Is Cryopreservation Frozen in Time?

Let’s break the ice on cryopreservation: much like many scientific breakthroughs, the field took off thanks to a mistake. In 1949, Christopher Polge returned from holiday to find unexpectedly good survival in his sperm-freezing experiments – only to realize he had accidentally frozen a sample containing glycerol, the compound he used to slow sperm during observation. That accident allowed the cells to survive freezing in liquid nitrogen.¹

Seventy-six years later, although the field has made progress, cryopreservation remains a bottleneck in both research and clinical practice. Will the next breakthrough also happen by accident, or can we find ways to accelerate it?

A crystal-clear problem

Cryopreservation works by cooling biological material to slow or pause cellular metabolism for long-term preservation. With slow-freezing methods, ice crystals can form inside and outside of cells causing mechanical damage or osmotic stress. With vitrification, ice formation is avoided during cooling, but can occur during warming, leading to recrystallization.

Historically, permeable cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO) have been used to reduce intracellular free water and limit ice formation. But this is a double-edged sword: DMSO, while effective, is cytotoxic.

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Until now, researchers have learned to live with it, given that “the core technology for preservation has changed little since the 1970's,” as explained by Prof. Allison Hubel from the Department of Mechanical Engineering, University of Minnesota. “The entire field needs improvement and there is hardly an aspect of cryopreservation that would not benefit from improvement,” Hubel added.

A bottleneck in and outside of research

In addition to being potentially cytotoxic, cryopreservation is generally slow and labor-intensive. “When we work with our primary human cells, we often generate 100+ vials while freezing. This takes 2–3 people working together to get them ready in record time and ensure room temperature does not damage them,” said Dr. Cynthia Hajal, assistant professor in the Department of  Mechanical and Industrial Engineering at Northeastern University.

As Dr. Hajal explained, currently “The main issues are post-thaw viability and phenotypic changes that occur due to freezing: if the background and behavior of the cells is affected, we cannot replicate our experiments well nor get accurate results.” Post-thaw viability also matters beyond research, in cell therapy, which often consists of injecting cells back into patients after modifying and cryopreserving them. Here, DMSO cytotoxicity damages both the cells and, if not fully removed, can even cause adverse reactions in patients.

If freezing cells reliably remains difficult, freezing tissues or organoids is even more challenging. Reliable preservation of patient- or animal-derived tissues is needed to enable asynchronous workflows between clinicians and researchers, so that tissues don’t always need to be shipped right away. Organ transplantation is also ripe for innovation: today, 25% to two-thirds of donated organs are discarded which could be largely prevented by increasing the time available to find suitable recipients through advancing preservation methods.² Similarly, shipping biologics that must be delivered far from their source remains a problem as often the only solution to maximize their viability during transport is to cryopreserve them.

Cool technologies to scale beyond research

In research, these issues are often circumvented with protocols optimized from one lab to the other. These challenges, however, still hold back larger-scale industrial or clinical applications. In some fields, such as in vitro fertilization, moving from slow freezing to vitrification has already dramatically improved outcomes, with survival rates improving by up to 30 points in some studies.³

But for many other applications, problems persist. This has spurred innovation, including the use of anti-freeze proteins (AFPs), which are naturally found in organisms that survive extreme cold. These non-toxic, bio-inspired proteins have improved the post-cryopreservation viability of human embryonic kidney cells when added both intra- and extracellularly.⁴ A group has even shown that fish AFPs extended the preservation time of transplanted rat hearts.⁵ To enter clinical practice, engineering more diverse AFPs, including membrane-permeating variants, will be essential.

DMSO-free CPAs also generate great interest: glycerol or trehalose have been used, but no single molecule seems to be capable of replacing DMSO. Instead, osmolyte mixtures could be transformative, according to Prof. Hubel, whose lab has tested them on dozens of cell types,⁶ as they combine solutes that stabilize proteins, maintain membrane integrity and reduce osmotic shock with far lower toxicity.

Another exciting direction is ice-free preservation through isochoric (rather than isobaric) conditions, where ~ 45% of the solution remains liquid. Biological samples can be stored in this liquid phase, protected from freezing damage while still benefiting from reduced metabolism at low temperature. Rat hearts were successfully cryopreserved this way for the first time in 2018.⁷ While energy-efficient, this method exposes tissues to higher pressures, which may introduce new risks.

Hydrogels, long used to mimic the cell’s microenvironment, may also improve cryopreservation by providing a protective barrier, inhibiting ice crystal growth during freezing and warming, and buffering CPA diffusion to prevent apoptosis from transiently high  CPA concentrations.⁸

Another line of innovation focuses on optimizing rewarming. Nanoparticles (NPs) can absorb energy from external physical fields and convert it into heat, enabling rapid and uniform heating of biological samples to inhibit crystal formation. However, NPs may be cytotoxic, and achieving uniform distribution remains challenging.

Since no single solution is perfect, studies have begun combining approaches. For example, Tian et al. microencapsulated mouse preantral follicles (PAFs) in alginate using a microfluidic device before mixing them with NPs. Microencapsulation separated the PAFs from the nanoparticles, eliminating potential toxicity.⁹ Their study reported a birth rate following fertilization of the vitrified PAFs comparable to the control group.

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Clearly, innovation is happening – but as Hubel noted: “The biggest obstacle to translation into clinical or industrial applications is the lack of knowledge of or expertise in cryopreservation. When people do not understand the scientific basis for cryopreservation, they do not understand how to improve outcomes or evaluate new technologies.” In other words, the field may need to return to first principles before it can meaningfully scale.

Not yet ready for prime time

Improving cryopreservation might seem like a logistical and incremental problem today – but once it’s solved, it could unlock disproportionately exciting possibilities. As Hubel explained: “We need to continue to raise awareness of the importance of cryopreservation for a variety of fields. It is a platform technology for a variety of fields, not just cell and gene therapy.” 

Longevity and transhumanism are one such area: if we cannot yet extend lifespan dramatically, could we at least halt post-mortem aging through cryogenics, preserving ourselves for a future revival? This is the incredible bet that hundreds of people have made since 1967 as they lie cryopreserved until future medical breakthroughs enable the possibility of safe thawing.

Once again, cell culture – the foundation of so much biomedical progress – stands to play a central role in shaping our medical future.