Cryopreservation: Applications and Advances

Cryopreservation has been the main method for preserving biological specimens for many years. It has allowed researchers to use rare or precious samples from decades ago to answer new research questions. Today it’s used to preserve the latest complex cell models for other scientists to use in the future and is essential for the storage and supply of biological treatments. In this article, we explore the challenges with cryopreservation and the emerging advances that could improve these methods.

What is cryopreservation?

Cryo is the Greek word for “frost” and cryopreservation literally involves freezing cells, tissues, organs or any other biological materials at very low temperatures.

There are a few ways of achieving this: the most common approach used in research labs is to freeze samples at –80 °C using solid CO₂ or −196 °C using liquid nitrogen. But a method called vitrification is used for freezing clinical samples such as sperm, fertilized eggs or ovarian tissue for long-term storage. The main difference is that traditional cryopreservation methods allow ice to form during the preservation process whereas in vitrification the whole solution is solidified without any ice crystallization.¹ In this article we will focus on conventional cryopreservation most typically used in labs.

Applications of cryopreservation

Traditional cryopreservation is a highly effective method for storing cells and tissues, which works by keeping cells in “suspended animation”, as Sameena Iqbal, bioresource manager at the ‎Wellcome Sanger Institute explains. “By freezing cells, it stops the metabolic activity and preserves the compounds within cells such as enzymes.”

Without cryopreservation you must keep cells and tissues alive in continuous culture – which means growing and splitting them to generate more cells (called passaging). But cells change as they multiply over time, and this can cause them to lose important characteristics. By freezing them, it reduces the heterogeneity which would otherwise be introduced by passaging them repeatedly.

“From our perspective, cryopreservation allows us to bank new cellular models for the research community which people can go back to for years to come,” says Dr Charlotte Beaver, a senior scientific manager at the Wellcome Sanger Institute who develops complex cell model systems such as organoids. “It means we can keep the model long term, but they're not dividing continually to the point where they've mutated to barely represent the cells you started with.”

One area where cryopreservation is becoming increasingly important is in the rapidly emerging field of cell-based therapies: for example, preserving mesenchymal stem cells for a transplant, or chimeric antigen receptor T cells (CAR T cells) for treating cancer. With CAR-T cell therapy, a patient’s T cells are removed, re-engineered to recognize antigens (such as tumor-associated antigens) and then returned to them. “You've got to do a lot of processing on these cells, potentially moving the cells between sites, getting them from the patient when they donate them to the lab, and then back to the patient again, and the cells can degrade really easily,” explains Professor Matthew Gibson, a biomaterial scientist at the University of Warwick. “Treatments like these ideally need freezing in a format that lets you quickly thaw them at the bedside and then administer them to patients. As soon as you have to do lots of processing in a hospital setting, that creates a barrier and all these steps introduce a large cost. These are extremely expensive therapies anyway, so anything to make that process more efficient and results in getting more healthy cells back after freezing is good for the patient.”

Cryogenically Storing Animal Cell Cultures

Maintaining healthy, growing cell cultures is a demanding task made more difficult by the ever-present risk of their loss through accidents or contamination. These problems can be reduced by using cryogenic preservation, a process that effectively puts the cells into true suspended animation. Download this guide to discover intracellular and extracellular events during cell freezing and the advantages of freezing cell cultures.

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Limitations of cryopreservation

One of the main limitations of current cryopreservation methods is the recovery rate of cells after freezing. And while on a research project you might have time to wait for your cells to grow in number, for clinical applications such as those mentioned above, this simply isn’t possible.

During the freezing process, ice crystals form within the sample and damage the cells and some of the cells never recover. Most lab and clinical protocols use cryoprotectants to protect the cells from ice crystals and/or controlled rate freezing and thawing to avoid shocking the cells with sudden changes in temperature.

Many different cryoprotectants are available, but the most common cryoprotectant used for mammalian cells and tissues is dimethyl sulfoxide (DMSO). Other options include glycerol for bacterial cells and red blood cells. But the cryoprotectants have their own drawbacks; DMSO is considered the best protectant but at certain concentrations it is toxic to cells. Glycerol is kinder to cells but less effective as a cryoprotectant compared with DMSO.² One way to mitigate potential toxicity problems is to use different combinations of cryoprotective agents, such as supplementing a lower concentration of DMSO with glycerol or polypropylene glycol. ²

There are also some primary cell types that just don't like being frozen in cryoprotectant solutions, which can present a challenge. “Acute myeloid leukemia peripheral mononuclear cells are much easier to freeze and recover than lymphoma cells, so if you are freezing samples where you don’t yet know what the malignancy is then you need to treat them very carefully,” explains Iqbal.  “Equally, different types of healthy blood cells freeze differently – T cells may recover well, although their functionality can be compromised, whereas granulocytes will not recover at all.”

“With novel complex models like organoids derived from a patient sample, we then have the danger of losing models,” explains Beaver. “One of the advantages of using organoids is that they are comprised of different cell populations representing the natural heterogeneity that you see within a tissue or tumor. Freezing puts them through a bottleneck and if that bottleneck is too harsh for some of the cells, you lose that heterogeneity and it's not such a good model anymore.”

Another consideration is the end use of the sample. For example, RNA is much more sensitive to temperature changes than genomic DNA, as Beaver says: “People have dug up skeletons from years ago and still managed to sequence all the DNA, whereas RNA will degrade if it’s in a – 80°C freezer that's been opened too many times!” This poses a problem for freezing samples if you don’t yet know how you plan to use them.

“If you've got very limited tissue, you've got to decide what the best way is to preserve this material as analysis methods improve,” says Iqbal. “I remember when I started out probably 18 years ago, we would get very large lymph node biopsies with more tissue than we could possibly use so were able to preserve in multiple ways, whereas now core biopsies are prevalent for diagnosis and so you've got very limited material to preserve for research purposes.” 

The Basics of Cellular Cryopreservation

Cryopreservation has become a routine practice in biomedical research and clinical medicine. When frozen and kept properly, specimens may remain in a state of suspended cellular metabolism indefinitely and can be thawed as needed. To reap the benefits of this process, it is important to have a thorough understanding of the key aspects of cryopreservation. Download this guide to the basics of cellular cryopreservation for research and clinical use.

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Advances in cryopreservation

There are two main advances that could improve cryopreservation, says Beaver. “Mid-range automation is what is missing from the market. Even if you're only freezing 30 to 50 vials of something, and not necessarily every day, it's still ergonomically a challenge for people in the lab.” And aside from robots, the other area to be improved is in finding alternatives to DMSO, which can be toxic to cells. Fetal bovine serum is often used with DMSO to provide proteins that further support the cells. But these are not ideal for freezing stem cells, for example. “There's no point in having live cells if they've differentiated down the route you don't want them to.  And if you're injecting these cells back into these people, e.g., for a cell transplant, then you would need a serum replacement that is not animal derived.”

“DMSO-based cryopreservation is a great method, but we really need to think about the efficiency of things,” agrees Gibson. “For a lot of cell types we normally freeze using standard methods we might only get between 20 and 60% of those cells back after freezing. If we can get that number higher, then we can either remove smaller batches from the freezer, or if using rare cells or primary cells which are harder to culture and expand, then the more you recover the faster you can do your research, or the more experiments you can do per batch.”

To address this need, Gibson is researching biomimetic materials, and one of the proteins they’ve been working to mimic is called the antifreeze protein, or an ice-binding protein.³ “These are proteins that somehow manage to selectively bind to ice. They can tell the difference between liquid water and frozen water- it's quite remarkable.” These ice-binding proteins are produced by a huge range of species, most famously fish found at the Earth’s poles. The proteins bind ice crystals to stop them growing so that the fish can survive at lower temperatures than fish without the proteins. “It turns out there's quite a few proteins and polysaccharides which either make or stop ice forming or control how and when it forms. If we can mimic that with synthetic polymers, then we could change how we freeze cells inspired by how nature protects itself during cold.” The team has already shown that they can use the polymers to reduce the amount of cryoprotectant while freezing bacterial cells.⁴

Another advance would be to be able to freeze cells already attached to tissue culture plates, says Gibson. “With cells in suspension, you have to put them onto plates and let them grow before you can use them. We are interested in how we can freeze cells on those plates so that you can take them out of the freezer and they're ready to use.” Gibson’s lab has developed some materials that work not by affecting the ice, but by effectively protecting cells during this freezing process.⁵ “We find these really remarkable levels of cell recovery. One of our most exciting results was by adding in polyampholyte to cells in monolayers, attached to tissue culture plastic, and we saw a dramatic increase from < 20% to > 80% of the cells being recovered.”⁶

This research into the science of freezing isn’t just limited to life sciences either – it’s being applied to everything from making ice cream taste creamier with less fat to avoiding freeze–thaw damage to concrete.⁷ 

“If you can make research any more efficient, that's a great outcome, but research like this is also helping to address basic questions about how these low temperatures are affecting life and how we can store things better, and more efficiently.”

References

1.       Tavukcuoglu S, Al-Azawi T, Khaki AA, Al-Hasani S, et al. Is vitrification standard method of cryopreservation. Middle East Fertil. Soc. J. 2012;17:152–156. doi: 10.1016/j.mefs.2012.07.007.

2.       Awan M, Buriak B, Fleck R, et al. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regenerative Medicine 2020; 15 (3) doi: 10.2217/rme-2019-0145

3.       Carpenter JF and Hansen TN. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc Natl Acad Sci USA. 1992;89(19):8953–8957. doi: 10.1073/pnas.89.19.8953

4.       Hasan M, Fayter AER, Gibson MI. Ice recrystallization inhibiting polymers enable glycerol-free cryopreservation of microorganisms. Biomacromolecules 2018;19(8):3371–3376. doi: 10.1021/acs.biomac.8b00660

5.       Stubbs C, Bailey TL, Murray K, Gibson M. Polyampholytes as emerging macromolecular cryoprotectants. Biomacromolecules 2020;21(1):7–17. doi: 10.1021/acs.biomac.9b01053

6.       Bailey TL, Stubbs C, Murray K, Tomás RMF, Otten L, Gibson MI. Synthetically scalable poly(ampholyte) which dramatically enhances cellular cryopreservation. Biomacromolecules 2019;20(8):3104–3114. doi: 10.1021/acs.biomac.9b00681

7.       Frazier SD, Matar MG, Osio-Norgaard J, Aday AN, Delesky EA, Srubar WV. Inhibiting freeze-thaw damage in cement paste and concrete by mimicking nature’s antifreeze. Cell Reports Physical Science 2020;1:100060. doi: 10.1016/j.xcrp.2020.100060.