How to Choose the Right Cryogenic Storage Vial for your Laboratory

What are Cryovials?

Cryogenic storage vials are small, capped and cylindrical containers designed for storing and preserving samples at ultra-low temperatures. Although traditionally these vials have been made from glass, now they are much more commonly made from polypropylene for convenience and cost reasons.  Cryovials have been carefully designed to withstand temperatures as low as -196℃, and to accommodate a wide variety of cell types. These vary from diagnosis stem cells, microorganisms, primary cells to established cell lines. Beyond that, there may also be small multicellular organisms that are stored within cryogenic storage vials, as well as nucleic acid and proteins which need to be stored at cryogenic storage temperature levels.

Cryogenic storage vials come in various different forms, and finding the correct type that fulfils all your needs will ensure that you maintain sample integrity without overpaying. Read through our article to learn more about the key buying considerations when selecting the right cryovial for your laboratory application.

Properties of Cryogenic Vial to Consider

External vs Internal Threads

People often make this choice based on personal preference, but there are in fact key functional differences to consider between to the two types of thread.

Many laboratories often opt for internally threaded vials to minimise tube storage space to allow a better fit into freezer boxes. Despite this, you may consider that the externally threaded option is the better option for you. They are considered to carry a lower contamination risk, due to the design which makes it is even harder for anything other than the sample to enter the vial.

Externally threaded vials are generally preferred for genomic applications, but either option is considered suitable for biobanking and other high throughput applications.

One last thing to consider on threading - if your laboratory uses automation, you may need to consider what thread can be used with the instrument grippers.

Storage Volume

Cryogenic vials are available in a variety of sizes to cover most needs, but mostly they range between a capacity of 1 mL and 5 mL.

The key is to make sure that your cryovial is not overfilled and that there is extra room available, in case the sample swells while freezing. In practice, this means that laboratories opt for 1 mL vials when storing samples of 0.5 mL of cells suspended in cryoprotectant, and 2.0 mL vials for 1.0 mL of sample. Another tip for not overfilling your vials is to make you use cryovials with graduated markings, which will make sure you prevent any swelling which could cause cracking or leaking.

Screw Cap vs Flip Top

The type of top you choose depends chiefly on whether you will be using liquid phase nitrogen or not.  If you are, then you will need screw capped cryovials. This ensures that they can’t pop open accidentally due to mishandling or temperature changes. Additionally, screw caps allow for easier retrieval from cryogenic boxes and more efficient storage.

However, if you are not using liquid stage nitrogen and need a more convenient top which is easier to open, then a flip top is the better option.  This will save you a lot of time as it much easier to open, which makes is particularly useful in higher throughput operations and those that use batch processes.

Seal Security

The best way to ensure a secure seal is to make sure that your cryovial cap and bottle are both constructed from the same material. This will ensure that they shrink and expand in unison. If they are made using different materials, then they will shrink and expand at different rates as the temperature changes, leading gaps and potential leakage and consequent contamination.

Some companies offer dual washers and flange for the highest level of sample security on externally threaded cryovials.  O-Ring cryovials are considered the most reliable for internally threaded cryovials.

Glass vs Plastic

For safety and convenience, many laboratories now use plastic, usually polypropylene, instead of heat-sealable glass ampules.  Glass ampules are now considered to be an outdated choice as during the sealing process invisible pinhole leaks may develop, which when thawed after storage in liquid nitrogen may cause them to explode. They are also not as suitable to modern labelling techniques, which is key to ensuring sample traceability.

Self Standing vs Rounded Bottoms

Cryogenic vials are available both as self-standing with star-shaped bottoms, or as rounded bottoms. If you need to place your vials on a surface then make sure to choose the self-standing

Traceability and Sample Tracking

This area of cryogenic storage is often overlooked but sample tracking and traceability is a crucial aspect to consider.  Cryogenic samples can be stored for many years, over which time period staff can change and without properly maintained records they can become unidentifiable.

Make sure to choose vials which make sample identification as easy as possible. Things you should look out for include:

  • Large writing areas to record sufficient details so records can be found if a vial is located in an incorrect location - usually cell identity, date frozen, and initials of the person responsible are adequate.

  • Barcodes to aid sample management and tracking systems

  • Coloured caps

A note for the future - ultra-cold-resistant chips are being developed which, when fitted within individual cryovials, could potentially store a detailed thermal history as well as detailed batch information, test results and other relevant quality documentation.

In addition to giving consideration to the different specifications of vials available, some thought also needs to be given to the technical process of storing cryovials in liquid nitrogen.

Storage Temperature

There are several storage methods for the cryogenic storage of samples, each operates at a specific temperature. Options and the temperature they operate at include:

  • Liquid phase LN2: maintain a temperature of -196℃

  • Vapour phase LN2: are capable of operating at specific temperature ranges between -135°C and -190°C depending on the model.

  • Nitrogen vapour freezers: -20°C to -150°C

The type of cells being stored and a researcher's preferred storage method will determine which of the three available options your laboratory uses.

However, due to the extremely low temperatures employed not all tubes or designs will be suitable or safe. Materials can become extremely brittle at extremely low temperatures, using a vial not suitable for use at your chosen temperature could cause the vessel to shatter or crack during storage or thawing.

Carefully check the manufacturers’ recommendations on proper use as some cryogenic vials are suitable for temperatures as low as -175°C, some -150°C others just 80°C.

It is also worth noting that many manufacturers state that their cryogenic vials are not suitable for immersion in the liquid phase. If these vials are stored in the liquid phase when returning to room temperature these vials or their cap seals may shatter due to a rapid build-up of pressure caused by small leaks.

If cells are to be stored in the liquid phase of liquid nitrogen, consider storing cells in suitable cryogenic vials heat-sealed in cryoflex tubing or storing cells in glass ampules that are hermetically closed.

Sterility & Regulatory Requirements

Clinical samples and some scaled-up processes can potentially become contaminated during storage and filling. Choose sterility-assured gamma-irradiated at a level of 10–6 according to ISO 1137 to ensure absolute sterility. Internally threaded vials are preferable to prevent contamination during filling.

Any cryogenic vials used to preserve cells for human therapy will need to meet any additional regulatory requirements including the absence of bioactive leachables and other particulates as well as closure integrity during storage.

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InsightsPierre Frizelle