Cryopreservation techniques & best practice freezing methods

Cryopreservation involves the preservation of cells, tissues, or other biological constructs by cooling them to extremely low temperatures. The preservation of cells at cryogenic temperature can be achieved using different techniques. This is far from a simple freezing process; it's a carefully controlled method that prevents the formation of ice crystals, thus maintaining the integrity of the biological material.

In this article, we will focus on the various techniques of cryopreservation. Whether a key tool in medical treatments or an essential part of scientific research, understanding the techniques of cryopreservation provides insight into a practice that is as complex as it is vital.

Historical development of cryopreservation techniques

Cryopreservation's historical roots can be traced back to early attempts to preserve biological materials at low temperatures. The initial methods were rudimentary, relying on simple freezing techniques that often led to the formation of damaging ice crystals.

These ice crystals posed a significant obstacle, causing mechanical injuries to cells and tissues. The early practitioners of cryopreservation were limited by the technology of their time, facing challenges such as inconsistent freezing rates and inadequate control over the thawing process.

The transition from early methods to modern advancements in cryopreservation represents a journey marked by technological innovation and scientific discovery. The introduction of controlled rate freezers, cryoprotectant agents, and precise temperature control mechanisms has revolutionized the field of cryogenic freezing. Modern techniques are now able to minimize the damaging effects of ice crystal formation, offering more reliable preservation of various biological constructs.¹ 

Vitrification

Vitrification is a transformative cryopreservation technique that rapidly cools biological materials to a glass-like state, bypassing the formation of damaging ice crystals. Vitrification avoids mechanical damage to cellular structures, making it an interesting tool for preserving delicate biological constructs.

The advantage of vitrification lies in its ability to freeze fast with -100°C per minute, keeping the integrity of the biological material. In specific cryopreservation scenarios, vitrification offers a method to ensure that the preserved material retains its functional properties.

Vitrification has the disadvantage that it cannot be applied to all compounds. Vitrification is only an option for certain types of cells, for example not suitable for mammalian cells like CHO cells. It is also a complex process and requires technical expertise. Due to increased complexities in larger volumes, only small volumes are commonly vitrified. Further, the choice of cryoprotectants must be right to avoid inconsistencies and reduced cell viability.^2 3 

Controlled Freezing

Freezing, also known as "slow freezing" due to the comparatively longer duration is primarily a controlled freezing process. While slow, uncontrolled freezing with static or blast freezing promotes cryoconcentration, it is important to freeze fast and controlled (but still slow compared to vitrification). Recommended is a steady freezing rate around 1-2°C per minute, which can be ensured with plate-freezing methods.

Freezing represents a more controlled approach to cryopreservation. This method allows for controlled ice crystal formation through a gradual cooling process. Freezing typically utilizes programmable freezers that enable precise control over the rate of cooling, thereby managing the ice crystal formation in a way that minimizes potential damage.

This technique has broader applicability and is commonly used for various cell types, including stem cells. The analytical comparison between freezing and vitrification reveals that the choice between these techniques hinges on the specific biological material being preserved. While freezing offers more control, vitrification might be preferable for preserving more elective structures.⁴ 

Controlled freezing in cryopreservation: Best practice

Single Use Support focuses on developing innovative solutions for freezing biopharmaceuticals. In the delicate handling of cells, rapid and secure processes take precedence. The freezing specialist’s objectives encompass achieving a high cell recovery rate and ensuring substantial cell viability after thawing.

Two techniques stand out for their efficacy in meeting these goals: cryogenic controlled rate freezing that reaches temperatures as low as -170°C, and plate-based freezing that descends to -80°C, complemented by subsequent storage in liquid nitrogen.⁵ 

Plate-based freezing

This two-stage process begins with an initial controlled cooling to -80°C / -112°F using specialized freezing plates. This stage ensures uniform cooling, thus maintaining consistency across the sample. Once this temperature is reached, the samples are then transferred to liquid nitrogen storage, where they can be kept stably at temperatures as low as -196°C.

The RoSS.pFTU lab scale by Single Use Support introduces an innovative approach to plate-based freezing, particularly applicable in clinical studies and laboratory environments.

• Controlled Freezing: Capable of achieving setpoint freezing down to -80°C / -112°F, this technology provides a controlled freezing environment. Depending on freezing recipe, the temperature will go down controlled and steady. Phase transition is shortened and cryoconcentration prevented. The system has been engineered to freeze substances with both speed and accuracy, allowing for the preservation of integrity.
• Protection: Utilizing protective single-use shells like ROSS® Shell and the option for small single-use bags RoSS.KSET, the design seeks to mitigate the risks associated with potential compromise during the freezing process. Protection can be provided for bioprocess containers of any manufacturer and size. Product loss is drastically reduced if not eliminated.

Future development in cryopreservation techniques

Emphasizing the importance of accuracy and control, these evolving techniques in cryopreservation aim to minimize damage and maximize viability.

Single Use Support's contributions in this area illustrate the alignment of industry practice with these future trends. The pioneering solutions offer tailored approaches to freezing different biopharmaceuticals, providing secure processes that prioritize high cell recovery rates and substantial cell viability after thawing the cells, notably in cell banking applications.