Development of a Quality System to Control DNA, Endotoxin and Particulates as part of an Extracorporeal Bioartificial Liver Medical Device

The Bioartificial Liver Devices (BAL) could provide treatment for acute liver failure by supporting patients awaiting transplantation or aid the process of liver regeneration. For use within a clinical setting, a number of regulatory criteria must be met, including controlling DNA, endotoxins and particulates. The aim of this thesis was to begin the development of a system to control plasma quality returning to the patient. Methods for DNA, endotoxin and particulates detection were established with human plasma to measure sensitivity for use with the BAL system. DNA detection by QPCR using Alu repeats were validated for use as an analytical method to demonstration DNA removal, achieving a Limit of Quantification (LoQ) of 0.1ng/ml DNA. Endotoxin analysis utilised a fluorescent derivative of the widely used LAL assay to increase sensitivity, enabling 2EU/ml to be detectable. Particulates down to 1μm were measured using laser light obscuration. Initially the removal of particulates from alginate as a starting material (alginate prior to encapsulation) was shown, using filtration by depth charge filter, sand bed filtration and gas solid cyclonic filtration. Encapsulated bead integrity, cell function and growth were compromised with all techniques of filtering alginate in solution, including depth charged and sand bed filtration. Conversely, gas solid cyclonic filtration maintained bead integrity, cell growth and function. Testing potential DNA levels in the large scale BAL system required the development of a scaled down model of the BAL treatment phase, replicating the large scale BAL system with cell number to plasma volume ratio. This provided an indication of the DNA challenge a removal system at a large scale would need to contend with, predicted to be 68ng/ml for a full scale BAL. A scaled down filtration model was then established to model the DNA removal capability of different 3M® Cuno® DNA depth charged filters. This established a requirement for a predicted surface area of 1300cm2 to achieve complete DNA removal. The volumetric capacity of the filters were calculated using established filter blockage models, in order to scale the capacity to the full BAL system size. Finally, the chosen depth charge filter was tested at a large scale with the extracorporeal BAL system, spiking human plasma with DNA and endotoxin, whilst measuring endotoxin and DNA removal over 8 hours of treatment. Pag

Development of small-scale fluidised bed bioreactor for 3D cell culture

Three-dimensional cell culture has gained significant importance by producing physiologically relevant in vitro models with complex cell-cell and cell-matrix interactions. However, current constructs lack vasculature, efficient mass transport and tend to reproduce static or short-term conditions. The work presented aimed to design a benchtop fluidised bed bioreactor (sFBB) for hydrogel encapsulated cells to generate perfusion for homogenous diffusion of nutrients and, host substantial biomass for long-term evolution of tissue-like structures and “per cell” performance analysis. The sFBB induced consistent fluidisation of hydrogel spheres while maintaining their shape and integrity. Moreover, this system expanded into a multiple parallel units’ setup with equivalent performances enabling simultaneous comparisons. Long term culture of alginate encapsulated hepatoblastoma cells under dynamic environment led to proliferation of highly viable cell spheroids with a 2-fold increase in cellular density over static (27.3 vs 13.4 million cells/mL beads). Upregulation of hepatic phenotype markers (transcription factor C/EBP-α and drug-metabolism CYP3A4) was observed from an early stage in dynamic culture. This environment also affected ERK1/2 signalling pathway, progressively reducing its activation while increasing it in static conditions. Furthermore, culture of primary human mesenchymal stem cells was evaluated. Cell proliferation was not observed but continuous perfusion sustained their viability and undifferentiated phenotype, enabling differentiation into chondrogenic and adipogenic lineages after de-encapsulation. These biological readouts validated the sFBB as a robust dynamic platform and the prototype design was optimised using computer-aided design and computational fluid dynamics, followed by experimental tests. This thesis proved that dynamic environment promoted by fluidisation sustains biomass viability in long-term cell culture and leads 3D cell constructs with physiologically relevant phenotype. Therefore, this bioreactor would constitute a simple and versatile tool to generate in vitro tissue models and test their response to different agents, potentially increasing the complexity of the system by modifying the scaffold or co-culturing relevant cell types

The effect of liver warm ischaemia reperfusion injury and modulation on bile composition evaluated by magnetic resonance spectroscopy

Orthotopic liver transplantation has become the preferred treatment for a variety of end-stage liver disease. As competency and survival rates increase, so does increasing demand, which puts greater strain on a static donor pool. This increases pressure to accept more marginal grafts, e.g. non-heart-beating and steatotic donors. However, graft dysfunction post-transplantation contributes considerably to postoperative morbidity and mortality. Proton nuclear magnetic resonance (1HNMR) spectroscopy is a powerful technique to explore the biochemical composition of biological fluids; it is rapid, non-invasive, nondestructive and it can detect metabolites at millimolar concentrations. In this study, 1HNMR assessed the changes in bile composition during liver ischaemia/reperfusion. The primary hypothesis was that bile composition changes during liver ischaemia reperfusion injury (IRI). The aims were to document these changes, identify biliary markers for liver IRI using 1HNMR, validate these markers using known modulators, determine if the same was true for steatotic livers and attempt to understand the mechanisms by which these changes happen in relation to the redox state of the liver. Materials and methods: A rabbit model of hepatic lobar IRI was used. In most experiments 3 groups were used (n=6): Sham group (laparotomy alone), I/R group (1hr ischaemia and 6hrs reperfusion), and a modulation group similar to I/R with the addition of N-acetylcysteine (I/R+NAC: 150mg/kg of NAC) and glycine (I/R+glycine: 5mg/kg glycine). Steatosis was induced by feeding with a 2% cholesterol diet for 8 weeks. Experiments were repeated on steatotic animals with Sham, I/R, I/R+NAC and IPC+I/R (5min ischaemia followed by 10min of reperfusion before prolonged ischaemia was induced) groups. The following parameters were measured: portal blood flow, bile flow (BF) and bile 1HNMR spectroscopy, hepatic microcirculation, intracellular tissue oxygenation, serum ALT, AST and ICG clearance were measured at 1, 2, 5 and 7 hours following reperfusion. Results: Bile spectroscopy demonstrated significant changes in bile composition following I/R and alterations with NAC, glycine and IPC. These changes are evident despite a constant post-reperfusion rate of BF. They were also present in steatotic livers, and were modulated by NAC and IPC. In experiment 1: BF, COX and biliary acetate decreased following I/R while AST, ALT, biliary PC and lactate increased along with PMN accumulation in sinusoids, KC hypertrophy, necrosis and apoptosis in normal livers. In experiment 2: BF, COX and biliary acetate decreased following I/R while ALT, PC, conjugated bile acids and lactate increased in the I/R group in normal livers. NAC administration attenuated the increase in ALT and lactate following I/R in the NAC+I/R group compared to the I/R group. Changes in conjugated bile acids seem to reflect changes in BF. In experiment 3: I/R+glycine was associated with increased BF, bile acid, acetate, pyruvate, glucose, acetoacetate, and decreased bile lactate and PC levels in normal livers. In experiment 4: NAC administration in steatotic livers reduced the extent of IRI, increased portal blood flow and liver parenchymal perfusion. NAC increased BF, biliary acetate and pyruvate and reduced acute liver injury, ALT and biliary PC. In experiment 5: IPC protected the steatotic liver from IRI and maintained hepatic oxygenation, tissue perfusion and mitochondrial redox state. COX activity was decreased by IRI in the fatty liver, but can was protected by IPC. Conclusions: This thesis has demonstrated changes in bile composition during warm normal liver I/R and its modulation with NAC and glycine. It has also demonstrated changes in bile composition during warm steatotic liver I/R and its modulation with NAC and IPC. It has noted several metabolites that are consistently changed, as well as the bile redox ratio of metabolites that provides a clearer indication of liver redox state than individual metabolites

Development of Human Liver Extracellular Matrix Hydrogel for Three Dimensional Cell Culture and Cell Transplantation

Introduction: It is increasingly evident that the currently available in vivo and in vitro methodologies for disease modelling are sub-optimal in recapitulating the complexity of human pathophysiology, as confirmed by the high failure rate of drug candidates due to lack of efficacy and safety. Moreover, hepatocyte transplantation has been tested as an alternative to liver transplantation for the treatment of liver diseases, but its applicability is hampered by the limited source of hepatocytes and poor hepatocyte engraftment. Aims: to develop human liver ECM hydrogels as novel in vitro platform for target identification/drug screening and for cell transplantation. Methods: Human livers unsuitable for transplantation were decellularized. The resulting ECM scaffold was then lyophilized and the resultant liver ECM powder was solubilised and mixed with three different biomaterials such as agarose, inert bio-ink or a synthetic thermo-responsive copolymer for hydrogel development. Samples were bioengineered with human hepatic cell lines (HepG2, LX2 or SNU-449), stem cells (IPSCs) or human primary hepatocytes. Validation of the hepatocellular carcinoma (HCC) model was investigated through treatment of SNU-449 samples with Sorafenib and TGF-β1. Furthermore, HepG2 bioengineered hydrogels were implanted for 3 weeks in immune-deficient mice. Samples were analysed by histology, immunofluorescence, immunohistochemistry, viability assays, gene expression and metabolic activity. Results: Bioengineered human liver ECM-based hydrogels with human liver cells showed an increase in cell survival, engraftment, proliferation and functionality compared to agarose, inert bio-ink or synthetic thermo-responsive copolymer. Viability assays of SNU-499 cells, upon Sorafenib treatment, revealed differences between 2D and 3D modelling in HCC. Implanted HepG2 ECM-hydrogels, retrieved from mice, showed that cells were still alive and engrafted. In vitro, ECM hydrogels combined with synthetic thermo-responsive copolymer showed the highest cell viability, better reproducibility, required less ECM volume and a smaller number of cells compared to ECM hydrogels combined with agarose or inert bio-ink. Conclusion: This study describes the development and the technical validation of human liver ECM hydrogels for in vitro and in vivo applications