Abstract
Background and Aims: A variety of potential Bioartificial liver (BAL) support systems have been developed over the years, to provide adequate conditions to support the liver cells required to replace liver function. A significant challenge for clinical utility is the preservation of the biomass with adequate viability during transport and set-up prior to use. Whilst cryopreservation proffers an ideal tool for maintaining frozen cells for unlimited periods until they are required, unfortunately this technique currently fails to optimally preserve organised tissues in a satisfactory manner. Oxygen carriers such as perfluorocarbons (PFCs) are routinely used for organ preservation before transplantation, and we have developed these as potentially ideal candidates for BAL preservation. We describe here the technique applied to our BAL support system, based on alginate encapsulated human liver cells (HepG2 line), with preserved function using PFC emulsion and antioxidants in the media during transport. Methods: Alginate encapsulated HepG2 cell beads are grown in a fluidised bed bioreactor into performance competent. Cells proliferate within ~450–500um size hydrogel beads generating spheroid-shaped tissue, including liver specific cell structures. During the transport cells are dispensed into polystyrene flasks containing Hepes (25mM) buffered media and oxygenated PFC. Catalase (500U/ml), N-acetyl cysteine (3mM) and Trolox (0.85mM) antioxidants are added to the standard media to prevent cell damage. Iterative experimentation defined the optimal ratio of alginate beads, PFC and media as 1:1:10. 50% of the media was replenished after 27hours. Following storage, viability was analyzed by vital dye staining (FDA/PI). Cell functionality after storage was evaluated. Results: The BAL biomass of ~2.5×1010 cells remained >90% viable 48 hours after storage and transportation. Analyses of the media showed lactate accumulation and sustained glucose consumption over the storage time, indicating persistence of low level metabolism, using normal HepG2 pathway of anaerobic glucose metabolism. After storage, cells were cultured in media and plasma, showing restored synthetic function. Conclusions: We describe a method for optimal maintenance of alginate entrapped biomass capable of preserving 2.5×1010 viable cells in a volume of ~1200ml alginate beads. The performance of this biomass is being tested in an animal ischemic model of acute liver failure.
Published Version
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