Massie, I.R.; (2012) Development of optimised cryopreservation protocol for encapsulated liver cell spheroids: towards delivery of a bioartificial liver. Doctoral thesis , UCL (University College London).

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Abstract

Acute liver failure (ALF) has a rapid and unpredictable onset with high mortality. The only current treatment is to transplant a donor organ but donor organ shortages exist. A bioartificial liver (BAL) could bridge the gap to transplant or buy time for spontaneous recovery. This BAL comprises HepG2 cells encapsulated within alginate that have formed tissue-like spheroids (ELS), which display upregulated function compared to monolayer cultures approximately 9 days after encapsulation. In order to treat ALF in a timely fashion, this thesis aimed to provide an off-the-shelf treatment via development of a cryopreservation protocol for ELS. A baseline recovery for ELS post-cryopreservation was established with particular attention paid to the clinically relevant time-course of recovery. Intracellular ice formation was identified as an injury mechanism causing large amounts of cell death. A strategy was devised to limit this and improved recovery was demonstrated. Other, more subtle, injury mechanisms were subsequently identified and steps taken to ameliorate their effects resulting in decreased apoptosis and improved ELS recovery. As ELS will be used to treat human patients as the cellular component of a BAL, a number of regulations must be complied with. The optimised cryopreservation protocol was modified to ensure mandates were met without a loss in ELS cell recovery. Using this knowledge, cryopreservation was then scaled-up near to volumes that would be required to treat an adult patient. Finally, these techniques were applied to cryopreservation of single cell suspensions of primary human hepatocytes. By doing so, it was determined whether or not a different cell type in a different culture format suffered injury via the same mechanisms as ELS and whether or not these could be ameliorated using the same methods. In conclusion, an optimised cryopreservation protocol has been developed for ELS which may be used within a BAL.

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