WHITE PAPER Thermo Scientific Sample Storage Tubes

The anatomy of a cryogenic tube

This paper will discuss best practices in sample storage and review the multiple features of a storage tube and how they can affect handling and overall long term sample integrity.

Considerations in selecting the right cryogenic tube

To ensure reproducible results and continuity when preserving valuable materials used in clinical, cell culture, biomedical and biobank laboratories, samples must be stored and handled with care to prevent contamination or sample degradation. Cryopreservation, or cryostorage, at extremely low temperatures maintains valuable biological organisms and sub-cellular components, such as nucleic acids and proteins, in stasis over long periods. Successful long term storage relies heavily upon the preparation of the specific sample. For example, tissue cultures are prepared and stored differently than bacterial stocks due to their complexity, and chemical compounds can even be stored at room temperature. As such, thoughtful consideration is needed when selecting the appropriate cryogenic vial to keep samples safe during their storage state.

With many features available, such as internal and external threads, round bottom or self-standing tubes, choosing the right storage tube that meets the needs of the application and personnel can prove challenging. Whatever the tube type selected, it is essential that it works within the parameters of the application. For example, if mammalian cells are stored in liquid nitrogen for a year, it is imperative the tube be rated for use at -196°C and have a reliable gasket to prevent any cross contamination from liquid nitrogen (LN2). It is likely that these considerations cross most researchers’ minds when it comes to sample storage. Careful selection of tubes or vials can prevent inconsistent results caused by contamination or sample degradation, minimize the costs associated with tube failures, and help facilitate good handling practices.

Sample storage methods and temperatures

Before looking in more detail at the anatomy of a cryogenic storage tube, it is important to review cold storage methods for some of the most commonly stored samples in life science (Table 1) as they will affect the type of storage tube selected.

Cryopreservation is comprised of techniques and technologies that are used to preserve life at sub-zero temperatures, slowing down, or even halting, the effects of enzymatic or chemical activity and time. If the correct storage equipment and cooling methods are used, samples, such as frozen cells, can remain viable for years, as demonstrated by the data in Figures 1 and 2. Some tissues, such as parathyroid glands, veins, cardiac valves, and aortic tissue have been successfully cryopreserved, in addition to human embryos, ova and sperm. Additionally, cell components such as DNA and RNA can be preserved to stock genetic libraries, chemical compounds, microorganisms and viruses for investigative research, and blood, plasma, urine and tissue can be collected in biobanks.

Table 1: Commonly stored samples in life science applications

Microorganisms (bacteria and archae)

Mammalian cells (including stem cells)

Viruses Embryos

Nucleic acids Proteins

Tissue Chemical compounds

Antibodies/hybridomas Cell lysates

Blood Plant cells

Serum Eggs

Sperm Urine

Saliva

Figure 1: Post-thawing cell viability after long term storage in Thermo Scientific™ Nunc™, Nalgene™ and Matrix™ storage tubes

Figure 2: Total cell recovery after long term storage in Thermo Scientific™ Nunc™, Nalgene™ and Matrix™ storage tubes

0102030405060708090

100

Cel

l Via

bili

ty (%

)

0-month1-month6-month1-year2-year

NuncUniversal

external threadtube

Nalgeneexternal thread

tube

Matrixscrew top

internal threadtube

Matrixopen top tube

with sterileclosure

Cells were prepared in 10% DMSO freezing media and stored in liquid nitrogen

60.0

70.0

80.0

90.0

100.0

110.0

120.0

To

tal C

ell R

eco

very

(%)

Nunc Universal external thread tubeNalgene external thread tubeMatrix screw top internal thread tubeMatrix open top tube with sterile closure

0 0.5 1 1.5 2

Storage Time (yrs)

Cells were prepared in 10% DMSO freezing media and storedin liquid nitrogen

There are various techniques available to stabilize living samples and the choice of method must be compatible with the intended use of the culture. Generally, the larger the cells, and the more complex the sample, the more important it is to freeze it slowly and uniformly. Most bacteria and spore-forming fungi can tolerate less than ideal cooling conditions, whereas protists, mammalian, and plant cells are particularly susceptible to damage during the phase change from water to ice and require uniform, controlled cooling.

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On the other end of the scale, multicellular structures such as embryos need even greater process control than single cells for successful cryopreservation and thawing.1 Long term recoverability of frozen biological samples demands storage temperatures of -130°C or less. Nucleic acids, proteins and cell lysates generally favor very quick flash freezing at -20°C for medium term and -80°C for long term storage. Chemical compounds should be stored in dimethyl sulfoxide (DMSO) at -20°C for the long term and 4°C or room temperature over the short term. Proper storage temperatures should be researched and tested to suit the need of unique samples, as there is some variability. Ultimately, a correct cryopreservation protocol will protect sample integrity, ensuring downstream analysis is not compromised.

Cryogenic Storage Tubes

One part of the storage process that can often be overlooked is the cryogenic or storage tube selection. Although a seemingly small decision to make, the tube can sometimes be a source of contamination, both from a poor quality material standpoint, and poor handling during the process. Selection of equipment and equipment supplier can have a significant impact on any cryogenic program. By choosing a supplier with expertise, users can trust the high quality products will be suitable for any cryostorage application needs.

When selecting a storage tube, several factors should be considered, including:• Cryotolerance and tube construction

• Ease of handling during stocking and retrieval, freezing and thawing

• Sterility of vials and extractable or leachable possibilities

• Types of sample to be stored and tube resin compatibility

• Identification and traceability

The order of consideration of the above will depend on the value of the samples being stored versus the workflow compatibility. Priority should be based on individual sample storage needs.

Cryogenic tubes protect the contents from the external environment during processing and cold storage. The choice of container should optimize the sample’s viability during storage, retrieval and handling. Any leaks would result in sample contamination and physical damage can occur during sample handing and transport. It is, therefore, critical to select a tube that has been constructed in such a way as to maximize robustness and seal reliability while maintaining ease of handling.

When storing material in LN2, tubes designed specifically for cryogenic conditions must be used. For temperatures above -100°C, a variety of tubes are appropriate, but gaskets should be taken into consideration as evaporation is a real threat regardless of storage temperatures. The rate of warming will also affect tube selection.

Tube construction and cryotolerancePolypropylene cryogenic tubes come in many varieties, shapes and sizes, such as the Thermo Scientific™ Nalgene™, Thermo Scientific™ Nunc™ and Thermo Scientific™ Matrix™ tubes and caps. Tube shape will affect storage and handling, and must be compatible with available cryogenic workstations, racks and trays. Flat bottomed vials will free-stand, while round and conical bottomed vials are designed to improve sample retrieval (Figure 3). The addition of self-standing feet will allow round and conical bottomed vials to free-stand and some base shapes are designed specifically to lock to the stand for more secure transport and handling. Tube design that aids sample identification and traceability to enable efficient retrieval of data is also an important selection consideration.

Tubes available with skirted bottom, starfoot, and round bottom, internal and external thread options, and available with graduations or white patches

Figure 3: Thermo Scientific Nalgene and Nunc cryotubes

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Internal vs External ThreadsTubes can be constructed with internal or external threads for caps to screw onto (Figure 4), or open top with no threads for septum caps to push into. For any storage below -80˚C, it is ideal to use a threaded tube with screw-on caps.

There has been some discussion to suggest that internally threaded cryogenic tubes offer an improved seal because they have equal outside dimensions for cap and vial which will, therefore, expand by equal amounts as the temperature changes. However, evidence demonstrates that contraction is so small when these containers are placed in LN2 that any change in size will not strain the seal.

The choice of internal or external thread construction is, in fact, entirely dependent on user preference and how tubes are manually handled by individuals. If aseptic best practices are applied, both models perform well. As demonstrated by Figure 1, cell viability remains at 80% or more after thawing from one month, six months, and one year storage, regardless of which closure type is used. All cryogenic tubes in this study were prepared, handled and stored (in vapor phase of liquid nitrogen) in the same way, which indicates that internal and external caps did not have significant effect on cell viability or total recovery after two years (Figure 2). There are no known differences in tolerance between these two designs and both types provide equally good seals. If over-torqued on closure, both designs can have issues with seal integrity. In automated workflows, any impact of cap and thread style is effectively eliminated through proper instrumentation settings. An internal study that questioned researcher preference and usage of cap closures found that 55.9% favored internal threaded closures and 44.1% favored external closures (Figure 5). The small spread in difference between cap types indicates that both closures are used in storage protocols without significant dominance of one in particular.

Cap gasketFor added seal integrity during storage, a gasket can be used between the tube and the cap to enhance the seal. The gasket is made from a soft material so it can compress and conform to the shape of the mating surfaces, filling the interface between cap and tube surfaces. This gasket can be either a silicone rubber O-ring that sits inside an external thread or outside an internal thread, or a thermoplastic vulcanizate (TPV) material molded directly onto the tube closure to produce a one-piece closure and gasket.

Figure 5: Do you prefer internal- or external-threaded closures?

InternalExternal

A Internal 33 55.9%

B External 26 44.1%

Researchers who perform sample storage and cryopreservationwere polled on preference of internal or external threaded closures. All answers were collected anonymously.

External thread Internal thread

Figure 4: Polypropylene cryogenic tubes can be manufactured with either an internal or external thread for the cap fitting

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Both gasket types require careful handling during closure. Overturning of the closure threads can cause excessive compression force and torsion simultaneously, causing the gasket material to extrude from the joint between cap and tube. Any exposed surface of the gasket is then subject to a higher risk of degradation, which could lead to leaking.

Silicone rubber O-rings provide an effective seal despite having two interfacing surfaces between the cap and the tube and have the advantage of being removable. Additionally, silicon-based polymers can cause increased gas permeability, which can result in sample dehydration if left at room temperature for long periods of time. Some tubes incorporate a specially designed lip inside the cap to prevent extrusion of the O-ring material and reduce gas permeation by ensuring a leak-proof seal.

A class of thermoplastic elastomer (TPE), TPVs offer the elastic and compressive properties of silicone rubber in combination with the ability to be molded like thermoplastics. This means TPV gaskets can be integrated with the plastic tube or cap to produce a single piece and reduce the number of compression-dependent mating surfaces in the cap/tube seal to one. This design also minimizes gasket extrusion, and reduces the risks associated with both overtightening and gas-permeability at room temperature.

Case study; seal integrity of TPV-sealed tubes4

A study carried out by Thermo Fisher Scientific investigated the seal integrity of TPV-sealed tubes. Four tube sizes (0.2, 0.5, 1.0 and 1.8 mL) were tested with both internal and external thread closures. New tubes and tubes aged in an oven to simulate five years of shelf life were tested. All tube capping was carried out using a Thermo Scientific 8-Channel Handheld Decapper for consistency. Tubes

were tested for leakage during at least one freeze/thaw cycle (Table 2) and evaporation during and after the thawing process following overnight in LN2 (Table 3). The goal of the study was to subject the tube materials to a worst-case scenario to maximize stress on the gasket and induce failures by exceeding product specifications. Less than 3.3% leakage failure was seen for all tube types (closure styles and sizes, new and aged tubes), indicating TPV gaskets are reliable and not prone to leakage of material out of the tube or from the environment into the tube.

The data also showed less than 0.93% evaporation in new tubes (not affected by aging or long term storage conditions) and less than 0.43% for aged tubes, which mimic the quality of storage tubes after long term exposure to storage conditions. Less than 1% evaporation is very small and shows that the TPV gasket type helps greatly reduce evaporation. It should be noted that whatever percentage of evaporation is witnessed in the study, it could also be partly attributed to any permeability through the polypropylene tube itself, as polypropylene is permeable to gases at some level.

Table 2: Tube failures in freeze/thaw leak test (%).

New tubes Aged tubes

Tube TPV TPV

Matrix 200 μL internal thread Screwtop Tubes 0% 0%

Matrix 0.5 mL internal thread Screwtop Tubes 1.6% 0%

Matrix 1.0 mL internal thread Screwtop Tubes 0% 3.3%

Nunc 1.8 mL external thread Tubes 0% 3.3%

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tube contents, it can cause cross-contamination when stored in LN2. To minimize the risk of contamination, it is recommended that all external surfaces are wiped with alcohol-soaked gauze.

Automated capping and decappingLike everything else in the laboratory, process control can not only improve efficiency but help reduce additional contamination caused by human error or the millions of particulates generated with every breath or movement. Sample integrity can be maintained by using a hands-free method of capping and decapping tubes, and even picking and moving around tubes with robotic arms.

For example, the Thermo Scientific™ Capit-All™ Screw Cap Tube Capper/Decapper is designed to eliminate any need to touch the cap with gloves, skin or a non-clean surface. Using capping and decapping equipment will eliminate virtually all risk for contamination. Moreover, automation does not discriminate between internal or external threads, so any preferences become less relevant. As an added benefit, equipment like the Capit-All capper is engineered to offer additional anti-contamination features. For example, unlike manual capping processes, the instrument can be used to inject any inert gas through the system to displace atmospheric water, vapors and air, all of which might also impact the integrity of the sample.

Tube SterilityTo avoid contamination, careful sterilization of tubes and vials at the manufacturing stage is essential. Look for tubes that have undergone gamma radiation or other guaranteed sterilization. Different sterility standards are available, so seek those suited to your needs. As a general rule, a Sterility Assurance Level (SAL) of 10-3 should be standard and SAL 10-6 should be used for highly valuable samples.

Table 3: Sample evaporation (mg) over 30 days

New tubes Aged tubes

Tube Average calculated weight loss (mg)

Average percent sample loss

Average calculated weight loss (mg)

Average percent sample loss

Matrix 200 μL internal thread Screwtop Tubes 1.86 0.93% 0.86 0.43%

Matrix 0.5 mL internal thread Screwtop Tubes 0.30 0.06% 1.47 0.29%

Matrix 1.0 mL internal thread Screwtop Tubes 0.77 0.08% 1.47 0.15%

Nunc 1.8 mL external thread Tubes 0.50 0.03% 1.96 0.11%

Tube handlingCareful, standardized, aseptic handling protocols are key to a well-managed storage program that aims to produce consistent results. Variation in processing and storage can have significant impact on biological samples. Careful tube filling and transport are essential to prevent overfilling or dampness around the tube threads, both of which can result in tube failure. Generally, a standard tube is designed to store between 0.5 and 2.0 mL of material but there are many sizes available. Whichever size is used, it is important not to fill the tube beyond, generally, 85% nominal capacity, to leave expansion room for aqueous components upon freezing, thus ensuring seal integrity is maintained.3 Also, while careful aseptic techniques are similar when using any model of cryostorage container, the choice of tube design can make a difference when accounting for the way each individual researcher holds the tube and cap during manual capping and decapping.

When tubes are filled, the thread can undergo stress and potential breakages depending on how it is handled by the user. If water is retained near the threads during the freeze/thaw cycle, ice expansion can cause tube fractures.4

A chief concern for all cryopreservation protocols is contamination. How a tube is handled has major influence on the potential for contamination. If the thread is inside the tube, there is automatically less risk of the inside of the tube being touched and therefore aseptic handling is easier. However, the user must take care when removing the cap, not to touch the thread on the cap or leave any materials on the cap thread.

When decapping externally threaded tubes, the user must be careful not to allow liquid onto the external threads. While excess liquid on the threads is no threat to the

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10-6 means that there is a one in a million chance that a purchased tube will be contaminated in some form from the manufacturer. If high value samples are being stored, care should be taken to ensure that the storage vials are of the utmost quality so that no byproducts could possibly alter, degrade, or damage the stored sample. Certifications such as RNase and DNase free, CE mark, endotoxin and pyrogen free should be considered for the tube, depending on the sample being stored.

Tube manufacturers should meet GMP and ISO Quality Standards, and be suitably certified. Tubes should be manufactured, assembled and packaged in an ISO 7 (Class 10,000) cleanroom, and transport and storage should be in tamperproof, resealable, safety-lock bags to ensure an unbroken chain of contamination protection prior to aseptic transferal to the cleanroom.

Thermo Scientific tubes are available with pyrogen-free, nuclease-free and endotoxin free certifications. Tubes are constructed using virgin grade polypropylene resins, which are resistant to many chemical solvents, bases and acids and naturally have few extractables and leachables due to their purity.

Identification and trackingWhile not always of major concern for sample integrity, sample tracking is a good lab practice that ensures samples are monitored through storage and correctly identified during retrieval.

Many tubes offer white marking areas and/or color coded caps and cap inserts to aid visual identification. Laser etched, unique two-dimensional barcoding provides highly reliable sample identification, while human readable one-dimensional barcodes remove the risk of selection errors during rapid manual identification for peace of mind (Figure 6).

Codes can be customized to include varying amounts of data for more sophisticated sample tracking. Furthermore, white patches for personalized labeling removes the risk of misidentifying samples within the cryobox.

2D barcode accompanied with human readable, alphanumeric

code on the bottom of tube

Non-proprietary, 12x12 Data-matrix with ECC200 Built-in

Error Correction

Figure 6: Variety of barcodes available on cryotubes for sample tracking

Linear barcode accompanied with human readable, numeric barcode

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Find out more at thermofisher.com/samplestorage

For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COXXXXXX 0016

Conclusion

Ultimately, selecting the best storage tube will be dictated by the complexity of the sample being stored, the scale of the experimental set-up, and the manual or automated process of the researcher. With so many tube features to consider that can maximize sample integrity during the storage process, it really is an important decision worth investing in early on in order to reduce risks to your samples later. Any downstream experiments will rely on that sample integrity to produce reliable and consistent results.

As a crucial, yet often overlooked part of an experimental workflow, there is no doubt that the quality and efficiency of the sample storage and cryopreservation process can have a huge effect on experimental outcome and data quality. Ultimately, sample preservation could be the deciding factor between experimental success and failure. For many scientists working with high-value, precious or rare samples and compounds, this is a risk that is just too great to take.

For 40 years, researchers have trusted the Thermo Scientific brand for safe sample storage, even in the harshest conditions. Each container within the extensive portfolio benefits from high purity resins, high sterility assurance levels and strict quality control standards. This provides users with products to meet their best practice preferences, as well as confidently maintaining sample integrity.

References 1. DNA Storage and Quality. http://www.ogt.com/resources/literature/403_dna_

storage_and_quality. Accessed April 27, 2016.

2. Scholz, EC. The Problem of Contamination: Open vs . Closed vs . Semi-Closed Vitrification Systems. www.intechopen.com

3. Cryogenic Preservation. 2012:1-16.

4. Scott, R, Granchelli, J. Gasket material and seal integrity in Storage Tubes.

40 What value do you put on trust?

Coming to the rescue in cryogenic storage for 40 years.