Cryopreservation- Introduction, Principle, Methods, Benefits, Significance

Introduction:

Cryopreservation is the method of preserving cells, tissues, or organs by freezing them at extremely low temperatures, often below -130°C, for long-term storage or future use. The goal of cryopreservation is to inhibit biological activity and metabolic reactions that can harm cells and tissues while preserving their viability and functionality after thawing.

Cryopreservation is widely employed in areas such as medicine, biology, and biotechnology. Cryopreservation is used in medicine to store human tissues and organs for transplantation, as well as reproductive cells and embryos for fertility preservation. Cryopreservation is used in biology to conserve endangered species as well as to keep biological samples for study and development. Cryopreservation is used in biotechnology to store microorganisms and cell lines for industrial uses.

Several factors influence cryopreservation success, including the type of cells or tissues maintained, the freezing and thawing techniques, and the storage conditions. Cryoprotectants such as glycerol or dimethyl sulfoxide (DMSO) are frequently used to protect cells and tissues during freezing and thawing. Nonetheless, cryopreservation can still cause cellular damage and viability loss, hence it is critical to optimize the methods and techniques employed for each application.

Principles:

The principles of cryopreservation involve four main stages: preparation, cooling, storage, and thawing. Each of these phases, including the choice of cryoprotectant, cooling rate, storage conditions, and thawing process, must be carefully optimized for successful cryopreservation. Various cryopreservation procedures and protocols may be required for different samples; therefore, it is critical to customize the approach to the individual sample being preserved.

Preparation

The cells, tissues, or organs to be preserved are processed for freezing prior to cryopreservation. Typically, a cryoprotectant solution is added to the sample to avoid ice formation and cellular damage during freezing. To alleviate stress and boost survival rates, the sample can also be pre-cooled.

Cooling

The sample is cooled to extremely low temperatures, often at a regulated rate. Depending on the type of sample and cryopreservation technology utilized, the cooling process might be performed slowly or quickly. The goal is to reduce ice formation while preventing cellular harm.

Storage

The cooled sample is then kept at extremely low temperatures, often in liquid nitrogen at -196°C. The sample remains in a state of suspended animation during storage, with all biological activity ceased.

Thawing

When the sample is suitable for use, it is thawed by a controlled warming procedure. Because rapid thawing can cause cellular damage, it is critical to thaw the sample gently and carefully. To avoid further damage, the sample must be handled carefully once it has been thawed.

Methods:

There are various cryopreservation procedures, each of which uses a unique methodology to cool and store cells, tissues, or organs. The type of sample being kept, the intended use of the sample after thawing, and the available equipment and resources all influence the method of cryopreservation used. Various methods may be more or less effective for different sorts of samples, so it is critical to select the best method for each application. Below tabulated are some of the most common methods:

Slow freezing: The sample is gradually cooled at a regulated rate using a programmable freezer in this procedure. The cooling rate can be modified to reduce ice crystal formation and protect cells and tissues.

Rapid freezing: This procedure involves rapidly cooling the sample, usually by immersing it in liquid nitrogen or using a specialized machine like a vitrification unit. Quick freezing can reduce the creation of ice crystals and increase the survival rate of specific cell types.

Vitrification: This is a form of quick freezing in which the sample is rapidly cooled so that it becomes a glass-like solid without the production of ice crystals. Embryos, oocytes, and other delicate cell types can be preserved by vitrification.

Freeze-drying: This procedure, also known as lyophilization, involves eliminating water from the sample via sublimation and then storing it at low temperatures. Bacterial and fungal cultures, as well as some plant tissues, are frequently freeze-dried.

Cryoprotectant:

A cryoprotectant is a chemical substance that is administered to cells, tissues, or organs prior to cryopreservation to protect them against freezing and thawing damage. Cryoprotectants act by preventing the production of ice crystals, which can cause cell injury by rupturing membranes and altering cell structure.

Table: Commonly used cryoprotective agents and their uses

Cryoprotectants come in a variety of forms, including sugars, alcohols, and glycols. Dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol are some popular cryoprotectants used in cryopreservation. These cryoprotectants are usually mixed into the solution or media in which the cells or tissues are immersed before being chilled and preserved.

The cryoprotectant used is determined by a number of criteria, including the type of cells or tissues being kept, the temperature at which they will be maintained, and the anticipated use of the sample after thawing. Cryoprotectants can be hazardous to cells if employed in high concentrations or for long periods of time, so it’s critical to optimize the cryoprotectant concentration and exposure time for each application.

Benefits of Cryopreservation:

Cryopreservation has numerous applications in medicine, research, and biotechnology. Overall, cryopreservation is a useful strategy for storing biological materials for future use, which can have huge consequences in health, research, and industry.

Long-term storage: Cryopreservation is the process of storing cells, tissues, and organs at low temperatures for years or even decades. This is important for storing biological materials for future use, such as research, clinical trials, or transplantation.

Preservation of genetic diversity: Cryopreservation can aid in the preservation of genetic diversity in endangered species as well as other rare or valuable biological resources. Biobanks can store samples for future use in genetics, disease research, and other fields.

Increased availability: Cryopreservation can aid to expand the availability of biological materials for transplantation, such as tissues and organs. Cryopreserved samples can be kept and delivered across great distances, making donor-recipient matching easier.

Cost-effective: Cryopreservation, particularly for large-scale operations, can be a cost-effective means of storing and transporting biological resources. It can also eliminate the need for ongoing maintenance of live samples, which can be both time-consuming and costly.

Improved experimental consistency: By lowering variability in biological materials, cryopreservation can aid to increase experimental consistency. Samples can be frozen at a certain time point and then used for research later on, maintaining uniformity and avoiding the need for repeated experiments.

Applications of Cryopreservation:

Preservation of Biological Samples: Cryopreservation allows the long-term preservation of biological samples, including cells, tissues, and organs, without compromising their integrity or function. This is particularly important for rare or fragile specimens that are difficult to collect or reproduce.

Research: Cryopreservation enables researchers to preserve samples and specimens for future use, allowing them to conduct experiments and studies on a wide range of biological materials, including stem cells, tissues, and viruses.

Agricultural Applications: Cryopreservation can be used to preserve the genetic material of plant and animal species, which can be used in breeding programs and genetic research. This helps maintain genetic diversity and can be used to develop new crop varieties or improve livestock genetics.

Medical Applications: Cryopreservation is used in the medical field to preserve blood cells, bone marrow, and other tissues for use in transplants and other treatments. It is also used to store sperm and embryos for in vitro fertilization.

Conservation: Cryopreservation is increasingly used as a tool for conservation, especially for endangered species. Cryopreserved genetic material can be used for assisted reproduction or reintroduction programs, helping to protect and restore threatened species.

References:

• Pegg DE. Principles of cryopreservation. Methods Mol Biol. 2007; 368:39-57.
• Jang TH, Park SC, Yang JH, Kim JY, Seok JH, Park US, Choi CW, Lee SR, Han J. Cryopreservation and its clinical applications. Integr Med Res. 2017 Mar;6(1):12-18.
• Mazur P. Cryobiology: the freezing of biological systems. Science. 1970; 168:939–949
• Hubalek Z. Protectants used in the cryopreservation of microorganisms. Cryobiology. 2003; 46:205–229