CRYOPRESERVATION

Introduction

The preservation of cells is an extremely important aspect of cell culture.  The only effective means of preservation of animal cells is by freezing, which can be accomplished with either liquid nitrogen or by employing cryogenic freezers.  The freezing process involves slowly reducing the temperature of prepared cells to -30 to -60°C followed by a transfer to temperatures less than -130°C.  Once at ultralow temperatures, the cells are biologically inert and can be preserved for years.

History

One of the most important early theoreticians of cryopreservation was James Lovelock. He suggested that damage to red blood cells during freezing was due to osmotic stress. During the early 1950s, Lovelock had also suggested that increasing salt concentrations in a cell as it dehydrates to lose water to the external ice might cause damage to the cell. In the mid-1950s, he experimented with the cryopreservation of rodents, determining that hamsters could be frozen with 60% of the water in the brain crystallized into ice with no adverse effects. Other organs were shown to be susceptible to damage.

Cryopreservation was applied to humans beginning in 1954 with three pregnancies resulting from the insemination of previously frozen sperm.Fowl sperm was cryopreserved in 1957 by a team of scientists in the UK directed by Christopher Polge. However, the rapid immersion of the samples in liquid nitrogen did not, for certain samples – such as some types of embryos, bone marrow and stem cells – produce the necessary viability to make them usable after thawing. Increased understanding of the mechanism of freezing injury to cells emphasised the importance of controlled or slow cooling to obtain maximum survival on thawing of the living cells. A controlled-rate cooling process, allowing biological samples to equilibrate to optimal physical parameters osmotically in a cryoprotectant (a form of anti-freeze) before cooling in a predetermined, controlled way proved necessary. The ability of cryoprotectants, in the early cases glycerol, to protect cells from freezing injury was discovered accidentally. Freezing injury has two aspects: direct damage from the ice crystals and secondary damage caused by the increase in concentration of solutes as progressively more ice is formed. During 1963, Peter Mazur, at Oak Ridge National Laboratory in the U.S., demonstrated that lethal intracellular freezing could be avoided if cooling was slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. That rate differs between cells of differing size and water permeability: a typical cooling rate around 1 °C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulphoxide, but the rate is not a universal optimum.

Temperature    

Storage at very low temperatures is presumed to provide an indefinite longevity to cells, although the actual effective life is rather difficult to prove. Researchers experimenting with dried seeds found that there was noticeable variability of deterioration when samples were kept at different temperatures – even ultra-cold temperatures. Temperatures less than the glass transition point (Tg) of polyol's water solutions, around −136 °C (137 K; −213 °F), seem to be accepted as the range where biological activity very substantially slows, and −196 °C (77 K; −321 °F), the boiling point of liquid nitrogen, is the preferred temperature for storing important specimens. While refrigerators, freezers and extra-cold freezers are used for many items, generally the ultra-cold of liquid nitrogen is required for successful preservation of the more complex biological structures to virtually stop all biological activity.

Preparation

Cryopreserving cultured cells differs from preserving bacteria and fungi in that higher viability is required.  Where a 1% survival rate of a microbial culture can be practical, such low viability is unacceptable with cultured cells.  High survival rates are clearly important for cell lines due to the expense and difficulty in preparation , slow relative rate of growth, and tendency to change with repeated passage in culture.  Consequently, methods used for cell culture cryopreservation must ensure high viability (i.e., >90%).

Factors that can affect the viability of cryopreserved cells include growth conditions prior to harvesting, the physiological state of the cells, the cell density, choice of cyroprotectant, and handling techniques.  Actively growing cells harvested from late-logarithmic to early-stationary phase cells usually yield the highest number of viable cells following freezing.  Once harvested, the desirable final concentration of cells should be between 106 to 107 cells/ml.  Higher densities are often useful with adherent cells since thawed cells can be diluted and plated at a desired density.  Cryoprotectants such as DMSO and glycerol are valuable to prevent cell dehydration during the freezing process.  The cell suspension is generally prepared at a concentration twice that desired for freezing so that an equal volume of 2X cyroprotectant can be added.  Gentle handling techniques during harvest and concentration will improve viability of the recovered cells.  Excessive enzymatic treatment, vigorous pipetting, and high-speed centrifugation should be avoided.

Cryoprotectants

The diffusion of cryoprotective agents such as glycerol or dimethylsulfoxide (DMSO) into a cell will result in a partial replacement of intracellular water and help to prevent dehydration (from ice formation) during freezing.  Glycerol is also known to stabilize proteins in their native states and to assist in the maintenance of critical macromolecular interactions at subzero temperatures.  The cryoprotectant should be prepared separately by combining the cryoprotective agent and the growth medium for the cells.  Cryoprotective agents are usually used individually in concentrations ranging from 5-15% (v/v) with the optimum varying with the cell type.   It is important that the cryoprotective agents be of highest possible quality and sterilized prior to use.   Glycerol may be sterilized by autoclaving for 15 minutes and should be stored in small aliquots to prevent introduction of contaminants.  DMSO should be sterilized by filtration with a 0.2 µm nylon syringe filter and stored at -20°C in small, single-use sealed aliquots.  Air oxidation of DMSO is relatively rapid and these products are toxic to cells.  DMSO should not be allowed to come into contact with the skin as it is rapidly absorbed and is a reported neurotoxin.   Preformulated cryoprotective media can also be purchased.

Equilibration

Cells mixed with the cryoprotectant require an equilibration time at room temperature prior to the onset of the cooling process.  This time generally ranges from 15 to 45 minutes and allows penetration of the cell by the cryoprotectant for maximum protective effect.

Cooling:

The rate of cooling controls the size of the ice crystals and the rate at which they are formed, both of which may affect cell recovery.  In most cases a slow, uniform cooling rate of -1°C per minute from ambient is effective.  Since programmable-rate freezing units are not generally available to the cell culturist, alternative methods have been developed.  Placement of the cryovials in a Styrofoam rack (from 15 ml centrifuge tubes) on the shelf of a -80°C freezer for 2-3 hours will result in a non-uniform cooling rate but is close to  -1°C per minute and satisfactory for a range of cell types.  Transfer should then be made to the storage temperature.

Storage

The temperature at which frozen cells are stored will affect their viability.  Storage at -80°C may permit slow chemical reactions (due to small amounts of unfrozen water), which will eventually result in cell death.  A temperature of less than  -130°C is required to completely stabilize cell preparations.  This is usually achieved by storage in liquid nitrogen (-196°C), liquid nitrogen vapor, or in an cryogenic freezer (-150°C).  All three methods are used with each presenting its own strengths.

Liquid nitrogen is a non-mechanical method of cryopreserving cells.  A large thermos-like container is used to house either racks or sleeves that hold cryogenic vials.  Cells stored in nitrogen can be placed above the liquid in a cold vapor phase or in the liquid nitrogen itself (-196°C).  Simple systems rely on a cycle of filling the tank and allowing the nitrogen to evaporate followed by refilling.  Liquid nitrogen storage systems do not require electricity, but rather a ready source of liquid nitrogen.  A small 50 liter tank will require filling every 5-6 weeks at a yearly cost of $900 to $1000.  Though liquid nitrogen is widely used for cell preservation, two problems exist with this storage method.  Cells stored in vapor phase can experience wide temperature fluctuations (i.e., -120 to -195°C), which can be potentially damaging to cells.  Secondly, capped vials submersed in the liquid can leak and pick up contaminants and also pose a risk of exploding when removed from the liquid.

Cryogenic freezers are an alternative to the traditional methods of cryopreserving animal cells.  Cryogenic freezers use high efficiency compressors to reach temperatures as low as -150°C.  No filling is necessary with freezers although back-up non-mechanical refrigerants are available for added security.  Additionally, freezers are generally easier to catalog than many liquid nitrogen systems.

Recovery

Unlike the freezing process, rapid thawing of frozen cells is necessary to maintain viability.  Certain precautions should be exercised when thawing cells.  Vials stored in liquid nitrogen, especially screw capped tubes, often fill with liquid nitrogen while submersed.  When these tubes are removed from the tank, the tubes may pressurize and burst.  Thus, a face shield or goggles should be worn while thawing cells.  Vials stored in cryogenic freezers are a reduced risk of bursting.  Directly after removal from storage, vials should be thawed with agitation (but not for fragile hybridoma cells) in a 37°C water bath.  As the last ice crystals are melting, the vial is removed from the water.  Wipe, spray, or submerse the vial with 70% ethanol before opening it in a biosafety hood.

It is prudent when working with an unfamiliar cell line to determine the percentage of viable cells recovered by Trypan Blue staining.  This may serve to uncover any deficiencies in the cryopreservation process.  Note that safety precautions must be taken when recovering vials from the liquid nitrogen.  Insulated gloves should be worn to protect against burns from the low temperatures.  Though specially-designed cryovials are used to store cells, a face shield and laboratory coat serve to protect against fragments of exploding vials caused by introduction of liquid nitrogen (an all too common occurrence with leaky vials).

Cryopreservation Protocol

Materials

cultured cells

hemocytometer and cover slip

2X cryoprotective medium (e.g., DMEM with serum and 15% DMSO or glycerol

cryovials or glass ampoules

propane torch (for glass ampoules)

cryogenic freezer (-150°C)

-80°C freezer

Protocol (Freezing)

If cells are a monolayer culture, gently trypsinize to detach.

Count an aliquot of cells in hemocytometer.

Adjust the density of cells in culture medium to 1 X 107 cells/ml.  Add an equal volume of 2X cryoprotective medium.  Allow the cells to sit at room temperature 15 min. so that the cryoprotectant can diffuse into the cell.

 Transfer 1 ml of cells to a plastic or glass vial.  For plastic vials it is necessary to use caps with O-rings that will allow for a tight fit.  Most plastic cryogenic tubes placed in liquid nitrogen do not form tight seals and will allow liquid nitrogen to seep into the tube if submersed.  This can be a concern since cultured cells, viruses, and bacteria, which may be present in the liquid phase of the nitrogen, can potentially contaminate a culture stored in a screw capped tube.  This is not a concern if cells are stored in only the vapor phase or in a cryogenic freezer.  To completely enclose the cells, glass ampoules can be used and sealed with a flame by rolling the neck of the ampoule in a flame until it becomes soft and pliable.  Using forceps, slowly pull the neck of the ampoule while continuing to roll the tube.  As the neck separates from the vial, roll the end of the vial in the flame to seal.  Once the vial is cooled, it can be submersed in a solution of Methylene Blue or Trypan Blue in order to ensure the vials are closed.  Wash the vials to remove the stain and examine the cell suspension for the dye.  Any dye on the inside of the vial means the vial was not sealed and should be discarded.

Clearly label the vials using permanent ink.  Include information on cell type and date.  This should be cross referenced to additional information on the cell line.  Cool the vials at a rate of 1°/min until they have reached -80°C.  Glass vials should be slanted while freezing so that  the liquid can expand without cracking the glass.  This can be done by placing the vials in a Styrofoam box and placing in a -80°C freezer overnight.

Transfer to a cryogenic freezer for permanent storage.

Protocol (Thawing)

Remove vials from the cryogenic freezer.  If cells are stored in liquid nitrogen, use tongs and insulated gloves, keeping in mind that pressure will build up inside the vials as the nitrogen expands into a gas.  The vial may shatter, thus wear goggles and a lab coat.

Thaw in a 37°C water bath with constant gentle shaking until completely thawed (<1 minute).  Carefully observe whether the glass vials have cracked during freezing or thawing.

Wash the vial with 70% ethanol.  Open the cryogenic tube or snap the ampoule aseptically.  Plate cells immediately into pre-warmed medium.

After attachment of monolayer cells, usually 1 to 10 hours, change the medium to remove the cryoprotectant.  If the cells are non-adherent, allow a sufficient recovery time (about 6 hours), then gently pellet (5 minutes at 400 X g) and resuspend in fresh medium.

Risks

Phenomena which can cause damage to cells during cryopreservation mainly occur during the freezing stage, and include: solution effects, extracellular ice formation, dehydration and intracellular ice formation. Many of these effects can be reduced by cryoprotectants. Once the preserved material has become frozen, it is relatively safe from further damage. However, estimates based on the accumulation of radiation-induced DNA damage during cryonic storage have suggested a maximum storage period of 1000 years.

Solution effects

As ice crystals grow in freezing water, solutes are excluded, causing them to become concentrated in the remaining liquid water. High concentrations of some solutes can be very damaging.

Extracellular ice formation

When tissues are cooled slowly, water migrates out of cells and ice forms in the extracellular space. Too much extracellular ice can cause mechanical damage to the cell membrane due to crushing.

Dehydration

Migration of water, causing extracellular ice formation, can also cause cellular dehydration. The associated stresses on the cell can cause damage directly.

Intracellular ice formation

While some organisms and tissues can tolerate some extracellular ice, any appreciable intracellular ice is almost always fatal to cells.

Main methods to prevent risks

The main techniques to prevent cryopreservation damages are a well established combination of controlled rate and slow freezing and a newer flash-freezing process known as vitrification.

1. Slow programmable freezing               

A tank of liquid nitrogen, used to supply a cryogenic freezer (for storing laboratory samples at a temperature of about −150 °C)

Controlled-rate and slow freezing, also known as slow programmable freezing (SPF), is a set of well established techniques developed during the early 1970s which enabled the first human embryo frozen birth Zoe Leyland during 1984. Since then, machines that freeze biological samples using programmable sequences, or controlled rates, have been used all over the world for human, animal and cell biology – "freezing down" a sample to better preserve it for eventual thawing, before it is frozen, or cryopreserved, in liquid nitrogen. Such machines are used for freezing oocytes, skin, blood products, embryo, sperm, stem cells and general tissue preservation in hospitals, veterinary practices and research laboratories around the world. As an example, the number of live births from frozen embryos 'slow frozen' is estimated at some 300,000 to 400,000 or 20% of the estimated 3 million in vitro fertilisation (IVF) births.

Lethal intracellular freezing can be avoided if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. To minimize the growth of extracellular ice crystal growth and recrystallization, biomaterials such as alginates, polyvinyl alcohol or chitosan can be used to impede ice crystal growth along with traditional small molecule cryoprotectants. That rate differs between cells of differing size and water permeability: a typical cooling rate of about 1 °C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulfoxide, but the rate is not a universal optimum. The 1 °C / minute rate can be achieved by using devices such as a rate-controlled freezer or a benchtop portable freezing container.

Several independent studies have provided evidence that frozen embryos stored using slow-freezing techniques may in some ways be 'better' than fresh in IVF. The studies indicate that using frozen embryos and eggs rather than fresh embryos and eggs reduced the risk of stillbirth and premature delivery though the exact reasons are still being explored.

2. Vitrification

Researchers Greg Fahy and William F. Rall helped to introduce vitrification to reproductive cryopreservation in the mid-1980s. As of 2000, researchers claim vitrification provides the benefits of cryopreservation without damage due to ice crystal formation. The situation became more complex with the development of tissue engineering as both cells and biomaterials need to remain ice-free to preserve high cell viability and functions, integrity of constructs and structure of biomaterials. Vitrification of tissue engineered constructs was first reported by Lilia Kuleshova, who also was the first scientist to achieve vitrification of woman’s eggs (oocytes), which resulted in live birth in 1999. For clinical cryopreservation, vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they decrease the freezing temperature. They also increase the viscosity. Instead of crystallizing, the syrupy solution becomes an amorphous ice—it vitrifies. Rather than a phase change from liquid to solid by crystallization, the amorphous state is like a "solid liquid", and the transformation is over a small temperature range described as the "glass transition" temperature.

Vitrification of water is promoted by rapid cooling, and can be achieved without cryoprotectants by an extremely rapid decrease of temperature (megakelvins per second). The rate that is required to attain glassy state in pure water was considered to be impossible until 2005.

Two conditions usually required to allow vitrification are an increase of the viscosity and a decrease of the freezing temperature. Many solutes do both, but larger molecules generally have a larger effect, particularly on viscosity. Rapid cooling also promotes vitrification.

For established methods of cryopreservation, the solute must penetrate the cell membrane in order to achieve increased viscosity and decrease freezing temperature inside the cell. Sugars do not readily permeate through the membrane. Those solutes that do, such as dimethyl sulfoxide, a common cryoprotectant, are often toxic in intense concentration. One of the difficult compromises of vitrifying cryopreservation concerns limiting the damage produced by the cryoprotectant itself due to cryoprotectant toxicity. Mixtures of cryoprotectants and the use of ice blockers have enabled the Twenty-First Century Medicine company to vitrify a rabbit kidney to −135 °C with their proprietary vitrification mixture. Upon rewarming, the kidney was transplanted successfully into a rabbit, with complete functionality and viability, able to sustain the rabbit indefinitely as the sole functioning kidney.

Applications of cryopreservation

The applications of cryopreservation can be categorized into the following areas: (1) cryopreservation of cells or organs5; (2) cryosurgery; (3) biochemistry and molecular biology; (4) food sciences; (5) ecology and plant physiology; and (6) many medical applications, such as blood transfusion, bone marrow transplantation, artificial insemination, and in vitro fertilization (IVF). Some suggested advantages of cryopreservation include the possible banking of cells for human leukocyte antigen typing for organ transplantation, the allowance of sufficient time for transport of cells and tissues among different medical centers, and the provision of research sources for identifying unknown transmissible diseases or pathogens. Furthermore, the long-term storage of stem cells is still the initial step toward tissue engineering, which holds promise for the regeneration of soft tissue esthetic function and for the treatment of known diseases that have currently no therapy option.

Oocytes and embryos

The first case of embryo cryopreservation for fertility preservation took place in 1996, with the application of a natural IVF cycle prior to chemotherapy in a woman diagnosed with breast cancer. Cryopreservation of mature oocytes is a proven technique for preserving the reproductive capacity. Results from a retrospective study of 11,768 cryopreserved human embryos that underwent at least one thaw cycle from 1986 to 2007 showed that there was no significant impact of the duration of storage on clinical pregnancy, miscarriage, implantation, or live birth rate, whether from IVF or oocyte donation cycles. Since oocytes are highly prone to chilling injury; cryopreservation of immature oocytes and ovarian tissue is a promising approach-with reports of live births-but the need for investigational improvements remain.

Sperm, semen, and testicular tissue

Germ cell depletion caused by chemical or physical toxicity, disease, or genetic predisposition can occur at any age. Fertility preservation is of great importance to guarantee the quality of life of patients facing chemo- and radiotherapy. Sperm and semen can be used almost indefinitely after proper cryopreservation. There are new trials for cryopreserving testicular tissues in the form of cell suspensions, tubular pieces, and entire gonads, but this technique is still premature. Overall, cryopreservation can be used as a first-line means of preserving fertility for men undergoing vasectomy or treatments that may compromise their fertility, such as chemotherapy, radiotherapy, or surgery.

Stem cells

Adult stem cells are capable of differentiating into multiple types of specific cells and can be obtained from various locations other than bone marrow, including fat tissue, the periosteum, amniotic fluid, and umbilical cord blood. Stem cells can be subdivided into embryonic stem cells, mesenchymal stromal cells, and hematopoietic stem cells, all of which are considered as goldmines for potential application in regenerative medicine. Clearly, the fields of tissue engineering, gene therapy, regenerative medicine, and cell transplantation are largely dependent on the ability to preserve, store, and transport these stem cells without modification of their genetic and/or cellular contents.

Hepatocytes

Primarily isolated hepatocytes have found important applications in science and medicine over the past 40 years in a wide range of areas, including physiological studies, investigations on liver metabolism, organ preservation and drug detoxification, and experimental and clinical transplantation.In addition, there is currently increasing interest in the applications of liver progenitor cells across a range of scientific areas, including both regenerative medicine and biotechnology, which raises the need for cryobanking.

Others

Although primary neuronal cells and cardiomyocytes are routinely used for neuroscience and cardiology research, a gold standard protocol for the preservation of these cells has not yet been developed. With the discovery of glucocorticoid-free immunosuppressive regimens, pancreatic islet transplantation may be considered as an alternative for the treatment of type 1 diabetes. For this reason, the development of islet cryopreservation methods has been ongoing, but results are still suboptimal, with a survival rate of less than 50%.

Limitations of cryopreservation

Although numerous usages of the cryopreservation technique exist, both in basic and clinical research, some limitations still exist. Cells metabolize almost nothing at low temperatures such as −196 °C (i.e., in liquid nitrogen), which has inevitable side effects, including a genetic drift toward biological variations of cell-associated changes in lipids and proteins that could result in the impairment of cellular activity and structure. If there were no limit to the amount of CPA that could be used, cells would be preserved perfectly. In conventional settings, however, CPAs themselves can be damaging to cells, especially when used in high concentrations. For example, there is a possibility that DMSO may alter chromosome stability, which can lead to a risk of tumor formation. Apart from endogenous changes in cells, the possible infection or contamination with cells such as tumorous ones should be prevented.