Cryopreservation: Purpose, process & applications

Cryopreservation describes methods to preserve living organisms and biological materials at very low temperatures. Central to this process is the use of liquid nitrogen, enabling successful cryogenic freezing and cryopreservation, opening possibilities for cell banking and other types of ex vivo preservation over extended periods of time.

In this article, we will discuss the purpose, process and applications of cryopreservation.

Definition: What is cryopreservation?

Cryopreservation refers to the technique of preserving living cells and tissues at extremely low temperatures to ensure their long-term storage and viability.¹ The process involves carefully cooling biological samples to sub-zero temperatures, typically using cryogenic substances like liquid nitrogen, and has paved the way for extensive cell banks, facilitating research and preserving valuable biological samples for future scientific investigations.

Key to the success of cryopreservation are cryoprotective agents (CPAs) such as glycerol and dimethyl sulfoxide (DMSO), which safeguard cells from damaging effects related to freezing and thawing.¹ 

Purpose of cryopreservation of cells

The primary goal of cryopreservation is to maintain the viability and functionality of living cells over extended periods of time. By carefully freezing cells and preventing the formation of ice crystals, cryopreservation ensures that cells can be stored for long durations without compromising their integrity. This is how cryopreservation has found entrance in clinical applications, particularly in cell therapies.

In biotechnology, cryopreservation is instrumental in preserving valuable cell lines and biological samples. This technique supports cutting-edge research and facilitates breakthroughs in various scientific disciplines, allowing to establish different cell banks for various applications. For instance, medical advancements like in vitro fertilization (IVF) and organ transplantation have been made possible, revolutionizing the world of regenerative medicine.

Cell preparation

Before cryopreservation, cells are carefully prepared to withstand freezing. Cryoprotective agents (CPAs), such as glycerol and dimethyl sulfoxide (DMSO), are introduced to protect cells from damage during freezing and thawing.

Subsequently, the cells are filled into cryogenic freezing containers, e.g. in single-use bags that can withstand these extremely low temperatures. In order to achieve consistent cell counts in bags, the filling process is accompanied by the homogenization of cells.

Cryoprotectant equilibration

Before the cells are frozen, cryoprotectant equilibration is performed. Mixing and homogenization techniques are carried out to substitute the water inside the cells with CPAs, with the aim to reduce the formation of ice crystals in the freezing process.

Cooling to cryogenic temperatures

The cells are cooled to cryogenic temperatures, typically using controlled-rate freezers or liquid nitrogen. The gradual cooling rate helps prevent ice crystal formation, which could harm the cell membrane.

Cryogenic storage

Once the cells reach their target temperature, they are stored in cryogenic containers filled with liquid nitrogen or other cryogenic substances. These extremely low temperatures halt all cellular activities, effectively preserving the cells in a dormant state.

Thawing

When needed, the cells are carefully thawed using specialized techniques. Rapid and controlled thawing is essential to prevent damage to the cells and maintain their viability, after which CPAs are removed.

Applications of cryopreservation

Cryopreservation plays a vital role in various fields, enabling the preservation of living cells and tissues for diverse applications. In the following, we will explore some of the key areas where cryopreservation has made a significant impact.

Cryopreservation in biotechnology

• Cell lines and biobanking: Cryopreservation facilitates the long-term storage of valuable cell lines and biological samples used in biotechnological research and drug development.
• Tissue engineering: Cryopreservation is crucial for preserving engineered tissues and scaffolds used in tissue engineering, enabling future implantation and therapeutic applications.

Cryopreservation in medicine

• Regenerative medicine: Cryopreservation plays a vital role in storing stem cells, such as embryonic stem cells and hematopoietic stem cells, which hold immense potential for regenerating damaged tissues and treating medical conditions.
• Cell therapies: Red blood cells, stem cells harvested from the bone marrow and other cell types can be preserved under cryogenic conditions, awaiting their therapeutic application.
• Organ transplantation: Cryopreservation allows the preservation of organs and tissues for transplantation, expanding the pool of available organs and potentially saving lives.
• Reproductive medicine: Cryopreservation of spermatozoa, ovary cells, ovarian tissue, and oocytes are frequently used procedures in IVF (in vitro fertilization), as well as embryo cryopreservation, offering fertility preservation options.

Other fields of application

Cryopreservation goes beyond medicine and biotechnology. It finds intriguing uses in other fields:

• Cryobiology and Research – preserving cell suspensions, microorganisms and mammalian cells for in-depth studies on cellular behavior at low temperatures
• Preservation of different cell types – ensuring the availability of diverse cell types for scientific research in various fields
• Food industry – preserving perishable foods like fruits, vegetables, and seafood to maintain quality and nutritional value for extended periods

Challenges in cryopreservation

Cryopreservation, despite its immense potential and wide-ranging applications, is not without its challenges. These hurdles must be carefully addressed to ensure the successful preservation of living cells and tissues.

One of the critical challenges lies in the formation of ice crystals during the freezing process. Ice crystals can inflict damage to cell structures and membranes, compromising cell viability. To counteract the loss of product quality, precise cooling techniques are necessary to minimize ice crystal formation and safeguard cell integrity.

Another significant challenge arises from the susceptibility of certain cells to intracellular ice formation. This phenomenon can prove detrimental to cell viability and requires specialized cryopreservation protocols and cryopreservation methods to protect cells from this potentially harmful occurrence. Moreover, excessive extracellular ice formation poses another obstacle, as it can lead to dehydration and further damage to cell membranes. Maintaining a delicate balance in ice formation is imperative for preserving cell survival.

The permeability of cryoprotective agents (CPAs) is a crucial consideration in cryopreservation. Ensuring the appropriate permeability allows for effective cell protection during freezing and thawing. However, using high concentrations of CPAs can be toxic to cells, impacting their viability and functionality. Striking the right balance of CPAs is vital to achieve low toxicity and a successful cryopreservation.

Thawing cells after cryopreservation presents its own set of challenges. Improper thawing techniques can result in reduced post-thaw cell viability and functionality, underscoring the importance of employing precise and controlled thawing methods.

Additionally, the successful long-term cryopreservation of cells and tissues requires careful monitoring and management. Continuous assessment of cell viability and functionality is essential to ensure sustained preservation quality over extended periods.

Furthermore, the availability and proper use of controlled rate freezers are critical for maintaining precise cooling rates during cryopreservation. These freezers play a vital role in achieving optimal results, ensuring consistency in the preservation process via controlled cryogenic freezing.³ 

Single-use technologies in cryopreservation

Single-use technologies have emerged as a promising approach in the field of cryopreservation, offering several advantages and addressing specific challenges. Single Use Support, a pioneering process solution provider, has established product platforms that further improve single-use technologies. Among the many advantages for its customers using advanced single-use systems – here are five of them:

1. Reduced variability: Single-use technologies provide consistent and standardized platforms for cryogenic freezing, reducing variability in freezing and thawing processes. This ensures more reliable and reproducible outcomes across different cell types and applications.
2. Ease of Use: Single-use technologies are user-friendly and come with a high degree of automation, requiring minimal preparation and cleaning and less manpower. This simplicity makes them ideal for various research, clinical, and biotechnological applications.
3. Scalability: Single-use technologies can be easily scaled up or down, making them suitable for both small-scale laboratory experiments and large-scale industrial applications.
4. Cost-Effectiveness: In many scenarios, single-use technologies are more cost-effective than traditional reusable systems, reducing the need for complex maintenance and sterilization processes.
5. Waste Reduction: Single-use technologies help reduce the use of water, energy and chemicals during manufacturing. Further, it lowers cross-contamination risks, as they are disposed of after each use, preventing potential contamination between different samples.

As cryopreservation continues to advance, Single Use Support can encompass these developments with exciting opportunities for standardization, improved efficiency, and cost-effectiveness. Enhanced control over the cryogenic freezing process paves the way for breakthroughs in various scientific and medical fields, shaping the future of cell banking and regenerative medicine.