Liquidus Tracking: a promising vitrification technique for large scale encapsulated 3-D cell culture preservation

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Liquidus Tracking: a promising vitrification technique for large scale encapsulated 3-D cell culture preservation

Puschmann, E; (2015) Liquidus Tracking: a promising vitrification technique for large scale encapsulated 3-D cell culture preservation. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Liver organ shortage is an increasing problem worldwide and many die each year waiting for a new liver. In case of acute liver failure a bioartificial liver (BAL) device could “buy” time until a donor liver is available or until the liver has spontaneously undergone self-repair. For the clinical application of a BAL large quantities of cells should be available immediately necessitating cryo-banking. However, cryopreservation of large volumes results in increased ice formation and increased cell death. Ice formation can be prevented by vitrification, but the high cryoprotectant agent (CPA) concentrations needed are normally toxic to mammalian cells. Short exposure time minimizes toxicity but can only be achieved in small samples where fast cooling rates can be reached. To reduce CPA toxicity a vitrification machine (Liquidus Tracker) designed by Planer plc was used, which provides the lowest toxic effect that can be established for a given CPA concentration by decreasing the sample temperature to just above the melting point of that particular mix. The CPA concentration is then gradually increased as temperature is decreased along the liquidus curve. The first aspect of this thesis was to standardise a rapid and reliable method to describe post-stress viability. A digital imaging system was used to evaluate membrane integrity and enzyme activity by quantifying the fluorescence signal of fluorescein and propidium iodide. To understand the Liquidus Tracking (LT) process but also to pre-test conditions for automatic LT, different methods to carry out manual LT were established, evaluated and improved. To further increase cell viability a low-toxicity CPA solution was developed with the requirement of low viscosity so that it may be used within the Liquidus Tracker. Finally improvements were applied to automatic Liquidus Tracking. The development of a new stirring system substantially increased post-warming viability. In conclusion, an optimised large scale slow cooling vitrification protocol was developed for alginate encapsulated liver cells which may be used in a BAL.

Type:	Thesis (Doctoral)
Title:	Liquidus Tracking: a promising vitrification technique for large scale encapsulated 3-D cell culture preservation
Open access status:	An open access version is available from UCL Discovery
Language:	English
UCL classification:	UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI:	https://discovery.ucl.ac.uk/id/eprint/1469902