The thesis describes the performance of the HepG2 cell line, a proliferating liver cell line, potentially to be used as the cellular component of a bioartificial liver (BAL). HepG2 cells in 3-Dimensional (3-D) spheroidal culture demonstrate dramatically improved function, compared to monolayer culture. The main aims of the thesis were to investigate the phenomenon in this culture system, whereby function in 3-D culture is optimal between Days 8-10, and thereafter there is a decline in function in spite of continued proliferation and viability. For optimal use in a BAL, it is necessary to increase function per cell either at later time points of 3-D HepG2 culture when cell numbers are greater, and/or increase cell number at times of peak function i.e. Days 8-10 of 3-D culture. The initial hypothesis was that the downregulation of activity observed from Day 11 occurred at the transcriptional level. Approaches to determine expression of liver-enriched transcription factors at times of peak and diminished function in 3-D culture and in monolayer culture were not conclusive. To understand the mechanisms underlying the temporal change in function in 3-D culture, the hypothesis that hypoxia and/or other forms of stress were responsible for the drop in function observed was explored. Microarray data and Western blot analysis highlighted the expression of genes and proteins known to be upregulated by hypoxia at Day 15. Ambient oxygen concentration was increased to attempt to increase cell performance but was ineffective. Genes and proteins implicated in oxidative stress were expressed. Results from assays to measure oxidative stress in HepG2 culture demonstrated an increase at Day 15 compared with Day 8 spheroidal cultures. Attempts to alleviate this stress by supplementing the culture medium with additional anti-oxidants at later times of 3-D culture did not enhance cell performance. Some stress-related proteins and genes were more strongly expressed in Day 8 cultures, as an adaptive response to increased metabolic activity during peak function, while others in Day 15 were turned off. These genes were investigated further at a functional level and results reflected the pattern observed. 3-D culture was manipulated with the addition of extracellular matrix (ECM) in order to enhance cell performance. Cell proliferation was measured by total nuclei quantification, incorporation of BrdU as a measure of DNA synthesis and Ki-67 labelling, as a measure of the total growth fraction. Positively labelled cells were seen throughout the spheroid indicating that even at the centre of the spheroids; there was maintenance not only of viability, but also the capacity to proliferate, indicating other microenvironmental changes may be responsible for the diminished function observed at Day 15. The thesis has highlighted the possible causes for the downregulation of function observed and modulation of the 3-D environment to overcome this, and emphasised the complex relationship between cell performance and the stress response, in order to improve a system which could provide the basis for the biological component of a BAL.

The harvesting of livers for transplantation involves assessment of the liver's suitability, including an examination of the colour and general appearance of the liver. If the organ is to be split for transplantation into two recipients, the vasculature of the liver must be studied and recorded. Remote assessment of livers and telesurgical guidance could save time and money. This thesis highlights the importance of colour in liver diagnosis, using animal and human models to examine the colour change associated with liver steatosis, which has implications for transplantation. It defines the magnitude and effects of colour distortions produced during remote imaging, and emphasises the need for calibration of any system where the capture and transmission of images is used to assess colour content. It also determines the maximum colour distortion permissible for diagnostic accuracy, with recommendations for viewing conditions, image compression and Internet transmission characteristics. The splitting of livers requires an important decision on where to cut, and it would be useful if a pre-planned incision plane could be viewed in relation to intraoperative landmarks. A system was developed utilizing a three-dimensional index block and multimedia database to allow an intraoperative ultrasound transducer to act as a pointer to pre-operative surgical planning images via image matching, with near real time display of the transducer position. Conclusions drawn include the importance of calibration of remote liver assessment viewing apparatus, and that the use of a multimedia database facilitated image matching so that an ultrasound transducer could be used as an intraoperative positional pointer to surgical planning information. This can be used to facilitate clinical trials of remotely guided liver splitting for multiple transplantations. Further work could include using colour enhancement to improve remote viewing of images, and a clinical trial of the index block system, with the possibility of using multi-modality images.

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Cold preservation is currently the basis of organ preservation. However, prolonged duration of cold storage or preservation of marginal organs leads to inferior outcome. Primary non-function of liver occurs in 3.5-6% of patients requiring retransplantation. Many marginal organs such as severely steatotic livers and non-heart-beating donor livers are either not retrieved or discarded after retrieval due to concerns regards viability. A better method of preservation could enable utilisation of many of these organs and increase the donor pool. This thesis describes and demonstrates successful preservation of livers at body temperature using normothermic perfusion. It aims to make preservation physiological, improve outcomes following transplantation and increase the number of donor livers that can be transplanted by allowing use of more marginal livers.

The liver is the metabolic powerhouse of the body and plays an important role in metabolism. The function of the liver includes drug detoxification, plasma protein synthesis and glycogen storage. Chronic liver failure (CLF) takes months or years to develop, whereas acute liver failure (ALF) may develop within 1-2 weeks. In the USA, 1 in every 10 persons is affected with a liver, biliary or gall bladder disease. The estimated medical and work loss cost per year due to Hepatitis B & Hepatitis 0 is more than $700 million & $600 million respectively. The accepted treatment of end-stage CLF is transplantation. However, the ability of the liver to regenerate has pointed to the development of temporary liver assist devices for the treatment of ALF. The purpose of this project is to review the state-of-the-art in liver assist devices and exciting developments in the field.

Liver transplantation (LT) has for decades been the most acceptable treatment for End-Stage Liver Disease (ESLD). Patient survival rates for LT are now approximately 91 % at one year, 77% at five years, and 59% at ten years post- LT. Furthermore, since LT has now been successful for several decades, there are a growing number of long-term survivors. Little is known about their quality of life (QOL). The aim of this thesis was to evaluate patient QOL and its correlates in new and long-term survivors following LT. A prospective longitudinal study of recent LT patients was conducted in tandem with a cross-sectional study of long-term LT survivors. Patients were recruited at King's College Hospital. New LT patients (N= 41) were recruited in hospital and were assessed at one month, three months, six months, and one year post-LT. Long-term survivors (N = 213) were recruited through the LT outpatient clinic. The questionnaire pack in both studies included measures of disease-specific and generic QOL, social support, optimism, emotional intelligence, anxiety, depression, and posttraumatic growth. Over the first year post-LT, patients experience improvements in physical QOL and disease-specific QOL, and decreases in anxiety. However, all patients reported impairment is physical QOL as compared to published UK results, yet no difference in mental QOL. Greater patient optimism and social support was associated with better QOL. There was an inverse relationship between anxiety and depression, and QOL scores. Patients experience a personal, positive growth post- LT, which is associated with greater emotional intelligence. The results of this thesis contribute to the understanding of QOL and psychological outcomes for patients over the first year post-LT, and in the long term. QOL varies over the first year post-LT, and patients benefit from social support and being optimistic. Similar to new LT recipients, long-term LT survivors have a significant impairment in their QOL compared with a UK sample. In both groups, clinicians need to continue monitoring patient QOL issues and anticipate that further patient support may be needed.

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