Maintaining a safe environment in a liquid nitrogen cryopreservation room

Author Gary Brown, LN₂ Product Specialist, Haier Biomedical, discusses the preservation and handling of precious biological materials.

 

Liquid nitrogen (LN₂) plays a crucial role in the world of assisted reproductive technology, as the go-to cryogenic agent for storing precious biological materials, including eggs, sperm and embryos. LN₂ ensures the long-term preservation of these delicate specimens by providing extremely low temperatures to maintain cellular integrity. However, handling LN₂ poses unique challenges, due to its extremely cold temperature, rapid expansion rate and the potential risks associated with oxygen displacement. Recent changes to UK fertility laws – extending the amount of time that gametes and embryos can be stored – will only increase the use of LN₂ to meet the growing demand for storage, making essential safety measures and best practices even more important to maintain a secure and efficient cryopreservation environment, safeguarding staff and the future of fertility treatments.

In modern fertility clinics, the storage of biological material primarily relies on LN_2, due to its established efficacy and reliability. Its ultra-low temperature – typically around -196 °C – halts all biological activity to preserve the viability and integrity of cells indefinitely. This is commonly achieved through vitrification, a rapid freezing technique that prevents the formation of ice crystals that could damage cellular structures. This ensures that embryos and gametes remain viable for future use, providing individuals and couples with the opportunity to achieve pregnancy at a time of their choosing, whether due to medical treatments, personal choice, or other circumstances.

Figure 1: Cryogenic tanks containing LN₂ provide long term- storage of eggs and gametes at ultra-low temperatures

 

The need to scale up

In the UK, the Health and Social Care Act 2022 included changes to fertility laws that extended the time that gametes and embryos can be kept from 10 years to 55 years – providing the patient reconsents every 10 years [1]. As a result, fertility clinics need to plan for significant increases in demand for storage, including expanding and modernising their LN₂ cryostorage solutions. Currently, a standard centre will typically use a number of medium-sized aluminium dewar in a cryopreservation room. Each dewar will contain locator racks and box system capable of holding up to 6,000 samples, and these containers require daily LN₂ top ups using a manual hose to ensure that samples are kept at appropriately low temperatures.

To increase storage, many clinics are now looking to scale up and consolidate sample storage into larger containers. Modern stainless steel storage containers come in various sizes, and can be capable of storing between 13,000 and 95,000 samples in 2 ml vials. For example, the YDD-370-326 from Haier Biomedical, can store around 2,000 individual patient samples in a customised racking system that clips goblets into columns for secure straw storage. These containers are designed to maximise storage capacity while minimising consumption of LN₂. Some are also capable of automated refilling from an attached replenishment tank, removing the need for manual daily top ups and minimising the risks associated with spending time in the cryopreservation room and handling liquid nitrogen. The development of smart liquid nitrogen management systems – using high precision temperature and liquid level sensors to ensure accurate measurements – is also helping to improve the safety of cryopreservation storage vessels. However, whatever adaptations are made to meet the increased storage, there is no doubt that the amount of storage – and therefore the amount of LN₂ – will have to increase. This will come with a greater need for continued education on the risks of handling LN₂, and the implementation of new safety procedures for its safe use.

 

Figure 2: Cryopreservation storage facility

 

Dangers associated with LN₂ handling

Handling LN₂ presents several risks, including explosion, asphyxiation, and cryogenic burns. LN₂ has a volume expansion ratio of about 1:700, meaning one litre of LN₂ vaporises into approximately 700 litres of nitrogen gas. This expansion can shatter glass vials, creating dangerous shards that could cause injury. With a vapour density of about 0.97, LN₂ is less dense than air and can pool at ground level, posing an asphyxiation hazard by depleting oxygen in confined spaces. The rapid release of LN₂ also forms vapor fog clouds, increasing asphyxiation risks. Exposure to LN₂ liquid, even briefly, can cause severe cold burns, frostbite, tissue damage, or permanent eye damage.

 

Minimising risks in the cryogenic room

Every fertility clinic should carry out an internal risk assessment of its cryogenic room, as outlined in the Codes of Practice (CP) publications from the British Compressed Gases Association [2]. In particular, CP36 is useful to advise on the storage of cryogenic gases onsite, and CP45 gives guidance on the design of a cryogenic storage room [3,4]. Factors including the layout of the room, safety procedures and maintenance should all be considered to reduce the risks for users of the storage facility

 

Layout

A cryogenic room should be located for maximum accessibility. The placement of the LN₂ storage container is crucial, as it requires filling from a pressurised vessel. Ideally, the liquid nitrogen supply vessel should be positioned outside the sample storage room in a well-ventilated and secure area. The supply vessel is often connected directly to the storage container via a cryogenic transfer hose for larger storage setups. If the building layout prevents external placement of the supply vessel, extra care must be taken when handling the LN₂, including conducting a detailed risk assessment with O₂ monitoring and extraction systems.

 

Ventilation

All cryogenic rooms must be well-ventilated to minimise the risk of asphyxiation. This can be achieved with an extraction system that prevents nitrogen gas build-up and protects against oxygen depletion. When choosing a system, it needs to be suitable for a cryogenically cold gas, and linked to an oxygen depletion monitoring system that can detect when the oxygen level drops below 19.5 per cent to initiate an increase in the air exchange rate. Typically, extract ducts should be located at low level, while depletion sensors must be placed approximately 1 meter above the floor level. However, exact positioning should be decided after a detailed site survey, as factors such as room size and layout will affect the optimal placement. An external alarm should also be installed outside of the room, to provide both audio and visual warnings that signify when it is unsafe to enter.

 

Personal safety

Employees who are required to access a cryostorage room should undergo training, including understanding the storage systems and equipment in the room, as well as the safe handling of LN₂. Staff should wear the appropriate personal protective equipment (PPE) to safeguard against cryogenic burns, including eye protection, gloves/gauntlets, suitable footwear and a lab coat. It is essential for all staff to undergo first aid training on how to deal with cryogenic burns, and it is ideal to have a supply of lukewarm water close by to rinse off the skin should a burn occur. Some clinics may also choose to equip employees with personal oxygen monitors, and employ a buddy system whereby people will only ever enter the cryogenic room in pairs, minimising the amount of time person is in the room on their own.

 

Maintenance

Because a pressurised vessel and LN₂ container have no moving parts, a basic annual maintenance schedule is all that is required. This should include inspecting the condition of the cryogenic hose, as well as replacing safety release valves as necessary. Staff should also continually monitor for areas of frosting – either on the container or on the feeder vessel – which could indicate an issue with the vacuum that would require further investigation. With careful consideration of all of these factors, and a regular maintenance schedule, pressurised vessels can last for up to 20 years.

Figure 3: A summary of LN₂ safe handling good practice for cryopreservation rooms.

 

Summary

Fertility clinics have never been busier, thanks to changing attitudes towards reproductive health and a drive to improve access to treatment. Recent changes to UK regulations mean that centres need to act now to avoid future strains on their cryostorage infrastructure, and scaling up LN₂ storage capacities is essential. With this comes an increase in the size and complexity of cryopreservation rooms, and the need to refocus on their safety. By adhering to best practices, and collaborating with trusted professionals, fertility clinics can maintain a secure cryopreservation environment, safeguarding both staff and the viability of precious reproductive materials.

 

References

[1] UK Government. Health and Care Act 2022. Available at: https://www.legislation.gov.uk/ukpga/2022/31/contents/enacted (Accessed 14 June 2024)

[2] British Compressed Gases Association. Codes of Practice. https://bcga.co.uk/pubcat/codes-of-practice/ (Accessed 14 June 2024)

[3] British Compressed Gases Association. Code of Practice 45: Biomedical cryogenic storage systems. Design and operation 2021. https://bcga.co.uk/wp-content/uploads/2021/11/BCGA-CP-45-Original-05-11-2021.pdf (Accessed 14 June 2024)

[4] British Compressed Gases Association. Code of Practice 36: Cryogenic liquid storage at users’ premises 2013. https://bcga.co.uk/wp-content/uploads/2021/09/CP36.pdf (Accessed 14 June 2024)

 

Acronyms

LN₂ = liquid nitrogen, PPE = personal protective equipment

 

Author biography

Gary Brown is a skilled LN₂ specialist at Haier Biomedical. He has over two decades of experience developing and implementing cryopreservation technologies for the biomedical and life sciences sectors, ensuring that technologies continue to evolve to meet the needs of modern IVF facilities.