for hepatocyte isolation, to compare inter-laboratory variability, to establish an efficient method for hepatocyte isolation and cryopreservation and to review whether anti-apoptotic compounds have any effect on cryopreservation outcomes. Method: A two-stage isolation protocol was used, with multiple washes and centrifugation compared with Percoll® purification pre- and post-cryopreservation and pre-incubation. A new cryopreservation media was validated and cells cryopreserved using this with the addition of potential anti-apoptotic agents. Demographic details were collected prospectively and histological samples collected where possible for analysis. Hepatocyte viability, yield per gram, hepatocyte loss, monolayer protein, visual attachment, phase I and II enzyme activity and measures of apoptosis were assessed. Results: Between October 2003-2005, 83 individuals donated tissue to UKHTB, with an additional 140 donors from elsewhere. 175 hepatocyte isolations were performed yielding more than 50 billion cells. Fresh cell culture was universally successful although purification led to significant cell loss. This was exacerbated during cryopreservation with especially poor porcine hepatocytes. None of the adjuncts to improved isolation or cryopreservation demonstrated reproducible improvement in outcome. There was only a weak negative correlation between histological injury and isolation outcome. Normothermic resuscitation prior to isolation conferred benefit across almost all measurable outcomes. Conclusion: There is significant inter-donor variability with regard to the outcomes of hepatocyte isolation and all purification methods cause unacceptable hepatocyte loss. A viable cell will attach and function and this is essential for many of the studies that are subsequently performed. Organs which are turned down for transplantation could be used for hepatocyte isolation.

Liver ischaemia-reperfusion (I/R) injury occurs in a number of clinical settings, including liver surgery, transplantation and haemorrhagic shock with subsequent fluid resuscitation. It is well recognised as a significant cause of morbidity and mortality and characterised by oxidative stress accompanied with depletion of antioxidants. Its pathophysiology is complex. This thesis investigates the effect of lobar liver I/R injury on liver microcirculation, hepatic oxygenation, cellular energetics and nitric oxide (NO) metabolism. It evaluates also the hypothesis that the antioxidant N-acetylcysteine (NAC) ameliorates liver I/R injury. Initially an experimental rabbit model was established where both phases (early and late) of liver I/R injury could be studied. New Zealand white rabbits underwent 60 min of lobar ischaemia followed by 7 h of reperfusion. It was found that cannulation of the femoral artery, for monitoring of the arterial blood pressure, induced remote liver injury, which could be avoided by use of the ear artery. Lobar liver I/R caused significant decrease in hepatic microcirculation, liver tissue oxygenation and cellular energetics. It also caused significant alterations in NO metabolism during the late phase (increase in plasma nitrites and plasma S-nitrosothiols, decrease in liver tissue nitrotyrosine) and significant oxidative stress. Intravenous administration of NAC (150 mg/Kg/h over the 15 min before reperfusion and maintenance at 10 mg /Kg/h during the 7 h reperfusion period) had a clear protective effect during the late phase of reperfusion injury in rabbits with normal and steatotic liver. This effect was associated with improved cellular energy metabolism (maintenance of cytochrome oxidase activity, increased acetates and decreased lactates in bile) and altered NO metabolism (reduced plasma nitrites, S-nitrosothiols and reactive nitrogen species). In conclusion, this thesis has shown that lobar hepatic I/R induces significant alterations on cellular energetics and NO metabolism. It provides also significant new information about the timing and the possible mechanism of the protective effect of NAC. It could form the basis for the performance of large scale prospective randomised clinical trials where the effect of thiols in clinical settings of liver I/R injury will be investigated.

No description available.

A promising method for growing functional liver tissue in vitro involves culturing hepatocytes as spheroidal cell aggregates. In this thesis, we develop mathematical models of cell aggregation, and use them to determine how hepatocytes' interactions with the extracellular matrix (ECM) on which they are seeded, and with stellate cells, affect the process. Chapters 2-4 focus on the effect that cell-ECM coupling has on the aggregation process. We use a novel formulation that couples a mechanical model for the ECM with a two-phase model for the cell-culture region. A combination of linear stability analysis and numerical simulations are used to identify parameter regimes in which aggregation occurs, and investigate the effect of changing key parameters. In Chapter 2, we assume a one-dimensional geometry, whereas in Chapters 3 and 4, the slender two-dimensional geometry is exploited to obtain two alternative one-dimensional models in which the mechanisms dominating aggregation are chemotaxis and surface tension. In Chapter 5, we focus on interactions between hepatocytes and stellates, neglecting the role of the ECM.We develop new non-local models to investigate the relative contributions of hepatocyte-heaptocyte and hepatocyte-stellate interactions in controlling spheroid formation. Comparison with experimental results suggests that the hepatocyte-stellate interaction is the stronger, in which case a 1:1 seeding ratio of hepatocytes to stellates is likely to be optimal for promoting swift aggregate formation.

It is important to account for all sources of uncertainty when evaluating the clinical or cost-effectiveness of health care technologies. Therefore, this thesis takes as its basis a cost-effectiveness study in liver transplantation and identifies two previously unexplored issues that can arise in clinical and cost-effectiveness studies. A literature review of studies evaluating the effectiveness, costs or cost-effectiveness of solid organ transplantation confirmed that these issues were important and relevant to other transplantation studies. The first issue concerns the selection of an appropriate method for estimating mean study costs in the presence of incomplete (censored) data. Twelve techniques were identified and their accuracy was compared across artificially created mechanisms and levels of censoring. Lin's method with known cost histories and short interval lengths is recommended for accurately estimating mean costs and their uncertainty. It is assumed that these findings are generalisable to any solid organ transplant study where censoring is an issue. The second issue explored in this thesis relates to methods for measuring uncertainty around survival, HRQL and cost estimates derived from prognostic models in the absence of observed data. Probabilistic sensitivity analysis is recommended for measuring prognostic model parameter uncertainty and estimating individual patient outcomes and their uncertainties, as it is able to incorporate the additional uncertainty from using prognostic models to estimate control group outcomes. This thesis shows the quantitative importance of these issues and the methodological guidance offered should enable decision makers to have more confidence in clinical and cost-effectiveness estimates. Providing decision makers with a fuller estimate of the uncertainty around clinical and cost effectiveness estimates will aid them in decisions about the necessity of conducting further research in to the clinical or cost-effectiveness of health care technologies.