Lintern, KB; (2013) Immobilisation of Lactate Oxidase and Deoxyribonuclease I for use within a Bio-Artificial Liver Assist Device for the Treatment of Acute Liver Failure. Doctoral thesis , UCL (University College London).

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Abstract

Constraints of cell supply indicate that proliferating cell lines are likely to be essential components of Bio-Artificial Liver support devices (BAL) for the treatment of acute liver failure. The Liver Group BAL employs clones of cells derived from the HepG2 cell line, which in common with many tumour derived cells, are predominantly dependent on anaerobic glycolysis for energy supply, leading to production of lactate within the bioreactor. The BAL system requires prolonged culture of alginate encapsulated HepG2 cells, and lactate accumulation presents a potential hazard in this system: at ~15 mM, accumulated lactate becomes toxic to the cells in the bioreactor, and also compromises alginate bead integrity by chelating the calcium ions necessary for alginate polymerisation. Furthermore, the tumour lineage of the cells could prove a potential threat to patient safety should any HepG2 DNA enter the patient’s system. It was hypothesised that inclusion of immobilised Lactate oxidase (LOx) to catalyse degradation of lactate into pyruvate could offset these limitations whilst simultaneously providing a potential energy source utilisable by HepG2 cells. In a similar fashion, immobilised Deoxyribonuclease I (DNase I) could be utilised to remove non-patient DNA during the treatment phase of the BAL system. Here it is demonstrated that functionalised glass beads are a feasible method of immobilising LOx and DNase I. Enzymatic activity was retained even after prolonged incubation at 37°C in the presence of human plasma, offering a means of reducing lactate levels during HepG2 culture, and potentially removing circulating DNA below practically detectable levels, thus facilitating cellular performance and BAL efficiency as a safe and effective potential therapy for acute liver failure.

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