Plan Your Cryogenic Storage Facility With Confidence

A Practical Guide to Planning a Cryogenic Storage Facility GUIDE azenta.com 2 Contents Introduction 3 Designing and Equipping Your LN2 Facility 4 Facility Considerations for LN2 Storage � � � � � � � � 4 LN2 Storage Equipment Types � � � � � � � � � � � � � � � � 6 LN2 Supply Systems � � � � � � � � � � � � � � � � � � � � � � � � � 7 Monitoring Your LN2 Facility and Storage Units 8 Installing and Maintaining LN2 Equipment 11 Installation and Setup � � � � � � � � � � � � � � � � � � � � � � � 11 Electrical Power � � � � � � � � � � � � � � � � � � � � � � � � � � � � 12 Calibration � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 13 Verification � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 13 Routine Maintenance � � � � � � � � � � � � � � � � � � � � � � � 14 Ensuring Safety in Your LN2 Facility 16 Oxygen Sensors � � � � � � � � � � � � � � � � � � � � � � � � � � � � 16 Personal Protective Equipment (PPE) � � � � � � � � � � 17 Training Ensures Safety and Specimen Quality � 18 Preparing Your LN2 Facility for an Emergency 19 On Call Staff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 19 Backup Capacity � � � � � � � � � � � � � � � � � � � � � � � � � � � 20 Other Best Practices for Risk Mitigation � � � � � � � 20 Conclusion 22 Streamline your Cryogenic Storage with Azenta Life Sciences � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 23 Azenta Life Sciences azenta.com 3 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 preserve 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 new guide, Azenta has brought together the most critical considerations following the best practices of The International Society for Biological and Environmental Repositories (ISBER)2 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 Introduction Azenta Life Sciences azenta.com 4 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: Designing and Equipping Your LN2 Facility Azenta Life Sciences azenta.com 5 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 Azenta Life Sciences azenta.com 6 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 either in vapor-phase or liquid form with vacuum insulation and are available in different sizes Automated Storage Units: These types of instruments typically use vapor-phase LN2 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, which can retrieve individual boxes without exposing the entire rack to ambient conditions, can better protect non-target samples from transient warming High-Efficiency Units: These storage units have an offset lid that provides access to stored space while maintaining a more vacuum-insulated surface compared to open-top units This means that temperatures can be maintained at a more stable level compared to units with open lids These instruments also have an interior turn tray, providing access to specimens Liquid Storage Units: Medium to large in size, 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 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 High Efficiency Units Automated Storage Systems Azenta Life Sciences azenta.com 7 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 LN2 Supply Systems LN2-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. Azenta Life Sciences azenta.com 8 Did you know that a proper LN2 monitoring system can mean the difference between life and death? Given that nitrogen is lighter than air, it can rapidly displace oxygen in a room, which can lead to asphyxiation To ensure the safety of your facility, below are six factors you should consider: 1. Oxygen Monitoring: Oxygen monitors need to be in place to notify staff of dangerous levels These systems should be in areas that are exposed to LN2, clearly explaining the nature of the alert and the associated risk In most systems, there are multiple notification settings for various oxygen levels to indicate slightly out-of-range conditions versus significantly out-of-range conditions However, if your oxygen monitor has only one alarm setting, it should be set at 195% oxygen, the point at which staff need to be evacuated immediately Monitoring Your LN2 Facility and Storage Units Azenta Life Sciences azenta.com 9 2. Curved Mirrors and Camera: Having curved mirrors and cameras installed in your LN2 facility makes it easier to perform visual inspections of LN2 storage units and identify staff during an emergency When positioned correctly, mirrors and cameras can help observe if anyone has collapsed in the facility 3. Specimen Integrity Monitoring: Monitoring systems are key to preventing specimen loss Having a specimen integrity monitoring system that will alert staff of urgent issues and provide insight into storage conditions, such as temperature and functionality, is necessary Make sure you have redundant sources of monitoring in place, both human and electronic, for added security 4. Temperature Monitoring: LN2 storage units need to have continuous temperature monitoring by a certified and traceable thermometer Even when specimens are completely submerged, monitoring can provide reassurance that a proper temperature is maintained if the LN2 level falls out of range In addition, you may want to place a redundant thermometer at the highest level of where the specimens are stored 5. LN2 Level Monitoring: The LN2 level in each storage unit should be monitored and recorded manually to the nearest quarter inch (025 inch or 6 mm) When performed manually, a plastic or metal yard stick is recommended Do not use a hollow tube, as LN2 can rapidly shoot out of the top once inserted into the cold liquid nitrogen When using an electronic component to measure LN2, the electronic level should be monitored and recorded The manually measured LN2 level should be compared to the electronic level within a predefined acceptable range If out of range, calibrate the electronic LN2 level to match the manually measured level Azenta Life Sciences azenta.com 10 SUMMARY A well-monitored and safe LN2 facility is critical to the long-term success of your cryogenic storage. By putting in place a comprehensive monitoring system, you can have peace of mind that your staff and specimens are properly protected. 6. LN2 Consumption Monitoring: LN2 consumption should be measured and recorded manually (for manual-fill LN2 storage units) and electronically (for auto-fill LN2 storage units) For manual-fill LN2 storage units, wireless remote, weight-based monitoring systems provide continuous measurements that can be precisely correlated to LN2 levels and used to determine evaporation rates On autofill LN2 storage units, the electronic monitoring system provides this information Since auto-fill LN2 storage units fill on an “as needed” basis, it can be difficult to determine LN2 usage manually A clear acceptable range of consumption with a calculated standard deviation should be included, as identified by the manufacturer’s guidelines and in accordance with the facility’s average usage Azenta Life Sciences azenta.com 11 Installing and maintaining liquid nitrogen equipment can be a daunting task, but it’s important to understand the basics if you want your facility to run smoothly To help you get started, below are key factors to consider Installation and Setup Installation and setup should be verified using manufacturer recommendations for electrical and LN2 supply as well as facility requirements Your storage unit should undergo a set of qualification procedures to ensure that it is installed correctly and meets all specifications: Installation Qualification (IQ): Oversight and verification of every physical aspect of the equipment (materials, dimensions, pressure ratings) and components (operational parameters, accuracy, voltage) Installing and Maintaining LN2 Equipment Azenta Life Sciences azenta.com 12 Operational Qualification (OQ): Testing each individual component, feature, and physical specification of the equipment Performance Qualification (PQ) or IQ/OQ/PQ: Testing the operational requirements of the equipment under real-world conditions Electrical Power While LN2-based cryogenic storage units are not dependent on electrical power for active cooling, it is required for alarms and monitoring A backup power system with sufficient monitoring and alarm capabilities should also be in place for when the facility is unattended LN2 Supply: The LN2 supply system should contain enough LN2 for at least 21 days of normal usage and replenishment intervals It’s a good idea to have a backup supply system in case the off-site provider is unable to re-supply within this time frame In addition, your bulk tanks or cylinders as well as LN2 supply volume and pressure should be verified regularly, and the LN2 connections should be routinely checked Secondary Monitoring: When using secondary systems for monitoring temperature and LN2 levels, make sure to install, calibrate, and verify before introducing samples Pressure Relief Valves: These devices are essential when working with LN2 Because nitrogen gas buildup can lead to an explosion, a risk mitigation plan must be in place Relief valves must be present on all storage units and components that could trap LN2, and pressure relief valves should be piped to the outside, verified, and replaced regularly Initial Fill: LN2 storage units must be filled with liquid nitrogen per manufacturer recommendations and stabilized up to 48 hours prior to introducing specimens An inventory rack system should also be installed at this time Azenta Life Sciences azenta.com 13 Calibration Instrument calibration is a key step in ensuring accurate readings Calibrated instruments are more reliable than those without any adjustments, and they also provide better protection against errors due to changes over time Records should include readings taken both before and after calibration In addition, a record that includes calibration date, name of who is performing the calibration, name/serial number of the device used, and reference to the SOP should be included Verification The installation and operation of cryogenic storage systems should be verified per manufacturer recommendations For LN2-based systems, this includes installation qualification and components Documentation: User manuals, certificates, maintenance logs, and factory acceptance documentation needs to be kept in a central location that is available to staff Personnel: Staff contact information should be made available and kept in a known location Training and SOPs: A training program that includes safety procedures, routine verification process, and emergency response plans should be in place for all staff members Temperature Mapping: Temperature mapping of the storage unit is typically conducted by the manufacturer as part of the design qualification To confirm the temperature within the unit, a secondary sensor should be used at the highest level that specimens can be stored Azenta Life Sciences azenta.com 14 Routine Maintenance The importance of performing system maintenance cannot be underestimated It is essential to be in close communication with manufacturers and service providers to develop appropriate maintenance and verification schedules LN2 Storage Units: Daily or weekly manual verification of storage temperature, LN2 level, and usage is suggested Be sure to keep records of manual verifications to watch for trends or changes in system performance Condensation, Frost, and Ice Buildup: Maintaining a dry environment can reduce condensation, frost, and ice accumulation, which impacts performance and usability To improve efficiencies and equipment life, be sure to routinely wipe surfaces and remove buildup Thaw, Clean, and Dry: Periodic rounds of thawing, cleaning, and drying may be required to maximize the efficiency and lifespan of storage units Precautions should be taken to ensure that samples are safely removed and stored securely in another unit during the process Backup Power: Given that batteries have a limited shelf-life and should be replaced per manufacturer’s recommendations, it is important to verify backup power Azenta Life Sciences azenta.com 15 Alarms and Alerts: All alarm systems should be routinely tested and verified The SOP should outline the process for conducting system verification, and results should be documented and made available for audits Steps, Handles, and Lids: Interface components should be routinely examined for deterioration and verified to ensure functionality Lid gaskets should be inspected for sealing and venting and replaced if damaged, as this can result in inefficiencies Plumbing: For auto-fill systems, plumbing should be regularly verified and replaced per manufacturer’s recommendations SUMMARY As the need for cryogenic storage grows in an organization, it’s important that stakeholders understand the value of proper installation and maintenance of LN2-based systems. By following the appropriate guidelines, repositories can ensure that these systems are operated safely and efficiently. Azenta Life Sciences azenta.com 16 Cryogenic storage facilities face many potential threats— from natural disasters to power outages and cyberattacks Exposure of specimens to warming, even if transient, can lead to degradation and significant loss of value Preparation is key to effectively address emergencies when they occur and minimize their impact to operations As such, an organization with LN2 facilities should have written emergency plans for disaster recovery, incident response, and business continuity Staff should be provided copies of the plans as well as the necessary training to execute them Oxygen Sensors Given that nitrogen can displace oxygen in an enclosed environment, oxygen level sensors should be used in LN2 storage units Normal levels of oxygen should be ~21% Many oxygen sensors have batteries or sensor cells that need Ensuring Safety in Your LN2 Facility Azenta Life Sciences azenta.com 17 to be replaced and re-calibrated as instructed by the manufacturer They will also have specific requirements as to the number of sensors needed based on the size of the room, placement in the room, and height of the wall mounting Alarms should be installed inside and outside of the room along with dedicated exhaust ventilation Another option to consider is having viewing windows so it’s easy to determine if the room is occupied during an alarm Fixed and mobile/personal monitors might be appropriate depending on the size of your facility If installed units indicate an alarm, it’s beneficial to use a personal monitor when entering the room in order to validate the alarm, especially if you can’t see inside the area Personal Protective Equipment (PPE) Any staff that are working with or around LN2 should wear the appropriate protective gear Safety goggles are required at all times and a face shield is necessary when working with large volumes of LN2 Cryogenic gloves that are designed for use in the vapor phase should never be immersed into liquid nitrogen and should be loosefitting for quick removal Disposable latex or nitrile gloves should also be used when personnel share insulated gloves A lab coat with long sleeves is required to minimize skin contact Pants should be worn on the outside of shoes to prevent them from filling with LN2 in case of a spill, and an apron is required when handling large amounts of liquid nitrogen When they are not in use, PPE should be stored properly to avoid damage and contamination Azenta Life Sciences azenta.com 18 Training Ensures Safety and Specimen Quality Training your staff is essential so they can perform their required tasks safely It also ensures quality in specimen handling It’s recommended that you document and repeat training on a yearly basis In general, all staff must be aware of occupational hazards and be fully trained in the procedures for usage, storage, and transportation of LN2 before they are allowed to handle it In addition, appropriate training for the safe handling of cryogenics should be provided Staff should be trained on how to respond to alarms and procedures, both for accurate recording as well as resolution The retrieval of specimens from cryostorage also requires specific training due to the risk of explosion For example, when liquid nitrogen enters an improperly sealed glass storage vial, the gas expands rapidly as the vials are brought to room temperature, resulting in the potential for explosions SUMMARY A safe and accident-free work environment is the top priority for any organization. While LN2-based systems can provide highly reliable storage for critical materials, there are serious risks that managers must address. Following these tips and guidelines will help promote safety and compliance in your liquid nitrogen facility. Azenta Life Sciences azenta.com 19 Cryogenic storage facilities face many potential threats— from natural disasters to power outages and cyberattacks Exposure of specimens to warming, even if transient, can lead to degradation and significant loss of value Preparation is key to effectively address emergencies when they occur and minimize their impact to operations As such, an organization with LN2 facilities should have written emergency plans for disaster recovery, incident response, and business continuity Staff should be provided copies of the plans as well as the necessary training to execute them On Call Staff Key personnel should be identified for responding to emergencies These individuals must be trained and qualified to handle situations associated with LN2-based specimen storage Emergency contact numbers should be in a central Preparing Your LN2 Facility for an Emergency Azenta Life Sciences azenta.com 20 location so they can be notified quickly if needed; this information should also be posted near notable areas such as at reception desks Staff should have a check list of procedures to follow during an emergency with telephone numbers for professional assistance They should also be familiar with how to operate key equipment during an emergency Backup Capacity In case of equipment failure, it’s recommended to have backup capacity for your LN2 units The extra-capacity equipment you have in your facility should be the same capacity as your largest single LN2 storage unit and maintained at operating temperature If space isn’t available, explore whether nearby facilities can accommodate temporary storage of specimens during an emergency In addition, LN2, dry ice, and/or portable freezers should be available at your facility to maintain specimens during the transfer It’s also important to have sufficient backup power for your storage and monitoring equipment LN2 cryogenic facilities should have written emergency procedures for transferring specimens from a failed/ malfunctioning unit as well as for returning those specimens to their original location once it is safe to do so Within these written procedures should be the LN2 storage unit name, number, and location of where specimens have been relocated Other Best Practices for Risk Mitigation Communication with Utility Companies: It’s important to be in close contact with local power companies to ensure that that the facility is considered “high priority” for power restoration following an emergency Azenta Life Sciences azenta.com 21 Emergency Drills: Where possible, emergencies should be simulated to ensure proper follow-through for the established emergency plan LN2 Refill: In the event of an impending need for evacuation, repositories that utilize LN2 bulk tanks should arrange to have them filled Storing Records On-Site: If inventory systems are housed on a server offsite, it may be wise to consider storing electronic records at the facility to ensure that they are accessible in case of an emergency Sample Collection Redundancy and Splitting: To ensure that the most valuable specimens are not lost, duplicates can be kept in different storage units or at separate sites Sample collections can also be divided across multiple units or locations to minimize the risk of losing an entire collection from one event SUMMARY Emergency planning is critical for any business, and this is especially true for those storing cryopreserved materials. In the event of an emergency, it’s imperative to have a plan in place so that you can minimize damage and get back up and running as soon as possible. Azenta Life Sciences azenta.com 22 Starting and running a cryogenic storage facility requires careful planning and robust operating procedures to ensure reliable long-term protection of critical materials Following the recommended measures mentioned here will significantly mitigate risk to your sample collection and staff Most LN2-based storage systems are operated manually: sample retrieval is performed by hand and inventory is managed with basic tools like spreadsheets This can lead to variability, human error, and inefficiencies4 Automation can address many of these drawbacks using robotics to retrieve samples and built-in software to track inventory, leading to better sample quality and simpler data management (see box to the right) Since cryogenic storage facilities require not only expertise but also significant investment in capital, staff, and training, many organizations decide to outsource Working with a capable external partner can greatly streamline ultracold storage and management, especially for regulated environments (see box to the right) Conclusion Automated Systems The CryoArc™ -190°C LN2-Based Automated Storage Systems offer the greatest sample protection and comprehensive inventory management and control with a superior user experience at an affordable price Targeted and nontargeted samples are protected from warming above Tg throughout sample retrieval Quality is assured by viewing user access, sample inventory, history, and audit-trail reports Emergency protection provides greater than 20 days of temperature stability in the case of energy or LN2 loss LEARN MORE → Ultracold Storage and Distribution of Cellular Products and Samples Managing sensitive materials like cellular therapies—including storage, transportation, and tracking—requires robust and adaptable systems to ensure safety, integrity, and regulatory compliance that are essential for FDA review We specialize in cGMP (Current Good Manufacturing Practice) controlled cold chain storage and logistics to help safeguard the integrity and quality of your sensitive products Our highly specialized team works with you to develop a comprehensive plan to receive, distribute, and manage your cellular products across the globe LEARN MORE → Azenta Life Sciences azenta.com 23 Streamline your Cryogenic Storage with Azenta Life Sciences Azenta Life Sciences has deep application experience, proven technologies, and a global infrastructure to protect the integrity of cryogenic materials and provide precise inventory accuracy and documentation If you’re in the process of planning your LN2 facility or want to store your samples at our certified facilities, we can help CONTACT AN EXPERT → References 1 Best Practices: Recommendations for Repositories (4th ed), Addendum 1: Liquid Nitrogen-Based Cryogenic Storage of Specimens ISBER 2019 References for “Streamline Your Cryogenic Storage with Azenta Life Sciences” 1 Hubel, A, Spindler, R & Skubitz, A P N Storage of Human Biospecimens: Selection of the Optimal Storage Temperature Biopreservation and Biobanking vol 12 165–175 (2014) 2 Best Practices: Recommendations for Repositories (4th ed) ISBER 2018 3 Best Practices: Recommendations for Repositories (4th ed), Addendum 1: Liquid Nitrogen-Based Cryogenic Storage of Specimens ISBER 2019 4 Why Automate? Explore the Benefits of Automated Sample Storage Azenta Life Sciences 2022 https://wwwazentacom/blog/why-automate-explore-benefits-automated-sample-storage azentacom © 2024 Azenta US, Inc. All rights reserved. All trademarks are property of Azenta US, Inc. unless otherwise specified. Illumina® is a trademark of Illumina, Inc. 23012-WE 1024