Cryopreservation

Introduction to What is Cryopreservation

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 which deals with cryopreservation is known as “cryobiology”. It can be done over the following temperature :

  • Solid carbon dioxide (at -79°C)

  • Low-temperature deep freezer (at -80°C)

  • In vapor phase nitrogen (at -150°C)

  • In liquid nitrogen (at -196°C)


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.


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.

  • Pollens 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.

FAQ (Frequently Asked Questions)

1. Is sperm banking necessary right now?

  • This is a common question men have during a cancer diagnosis about whether they can store their sperms in the sperm backs.

  • However, even a single dose of chemotherapy or testicular radiation can affect the quality of your sperm. 

  • Sperm banking can take some time and coordination from a specific person. Therefore it should be done before the beginning of cancer treatments. 

  • The decision to store sperm should be performed soon after diagnosis as the procedure can be long and tiring. 

  • Doctors and health experts recommend sperm banking for the purpose of storing better quality of sperms to male individuals.

2. What are the major challenges in cryopreservation?

  • In some cases, information is still an issue and can cause damage. 

  • Another major challenge is that cryoprotectant agents have toxic effects on human tissues with prolonged exposure, through various different kinds of mechanisms. 

  • For large organs, vitrifying can also result in fractures due to different cooling rates in the different parts of the organ. 

  • To a certain extent, the cryoprotectant is used to reduce the chances of freezing.

  • Apart from all this cryopreservation also creates a drastic effect on Tregs which further leads to a reduction in the liability of the Tregs which leads to circumstances of causing abnormal cytokine secretion.