Frozen in Time: Examining the Latest Frontiers in Cryopreservation Technology
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Cryopreservation has underpinned biomedical research for many decades – and offers many exciting new opportunities ahead, including the long-term storage of cells, tissues and organs for therapeutic applications.
“Cryopreservation is the preservation of biological materials at very low temperatures,” describes Dr. Roman Bauer, a lecturer at the University of Surrey who is developing computational and statistical models of how tissue changes during processes such as cryopreservation.
The ability to store biological materials such as molecules, cells or tissues at sub-zero temperatures over extended lengths of time has far-reaching applications throughout biology and medicine.
“There are three major areas that are currently driving advances in cryopreservation technology,” says Professor Matthew Gibson from the Department of Chemistry at the University of Warwick. “One is the development of cell-based therapies, which have shown huge promise in cancer treatment. Another is in the storage and transportation of mRNA vaccines, which came to the forefront during the pandemic. And the final area is the use of cryopreservation to facilitate biomedical research, particularly with helping to reduce the use of animals for drug testing.”
Looking further ahead, advanced cryopreservation techniques also offer the potential to help solve a major challenge in medicine.
“The holy grail would be organ transplantation,” says Bauer. “It could save a lot of lives if you could store donated tissues or organs until a suitable recipient is found.”
But there are huge challenges that will need to be overcome to unleash the full potential of cryopreservation. These include the scaling up of protocols to larger volumes and mitigating the toxic effects of the cryoprotective agents (or “cryoprotectants”) used during the cooling and rewarming process.
A balancing act
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Several challenges occur when cells are cooled for cryopreservation and during the rewarming process. Much of the damage from transitioning between different temperatures is driven, either directly or indirectly, by the formation of ice crystals.
“You’ve got to get things cold, keep them cold and then you’ve got to warm them up – and in each of those stages the stressors the cells are under are different,” states Gibson.
Currently, there are two main approaches for cryopreservation: freezing and vitrification.
“In freezing, you have to deal with ice crystal formation, which can be fatal,” says Bauer. “But in vitrification – which involves the solidification of the sample into a glass-like state – you need to use relatively high concentrations of cryoprotectants, which are often toxic.”
During classical freezing, the sample is slowly cooled at a controlled rate, which causes ice crystals to form in the extracellular spaces of tissues, which can damage cell membranes or extracellular structures. But water flows out of the cells as the sample cools, preventing intracellular ice formation.
“Using cryoprotectants can help to circumvent any damage caused by ice crystals forming outside of the cells during freezing,” describes Bauer. “And the chance of intracellular ice crystal formation is greatly reduced because of the dehydration process.”
Rewarming samples after cryopreservation also needs to be carried out in a carefully controlled way to prevent damage to the cells or tissues, which can occur after both freezing or vitrification. During this process, ice recrystallization can occur, where ice crystals grow and cause cell damage – and exposure to cryoprotectants can be problematic.
“If you expose your sample to cryoprotectants for too long, they can lead to toxicity,” says Gibson. “There’s a delicate balance between its effectiveness against protecting against the cold versus the damage it might cause at higher temperatures.”
The challenge of scale
The most commonly used cryoprotectants are dimethyl sulfoxide (DMSO) and glycerol, which were discovered to have cryoprotective effects over 60 years ago and have since proven to be exceptionally effective in both research and clinical applications.
“The reason both these molecules are still used so widely today is because they work,” enthuses Gibson.
DMSO is routinely used for the storage of mammalian cells – including immortalized cell lines, stem cells and cell-based therapeutics – while glycerol tends to be used more for the cryopreservation of red blood cells, proteins, microorganisms, or eggs and sperm for fertility treatments. But despite their usefulness, some cell types remain challenging to cryopreserve and recover – including human embryonic stem cells. And several challenges still need to be overcome with the preservation of larger, more complex samples – such as advanced cell models, tissues and organs.
“With cells or small cellular aggregates, your cryoprotectants can diffuse in and out relatively easily”, says Gibson. “But that’s much harder to achieve when you’re dealing with bigger volumes.”
Thermal gradients are another major challenge, especially with larger samples.
“If you put an ice lolly in the bath, it’s going to melt from the outside in – and the middle will stay frozen for quite a long time,” explains Gibson. “You face the same problem with thermal gradients when you’re trying to rewarm an organ – and it’s also the same when you’re trying to freeze it.”
Another long-standing challenge is the control of ice nucleation – as aqueous solutions tend to supercool to a temperature below their optimal melting point, especially in smaller volumes – increasing the chance of fatal intracellular ice formation.
“People are beginning to think about the whole gamut of problems which cells face in this freezing process and the biophysical processes involved,” says Gibson. “Some of the challenges are really quite significant.”
Innovations in cryopreservation
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In recent years, researchers have started to employ rational approaches to design, discover and apply new chemical tools for cryopreservation that can help mitigate damage pathways not addressed by common cryoprotectants like DMSO. A variety of innovative agents are being developed – including ice recrystallization inhibitors, macromolecular cryoprotectants and apoptosis inhibitors. Other substances may help reduce the concentrations required and/or neutralize the toxicity of some cryoprotectants.
“Around a decade ago, people started to ask whether we can design molecules with advanced functions that traditional approaches don’t address,” describes Gibson.
Gibson’s team has developed a synthetic macromolecular cryoprotectant that enables the routine cryopreservation of cells grown on multiwell plates, which can be thawed and “assay ready” within 24 hours – offering the potential to transform cell culture for drug discovery and testing, reducing the need for animals in biomedical research. In another study, they showed that using soluble extracellular chemical nucleators can dramatically improve the cryopreservation outcomes of advanced cell models by reducing intracellular ice formation.
Nanowarming is another innovative novel method that employs magnetic nanoparticles to enable even and rapid rewarming after vitrification – showing promising results for improving the outcomes of rewarming vitrified cryogenic storage of tissues in larger sample volumes.
But optimizing cryopreservation protocols is still largely dependent on a trial-and-error experimental process, which is both time-consuming and costly.
“The problem grows exponentially when you consider the number of parameters involved – including the cooling rate, selecting which cryoprotectants to use at what concentrations, and when to administer them,” explains Bauer.
To address this challenge, researchers are looking towards applying sophisticated computer-based simulations to generate hypotheses that can then be tested in experimental systems.
“These programs will iteratively be refined until ultimately, we can generate more powerful cryopreservation protocols,” says Bauer.
Transformative potential
Cryopreservation remains an essential tool for biomedical research – but many challenges still need to be overcome to protect precious biological samples for a variety of applications.
“Cryopreservation is such a fundamental part of the supply chain – from cell-based therapies, mRNA vaccines to drug discovery,” says Gibson. “But if we take a few steps back, any benefits or simplifications we can introduce to these processes along the way can deliver big benefits. That’s why I find it exciting.”
But addressing the challenges that arise from the complexities of cryopreservation techniques requires an interdisciplinary approach between medicine, biology, bioinformatics, chemistry and physics.
“I’m a computational biologist working at the interface between experimental and theoretical biology,” says Bauer. “Computational approaches have revolutionized many other domains of biology – and I think we can now do the same thing in cryopreservation.”
