Part One of our A Practical Guide to Planning a Cryogenic Storage Facility Series
Storing biological materials, especially living cells and tissues, can be a complicated process. Improper handling or storage conditions can damage cells, reducing viability and compromising their utility in downstream applications. Keeping specimens below -135°C, the glass transition temperature (Tg) of water, stops all metabolic activity and safely preserves cells indefinitely. This process, known as cryopreservation, is key to maintaining the integrity of sensitive biological materials for extended periods of time. With a boiling point of -196°C, liquid nitrogen (LN2) is the gold standard for cryopreservation1. Specimens can be stored in either the liquid or vapor phase of LN2 to keep them below Tg. LN2-based storage units operate very differently than conventional freezers, requiring specific guidelines to be followed when housing them in a storage facility.
In this 5-part blog series, Azenta has brought together the most critical considerations following the best practices of The International Society for Biological and Environmental Repositories (ISBER). These documents provide comprehensive recommendations for setting up and maintaining an LN2 storage facility, including good design and robust operational procedures to reliably protect the integrity of cryogenic materials as well as the safety of users.
LN2 is the ideal preservation medium for biological material because it can be stored indefinitely without loss of quality and doesn’t rely heavily on mechanical freezers, which can be unreliable. To set your facility up for success, there are several key considerations for selecting the appropriate space and equipment.
Facility Considerations for LN2 Storage
The design and construction of a repository is critical – from ensuring security of stored material to developing a safe and optimal working environment for staff. As a result, it is necessary to consider the following:
- Space: You need to provide enough space for your cryogenic equipment with adequate room for operations, verifications, maintenance, and cleaning. The width and height of doorways, hallways, elevators, storage space, and outdoor areas should also be considered to ensure there is room for installation and relocation of this equipment.
- Air Flow, Circulation, Temperature, and Humidity: Ensuring sufficient air circulation is necessary to prevent excess moisture and condensation. Excess humidity can lead to fungal growth, impacting specimen integrity and causing health problems for your staff. Air circulation should be managed so that the air passing over the opening of a storage unit is minimized, as this can accelerate nitrogen boil off. Adequate ventilation and monitoring are also critical to ensure proper oxygen levels are maintained.
- Network Reception: Cellular and/or wireless reception can be considered but using wired connections are recommended, as they can ensure a reliable network connection.
- Electrical Power: While LN2-based storage units do not rely on electrical power for active cooling, electricity is needed for monitors, alarms, and replenishment of LN2 for auto-fill systems. Having a backup power system to provide monitoring and alarm capabilities if a facility is unattended is recommended. Your battery backup should be capable of providing one complete filling cycle.
- Flooring and Structural Support: Flooring should be easy to clean. The weight and vibration of large cryogenic units is also important to evaluate, as over time this will cause wear on floors. In addition, the weight of such equipment must be taken into consideration when relocating or designing a new facility.
LN2 Storage Equipment Types
LN2-based storage units are categorized into two groups: small aluminum Dewars and large stainless steel or plastic storage units. Both are designed to hold LN2 efficiently with vacuum insulation for long lasting durability and are available in different sizes.
- Dewars: Small, efficient, and transportable, these containers provide a stable storage temperature and low LN2 usage. While auto-fill models are available, most require manual filling of LN2 to maintain temperature. They typically do not have full monitoring and LN2 level control options, so you will need a higher level of manual management and verification.
- Open-Top Storage Units: Despite being less expensive to purchase, these storage units have higher operating costs and LN2 usage due to a large lid opening and additional heat input. The larger lid opening allows for easier access to your specimen, which is especially useful for a high-throughput workflow.
- Dry Storage Units: These types of instruments do not use liquid in the specimen storage area, thus reducing contamination and safety risks associated with handling liquid nitrogen, while still maintaining the steady temperature associated with LN2-based storage.
- Automated Units: Retrieve individual boxes without exposing the entire rack to ambient conditions, and better protect non-target samples from transient warming with this type of unit.
- Liquid Storage Units: Medium to large sized, these units are designed for long-term preservation of biological samples. Many have auto-fill capabilities with temperature, LN2 level and usage monitoring. While these instruments provide convenience and consistency, they need to be manually verified at regular intervals to ensure specimen integrity.
- High-Efficiency Units: These storage units have an offset lid that provides access to stored space while maintaining more vacuum-insulated surface compared to open-top units. This means temperature performance is independent of LN2 levels and lid openings with low use of LN2. These instruments also have an interior turn tray, providing access to specimens.
LN2 Supply Systems
The following supply systems should also be considered when designing your LN2 facility:
- Bulk Supply Systems: Vacuum-jacketed withdrawal, piping, and valves are recommended to improve efficiency. They can also help increase safety by avoiding cold surfaces, condensation, and wet floors. These systems require relief valves to prevent pipe and bulk tank ruptures in the event of overpressure. A line pressure gauge should be used as a reference for pressure build events and troubleshooting.
- Portable Supply Tanks: These tanks require regular delivery depending on your LN2 storage unit. LN2 volume and pressure need to be continuously monitored to ensure adequate supply. These tanks also require the LN2 storage unit transfer house to be routinely disconnected and reconnected.
Summary
Designing and equipping a cryogenic storage facility can be a complicated process. While there are many factors to consider, with careful planning and execution, you can ensure the safety of your specimens as well as your staff.
Interested in learning more about cryogenic storage and how to set up your own facility? Download our full guide.
References
1. Best Practices: Recommendations for Repositories (4th ed), Addendum 1: Liquid Nitrogen-Based Cryogenic Storage of Specimens. ISBER. 2019.