Cryopreservation

Cryopreservation is a technique of preserving and storing viable biological samples in a frozen state over extended periods of time. The preservation of animal cell has become dependent upon using low temperature to render the cell metabolically inert. The term ‘cryopreservation’ refers to ‘a method of cold storage.’ The optimum temperature for preserving animal germplasm is liquid nitrogen temperature of -196 degrees Celsius. Temperatures from -70 to -80 degree Celsius may be sufficient enough to maintain cell viability for a few months. Cryobiology is the study of the effects of extremely low temperatures on biological systems such as cells or organisms.

Definition

Cryopreservation is the process of cooling and storing cells, tissues and organs at very low temperatures to maintain their viability. Spermatozoa were the first mammalian cells to be cryopreserved successfully (Polge et al 1949).

Cryopreservation methods

The two most commonly used cryopreservation methods for animal germplasm are slow programmable freezing and vitrification.

Slow programmable freezing – In slow freezing, cells in a medium are slowly cooled to below freezing point. Slow cooling is needed in order to increase osmotic strength, which causes an efflux of water from the cells. Machines are used to freeze biological samples  using programmable sequences i.e. ‘freezing down’ a sample to better preserve it for eventual thawing before it is frozen or cryopreserved in liquid nitrogen. Biological samples such as oocytes, skin, blood products, embryo and stem cells are preserved by slow programmable freezing machines.

Vitrification – The term ‘vitrification’ refers to any process resulting in ‘glass formation’(‘arrested liquid state’) the transformation from a liquid to a solid in the absence of crystallization (solid-liquid).  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.

Stroage temperature

Cells can be stored for varying lengths of time at temperatures between -70 to -196 degrees Celsius. For short –term storage (few weeks) cells can be preserved at -70 degrees Celsius in standard mechanical freezers. For long-term storage, cells can be preserved at liquid nitrogen containers.

Liquid nitrogen containers

Liquid nitrogen containers are basically robust, heavily insulated vessels into which liquid nitrogen is poured at regular intervals in order to maintain the required temperature. Cells can be stored in either vapour phase – containers at -120 degrees Celsius or liquid phase containers at -196 degrees Celsius of liquid nitrogen. Storage at -120 degrees Celsius does not significantly reduce cell viability. But indefinite storage of cells requires liquid phase containers.

Cryoprotective agents (CPAs)

Glycerol and DMSO are the most commonly used cryoprotective agents. Fetal bovine serum (FBS) is employed in mammalian cryopreservation solutions, but it is not a cryoprotective agent. Dextrans, sorbitol, trehalose, polyethylene glycols, starches, sugars, and polyvinylpyrrolidone provide considerable cryoprotection in a variety of biologic systems (Mazur 1981).  Salts, such as magnesium chloride, have been reported to be cryoprotective agents (Karow and Carrier 1969). Cryoprotectants protect slowly frozen cells by one or more of the following mechanisms: suppressing high salt concentrations; reducing cell shrinkage at a given temperature;reducing the fraction of the solution frozen at a given temperature and minimizing intracellular ice formation. Combinations of cryoprotectants may result in additive or synergistic enhancement of cell survival (Brockbank and Smith 1993, Brockbank 1992). Intracellular cryoprotectants of low molecular weights permeate cells e.g. glycerol, dimethyl sulfoxide. Extracellular cryoprotectants with relatively high molecular weights do not penetrate cells e.g. polyvinylpyrrolidone, hydroxyethyl starch.

Advantages of cryopreservation

Germplasm cryopreservation of the sperm, eggs and embryos contributes directly to animal breeding programmes. Germplasm cryopreservation also assist the ex situ conservation for preserving the genomes of threatened and endangered species. Cryopreserved sperm, oocytes and embryos are used for artificial insemination and embryo transfer in the livestock industry. Cryopreservation also has enormous applications in the artificial propagation of widely diverse aquatic organisms. Cryopreservation of  sperm and embryonic cells has been successful in a number of teleosts and invertebrate species.  The establishment of germplasm banks using cryopreservation can contribute to conservation and extant populations in the future. Cryopreservation provides a continuous source of tissues and genetically stable living cells for a variety of purposes including research and biomedical processes. Cryopreservation reduces the risk of microbial contamination or cross contamination with other cell lines. Cryopreservation reduces the risk of morphological or genetic changes. It also reduces costs of maintenance of cells, tissues or organs.

Safety considerations

The main dangers come from explosions of ampoules or cryotubes. The technician must wear appropriate protective clothing, protective goggles, face mask, insulated gloves etc. Careless handling of cold containers can also cause burns, so it is important to use handling tongues. Nitrogen gas is colorless, odourless and tasteless. It cannot be detected by the human senses. So liquid nitrogen can be used only in well ventilated areas.

Stages in the cryopreservation

Selection of cell lines -à cultivation of cells------à Screening of cells -----à preparation of cells-----à freezing of cells ---à Evaluation of viability.

Preparing cells for cryopreservation

Cells are prepared by trypsinization (0.25% w/v trypsin) to detach adherent cell from flask surfaces. Cells in suspension are centrifuged at around 100xg for 5to 10 minutes and the pellet is suspended in a small volume (1-2 ml) of storage medium.

Storage medium

The cryopreservation media generally consists of a base medium, protein source and a cryoprotective agent. The cryoprotective agent protects the cells from mechanical and physical stress and reduces water content within the cells. Cryoprotective agent minimizes the formation of cell-lysing ice crystals. The formation of ice crystals may disrupt the cell membrane leading to the death of the cells. The composition of the medium used to suspend cells contains a cryoprotectant (usually glycerol or dimethyl sulphoxide, DMSO) and a high protein concentration (serum 20% v/v) cryprotectant = 7-10 % (v/v); serum – 20% (v/v).

Freezing

Since freezing is stressful, the rate of cooling from 0 to 50 degrees Celsius must be slow and controlled. A fall of about 1 degree Celsius per minute is optimal. Rapid chilling results in thermal shock and leads to cell death or injury.  Once a temperature of -50 degrees Celsius is reached, the cells must be cooled rapidly to the final holding temperature.

Thawing of frozen cells

Cells retrieved from storage must be thawed rapidly to ensure maximum survival. The ampoule should be plunged into a beaker of water at 37 degrees Celsius. The cell suspension can be transferred drop wise into a container holding about 20 ml of pre-warmed growth medium supplemented with 10% fetal calf serum.

Conclusion

Cryopreservation of gametes, embryos and embryonic cells has become of immense value in animal biotechnologies which provide an important tool for protecting the endangered species and genetic diversity. Cryopreservation protocols have been introduced as techniques for germplasm preservation of vegetatively propagated horticultural and staple food crops. The establishment of cryobanks to utilize the cryopreservation worldwide would be a significant and promising task in the future.