Cryopreservation: Application and Advantages

Introduction:

Cryopreservation term comes from the Greek word “kayos,” meaning frost. Therefore cryopreservation means preservation in a frozen state. In this process, the body tissues, cells, or organs are at a very low temperature to retain their viability. Therefore in this way, tissues can be preserved for a very long duration, and the science that deals with cryopreservation is known as cryobiology.

What Is Cryopreservation?

Cryopreservation is the technique of keeping living cells, tissues, and other biological materials in a deep freezing state at subzero temperature for their preservation. The temperature is mostly kept at -196 degrees Celsius (°C). At such low temperatures, all the biological activities of the cells seize, and the cell dies. Therefore cryopreservation helps the cells to survive freezing and thawing.

The main aim of the cryopreservation technique is to achieve low temperatures without causing any harm to the tissues due to crystal formation during freezing. The biological materials, such as organelles, cells, tissues, extracellular matrix, and organs that are vulnerable to harm from uncontrolled chemical kinetics, are preserved by cooling to extremely low temperatures.

Under the following temperatures, cryopreservation can be done -

• Solid carbon dioxide (at -79 degrees Celsius).

• Low-temperature deep freezer (at -80 degrees Celsius).

• In vapor phase nitrogen (at -150 degrees Celsius).

• In liquid nitrogen (at -196 degrees Celsius).

Normally used temperatures are solid carbon dioxide -80 degrees Celsius or liquid nitrogen -196 degrees Celsius.

What Is the Principle of Cryopreservation?

• Cryopreservation is the method of preserving cells, tissues, organs, or other biological materials from any potential harm by keeping them at very low temperatures. This helps in preserving cell integrity and maintain its viability.

• This process is based on the principle of conversion of water present in the cells to a solid state. During this process, when the temperature falls below 0°C, the biological functions are seized by the freezing water, which constitutes 80 percent of the tissue mass.

• However, the cell walls require a temperature of below -68 °C due to the presence of salts and organic molecules in the cells as compared to water which has a freezing point of around 0°C.

• After this, all the metabolic processes and biological divisions in the cells and tissues are stopped at lower temperatures.

What Are the Steps of Cryopreservation?

The steps involved in the process of cryopreservation include -

1. Harvesting or Selection of Material - Any tissue, such as an embryo, ovules, or plant seeds, can be selected for cryopreservation. A few important criteria should be considered while selecting biological materials, such as volume, pH, density, morphology, and without any damage.

2. Addition of Cryo-protectant - A chemical material is needed to prevent any damage during cryopreservation. Cryoprotective agents such as glycerol, FBS, salts, sugars, glycols some amino acids like proline and dimethyl sulfoxide are added to the samples. These agents help in reducing the freezing point of the medium and also inducing a slower cooling rate that helps in lowering the risk of crystallization. Usually, two cryoprotectants are recommended to be used together as they are more effective.

3. Freezing - There are different ways of freezing in cryopreservation to protect the cells from damage and cell death due to exposure to warm solutions. The methods include -

• Slow Freezing Method - In this method, the tissue or plant material is frozen slowly.

• Rapid Freezing Method - In this, liquid nitrogen is used, and the temperature is decreased from -300 to -1000 degrees rapidly.

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

4. Storage in Liquid Nitrogen - The cryopreserved samples are advised to store at extremely cold temperatures between - 70 to - 196°C. For a prolonged duration, they are stored at -196 °C in liquid nitrogen, and a continuous supply of liquid nitrogen is needed to prevent any damage.

5. Thawing - In this process, the biological samples are warmed to control the rate of cooling and to prevent damage from crystallization.

6. Washing and Reculturing - In this stage, the preserved material is washed, cryoprotectant is removed, and the medium is recultured again in the fresh medium.

7. Measurement of Viability - The viability or survival rate of frozen material can be measured at any stage of cryopreservation, mostly done after the thawing or re-culture stage. This can be calculated by the formula - (number of cells growing/number of cells thawed)X100.

8. Regeneration - This is the last stage in which the regeneration of desired plant or tissue can be done after careful growth, along with maintaining proper environmental conditions.

What Are the Applications of Cryopreservation?

• Cryopreservation has gained popularity in human medicine as it is used in the treatment of infertility. Human sperms were the first frozen reproductive cell and remained one of the easiest biological substances to freeze due to their tiny cytoplasm and low water content. Moreover, the nuclear material of sperm is compressed and protected from damage. Therefore the cryopreservation of sperm cells is frequently done procedure in human medicine nowadays.

• In recent years embryos have also been preserved using cryopreservation. Human oocytes and ovarian tissues can also be preserved with this technique.

• In addition to that, cryopreservation of the cornea, umbilical cord, and hematopoietic cells, as well as sperm banking, is being done commonly.

• In the case of animals, their semen can also be preserved and can be used later on to reproduce rare and threatened species.

• Plants' genetic material can be preserved by this technique and can be used to improve domestic varieties and adaptability to environmental changes.

What Are the Advantages of Cryopreservation?

• Cryopreservation assists in preserving and boosting the efficiency of assisted reproductive treatments by keeping the fertilized cells for future use.

• Embryos can be preserved for future treatment, and it also helps couples to conceive in their first treatment cycle.

• This tool plays an important role in preserving endangered species.

• Once any material is preserved at a specific temperature, it can be preserved indefinitely.

• There is no risk of contamination by bacteria or fungus during or after the storage process.

• Minimal space and labor are required to perform this procedure.

What Are the Risks Associated With Cryopreservation?

This a safe procedure, but there are still some risks associated with it which include -

• Solution Effects - During freezing, there is a chance of forming a high concentration of solutes which can be harmful to the tissue.

• Extracellular Ice Formation - In some cases, the water during the freezing process may migrate out of the cells, and the ice forms in the extracellular space. This can cause damage to the cell membrane.

• Dehydration - This migration of water out of the cell and causing extracellular ice formation can lead to cellular dehydration and can be very harmful to the preserved material.

• Intracellular Ice Formation - In some cases, there may be intracellular ice formation that can be fatal to the cells.

Conclusion:

Cryopreservation is an amazing tool by which cells or tissues can be preserved for a very long duration. It has a greater clinical significance in medical science, and understanding the principle behind the chemistry of freezing and thawing can help in the development of more efficient procedures for cryopreservation. The successful technique of cryopreservation depends upon multiple factors which should be assessed before doing the procedure. This technique shows a promising future in the field of medical sciences and more focus should be put on the make this procedure more safe and reliable.