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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 Liquidus Tracking: a promising vitrification technique for large scale encapsulated 3-D cell culture preservation A thesis submitted for the degree of Doctor of Philosophy (PhD) Eva Puschmann 2015 UCL Division of Surgery & Interventional Science Royal Free Campus Department of Surgery (in collaboration with) UCL Institute for Liver and Digestive Health Royal free Campus Devision of Medicine 1 Declaration I, Eva Puschmann confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in this thesis. 2 2 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. 3 List of Contents COVER PAGE ----------------------------------------------------------------------------------------------------------------- 1 DECLARATION --------------------------------------------------------------------------------------------------------------- 2 ABSTRACT -------------------------------------------------------------------------------------------------------------------- 3 LIST OF CONTENTS ---------------------------------------------------------------------------------------------------------- 4 LIST OF FIGURES ------------------------------------------------------------------------------------------------------------11 LIST OF TABLES -------------------------------------------------------------------------------------------------------------14 ABBREVIATION -------------------------------------------------------------------------------------------------------------15 ACKNOWLEDGMENTS -----------------------------------------------------------------------------------------------------18 CHAPTER 1: GENERAL INTRODUCTION --------------------------------------------------------------------19 1.1 THE LIVER -------------------------------------------------------------------------------------------------------19 1.1.1 Liver functions ----------------------------------------------------------------------------------- 20 1.1.1.1 Metabolic functions ------------------------------------------------------------------------ 20 1.1.1.2 Detoxification ------------------------------------------------------------------------------ 20 1.1.2 Liver regeneration -------------------------------------------------------------------------------- 21 1.2 LIVER FAILURE -------------------------------------------------------------------------------------------------22 1.2.1 Acute Liver Failure ------------------------------------------------------------------------------ 22 1.2.2 Acute on-chronic Liver Failure ----------------------------------------------------------------- 23 1.3 LIVER DONOR SHORTAGE --------------------------------------------------------------------------------------23 1.4 EXTRACORPOREAL LIVER DEVICES -------------------------------------------------------------------------24 1.4.1 Artificial Liver Devices ------------------------------------------------------------------------- 24 1.4.2 Bioartificial Liver Devices ---------------------------------------------------------------------- 25 1.5 LIVER GROUP BAL ---------------------------------------------------------------------------------------------26 1.5.1 HepG 2 cell line ---------------------------------------------------------------------------------- 27 1.5.2 Alginate encapsulation -------------------------------------------------------------------------- 27 1.5.2.1 HepG2 cell growth in alginate beads --------------------------------------------------- 29 1.5.3 The fluidized bed bioreactor system ----------------------------------------------------------- 30 1.6 BIOARTIFICIAL LIVER PRESERVATION -----------------------------------------------------------------------30 1.6.1 Short-term preservation ------------------------------------------------------------------------- 31 1.6.2 Short-term Bioartificial Liver preservation --------------------------------------------------- 32 1.7 CRYOPRESERVATION: LONG-TERM PRESERVATION ------------------------------------------------------32 1.7.1 Extracellular ice formation ---------------------------------------------------------------------- 33 1.7.2 Intracellular ice formation ---------------------------------------------------------------------- 33 1.7.3 Intercellular ice formation ---------------------------------------------------------------------- 34 1.7.4 Osmotic effects and dehydration --------------------------------------------------------------- 34 1.7.5 The Equilibrium Melting temperature of aqueous solutions -------------------------------- 35 1.7.6 Cryoprotectant agents --------------------------------------------------------------------------- 36 1.7.6.1 Penetrating CPAs -------------------------------------------------------------------------- 36 4 1.7.6.2 Non-penetrating CPAs -------------------------------------------------------------------- 37 1.7.7 Slow cooling protocols -------------------------------------------------------------------------- 37 1.7.7.1 Supercooling and Ice Nucleation -------------------------------------------------------- 38 1.7.7.2 Warming process -------------------------------------------------------------------------- 39 1.7.8 Vitrification --------------------------------------------------------------------------------------- 39 1.7.9 Cooling and warming of bulky samples ------------------------------------------------------- 40 1.7.10 Liquidus Tracking -------------------------------------------------------------------------------- 41 1.8 AIM OF THE PROJECT -------------------------------------------------------------------------------------------44 CHAPTER 2: GENERAL METHODS AND MATERIALS --------------------------------------------------46 2.1 CELL CULTURE -------------------------------------------------------------------------------------------------46 2.1.1 Preparation of complete media for monolayer cell culture --------------------------------- 46 2.1.2 Trypsinising monolayer cells ------------------------------------------------------------------- 47 2.1.3 Cell Count and viability using Trypan blue exclusion test---------------------------------- 48 2.1.4 Culture of Encapsulated HepG2s -------------------------------------------------------------- 48 2.2 ENCAPSULATION OF HEPG2 CELLS INTO ALGINATE -------------------------------------------------------49 2.3 CELL COUNT USING THE CHEMOMETECT NUCLEO COUNTER --------------------------------------------52 2.4 FLUORESCIN DIACETATE/PROPIDIUM IODIDE STAINING ---------------------------------------------------54 2.4.1 Viability assessment in beads – Image analysis ---------------------------------------------- 54 2.5 METHYLTHIAZOLYLDIPHENYL-TETRAZOLIUM BROMIDE (MTT) ASSAY -------------------------------55 2.6 QUANTIFICATION OF HEPATO-SPECIFIC PROTEINS SYNTHESISED AND SECRETED IN CULTURE -----56 2.7 USE OF CONTROLLED RATE FREEZERS -----------------------------------------------------------------------58 2.8 STORAGE OF CRYOPRESERVED SAMPLES --------------------------------------------------------------------59 2.8.1 Temperature measurements during cryopreservation --------------------------------------- 59 2.9 STATISTICAL ANALYSIS ----------------------------------------------------------------------------------------60 CHAPTER 3: VALIDATION OF IMAGE ANALYSIS FOR CELL VIABILITY QUANTIFICATION ----------------------------------------------------------------------------------------------------61 3.1 INTRODUCTION --------------------------------------------------------------------------------------------------61
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