Why Is Cryopreservation Important in Modern Biology?
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 -79oC)
Low-temperature deep freezer (at -80oC)
In vapor phase nitrogen (at -150oC)
In liquid nitrogen (at -196oC)
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:
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.
Rapid Freezing Method - The vials are plunged in liquid nitrogen. In this process, the temperature decreases from -300 to - 1000 degrees rapidly.
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)×100Regeneration 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.
FAQs on Cryopreservation: Definition, Steps & Applications
1. What is cryopreservation?
Cryopreservation is a scientific process of preserving living cells, tissues, organs, or any other biological material by cooling them to very low temperatures, typically -196°C (the temperature of liquid nitrogen). The primary goal is to halt all biological activity, including metabolic processes, to maintain the material's viability for an indefinite period upon thawing.
2. What are the main steps involved in the cryopreservation process?
The cryopreservation process generally involves several critical steps to ensure the survival of the biological sample:
Selection and Preparation: Healthy and viable biological material is selected and prepared for the process.
Addition of Cryoprotectant: A cryoprotective agent (CPA), like glycerol or DMSO, is added to protect the cells from damage caused by ice crystal formation.
Controlled Cooling: The sample is cooled at a specific, controlled rate. This slow cooling is crucial to minimise intracellular ice formation.
Storage: Once frozen, the material is stored in liquid nitrogen at -196°C for long-term preservation.
Thawing: When needed, the sample is warmed rapidly and carefully to return it to a physiological temperature, and the cryoprotectant is removed.
3. What is the role of a cryoprotectant in cryopreservation?
A cryoprotectant is a substance that acts like a biological antifreeze. Its main role is to protect cells and tissues from damage during the freezing process. It achieves this by increasing the solute concentration inside and outside the cells, which lowers the freezing point and prevents the formation of large, sharp ice crystals that could otherwise puncture cell membranes and destroy organelles.
4. What are some key applications of cryopreservation in biology and medicine?
Cryopreservation has numerous important applications across various fields:
Fertility Preservation: Storing sperm, eggs (oocytes), and embryos for future use in assisted reproductive technologies (ART).
Conservation Biology: Preserving gametes and embryos of endangered species in 'frozen zoos' and maintaining seed banks and pollen banks (ex-situ conservation).
Medical Field: Long-term storage of stem cells, red blood cells, tissues like heart valves, and umbilical cord blood.
Scientific Research: Preserving cell lines, microbes, and other biological samples for laboratory use, ensuring genetic stability over time.
5. What is the main difference between simple freezing and cryopreservation?
The main difference lies in the prevention of cell damage. Simple freezing, like putting food in a home freezer, causes water inside cells to form large ice crystals, which rupture cell membranes and lead to death. Cryopreservation, however, uses cryoprotective agents and a controlled cooling rate to minimise ice crystal formation. This process often leads to vitrification—a glass-like, non-crystalline solid state—which keeps the cellular structure intact and viable upon thawing.
6. Why is liquid nitrogen used for long-term storage in cryopreservation?
Liquid nitrogen is used because its extremely low temperature of -196°C is well below the 'glass transition temperature' of water. At this temperature, virtually all metabolic and biochemical reactions within the cells cease. This state of suspended animation ensures that the biological material can be stored for decades or even centuries without any significant degradation or genetic alteration, preserving its viability for future use.
7. What are the major challenges or risks associated with cryopreservation?
Despite its benefits, cryopreservation has several challenges:
Ice Crystal Damage: If the cooling rate is not perfectly controlled, both intracellular and extracellular ice crystals can form, causing lethal damage to cells.
Cryoprotectant Toxicity: The chemicals used as cryoprotectants can be toxic to cells at higher concentrations or with prolonged exposure at warmer temperatures.
Osmotic Stress: The addition and removal of cryoprotectants can cause cells to swell or shrink rapidly, leading to osmotic shock and damage.
Fracturing: Larger tissues and organs can fracture during cooling or warming due to thermal stress.
8. How does cryopreservation contribute to the conservation of endangered species?
Cryopreservation is a vital tool for the ex-situ conservation of endangered species. It allows for the creation of 'frozen zoos' or gene banks where genetic material—such as sperm, eggs, and embryos—from threatened animals is stored at ultra-low temperatures. This preserves the genetic diversity of a species. If a species becomes extinct in the wild, this cryopreserved material can be used in the future for artificial insemination, in-vitro fertilisation (IVF), or cloning to help re-establish populations and prevent total extinction.
9. How is cryopreservation technology used to maintain viability in pollen banks and seed banks?
In agriculture and botany, cryopreservation is used to store seeds (especially those that do not survive drying) and pollen in a viable state for extremely long periods. By freezing them in liquid nitrogen, their metabolic activity is halted, preventing germination or degradation. This method of ex-situ conservation is crucial for preserving the genetic diversity of important food crops and rare plant species, safeguarding them against diseases, climate change, and habitat loss for future research and cultivation.
