Advancing best practices in cryogenic cold chain solutions
Sharing fundamental research, education, discussion and best practices for cryogenic cold chain management, biobanking, glass transition; biosample storage, preparation, planning and recovery and associated issues.
John Fink is marketing manager for cryogenic solutions at Brooks Life Science Systems, the global leader in automated cold-chain sample management for drug discovery and biostorage applications and a division of Brooks Automation, Inc. if you have any questions or ideas for future blog posts.
For over a year we stored Jurkat CAR-T cells in four different conditions to understand what temperature and cryoprotectant media performs best. The two temperature conditions were -80°C and -190°C and the two medias were a home-brew cocktail of FBS/DMSO 95%/5% and cGMP manufactured CryoStor5. At ISBER earlier this year we presented the results [download poster here].
In summary, the highest performing group was the Jurkat T-cells prepared with CryoStor5 media and stored at -190°C. Their post-thaw viability and metabolic activity after 410 days in storage was nearly identical to the non-shipped control. The group stored at -190°C with home-brew media had reduced viability and both groups stored at -80°C had reduced viability and reduced metabolic activity.
This brings to mind that to keep your cells safe while frozen and thus, viable upon thawing, both temperature and cryoprotectant media are important.
We kept the experiments running and at ISCT 2017 in London last month we published another poster of the same cells and study, now after 70 exposures and 13 months storage. The results were similar; the variable group (subjected to TWEs) had lower viability, lower recovery and lower proliferation than the control group that never warmed above Tg.
The “But…” is that statistically, the TWE variable group was no worse after 13 months and 70 exposures than vs. 3 months and 20 exposures. This leads us to believe that the damage from TWEs happened in less than 20 exposures. How soon? We do not know yet, but I hope to set up some new experiments with partners to determine just that.
The effects of transient warming events (TWE) are not widely known yet, but thanks to this type of research we are getting the word out.
Some further discussion: an interesting trend we saw on this new data after 13 months storage is a noticeable change in some of the antigen surface markers. Although they tend to up/down express the same on all batches, there is a difference between the -190°C control and TWE variable group this time on CD19 & CD34. The other interesting bit on the -80°C experiment group is the -80°C to -190°C variable batch (think LN2 to dry ice and back) were nearly all dead (<4% viability) whereas last year’s same variable batch had similar results to the -80°C control batch (83% viability).
Our good friends at BioCoR asked me to write an article summarizing automation then and now. Please read it in the BioCoR February 2017 Newsletter and be sure to visit their website as it has much valuable information around preservation.
In a post last June we discussed a Poster we presented with BioLife Solutions around standards and best practices for cryopreservation, transport and storage. Since we had strong interest in the data, we gathered back together and wrote the data into a white paper with far more detail and depth. If you would like to read it, please download it here.
Note: Stay tuned as we have more original samples of these vials chilling at -190°C nearing the one year storage time. We plan to run the same methods/experiments to see how best vs. status-quo practices affect the cells after 1 year of storage and then compare vs. the original 3 months storage.
Frost is generally a sign of poor sample handling and/or controls. Frost accumulates from samples (vials, boxes) being exposed to an ambient environment where moisture in the air condenses on them and freezes to frost when back in the freezer. Some frost is nearly unavoidable (unless you are in a hermetic environment), but excessive frost is normally a sign of samples being out of the freezer too long or with -80°C ULT freezers, the door being open too long. In both circumstances, innocent samples are warming! Frost can cause a few other problems, for example in fully automated systems frost can prevent reading barcode labels and may cause the vials to freeze to trays causing picking problems.
We moved the vials to a CryoPod and wearing cryo gloves, easily wiped the frost off the vials. Also of note was a frost bed in the bottom of the cryobox of about 1cm deep which we dumped out. We then put the vials and cryobox back into the LN2 freezer and after allowing a day to re-equilibrate, monitored the next several warming exposures and the results were surprising.
The frost actually slowed the warming rate of the samples!
Why is this? The frost is snow/ice, and when exposed to the ambient room it absorbs some of the energy (heat) keeping it from entering the sample vial. Of course the sample will still warm and risk crossing Tg, but at a slightly slower rate.
Bottom Line – We do not advocate allowing frost to accumulate on your sample vials, we actually recommend you do what is possible to minimize frost as frost means exposures (warming!). We just thought this is interesting information to pass along.
There is good understanding that colder temperature storage yields better preservation of biological material. Generally, with proper cryo preservatives, freezing and thawing protocols, what you put into a LN2 vapour freezer is what you will get back out – including high recovery, viability and functionality of your cells.
But what if you do everything right and still have poor recovery post thaw? Many experts have pointed to the concern of transient warming – that brief time all innocent vials are exposed while the rack is out of the freezer. Even though just 1 vial may be retrieved, 1,000+ others are exposed to 200°C+ warmer temperatures, possibly daily/weekly over months or years of storage.
What is the effect on the cells due to this transient warming?
We have already published data on the rate of warming of different individual vials, vials in cryoboxes and even that vials in cryoboxes still warm when back in the freezer! But we did not have any (and could not find any published) about the effect of transient warming on cells in a typical LN2 freezer workflow (ie. exposed few times a week, always allowed time to re-equilibrate).
We partnered up with Cook Regentec who are focused on developing research and clinical tools for Cell Therapy. They sourced and cryo preserved mesenchymal stem cells and shipped to us in LN2 dry shippers. We divided them into four batches and stored two of the batches in a -190°C LN2 vapour freezer. One batch was left in the freezer and never touched, the other was pulled up in the rack 20 times over 67 days for just long enough to allow the cells to cross Tg (glass transition). After the 67 days we sent back three samples from each batch to Cook Regentec who performed viability and recovery testing. The results were surprising…
Comparing the control batch (never warmed to Tg) vs. the variable batch (warmed above Tg), the variable batch’s viability was only about 6% lower, but its recovery was approximately 50% lower. This means that approximately 50% of the cells exposed to transient warming above Tg failed to live and even be detectable after thawing/washing.
This study is ongoing and we plan to pull more samples and run the same tests after one year of storage.
One thing is clear thus far, no matter how you store and retrieve your samples from LN2 storage (automated or manual), be sure all exposures are quick and controlled enough to avoid crossing Tg.
People often think the path to automation is a very steep incline costing a lot of time and money, but it doesn’t have to be.
In this poster from the Mayo Clinic they describe how they are moving a legacy cryo vial collection towards automation through small steps at much less cost and complexity than if done in one big step.
Their steps to automation are:
Purchase an Automation-Ready LN2 Freezer (able to be upgraded to full automation).
Much less expensive than purchasing full automation up front.
The legacy collection is all hand written IDs. Therefore, they are moving legacy unbarcoded vials into barcoded cryoboxes that will enable tracking in LIMS going forward.
Moving legacy samples into barcoded vials would thaw the samples.
These new barcoded cryoboxes will go into the Automation Ready LN2 Freezer and the inventory can be recorded.
Going forward, new samples will be stored in barcoded vials and cryoboxes which will be stored in the Automation Ready LN2 Freezer.
When sample demand increases they plan to upgrade their Automation Ready LN2 Freezer to a full BioStore III Cryo storage system.
For labs interested in cryo automation, it is good to know you can step into it over time rather than in one giant leap.
Continuing on with our research of best practices and now adding cell viability into the mix, we partnered up with BioLife Solutions to do experiments and present a poster at ISCT 2016. These experiments focused on best vs. status-quo practices involving cryoprotectants (CPAs) and dry ice shipping containers.
BioLife prepared T-Cells (Jurkat cells) with two different cryoprotectants and packed in two different shipping containers. They shipped the cells to our office in Chelmsford, MA where we stored them in a BioStore III Cryo -190ºC to ensure restricted access, temperature and to replicate an offsite storage operation. After 45 days of LN2 storage we shipped the cells back in the same containers as they arrived. Upon receipt, BioLife thawed and performed viability and testing on the cells.
The results showed that using BioLife’s commercially available cryoprotectant CryoStor®, and -80ºC evo™ smart shipper gave higher post-thaw cell viability and 24-48hr functionality vs. the typical home-brew methods of DMSO and EPS shippers.
What does all this mean? It brings focus on the importance to adopt standardized equipment and processes. Whereby some scientists may prefer their home brew processes and equipment, do those processes and equipment protect the samples as much as standardized technologies?
The International Society of Biobanking and Biorepository Annual Conference (ISBER) was held in April in Berlin, Germany. We conducted some new experiments and published a new poster at ISBER that compared typical workflow, time and temperature of manual vs. automated LN2 storage.
The Manual workflow, as you would expect, included racks pulled out by hand into the air. For the automated system we used the BioStore III Cryo storage system.
As one would expect with any automation, the user steps are greatly reduced and any time you can reduce manual intervention you will reduce the risk of human errors. Also ergonomics and user safety were improved.
On the temperature side, the results were interesting. Keep in mind that we previously found that convection was the primary source of innocent sample warming. Because of these findings, the BioStore III Cryo was designed to pull racks into an insulated sleeve. Our test data showed a 70% reduction in innocent sample warming during the first 30 seconds of exposure using the BioStore III Cryo vs. Manual.
With both Manual and BioStore III Cryo workflows, the top box innocent sample warmed the quickest. During the first 30 seconds of manual workflow a vial in the top box warmed at about 20°C/min, whereas in the BioStore III Cryo workflow it was near 7°C/min. Keep in mind that shelf height, sample volume, vial location and the presence of surrounding vials all affect the rate of warming. There is no one rate-of-warming for all situations.
Furthermore, a common manual workflow practice is to leave the rack out of the LN2 freezer while picking vials. Testing this workflow we found the top box innocent sample in a manually lifted rack to cross Tg (-135°C) in approximately 2 mins, whereas with the BioStore III Cryo it takes over 4 mins. Again, keep in mind that samples continue to warm after returned to the freezer, so if you returned your samples before 2 mins at -140°C they would warm far above Tg during the next few minutes of being back inside the freezer.
Have you ever wondered where liquid nitrogen (LN2) comes from? Just look around you and take a deep breath…
We often just hook our cyro freezers up to a metal cylinder, which a truck dropped off or filled, and magically our freezers stay unbelievable cold. But where does all that super cold fuel come from..?
LN2 boils from a liquid to gas at -195.8°C @ 1ATM (sea level). When in a freezer, the LN2 absorbs the surrounding heat, boils, and thus cools the environment. You can think of the LN2 in your freezer as a fuel source, a sacrificial refrigerant that absorbs heat and in doing so, changes back nitrogen gas (N2).
Converting back to a gas is key here. Nitrogen is not naturally in liquid form (on earth at least, Pluto is another story). So the liquid form of nitrogen we use has to be manufactured.
On earth, nitrogen exists all around us and approximately 78% of the air we breathe is nitrogen. This is the nitrogen source used to create liquid nitrogen.
Liquid nitrogen is created (or changed) from nitrogen gas in a process called Air Separation or Fractional Distillation. It is a complicated process, but essentially takes regular air, purifies, compresses and cools it to clean it up and remove the water and then the fractionation begins. In this process the purified air is significantly cooled, compressed and decompressed using heat exchangers and pressurized tanks. During this process the elements in the air turn to liquid and are then captured and separated.
LN2 isn’t the only liquid separated from the air. More valuable are the other elements in the air, such as oxygen and argon. Some even consider liquid nitrogen a by-product of air separation where more valuable gases are needed (worth more money) and LN2 happens to be created in the same process.
Once liquid nitrogen has been separated, it is kept under very high pressure to keep it in a liquid phase and is transferred to storage tanks, trucks and cylinders. This allows it to be easily transported to where it is needed.
The next time your LN2 freezer tops up you can take a deep breath and ponder that the air all around you is just a phase change away from being the cryo fuel source in your freezer.
We have focused on cryogenic temperatures and workflows (below -150ºC) on this blog thus far, but what happens upstream of cryo storage?
Obviously bio-samples start fresh and are later frozen to preserve them over time. Fresh cells specifically, should never be placed directly into an LN2 vapour freezer. They are normally prepared with a cryoprotectant and then frozen in a controlled manner to minimize cell damage during freezing and subsequent thawing. Without these precise freezing protocols, post thaw recovery would be poor.
The controlled freezing may be done in an automated device or a passive device (i.e. the CoolCell®) placed in a ULT mechanical freezer, but in both these cases the freezing device is separate (and may be far, far away) from the LN2 storage freezer.
So what happens between the freezing device and the LN2 freezer? How are the samples handled and moved? There will be transient warming during any sample movement outside of a controlled environment. And what are the variables that affect warming during this move?
At ESBB in London I presented a poster that demonstrated an example workflow of moving samples at -80ºC to an LN2 freezer. During the transfer we monitored the sample temperature and some variables that increased or decreased the rate of warming.
What we found is the rate of warming at -80ºC is less than half compared to the rate of warming at -190ºC, approx 0.5ºC/sec. We also found that transferring the -80C samples into a cryobox already at LN2 temperature stopped the transient warming (for how long though, we did not test). Lastly, the rate of cooling once inside the LN2 freezer is affected by the presence of surrounding samples.
The take-away from these experiments is if you are concerned with controlled rate freezing of your samples, you should also be concerned with how you transfer these samples to the LN2 freezer, lest you get uncontrolled warming.
Best practices that I recommend after performing these experiments are:
Minimize the time the samples are outside of a controlled environment.
Best case is to locate or take the -80ºC environment close to the LN2 freezer. Alternatively, bring the LN2 environment to the ULT freezer (i.e. CryoPod).
Transfer the -80ºC sample(s) quickly to an already LN2-frozen cryobox.
We have talked about the rate of warming of samples, the danger of crossing Tg and best practices to avoid and control warming, but we have not performed any complete workflow experiments while monitoring sample temperature.
At the ESBB Conference in London last month I presented a poster that did just this. We stored a sample in the BioStore™ III Cryo storage system (B3Cryo), retrieved it, moved it to a CryoPod Carrier, moved the vial to simulate a picking/identifying operation, and finally stored the vial back into the B3Cryo system. We monitored and logged the sample temperature throughout all the operations.
The good news is that at all times the sample stayed below -135°C (Tg). The cautionary news is that timing is critical during all transient exposures.
The purpose of the experiments and poster was to ensure samples did not cross Tg during a typical workflow when using new cryo technology products.
When it comes to cryogenic cold chain management, there are continued learnings and best practices we can take from these experiments that also apply to manual use.
Timing is critical during any and all exposures outside of the initial cryogenic environment.
When the vial was lifted out of the CryoPod (from -183°C) it warmed at 1.2°C/second. We’ve seen this warming rate before and it is encouraging to see it is repeatable.
Conduction heat transfer improves the rate of cooling
The vial in a cryobox cooled quicker (re-equilibrated) when placed back into the CryoPod than when put into an LN2 freezer. This is interesting, because the LN2 freezer is actually colder (-190°C vs -183°C). The reason for this is the addition of conductive heat transfer. The vial sat in a cryobox on the metal bottom of the CryoPod basket which has liquid nitrogen in an absorber underneath it. Therefore, rather than primarily cooling from convection (like in an LN2 vapour freezer), the vial experienced conductive cooling in addition to convective cooling. This resulted in much faster sample temperature recovery, as you can see in Figure 3 in the poster.
Innocent samples offer insulation (both good and bad)
When the vial was placed back into the LN2 freezer we placed one vial by itself and another in the middle of surrounding vials (that were already in the LN2 freezer). The surrounded vial warmed less, but cooled slower, whereas the alone vial warmed more, but cooled faster (interesting!). This is another example of a variable that effects warming/cooling and should be considered in an SOP. [Figure 4]
We like to think that when we put samples back into the freezer, they immediately begin to cool. That “Phew, they are safe again” feeling, but this is not the case. It turns out that samples continue to warm for several minutes after being returned to a -190°C environment before they slowly cool again. Furthermore, the samples can actually warm more when back in the freezer than when outside the freezer!
At ISCT in Las Vegas this past May, Julian Warhurst presented experimental data which demonstrated in all scenarios that when racks are exposed to ambient conditions then promptly returned back into a ‑190°C LN2 vapour freezer, the samples inside the cryoboxes continued to warm. There are several variables that effect how much and for how long they warm, such as cryobox location in the rack, vial location and population density.
How does this relate to cryogenic cold chain management? The important learnings from this research are to understand and plan for the warming that is happening to samples after they are returned to the freezer and also the long time it then takes for those samples to equilibrate to the freezer’s temperature.
For example a prudent protocol may account for sample warming in the freezer and estimate a two minute exposure raises sample temperatures by 50°C to -140°C, but does the protocol also account for a repeat exposure several hours later where another two minute exposure may warm the samples above Tg?
Aside from an inclusive SOP, pen, paper and stopwatch, developing intelligent software may be the best way to record, predict and manage sample access to ensure there is no risk of crossing Tg in any scenarios.
In order for scientific breakthroughs to be effective, they must be reproducible. This isn’t always simple though, something I learned early on in grade school science labs…
At the professional level however, there are reported instances where published research was not able to be reproduced by other scientists. Some estimate this irreproducibility as high as 50%. What is the cost of this irreproducible research? According to NPR’s “All Things Considered”, experts estimate the potential cost to be as high as $28 billion (that’s billion with a “B”). And although, some believe this dollar value is sensationalized, there is always room for improvement. In fact, GBSI is pushing for standardizations for cellular research and subsequent publications.
How does this relate to cold chain management?
Cold space storage of samples or product materials is a function with a significant quality aspect, because any variability in storage is carried forward to all future use of the stored material – any changes are irreversible.
The old adage garbage-in, garbage-out holds true for cryogenic storage, what you put into the freezer is what you will get back out. However, if your cold chain management standards are not consistent and of a high quality you may have gold-in, garbage-out and thus, non‑reproducible research.
It’s important to understand sample warming at all levels of your workflow and with all types of consumables to truly manage your cold chain. At ISCT in Las Vegas, Dr. Salvetti presented a poster on thermal excursion research at the vial level — which nicely complements his ISBER cryobox warming research poster.
In every workflow, individual vials are accessed (picked) and when they are separated from their cryo surroundings, they warm extremely quickly: From -173°C to Tg of water (-135°C) in as few as 9 seconds! Additionally, in every experiment tested here, the vials warm faster to -90°C on dry ice than in ambient. This phenomenon warrants a separate conversation (and future posting) which I plan to have with Dr. Salvetti to dig deeper into why this is happening.
In summary, understanding how fast vials will cross glass transition (Tg) allow scientists to create SOPs to ensure valuable samples stay well frozen (or should I say “glassy”). Understanding the individual variables effecting warming is also important. Obviously, the temperature delta is most critical, but the fill volume and touching the vial lid has minimal increased warming effect.
Good biological cryogenic practices tell us to store as cold as possible, minimize freeze-thaw cycles and always protect innocents from warming above Tg (the glass transition temperature, -135°C for H2O). But just how quickly do samples warm? Are there variables that speed or slow warming?
Dr. Matteo Salvetti, Sr. Product Engineer, presented data at ISBER 2015 which discussed the thermal excursions of different types of vials within different types of cryoboxes. The results are surprising. For example, samples can warm as quickly as 70°C/minute and a sample can warm from -180°C to -135°C (Tg) in less than 40s. A vial’s location and its surrounding vials have a huge effect on its warming rate.
And did you know that when using a cryobox with holes in the bottom, its samples will warm faster when placed from -180°C onto dry ice than when placed in an ambient environment? Quite counterintuitive, but likely due to increased convection from the sublimation of the dry ice.
The International Society for Biological and Environmental Repositories’ (ISBER) annual meeting in Phoenix in May was particularly exciting and there were three major product announcements — two planned and one surprise — and all of which marked exciting developments for cryogenic cold chain management.
First, BioCision and Brooks Life Science Systems announced the industry’s first portable cryogenic biospecimen transport system with integrated real-time temperature monitoring. With over 4hrs of cryogenic hold time, the liquid nitrogen-based carrier appears ideal to safely move samples around a lab or campus while keeping them well below Tg. And the automated filling station removes the risk and complexity of dealing with liquid nitrogen filling.
The second announcement was, as expected, Brooks and Chart Industries’ introduction of the new BioStore™ III Cryo — a -190°C (vapor phase) automated cryogenic sample management system. BioStore III Cryo introduces automation to users currently using manual LN2 storage freezers. It incorporates box-level automation, sample inventory, and has built in features to help protect innocents and Tg, plus it maintains all of the benefits of a manual LN2 freezer. No more climbing up steps, lifting out heavy racks and prying cryoboxes out!
Then — the surprise — as ISBER named the CryoPod carrier as its 2015 New Product of the Year — calling it “clearly the most innovative product among this year’s entries,” as well as “elegant, unique, game changing, and versatile.” Read the announcement here.
Pictured right: Hugh Douglas, Chief Operating Officer of BioCision and Dusty Tenney, President of Brooks Life Science Systems accept the 2015 New Product of the Year award.
Who remembers the old saying: “Prior Planning Prevents Poor Performance?” Though well-worn, the truism may have relevance when it comes to — of all things — next-generation biological repositories.
As Ian Pope notes, an NIH estimate of up to 70% of all of the sample material stored at -80°C and below is unusable because of its lack of provenance.
The preferred alternative “effective planning” Pope says will provide two benefits: sample value and viability — and cost reduction. To that end, he gives us five key steps to use in developing an archival sample storage strategy: Storage Objective, Biological Viability, Thermal Performance, Environmental Impact, and Cost. A good read for those in the biobanking, biorepository space.
Anyone moving to cryogenic storage may find themselves facing an element that they have not deal with previously — Liquid Nitrogen (LN2). It’s something that safety requires special safety considerations.
Briefly, the warnings: at atmospheric pressures, LN2 boils at -196°C, therefore it can easily burn skin upon contact. It is colorless, odorless, tasteless (though I don’t know who ever tasted it…) — and can displace breathable oxygen from the air by expanding 1:694 (ratio of liquid LN2 to gas N2) — creating a potentially life-threatening situation through asphyxiation.
Although there are safety concerns, LN2 is widely used as the fuel for cryogenic temperature storage due to its low costs and ease of access. A common use is cryogenic biological storage.
For best practices regarding the safe storage, handling, use of LN2, and information on the specialized personal protection equipment that’s needed, please refer to these example resources:
We’ve been hearing a fair bit of excitement and speculation as to the timing of the first results of the collaboration on a new -150°C automated cryogenic storage solution from Brooks Automation and Chart Industries. The announcement, made back in January, targeted “mid-year” for information on a “joint product.” Stay tuned for breaking news.