Cryopreservation in Biology Principles and Applications

The word cryo comes from the Greek word "kayos" meaning "frost". It means preservation in a "frozen state". It is the process of cooling and storing cells, tissues, or organs at very low temperatures to maintain their viability. Cryopreservation is a technique in which low temperature is used to preserve the living cells and tissue. In this technique, tissues can be preserved for a very long time. The science that deals with cryopreservation is known as “cryobiology”. It can be done over the following temperature :

• Solid carbon dioxide (at -79^oC)

• Low-temperature deep freezer (at -80^oC)

• In vapor phase nitrogen (at -150^oC)

• In liquid nitrogen (at -196^oC)

Organelles, cells, tissues, extracellular matrix, organs, and other biological structures that are vulnerable to harm from uncontrolled chemical kinetics are maintained by cooling to extremely low temperatures. Cryo-preservation or cryo-conservation is the term for this process.

The temperature which is normally used is:

Using solid carbon dioxide −80 ^oC or Using liquid nitrogen −196 ^oC

The main aim of the Cryopreservation technique is to achieve low temperatures without incurring further harm due to ice crystal formation during freezing.

In the past, Cryopreservation was based on coating the material to be frozen with cryoprotectants. Due to the intrinsic toxicity of many cryoprotectants, new techniques are being studied and worked upon.

Steps of Cryopreservation

The technique followed by the regeneration of plants involves the following steps.

• Selection of Material: For cryopreservation, the selection of proper plant material is important. Two important factors depend on it such as nature and density. Any tissue can be selected for this purpose, for example embryo, meristem, ovules seeds, etc. The density should be high. 

• Addition of Cryoprotectant: The chemical material is important as it prevents cryo destruction. Some examples of cryoprotectants are alcohol, some amino acids like proline, and dimethyl sulfoxide. Mainly two cryoprotectants should be used together instead of a single one as they are considered to be more effective.

• Freezing: Different species of plants show different types of sensitivity to low temperatures. They are different types of methods:

1. Slow Freezing Method- In this process, the tissue or plant material is slowly frozen at a slow cooling rate. The major advantage is that the plant cells are partially hydrated and serve in a better manner.

2. Rapid Freezing Method -  The vials are plunged in liquid nitrogen. In this process, the temperature decreases from -300 to - 1000 degrees rapidly.

3. Dry Freezing Method - In this method hydrated cells and seeds are stored.

• Storage in Liquid Nitrogen: It is also important for the maintenance of the sale or material at a specific temperature. In general, the temperature is kept - 70 to - 196°C. Prolonged storage is done at the temperature of -196 °C in liquid nitrogen. A continuous supply of nitrogen is needed to prevent damage.

• Thawing: The thawing process is usually carried out by plunging the vials into a warm water bath with vigorous swirling. It also causes the vials to get transferred or move to another bath at 0 °C 

• Washing & Reculturing: The preserved material is washed to remove the cryoprotectant. Furthermore, the material is recultured in a fresh medium.

• Measurement of Viability: Due to storage stress, there is a possibility of cell death. The presence of viability can be seen in most cases.

•  It is calculated by the formula:
  (no of cells growing/no of cells thawed)×100

• Regeneration of Plants: After that, the viable seeds are cultured on a non-specific growth medium. Suitable environmental conditions are maintained.

Steps, Applications and Advantages

Steps

The major steps in Cryopreservation are 

• The process of combining CPAs with cells or tissues before cooling

• The freezing of cells or tissues at a low temperature, followed by their storage

• The process in which cells or tissues are being warmed up

• After freezing, the process of removal of CPAs from cells or tissues 

Applications

In Medical sciences

Cryopreservation gained prominence in human medicine after its use in infertility treatment. Since then, gamete cryopreservation has been developed to combat infertility.

Sperm was the first successfully frozen reproductive cell and remains the easiest to freeze due to its tiny cytoplasm and thus low water content. Also, sperm nuclear material is compressed and protected from damage. For these reasons, cryopreservation of sperm cells is frequently used in human medicine today.

Live births from assisted reproductive cycles employing frozen semen or embryos have been observed in recent years. Human oocytes and ovarian tissues have also been cryopreserved. Studies and research on immunological memory lymphoid cells, aortic root allografts, and osteoblasts for bone banking are still going on.

Human medicine is also now commonly performing cryopreservation of cornea, umbilical cord, and hematopoietic cells, as well as sperm banking.

Cryopreservation of bull semen has been used to reproduce rare and threatened species. Every year, more than 25 million bovine calves are artificially impregnated with frozen-thawed bull sperm. Tissues, cell lines, DNA, and serum samples can also now be kept in cryogenic banks.

In Biological sciences

Cryopreservation is one of the most reliable strategies for preserving plant genetic resources for the long term.

In agriculture, germplasm cryopreservation is used to improve domestic varieties' genetics and adaptability to environmental changes. While the practice of preserving plant germplasm in cryogenic temperatures is relatively new, scientists have been developing cryopreservation procedures for plant cells and tissues for over 40 years now.

These strategies can now be used for plant genotypes also. New cryogenic methods utilizing cryoplates (V and D) have recently been developed. These technologies have advantages such as ease of application and excellent regeneration rates after cryopreservation.

Aquatic biotechnologies rely on cryopreservation of gametes, embryos, and embryonic cells to propagate economically significant species, safeguard endangered species, and maintain genetic variety.

The results of studies show that marine fish sperm cryopreservation is more successful than freshwater fish cryopreservation and that fertilization rates are similar to mammalian species.

Advantages

• Cryopreservation boosts the efficiency of assisted reproductive treatments by allowing all extracted and/or fertilized cells to be kept for future use.

• By freezing embryos between cycles, ovarian stimulation is not required each time, and if the woman's ovaries are overstimulated, implantation can be postponed without squandering retrieved oocytes.

• Cryopreservation allows couples who conceive in their first treatment cycle to contribute their unused frozen embryos to research.

• It is currently common to implant only one or two embryos, with any remaining embryos being cryopreserved for future treatment cycles.

• Cryopreservation allows people who are losing their fertility to keep their reproductive cells and maybe conceive via aided methods in the future. Women who want to delay childbearing or have a family history of early menopause may use it.

• Cryopreservation is a powerful tool for preserving endangered species' germplasm. It can also help to maintain plant fertility.

Applications of Cryopreservation

• It is an ideal method for long term conservation of material.

• Disease-free plants can be conserved and propagated and recalcitrant seed can be maintained for a long time.

• Endangered species can be maintained.

• Pollen can be maintained to increase longevity.

Advantages of Cryopreservation 

• Once the material is successfully conserved at a particular temperature, it can be preserved identifiably.

• No change or contamination of fungus or bacteria takes place after the storage process is completed and material is preserved.

• Minimal space is required for the purpose of cryopreservation.

• Minimal labor is required for the purpose of cryopreservation.

Cryopreservation of Animal Cells

The development of animal cell lines is expensive, time-consuming, and labor-intensive. 

The continuous cell line has several advantages of over fertilizers cell lines such as:

• They survive indefinitely. 

• They grow more rapidly. 

• They can clone more easily.

Cryopreservation of Plant Cell

Due to the gradual disappearance of economic and rare species the necessity for storage of genetic resources increases. The convent journal method of the storage fails to prevent losses caused by:

• Attack of pathogen and pest

• Climatic disorders

• Natural disorder

• Political and economic causes

The material to be preserved is stored at low temperatures due to which growth rate of cells retards. Consequently, biological activities are reserved for a long period of time.