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Title: Alginate-encapsulated human hepatocytes for transplantation
Author: Jitraruch, Suttiruk
ISNI:       0000 0004 5368 0441
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2014
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Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure by providing short-term support to bridge patients to liver transplantation or allow native liver regeneration. Its success depends on mechanical stability, optimal permeability, and biocompatibility of the encapsulated cells to provide good viability and function. The main aims of this study were to optimise protocols for production and cryopreservation of human hepatocyte microbeads (HMBs) for future clinical use. HMBs were prepared using sterile ultrapure materials. Physical integrity of HMBs was evaluated using an osmotic stress test. A polymerisation of 15min provided mechanical stability with well-maintained cell viability and function. Cell content in HMBs was optimised and a density of 3.5x106cells/ml alginate provided the highest cell viability with hepatocytes evenly distributed within microbeads. Permeability of optimised HMBs was measured and the largest protein which could diffuse out of microbeads had a molecular weight of 164kDa. Immune activation of human peripheral blood mononuclear cells was assessed by flow cytometry after co-culture with empty microbeads and HMBs. No evidence of cellular immune activation was observed. Optimisation of hepatocyte microbead cryopreservation was performed using rat and human hepatocytes. Improved outcome of cryopreservation was found when hepatocyte microbeads were cryopreserved in UW solution containing 10% DMSO, 5% glucose and 60μM benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVAD). Safety, therapeutic effects, and immunogenicity of hepatocyte microbeads produced were confirmed in immunocompetent rats with and without acute liver failure. HMBs showed functionality with no inflammatory response in rats over 7 days. Transplantation of rat hepatocyte microbeads was safe and able to support the failing liver. Cryopreserved microbeads showed an encouraging outcome, but may require greater cell numbers compared to fresh microbead. In conclusion, optimised protocols for production and cryopreservation of GMP grade alginate-encapsulated hepatocytes were established. These high quality microbeads are suitable for clinical transplantation.
Supervisor: Hughes, Robin David ; Dhawan, Anil ; Mitry, Ragai Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available