Cryopreservation Techniques and Cell Survival

• Cryopreservation involves the validation and freezing of stock cultures for safe use in research
• Cryoprotectants are used with suitable cooling apparatus and procedures with cryofreezers to maintain cell viability for long-term storage
• Freezer records are essential for tracing a cell lines history.
• Thawing should be done rapidly in an aseptic environment to ensure cell viability and sterility
• Stock maintenance should follow SOPs to prolong usage
• Cell banks maintain and sell cell lines to researchers
• Cells can be transported in a frozen state or at mid-log phase of growth, and requires strict adherence to proper packaging

Learning Outcomes

• The core learning objective is to define cryopreservation
• How to describe the various equipment and apparatus used in cryopreservation of cell lines
• Explain the basic principles and concepts in the cryogenic preservation of cell lines

Rationale for Cell Freezing

• Genotypic drift can occur due to genetic instability
• Cell lines can undergo senescence and face extinction
• Growth characteristics may transform, acquiring malignancy-associated properties
• Phenotypic instability can happen due to selection and dedifferentiation
• Cell lines risk contamination by microorganisms or cross-contamination from other cell lines
• Misidentification can result from careless handling
• Incubator failure can occur
• Freezing saves time and materials by reducing the need to maintain lines not in current use
• Distribution of cells to other users necessitates freezing

Theoretical Background to Cell Freezing

• Slow freezing allows water to exit the cell wile avoiding encouragement to the growth of ice crystal growth.
• Hydrophilic cryoprotectants are used to sequester water
• Cells should be stored at the lowest possible temperature to minimize the effects of high salt concentrations on protein denaturation in micelles within the ice
• Rapid thawing minimizes ice crystal growth and the generation of solute gradients formed as the residual intracellular ice melts

Cell Concentration

• Cells survive freezing best at high concentrations
• Reduced viability upon thawing requires higher seeding concentrations
• Survival improves at high cell concentrations in leaky cells, due to cryogenic damage
• High concentrations at freezing allow sufficient dilution of cryoprotectants upon reseeding after thawing, eliminating the need for centrifugation in most cases

Freezing Medium

• Freezing medium comprises media, serum, and a cryoprotectant
• Common cryoprotectants include glycerol and dimethyl sulfoxide (DMSO)
• Dimethyl sulfoxide is more effective and penetrates cells better
• Concentrations between 5% and 15% are effective, with 7.5% or 10% concentrations being more commonly used
• In hematopoietic cells, dimethyl sulfoxide may be toxic or may cause induce cells to differentiate after they are thawed
• In such cases, glycerol is preferable
• Many laboratories increase the serum concentration in freezing medium to reach up to 40%, 50%, or even 100%

Cooling Rate

• Cells are cooled at a rate of 1°C per minute
• The cooling curve depends on ambient temperature, insulation (including the ampoule), specific heat and volume of the ampoule contents, and latent heat absorption during freezing

Ways to Control Cooling Rate

• Cotton wool and polystyrene foam boxes can be used to adjust the rate of cooling
• Ampoule canes placed in tubular foam pipe insulation
• Using a freezer neck plug
• Utilizing a Nunc™ freezing container
• Using a controlled-rate programmable freezer
• If recovery is low, adjust the cooling rate by using more or less insulation

Considerations When Cooling

• Cooling rate is directly proportional to the temperature variance between ampoules and the ambient air
• At -70°C, cells cool rapidly to around -50°C, after at this point, the cooling rate falls off significantly
• The projected cooling rate of 1°C/min should not be relied upon, as the bottom of the curve is asymptotic
• It is recommended to leave ampoules at -70°C overnight before transferring them to liquid nitrogen
• Upon removal of cells, will heat up at a rate of around 10°C/min
• Ensure they do not warm above −50°C
• Transfer to the liquid nitrogen freezer occurs in two minutes

Ampoules

• Plastic (polypropylene) ampoules are safer than glass.
• Alcohol and low-temperature-resistant markers are used for labeling
• Labeling with different colored caps aid in easy identification
• A reference record must be maintained
• Plastic ampoules risk leakage if sealed improperly (too loose or too tight)
• Inexperienced users should avoid glass ampoules due to the risk of explosion upon thawing
• If using glass ampoules, ensure they are perfectly sealed and use vapor phase cooling instead of a -70°C freezer

Cryofreezers (Liquid Nitrogen Containers)

• Cells are rapidly transferred cells into a cryofreezer post-cooling
• Storage Systems include either canisters or trays
• Cells can be stored submerged in the liquid phase (-190°C) or suspended in the vapor phase (-110°C)
• Weekly monitoring with a dipstick is required
• Since liquid-phase storage poses an potential explosion hazard it's important that wear face shield/goggles
• If liquid nitrogen storage is unavailable, cells may be stored in a conventional freezer at -70°C
• At-70°C, A deterioration rate of 5-10% per annum may occur

Freezing Cells Procedure

• Trypsinize monolayer and resuspend cells in medium at 1-10 x 10^6 /ml
• Add cryoprotectant, dimethyl sulfoxide or glycerol, to a concentration of 10% V/V
• Introduce cells into prelabeled ampules
• Affix the ampules onto an aluminum cane and insert into a cardboard tube
• Place the assembly into an insulated foam tube
• Transfer to a -70°C freezer for a minimum of 4 hours

Freezing Flasks/Plates

• Flasks can be frozen by growing cells to late log phase, adding 5 - 10% DMSO to the minimal volume to cover monolayer and then plating the flask in an expanded polystyrene container with a wall thickness of 15mm
• The insulated container is placed in -70 degrees celcius freezer, where it will freeze approximately 1 degrees celcius per minute
• Survival after freezing is good for several months as long as the flask its container is not removed from the freezer
• Twenty-four-well plates can be frozen the same way with 150 microliters of freezing medium per well and used used to store clones if evaluating procedures

Vitrification

• Most cultured lines are freeze with slow cooling methods for optimal effect
• Some cells, like preimplantation embryos and humna embryonal cells are best done with rapid cooling and vitrification
• Those cells have a three dimensional structure
• Vitrification is the transformation of a liquid into a glass with supercooled liquid
• Plunging the cells in a plastic capillary tube or straw into liquid nitrogen to complete
• Cryoprotectants used are Dimethyl Sulfoxide, ethylene glycol and sucrose

Cell Banking

• Various cell banks exist for validated cell lines in secure storage
• Many cell lines are genetically modified and restricted for hybridomas so patent repositories access is restricted
• As a rule, obtain initial stock seed from reputable cell bank
• Furthermore it is essential, highly recommended that you submit all valuable culutres to the cell bank as this will safeguard against loss and allow you to share with others
• You can set restrictions to specify parameters for shipping