Cryopreservation and Freeze-Drying Protocols

Dedication
Preface
Contents
Contributors
Part I: Fundamental Aspects of Cryopreservation and Freeze-Drying
	Chapter 1: Principles Underlying Cryopreservation and Freeze-Drying of Cells and Tissues
		1 Nature´s Way to Preserve Life
		2 Methods for Preservation of Cells and Tissues
		3 Freezing and Drying Injury
			3.1 Freezing Injury
			3.2 Drying Injury
		4 Mode of Action of Protectants
			4.1 Cryoprotective Agents
			4.2 Lyoprotective Agents
		5 Water and Solute Transport Across Cellular Membranes and Loading Cells and Tissues with Protective Agents
			5.1 Water Transport Across Cellular Membranes
			5.2 Loading Cells with  CPAs
			5.3 Loading Cells with Lyoprotective Agents
			5.4 Loading Tissues with Protective Agents
		6 Cryopreservation
			6.1 Types of Cryoprotective Agent and CPA Toxicity
			6.2 Slow Cooling Cryopreservation and Optimal Cooling  Rate
			6.3 Cryopreservation by Vitrification
		7 Lyopreservation by Freeze-Drying
			7.1 Freeze-Drying
			7.2 Formulations for Freeze-Drying of Cells
			7.3 Formulations for Freeze-Drying of Tissues
		References
	Chapter 2: Principles of Ice-Free Cryopreservation by Vitrification
		1 Introduction and General Orientation
			1.1 Overview
			1.2 Basic Terminology and Concepts
			1.3 Vitrification and Molecular Stability at Low Temperatures
			1.4 Cryopreservation by Vitrification: A Conceptual History
				1.4.1 Beginnings: 1930-1958
				1.4.2 Information Development, 1965-1972: Supercooling and Vitrification Tendencies of Cryoprotectant-Water Solutions
				1.4.3 Cryoprotectant-Enabled Vitrification: 1977-1986
				1.4.4 Further Developments: 1985-Present
			1.5 Advantages and Disadvantages of Vitrification
			1.6 Vitrification in Nature
			1.7 Vitrification During Freezing of Living Cells
		2 The Physical Principles of Vitrification
			2.1 Vitrification Depends on the Solute Concentration of Aqueous Solutions
			2.2 The Physical Nature and Basis of Vitrification
			2.3 Ice Nucleation
			2.4 Kinetic Aspects of Ice Formation in Vitrification Solutions During Cooling
			2.5 Devitrification and Recrystallization
			2.6 Antinucleation and Specific Ice Growth Inhibition
			2.7 Thermally Induced Volume Changes, Strain, and Fracture Formation
		3 The Biological Principles of Vitrification
			3.1 Are Cryoprotectants Necessary for Vitrification?
			3.2 ``Vitrification´´ into Doubly Unstable Glasses and ``One-Way´´ Vitrification
			3.3 Carrier Solutions and Cryoprotectants
			3.4 Osmosis, Osmotic Limits, and Osmotic Protocols
			3.5 Procedures for Avoiding Cryoprotectant Toxicity
			3.6 Extracellular Agents in Vitrification
			3.7 Cryoprotectant Toxicity Neutralization
			3.8 Mechanisms of Cryoprotectant Toxicity
			3.9 Chilling Injury and Its Modification or Avoidance
			3.10 Storage in the Vitreous or Near-Vitreous State
			3.11 Proteins at Low Temperatures
		4 Summary and Conclusions
		References
	Chapter 3: The Principles of Freeze-Drying and Application of Analytical Technologies
		1 Introduction
			1.1 General Overview
			1.2 History
			1.3 Applications of Freeze-Drying in the Pharmaceutical and Biological Sectors
		2 The Process of Freeze-Drying
			2.1 Operational Principles
			2.2 Product Freezing
				2.2.1 Controlled Nucleation
				2.2.2 Shelf-Cooling Rate
				2.2.3 Ice Structure and Freeze Consolidation
				2.2.4 Solute Freezing Behavior
				2.2.5 Freezing in Practice
			2.3 Primary and Secondary Drying
				2.3.1 Primary Drying (Sublimation)
					Sublimation Rate and Chamber Pressure Conditions
					Vapor Differential Pressure and Drying Efficiency
					Heat and Mass Transfer
					Cooling and Warming the Product
					The Sublimation Interface
				2.3.2 Secondary Drying
			2.4 Stoppering the Product
			2.5 Reconstituting the Product
		3 Thermoanalytical and Microscopic Methods
			3.1 Freeze-Drying Microscopy (FDM)
			3.2 Modulated Differential Scanning Calorimetry (mDSC)
		4 Scale-Up Factors and PAT
		5 Factors Affecting Freeze-Dried Products
		References
	Chapter 4: Mathematical Modeling and Optimization of Cryopreservation in Single Cells
		1 Introduction
		2 Model Selection
			2.1 Cell Volume
			2.2 Boyle van´t Hoff
			2.3 Osmolality and Chemical Potential
				2.3.1 Application of Osmolality Models
				2.3.2 Chemical Potential
			2.4 Membrane Transport Models
				2.4.1 Chemical Potential Approximations
				2.4.2 Temperature Dependence
				2.4.3 Nondimensional Model
				2.4.4 Reparametrization for Stiff Solutions and Analytic Solution
				2.4.5 Effects of the Selection of Chemical Potential Approximation
			2.5 Model Fitting
				2.5.1 Volume Measurement
				2.5.2 Curve Fitting
			2.6 Ice Formation Models
		3 Optimization
			3.1 CPA Equilibration Protocols
				3.1.1 Classical CPA Equilibration Optimization Approach
			3.2 Cooling Rate
				3.2.1 Mazur Model
			3.3 Warming
		4 Conclusions
		5 Notes
		References
	Chapter 5: Mathematical Modeling of Protectant Transport in Tissues
		1 Introduction
		2 Tissue Modeling Considerations
			2.1 Mass Transfer in the Extracellular Space
			2.2 Tissue Size Changes Due to CPA Exposure
			2.3 Fixed Charges
			2.4 Coupling Between Cell Membrane Transport and Mass Transfer in the Extracellular Space
		3 Tissue Mass Transfer Models
			3.1 Models That Assume a Constant Tissue  Size
				3.1.1 Fick´s Law of Diffusion
				3.1.2 Interstitial Diffusion with Coupled Cell Membrane Transport
				3.1.3 Maxwell-Stefan Diffusion
			3.2 Models That Account for Changes in Tissue  Size
				3.2.1 Islet Model of Benson et al.
				3.2.2 Network Thermodynamic Model
				3.2.3 Non-dilute Biomechanical Transport Model
		4 Conclusions and Future Directions
		References
Part II: Technologies and Methods to Study Freezing and Drying
	Chapter 6: Freezing Technology: Control of Freezing, Thawing, and Ice Nucleation
		1 Introduction
		2 Materials
			2.1 Cooling
			2.2 Ice Nucleation During Cooling
			2.3 Storage and Transfer
			2.4 Thawing
		3 Methods
			3.1 Cooling
				3.1.1 Passive Coolers
				3.1.2 Controlled-Rate Freezers
			3.2 Ice Nucleation During Cooling
			3.3 Storage and Transfer
			3.4 Thawing
				3.4.1 Water  Bath
				3.4.2 Dry Thawing Systems
		4 Notes
		References
	Chapter 7: Microwave- and Laser-Assisted Drying for the Anhydrous Preservation of Biologics
		1 Introduction
			1.1 General Introduction
			1.2 Microwave-Assisted Drying
			1.3 Laser-Assisted Drying
			1.4 Optical and Mechanical Characterization of Dehydrated Samples
				1.4.1 Polarized Light Imaging (PLI)
				1.4.2 Scanning White Light Interferometry (SWLI)
				1.4.3 Profilometry
				1.4.4 Raman Spectroscopy
				1.4.5 Bright-Field and Fluorescence Microscopy
		2 Materials
			2.1 Microwave-Assisted Drying of Gametes
			2.2 Laser-Assisted Drying of Proteins
			2.3 Characterization of Dried Samples
		3 Methods
			3.1 Microwave-Assisted Drying of Gametes
				3.1.1 Assessment of Microwave-Assisted Desorption Kinetics of Trehalose Buffer Solution
				3.1.2 Microwave-Assisted Drying, Storage, and Rehydration
			3.2 Laser-Assisted Drying of Proteins
				3.2.1 Laser-Assisted Drying and Storage of Proteins
				3.2.2 Characterization of Dried Samples
		4 Notes
		References
	Chapter 8: High-Speed Video Cryomicroscopy for Measurement of Intracellular Ice Formation Kinetics
		1 Introduction
		2 Materials
			2.1 High-Speed Imaging System and Accessories
			2.2 Microscope and Accessories
			2.3 Temperature-Control System and Accessories
			2.4 Computer System and Electronic Accessories
			2.5 Tools
			2.6 Materials and Supplies
		3 Methods
			3.1 Trigger Interface Cable
			3.2 Instrumentation Setup
			3.3 Experimental Setup
			3.4 Initial Configuration of High-Speed Imaging System
			3.5 Sample Preparation
			3.6 Sample Loading
			3.7 Freezing Experiment
			3.8 Experiment Shutdown
			3.9 Data Analysis
		4 Notes
		References
	Chapter 9: Use of Ice Recrystallization Inhibition Assays to Screen for Compounds That Inhibit Ice Recrystallization
		1 Introduction
			1.1 General Background
			1.2 Examples of Ice Recrystallization Assays
		2 Materials
			2.1 Standard Solutions
			2.2 IRI Sample Testing
			2.3 Image Analysis
		3 Methods
			3.1 IRI Sample Preparation
			3.2 Instrument Setup
			3.3 IRI Sample Testing via Splat Assay
			3.4 IRI Sample Testing via Sucrose Assay
			3.5 Image Analysis
		4 Notes
		References
	Chapter 10: DSC Analysis of Thermophysical Properties for Biomaterials and Formulations
		1 Introduction
			1.1 Calorimetrical Measurement
			1.2 Interpretation of a Calorimetrical Thermogram
		2 Materials
		3 Methods
			3.1 DSC Verification
			3.2 Operation Procedure
			3.3 Data Analysis
		4 Specific Measurements of Thermal Properties for Biomaterials and Protectant Solutions
			4.1 Unfrozen Water Content in Frozen Materials or Solutions
			4.2 T′g or Te of Frozen Materials or Solutions
			4.3 Vitrification (Tg) and Devitrification (Td) of Cryopreservation Solutions
			4.4 Ice Damage Upon Cryopreservation
			4.5 Stability of Frozen Cells at Subzero Temperatures
			4.6 Molecular Mobility and Phase Separation
		5 Notes
		References
	Chapter 11: Osmometric Measurements of Cryoprotective Agent Permeation into Tissues
		1 Introduction
		2 Materials
			2.1 Tissue Preparation
			2.2 Cryoprotective Agent Permeation into Tissue and Osmometric Measurement
			2.3 Chemicals
		3 Methods
			3.1 Preparation of Articular Cartilage
			3.2 Setup of Holding Baths
			3.3 Setup of Osmometer (e.g., Micro-Osmette Osmometer)
			3.4 Cryoprotective Agent Permeation into Tissue
			3.5 Cryoprotective Agent Efflux from Tissue and Osmometric Measurement
			3.6 Mathematical Procedures
		4 Notes
		References
	Chapter 12: Use of X-Ray Computed Tomography for Monitoring Tissue Permeation Processes
		1 Introduction
		2 Materials
			2.1 CT Imaging
			2.2 Cooling Equipment for CT Measurement of Cryopreserved Samples
			2.3 Solutions and Containers
		3 Methods
			3.1 CT Calibration at Room Temperature
			3.2 CT Calibration for Samples Imaged Below -140 C
			3.3 CT Acquisition of the Biological Samples After Cryopreservation
			3.4 CT Acquisition of the Biological Samples After Rewarming
			3.5 Quantification of Me2SO Concentration in the Biological Samples
		4 Notes
		References
	Chapter 13: Use of In Situ Fourier Transform Infrared Spectroscopy in Cryobiological Research
		1 Introduction
		2 Materials
			2.1 Fourier Transform Infrared Spectrometer
			2.2 Attenuated Total Reflection Accessory and Setup for Diffusion Measurements
			2.3 Attenuated Total Reflection Accessory and Setup for Controlling the Sample Humidity
			2.4 Temperature-Controlled Sample Holder for Transmission Spectra Acquisition
		3 Methods
			3.1 Setting Up and Background Spectrum Acquisition
			3.2 Acquisition of Spectra During Permeation of Molecules into a Tissue, Using an ATR Device
			3.3 Acquisition of Spectra During Drying of a Sample Under Controlled Humidity Conditions, Using the ATR Device
			3.4 Acquisition of Transmission Spectra Using the Temperature-Controlled Sample Holder
			3.5 Spectral Analysis: Diffusion Coefficients of Protective Molecules Permeating into Tissues
			3.6 Spectral Analysis: Membrane Phase State and Phase Behavior
			3.7 Spectral Analysis: Water-to-Ice Phase Transition
			3.8 Spectral Analysis: Protein Secondary Structure and Heat Denaturation
			3.9 Spectral Analysis: Drying Kinetics
			3.10 Spectral Analysis: Glass Transition Temperature of Amorphous Systems
		4 Notes
		References
	Chapter 14: Raman Cryomicroscopic Imaging and Sample Holder for Spectroscopic Subzero Temperature Measurements
		1 Introduction
		2 Materials
			2.1 Confocal Raman Microscopy/Spectroscopy
			2.2 Temperature-Controlled Cooling Stage and Sample Handling
			2.3 Setup for Preventing Condensation on the Sample
			2.4 Cell Culture
		3 Methods
			3.1 Freeze Cells Using the Cooling Stage
			3.2 Raman Imaging of a Single  Cell
			3.3 Raman Spectra and Image Analysis: Intracellular Ice Formation
			3.4 Raman Spectra and Image Analysis: Distribution of Cryoprotectants
		4 Notes
		References
Part III: Cryopreservation and Freeze-Drying Protocols
	Chapter 15: Cryopreservation of Semen from Domestic Livestock: Bovine, Equine, and Porcine Sperm
		1 Introduction
		2 Materials
			2.1 Materials for Processing of Raw Semen and Dilution
			2.2 Materials and Equipment for Cooling, Cryopreservation, and Thawing of Sperm Samples
		3 Methods
			3.1 Preparation of (Clarified) Egg  Yolk
			3.2 Preparation of Diluents (INRA-82) With(out) Cryoprotective Agents for Equine Semen
			3.3 Preparation of Diluents (TEY) With(out) Cryoprotective Agents for Bovine Semen
			3.4 Preparation of Diluents (BTS, LEY) With(out) Cryoprotective Agents for Porcine Semen
			3.5 Semen Collection and Dilution with Primary Extender
			3.6 Centrifugation Processing for Removal of Seminal Plasma and to Concentrate the Sample
			3.7 One-Step Approach for Diluting Sperm with Freezing Extender
			3.8 Two-Step Approach for Diluting Sperm with Freezing Extender
			3.9 Slow Cooling and Freezing of Sperm Packaged in Straws
			3.10 Thawing of Cryopreserved Samples
		4 Notes
		References
	Chapter 16: Cryopreservation of Avian Semen
		1 Introduction
			1.1 Historical Perspective
			1.2 Background
			1.3 Extenders and Freezing Media
			1.4 Cryoprotective Agents
			1.5 Other Stabilizing Additives
			1.6 Freezing Methods
		2 Materials
			2.1 General Laboratory Materials and Equipment
			2.2 Diluents
			2.3 Freezing Media
		3 Methods
			3.1 Semen Collection and Processing
			3.2 Quality Assessment
			3.3 Fresh Storage
			3.4 Semen Freezing
				3.4.1 Method DMA in Straws
				3.4.2 Method DMA in Pellets
				3.4.3 Method Glycerol in Straws
		4 Notes
		References
	Chapter 17: Cryopreservation of Mouse Sperm for Genome Banking
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Mouse Sperm Freezing Solution
			3.2 Preparation of FHM Medium
			3.3 Preparation of FERTIUP Medium
			3.4 Preparation of HTF Medium
			3.5 Sperm Collection
			3.6 Sperm Freezing
			3.7 Thawing Procedure
		4 Notes
		References
	Chapter 18: Cryopreservation of Marine Invertebrates: From Sperm to Complex Larval Stages
		1 Introduction
			1.1 General Introduction
			1.2 Cryopreservation: Why Is It Useful?
			1.3 Target Species
		2 Materials
			2.1 Cryopreservation of Sea Urchin (Paracentrotus lividus) Sperm
			2.2 Cryopreservation of Sea Cucumber (Holothuria forskali) Sperm
			2.3 Cryopreservation of Mussel (Mytilus galloprovincialis) Sperm
			2.4 Cryopreservation of Oyster (Crassostrea angulata) Sperm
			2.5 Cryopreservation of Sea Urchin (Paracentrotus lividus) Embryos
			2.6 Cryopreservation of Mussel (Mytilus galloprovincialis) D-Larvae
		3 Methods
			3.1 Cryopreservation of Sea Urchin (Paracentrotus lividus) Sperm
			3.2 Cryopreservation of Sea Cucumber (Holothuria forskali) Sperm
			3.3 Cryopreservation of Mussel (Mytilus galloprovincialis) Sperm
			3.4 Cryopreservation of Oyster (Crassostrea angulata) Sperm
			3.5 Cryopreservation of Sea Urchin (Paracentrotus lividus) Embryos
			3.6 Cryopreservation of Mussel (Mytilus galloprovincialis) D-Larvae
		4 Notes
		References
	Chapter 19: Aseptic Cryoprotectant-Free Vitrification of Human Spermatozoa by Direct Dropping into a Cooling Agent
		1 Introduction
		2 Materials
			2.1 Media for Spermatozoa Preparation
			2.2 Medium for Spermatozoa Vitrification
			2.3 Reagents for Spermatozoa Assessment
			2.4 Equipment
		3 Methods
			3.1 Spermatozoa Preparation
			3.2 Spermatozoa Cooling
			3.3 Spermatozoa Warming
		4 Notes
		References
	Chapter 20: Cryopreservation of Mammalian Oocytes: Slow Cooling and Vitrification as Successful Methods for Cryogenic Storage
		1 Introduction
		2 Materials
			2.1 Slow Controlled Rate Cooling
			2.2 Vitrification
		3 Methods
			3.1 Slow Controlled Rate Cooling
			3.2 Vitrification
		4 Notes
		References
	Chapter 21: Vitrification of Porcine Oocytes and Zygotes in Microdrops on a Solid Metal Surface or Liquid Nitrogen
		1 Introduction
		2 Materials
			2.1 Media
			2.2 Tools for Oocyte/Zygote Vitrification and Storage
			2.3 Tools for Warming Vitrified Samples
			2.4 Tools for Subsequent Culture After Warming and Evaluation of Live/Dead Status of Oocytes/Zygotes
		3 Methods
			3.1 Setting Up for Vitrification
			3.2 Vitrification and Storage
			3.3 Setting Up for Warming
			3.4 Warming
			3.5 Subsequent Culture and Evaluation of Live/Dead Status
		4 Notes
		References
	Chapter 22: Cryopreservation and Transplantation of Laboratory Rodent Ovarian Tissue for Genome Banking and Biomedical Research
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Tyrode´s Lactate HEPES (TL-HEPES) Medium
			3.2 Preparation of Freezing Solution
			3.3 Ovarian Tissue Donors and Recipients
			3.4 Isolation of Donor Ovaries for Cryopreservation
			3.5 Cryopreservation Procedure
			3.6 Thawing
			3.7 Ovarian Tissue Transplantation
			3.8 Determination of Reproductive Cycle Via Vaginal Cytology
			3.9 Vaginal Smears
			3.10 Vaginal Cytology and Corresponding Estrus Cycle Stages
		4 Notes
		References
	Chapter 23: Cryopreservation and Thawing of Human Ovarian Cortex Tissue Slices
		1 Introduction
		2 Materials
			2.1 Medium for Transportation, Preparation, and Cryopreservation of Ovarian Tissue
			2.2 Materials Needed for the Cryopreservation Procedure
			2.3 Materials Needed for the Thawing Procedure
			2.4 Viability Assessment (Calcein Staining)
			2.5 Requirements of an Ovarian Tissue Bank for Preparation, Cryopreservation, and Storage of Ovarian Tissue
		3 Methods
			3.1 Removal of Ovarian Tissue for Cryopreservation
			3.2 Preparation of Medium for (Overnight) Transportation
			3.3 Preparation of Medium for Cryopreservation of Ovarian Tissue
			3.4 Preparation, Cryopreservation, and Storage of Ovarian Tissues
			3.5 Thawing Procedure
				3.5.1 Preparations of Warming/Thawing Solutions
				3.5.2 Thawing and Removal of Cryoprotectants, Restoration of Cell Volumes, Washing, and Equilibration
			3.6 Determination of Ovarian Cortex Potential Before and After Cryopreservation
		4 Notes
		References
	Chapter 24: Vitrification: A Simple and Successful Method for Cryostorage of Human Blastocysts
		1 Introduction
			1.1 General Background
			1.2 Blastocyst Vitrification in the Fertility Centers of Illinois
		2 Materials
			2.1 Materials
			2.2 Reagents
			2.3 Equipment
		3 Methods
			3.1 Stepwise Blastocyst Vitrification Procedure
			3.2 Stepwise Blastocyst Warming Procedure
		4 Notes
		References
	Chapter 25: Vitrification of Equine In Vivo-Derived Embryos After Blastocoel Aspiration
		1 Introduction
		2 Materials
			2.1 General Materials
			2.2 Holding Medium, Vitrification, and Warming Solutions
		3 Methods
			3.1 Embryo Collapse
			3.2 Vitrification
			3.3 Warming
		4 Notes
		References
	Chapter 26: Frozen Blood Reserves
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
			2.3 Disposables
		3 Methods
			3.1 Glycerolization and Freezing of Red Blood Cell Concentrates
				3.1.1 Removal of Additive Solution
				3.1.2 Warming Procedure
				3.1.3 Glycerolization Procedure
				3.1.4 Removal of Supernatant Glycerol
				3.1.5 Freezing the  Unit
			3.2 Thawing and Deglycerolization of Red Blood Cell Concentrates
				3.2.1 Thawing the  Unit
				3.2.2 Deglycerolization Procedure
			3.3 Deglycerolization of LGM Frozen Units
			3.4 Quality Control and Post-thaw Storage
		4 Notes
		References
	Chapter 27: Isolation, Cryopreservation, and Characterization of iPSC-Derived Megakaryocytes
		1 Introduction
		2 Materials
			2.1 Cell Culture Maintenance Equipment and Consumables
			2.2 Cell Culture Maintenance Medium, Reagents, and Cell Lines
			2.3 Cryopreservation Equipment, Consumables, and Reagents
			2.4 Equipment, Consumables, and Reagents for Microscopic Analysis
			2.5 Equipment, Consumables, and Reagents for Flow Cytometry Analysis
		3 Methods
			3.1 Production and Harvesting of iPSC-Derived  MKs
			3.2 Cryopreservation of iPSC-Derived  MKs
			3.3 Thawing of iPSC-Derived  MKs
			3.4 Evaluation of Survival of iPSC-Derived MKs After Cryopreservation with Trypan Blue Exclusion  Test
			3.5 Evaluation of Survival with Distinguishing of Apoptotic Processes in iPSC-Derived MKs After Cryopreservation with Annexin ...
			3.6 Characterization of iPSC-Derived MKs for Typical MK Markers
			3.7 Characterization of iPSC-Derived MKs for Polyploidy with Fluorescence Microscopy
			3.8 Characterization of iPSC-Derived MKs for Polyploidy with Flow Cytometry
			3.9 Characterization of iPSC-Derived MKs for the Capability to Form ProPLTs After Cryopreservation
		4 Notes
		References
	Chapter 28: Chemically Defined, Clinical-Grade Cryopreservation of Human Adipose Stem Cells
		1 Introduction
		2 Materials
			2.1 Materials and Equipment
			2.2 Reagents and Media
		3 Methods
			3.1 Isolation of Human ASCs from Lipoaspirate
			3.2 Magnetic Cell Sorting (Optional)
			3.3 Controlled-Rate Freezing of Human  ASCs
			3.4 Thawing Human  ASCs
		4 Notes
		References
	Chapter 29: Chemically Defined and Xeno-Free Cryopreservation of Human-Induced Pluripotent Stem Cells
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Disposables
			2.3 Reagents, Solutions, and Media
		3 Methods
			3.1 Coating Culture Plates with Recombinant Vitronectin
			3.2 Culturing hiPSCs
			3.3 Passaging of hiPSCs
			3.4 Freezing of hiPSCs
			3.5 Thawing of hiPSCs
		4 Notes
		References
	Chapter 30: Protocol for Cryopreservation of Endothelial Monolayers
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Supplies and Reagents
		3 Methods
			3.1 Cell Cultures on Rinzl Plastic Coverslips
				3.1.1 Preparing Cover Slips
				3.1.2 Seeding Endothelial Cells on Cover Slips
			3.2 Freezing Cell Monolayers
			3.3 Thawing Cell Monolayers
			3.4 CPA Removal by Single Wash or Serial Dilution
		4 Notes
		References
	Chapter 31: Vitrification of Heart Valve Tissues
		1 Introduction
		2 Materials
			2.1 Preparation of EuroCollins Solutions
			2.2 Preparation of Vitrification Solutions
			2.3 Preparation of Addition and Removal Solutions
		3 Methods
			3.1 Protocol 1
			3.2 Protocol 2
			3.3 Protocol 3
			3.4 Protocol 4
		4 Notes
		References
	Chapter 32: Cryopreservation of Algae
		1 Introduction
		2 Materials
		3 Methods
			3.1 Standard Method for Cryopreserving Microalgae (Used at the RCC and CCAP)
			3.2 Cryopreservation of Microalgae of Interest in Aquaculture: Chaetoceros neogracilis (RCC2278), Nannochloropsis gaditana (CS...
			3.3 Cryopreservation of Hypersaline Chlorophyte Strains of Interest in Biotechnology: Dunaliella salina, D. minuta, D. tertiol...
			3.4 Cryopreservation of Macroalgae
			3.5 Recalcitrant Species
			3.6 Cryopreservation, Long-Term Storage, and Viability
		4 Notes
		References
	Chapter 33: Cryopreservation of Fern Spores and Pollen
		1 Introduction
		2 Materials
			2.1 Fern Spore Collection and Cleaning
			2.2 Drying of Fern Spores and Desiccation-Tolerant Pollen
			2.3 Cryopreservation of Fern Spores and Desiccation-Tolerant Pollen
			2.4 Cryopreservation of Desiccation-Sensitive Pollen (e.g., Zea mays)
		3 Methods
			3.1 Fern Spore Collection and Cleaning
			3.2 Fern Spore Drying
			3.3 Fern Spore Cryopreservation (Cooling and Warming)
			3.4 Drying and Cryopreservation (Cooling and Warming) of Desiccation-Tolerant Pollen
			3.5 Pollen Cryopreservation for Pollen Sensitive to Desiccation (e.g., Zea mays)
		4 Notes
		References
	Chapter 34: Cryopreservation of Plant Cell Lines Using Alginate Encapsulation
		1 Introduction
		2 Materials
		3 Methods
			3.1 Immobilization and Pre-culture
			3.2 Cryoprotection
			3.3 Freezing
			3.4 Recovery
		4 Notes
		References
	Chapter 35: Cryopreservation of Plant Shoot Tips of Potato, Mint, Garlic, and Shallot Using Plant Vitrification Solution 3
		1 Introduction
		2 Materials
			2.1 In Vitro Culture Establishment, Maintenance, and Propagation
				2.1.1 General Equipment
				2.1.2 Surface Sterilization of Plant Material Derived from the Field or Greenhouse
				2.1.3 In Vitro Maintenance and Propagation
			2.2 Cryopreservation
				2.2.1 Equipment for Cryopreservation
				2.2.2 Plant Preparation and Cryopreservation Treatment
				2.2.3 Media and Solutions for Cryopreservation
		3 Methods
			3.1 Preparation and Sterilization of the Culture Media
			3.2 Plant Material for Establishment of In Vitro Cultures
				3.2.1 Potato
				3.2.2 Mint
				3.2.3 Garlic Bulbs
				3.2.4 Garlic Bulbils
				3.2.5 Garlic Inflorescences
				3.2.6 Shallots
			3.3 Surface Sterilization
			3.4 Culturing of In Vitro Plants and Cold Hardening
				3.4.1 Potato In Vitro Plant Propagation and Cold Hardening
				3.4.2 Mint In Vitro Plant Propagation and Cold Hardening
				3.4.3 Garlic and Shallot In Vitro Plant Propagation and Cold Hardening
			3.5 Shoot Tip Preparation and Pretreatment for Cryopreservation
				3.5.1 In Vitro Plantlets
				3.5.2 Garlic Bulbils, Cloves, and Shallot Cloves
				3.5.3 Garlic Inflorescences
			3.6 Cryopreservation
			3.7 Rewarming and Regeneration
			3.8 Explant Regeneration
		4 Notes
		References
	Chapter 36: Cryopreservation of Seeds and Seed Embryos in Orthodox-, Intermediate-, and Recalcitrant-Seeded Species
		1 Introduction
		2 Materials
			2.1 Drying of Orthodox Seeds for Cryopreservation
			2.2 Drying of Intermediate Seeds for Cryopreservation
			2.3 Cryopreservation and Warming of Orthodox and Intermediate Seeds
			2.4 Excision of Embryonic Axes of Recalcitrant Seeds
			2.5 Partial and Rapid (``Flash´´) Drying of Embryonic Axes of Recalcitrant Seeds
			2.6 Moisture Content Determination
			2.7 Cooling and Cryopreservation of Recalcitrant Seeds
			2.8 Warming and Rehydration of Embryonic Axes of Recalcitrant Seeds
			2.9 Sterilization of Embryonic Axes and In Vitro Culture
		3 Methods
			3.1 Drying Orthodox Seeds for Cryopreservation
			3.2 Use of a ``Rotronic´´ Water Activity Sensor
			3.3 Packing and Cryopreservation of Orthodox Seeds
			3.4 Removing Orthodox Seeds from  LN
			3.5 Cryopreservation of Fagus sylvatica Whole Seeds
			3.6 Cryopreservation of Whole Seed of Coffea sp. in  LN
			3.7 Cryopreservation of Whole Seed of Citrus sp. in  LN
			3.8 Melting Lipids After Cryo-Storage
			3.9 Excision of Embryonic Axes (Quercus sp. and Aesculus sp.)
			3.10 Rapid (Flash) Drying of Embryonic Axes of Recalcitrant Seeds
			3.11 Cryopreservation of Embryonic Axes of Recalcitrant Seeds
			3.12 Thawing the Cryopreserved Embryonic  Axes
			3.13 In Vitro Culture and Regeneration of Embryonic  Axes
		4 Notes
		References
	Chapter 37: Freeze-Drying of Proteins
		1 Introduction
			1.1 Theory of Freeze-Drying
			1.2 Impact of Freeze-Drying on Proteins
			1.3 Stabilizers for Cryo- and Lyoprotection of Protein
		2 Materials
			2.1 Freeze-Drying Systems
			2.2 Formulations for Freeze-Drying of Proteins
		3 Methods
			3.1 Preparation of the Materials
			3.2 Filling Container
			3.3 Steps of a Freeze-Drying Protocol
			3.4 Freeze-Drying Cycle Design
			3.5 Freezing
			3.6 Primary Drying (Sublimation Drying)
			3.7 Secondary Drying (Desorption Drying)
			3.8 Conditioning-Packing and Storage
		4 Notes
		References
	Chapter 38: Freeze-Drying of Lactic Acid Bacteria: A Stepwise Approach for Developing a Freeze-Drying Protocol Based on Physic...
		1 Introduction
		2 Materials
			2.1 Differential Scanning Calorimeter (DSC)
			2.2 Freeze-Drying Microscopy System
			2.3 Freeze-Dryer
			2.4 Formulations for Freeze-Drying Bacteria
			2.5 Water Activity and Water Content Measurement of the Freeze-Dried Product
		3 Methods
			3.1 Determination of the Effect of Cells and Supernatant on the Physical Properties (Tcoll and Tg′)
			3.2 Adjustment of the Composition of the Protective Solution (PS)
			3.3 Freeze-Drying Protocol
			3.4 Characterization of the Freeze-Dried Product
		4 Notes
		References
	Chapter 39: Preservation of Mammalian Sperm by Freeze-Drying
		1 Introduction
		2 Materials
		3 Methods
			3.1 Freeze-Drying
			3.2 Rehydration and Preparation of Freeze-Dried Sperm for  ICSI
		4 Notes
		References
	Chapter 40: Freeze-Drying of Decellularized Heart Valves for Off-the-Shelf Availability
		1 Introduction
		2 Materials
			2.1 Decellularization and Freeze-Drying of Heart Valves
			2.2 Histological Evaluation of Tissue Structure
		3 Methods
			3.1 Preparation of Heart Valves and Decellularization
			3.2 Loading Decellularized Heart Valve Tissue with Lyoprotectants and Freeze-Drying
			3.3 Histological Analysis
		4 Notes
		References
Index