Development of Cryopreservation Protocol for Liver Cell Spheroids - Isobel Massie, UCL

Development of an Optimised Cryopreservation Protocol for Encapsulated Liver Cell Spheroids: towards delivery of a Bioartificial Liver A thesis submitted for the degree of Doctor of Philosophy (PhD) Isobel Ruth Massie 2011 UCL Centre for Hepatology Royal Free Campus Department of Medicine University College London 1 Declaration I, Isobel Ruth Massie 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 the thesis. 2 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. 3 List of Contents TITLE PAGE __________________________________________________________________________1 DECLARATION _______________________________________________________________________2 ABSTRACT ___________________________________________________________________________3 LIST OF FIGURES _____________________________________________________________________15 LIST OF TABLES ______________________________________________________________________19 ABBREVIATIONS _____________________________________________________________________20 ACKNOWLEDGMENTS ________________________________________________________________23 CHAPTER 1: INTRODUCTION ___________________________________________________________24 1. INTRODUCTION TO THE LIVER _______________________________________________24 1. Structure of the liver_ ___________________________________________24 1.

Cells of the liver ________________________________________________25 1. Functions of the liver____________________________________________26 1. Acute liver failure _________________________________________________________27 1. Artificial liver devices ____________________________________29 1.

Molecular adsorbent recirculating system _________29 1. Bioartificial liver devices (BALS) ____________________________30 1. Extra-corporeal liver assist device _______________30 1. The Liver Group BAL _______________________________________________________31 1.

Fluidised bed bioreactor culture ___________________________________32 1. Liver Group bioartificial liver principle ______________________________32 1. Biopreservation for BAL as long- or short-term strategies _________________________32 1. Short-term preservation _________________________________________33 4 1.

Tissue-engineering construct preservation ___________________34 1. Storage at intermediate temperature with oxygenated support (perfluorodecalins) ______________________________________34 1. Long-term cryopreservation ______________________________________34 1. Cooling rates during cryopreservation ______________________________35 1.

Cell death at slow-cooling rates – solute toxicity _______________36 1. Cell death at rapid cooling rates ____________________________37 1. Methods to avoid supercooling _________________38 1. Thawing/warming protocols ______________________________________41 1.

Warming of slowly cooled cells ____________________________41 1. Warming of rapidly cooled cells ____________________________41 1. Addition and removal of cryoprotectants ____________________42 1. Non-penetrating CPAs ___________________________________43 1.

Cryopreservation of hepatocytes __________________________________43 1. Cryopreservation of alginate-encapsulated cells ______________________44 1. Cryopreservation of interconnecting cells ___________________________44 1. Intracellular ice formation ________________________________45 1.

Intercellular ice formation ________________________________45 1. Assessment of success following cryopreservation ____________________46 1. Aim and hypothesis ________________________________________________________48 CHAPTER 2: GENERAL METHODS _______________________________________________________50 2. HepG2 cell line culture _____________________________________________________50 2.

Culture medium preparation _____________________________________50 2. HepG2 culture medium (complete culture medium) ___________50 2. Culture medium for HepG2 cells encapsulated in alginate using the Inotech syringe pump system (high glucose medium) ___________50 2. Culture medium for HepG2 cells encapsulated in alginate using the JetCutter system (FFP medium) ____________________________51 2.

Preparation of fresh frozen plasma ______________51 2. Preparation of FFP media ______________________51 5 2. Culture of HepG2 cell line as monolayer cultures of adherent cells _______51 2. Determination of cell number and viability ___________________52 2.

Cryopreservation and revival of HepG2 cell line _______________52 2. Cryopreservation for maintaining HepG2 cell stocks for monolayer cultures ___________________________53 2. Alginate encapsulation and 3D culture ________________________________________53 2. Encapsulation of HepG2 cells using the Inotech syringe pump system _____53 2.

Preparation of 2% alginate solution _________________________55 2. Preparation of polymerization buffer _______________________55 2. Preparation of alginate cell suspension ______________________55 2. Cell encapsulation and bead collection ______________________55 2.

Static culture of Inotech encapsulated HepG2 cells ____________56 2. JetCutter cell encapsulation ______________________________________56 2. Cell encapsulation and bead collection ______________________58 2. Culture of JetCutter encapsulated cells ______________________58 2.

Fluidised bed bioreactor _______________________58 2. Preparation for bioreactor setup _________59 2. Rotating cell culture system ____________________60 2. Evaluation of cell numbers from 3D cultures ____________________________________61 2.

Removing cells from alginate _____________________________________61 2. Methods for beads maintained in 6-well plates _______________61 2. Methods for beads maintained in FBB or RCCS ________________61 2. Quantification of cell number using the Nucleocounter system __________62 2.

Calculation of viable cell number _____________________________________________63 2. Evaluation of cell viability and basal metabolic activities __________________________63 2. Qualitative assessment in beads – fluorescein diacetate/propidium iodide staining ______________________________________________________63 2. Quantitative assessment in beads – image analysis ____________________64 2.

Quantitative assessment – tetrazolium salt reduction assay _____________65 2. Quantification of total protein content using bicinchoninic acid protein assay _________66 2. Quantification of hepato-specific proteins synthesized and secreted in culture ________66 2. Quantification of broad-spectrum cytochrome P450 activity _______________________68 2.

Induction using indirubin ________________________________________69 2. Cryopreservation of alginate-encapsulated liver cell spheroids _____________________70 2. Use of controlled rate freezers ____________________________________70 6 2. Filling the CRF nitrogen dewar _____________________________70 2.

Entering and running a programme _________________________70 2. Cryopreservation and thawing of ELS _______________________________71 2. Addition of CPA _________________________________________71 2. Starting cryopreservation protocol _________________________72 2.

Storage of cryopreserved samples _________________________72 2. Thawing of ELS following cryopreservation ___________________73 2. Temperature measurements during cryopreservation _________________73 2. Differential scanning calorimetry _________________________________________74 2.

Evaluation of oxidative stress ____________________________________________76 2. Reactive oxygen species assay ____________________________________76 2. Method for 3D cultures __________________________________76 2. Method for monolayer culture _____________________________77 2.

Oxidative damage assay _________________________________________77 2. Statistical analysis _____________________________________________________78 CHAPTER 3: COOLING RATES, SUPERCOOLING AND NUCLEATION _____________________________79 3. Time over which ELS recovery should be assessed in post-thaw cultures ___79 3. Cooling rates, supercooling and warming rates _______________________79 3.

Determination of optimal cooling rates _____________________________81 3. Determination of supercooling ____________________________________81 3. Determination of equilibrium melting point by mDSC __________________83 3. Silver iodide bead formation ______________________________________84 3.

Assessing recovery in post-thaw cultures ____________________________86 3. Recovery in the first 6 hours following cryopreservation _______________88 3. Qualitative assessment of viability __________________________88 3. Quantitative assessment of viability ________________________89 7 3.

Recovery between 24 and 72 hours following cryopreservation __________90 3. Qualitative assessment of viability __________________________90 3. Quantitative assessment of viability ________________________90 3. Supercooling without nucleators ___________________________92 3.

Effect of nucleators ______________________________________92 3. Silver iodide bead formation and effect on supercooling ___________________________________________92 3. Effect of cholesterol on supercooling _____________93 3. Effect of minimizing supercooling using cholesterol on ice formation intra- and extracellularly ______________________________________________95 3.

Ice formation in the absence of DMSO ______________________95 3. Ice formation in presence of DMSO _________________________96 3. Ice formation in presence of DMSO and cholesterol ____________97 3. Effect of minimizing supercooling using cholesterol on ELS recovery in post- thaw cultures __________________________________________________97 3.

Effect of cholesterol on viability in post-thaw cultures __________98 3. Effect of cholesterol on viable cell numbers in post-thaw cultures _98 3. Effect of cholesterol on function in post-thaw cultures _________99 3. Hepato-specific protein synthetic function _______ 101 3.

Effect of warming rate on ELS recovery in post-thaw cultures ___ 107 3. Viable cell numbers __________________________107 3. Conclusions _____________________________________________________________121 CHAPTER 4: CRYOPRESERVATION STRESSES _____________________________________________122 4. Approaches to limit onset of apoptosis as a result of cryopreservation ____________________________123 4.

Cryopreservation of ELS ________________________________________126 4. Dose response to antioxidants ____________________________126 4. Inclusion of antioxidants during and after cryopreservation _____126 4. Inclusion of 1K1 during and after cryopreservation ____________126 4.

Assessing necrosis in post-thaw cultures ___________________________128 4. Assessing apoptosis in post-thaw cultures _________________________129 4. Assessment of oxidative injury ___________________________________131 4. Lipid peroxidation assay _________________________________131 4.

Reactive oxygen species assay ____________________________131 4. Measuring effect of 1K1 on ELS cell growth _________________________132 4. Cryopreservation of ELS with optimised CPA media __________________132 4. Assessment of ELS recovery in post-thaw cultures ___________________132 4.

Viable cell numbers ____________________________________132 4. Hepato-specific protein synthesis and secretion ______________132 4. Time course of necrosis in post-warming culture ____________________133 4. Time course of apoptosis in post-warming culture ___________________133 4.

Effect of catalase during cryopreservation __________________________135 4. Viable cell number _____________________________________135 4. Effect of Trolox during cryopreservation ___________________________136 4. Viable cell number _____________________________________137 4.

Does hypothermia cause oxidative stress? _________________________138 4. Storage at 4°C for 20 hours ______________________________138 4. Reactive oxygen species production ____________138 4. Storage at -4°C for 2 hours _______________________________139 4.

Reactive oxygen species production _____________139 4. Does cryopreservation cause oxidative stress? ______________________141 4. Reactive oxygen species production _______________________141 4. Can 1K1 protect against staurosporine-triggered apoptosis? ___________142 4.

Can 1K1 protect against apoptosis caused by cryopreservation? ________144 9 4. Effect of 1K1 when included during cryopreservation __________144 4. Effect of 1K1 when included during cryopreservation and in post- warming cultures ______________________________________144 4. Does 1K1 improve ELS recovery following cryopreservation? ___________146 4.

Does 1K1 affect cell growth rates? ________________________________146 4. Recovery of ELS cryopreserved using optimal CPA media ______________147 4. Conclusions _____________________________________________________________156 CHAPTER 5: SCALE UP _______________________________________________________________157 5. Encapsulated liver cell spheroid cell density ________________________157 5.

Cryopreservation of ELS at different cell densities ____________________160 5. Cryopreservation of cell-dense ELS at slower cooling rates _____________161 5. Cryopreservation of ELS with different volumes of cryoprotectant medium ____________________________________________________________161 5. Cryopreservation in cryobags ____________________________________162 5.

Measuring temperature profiles in cryobags during cooling and warming _____________________________________________162 5. Comparison between cryopreservation in cryobags and cryovials _____________________________________________________163 5. Cryopreservation in culture chamber ______________________________163 5. Cooling of culture chamber ______________________________164 5.

Warming of culture chamber _____________________________164 5. Standard warming of culture chamber ___________164 5. Improved warming of culture chamber __________165 5. Measuring temperature profiles in culture chamber during cooling and warming _________________________________________166 5.

Effect of ELS cell density on recovery following warming ______________167 5. Effect of slower cooling rates for cell-dense ELS _____________________169 5. Viable cell number _____________________________________169 5. Effect of different ELS:CPA ratio on ELS recovery following warming _____170 5.

Viable cell number _____________________________________170 5. Cryopreservation in cryobags ____________________________________172 10 5. Viable cell number __________________________173 5. Cryopreservation in culture chamber ______________________________175 5.

ELS recovery following cryopreservation in culture chamber using standard thawing methodology ___________________________177 5. ELS recovery following cryopreservation in culture chamber using optimised thawing methodology __________________________178 5. Conclusions _____________________________________________________________189 CHAPTER 6: REGULATORY CONSIDERATIONS ____________________________________________190 6. Use of fresh frozen plasma in place of foetal calf serum during cryopreservation ______________________________________________190 6.

Use of DMSO during cryopreservation _____________________________191 6. Use of GMP-compliant preservation media during cryopreservation _____192 6. Use of liquid nitrogen during cryopreservation ______________________193 6. Avoiding the use of nitrogen during cooling _________________193 6.

Avoiding the use nitrogen during long-term storage at low temperatures _________________________________________194 6. Effect of FFP on ELS recovery ____________________________________197 6. Reducing DMSO concentration during cryopreservation _______________197 6. Use of GMP-compliant CPA media ________________________________197 6.

Avoiding the use liquid nitrogen during cooling _____________________198 6. Can the EF600 perform a multi-step cooling profile? __________198 6. Can the EF600 apply a multi-step cooling profile to cryovials? ___198 6. Does cholesterol function as a heterogeneous nucleator in the EF600? _______________________________________________199 6.

How does the EF600 compare to a cryogen cooler? ___________199 6. Can ELS be stored without nitrogen for extended time periods? ________199 11 6. Differential scanning calorimetry __________________________200 6. Effect of fresh frozen plasma on cell viability, number and function in post- warming cultures _____________________________________________201 6.

Effect of reducing DMSO concentration during cryopreservation ________202 6. Use of different GMP-compliant cryopreservation media ______________205 6. Use of EF600 during cooling _____________________________________206 6. Can the EF600 perform a multi-step cooling profile? __________206 6.

Can the EF600 apply cooling to cryovials? ___________________206 6.