HMN 2026: How High-pressure freezing boosts cell survival with less cryoprotectant

A high-pressure method of instantaneously freezing cells has proven to be effective in the first empirical validation of its kind. Through further development, the method holds promise in finding broad applications in regenerative medicine research. The results are published in the journal PNAS Nexus.

Cryopreservation—the process of cooling and storing cells and tissues at extremely low temperatures, to maintain function for future use—is not a new technology, but there is still much to explore and perfect in the field. Current methods use slow freezing, a process that is conducive to ice formation, cell dehydration and an increase in cryoprotective agents (CPAs), which guard cells against the harmful effects of freezing. These are not ideal circumstances for achieving immaculately cryopreserved cells.

Researchers from the University of Tokyo use vitrification, a process that transforms a substance into a noncrystalline solid by rapid cooling. This cooling yields favorable outcomes in biological samples, even those that are typically difficult to freeze and thaw successfully. Despite some challenges within this method, the future of regenerative medicine research may be greatly and positively impacted by the use of vitrification for cell cryopreservation.

Vitrification uses approximately 2,000 times higher pressure than atmospheric conditions to rapidly cool the cells while inhibiting ice crystal formation. This requires a certain percentage of the volume concentration to be cryoprotective agents to prevent ice crystal formation. However, CPA cytotoxicity (damage to tissues or cells caused by the agents) is an issue that has to be considered under the vitrification process.

“In vitrification, a trade-off lies between the CPA’s cytotoxicity and its ice-inhibiting ability,” said Associate Professor Masaki Nishikawa from the Department of Chemical System Engineering at the University of Tokyo, who led the study. “Lowering CPA concentration typically requires higher cooling and warming rates to prevent ice crystal formation. The major challenges in vitrification include low sample viability caused by CPA cytotoxicity and ice crystal damage, and limitations in scaling up sample volumes.”

Researchers were able to reduce the CPA percentage required to 20–30%, down from the typical 30–50% volume seen in conventional cryopreservation through vitrification, by using high pressure during the process. When comparing the effects of normal-pressure freezing (NPF) to high-pressure freezing (HPF), cell viability and metabolic activity were lower than those treated under HPF, and ice crystals were much more of an issue under NPF.

Additionally, success was also found in freezing challenging cellular formats, such as spheroids and monolayers. The high-pressure freezing process proved to be a viable method for cryopreservation, though designs specifically made with the freezing process in mind appear necessary for success. Samples treated with HPF exhibited a transparent and fracture-free morphology, indicating that the ice-crystal mitigation strategy works.

More research into the impacts of recrystallization is a priority, given that recrystallization can be triggered during thawing under these conditions in amorphous (shapeless) ice. The high pressure used for this method of vitrification helps to form high-density amorphous ice, whose structure is not as hospitable for crystal formation. The effect of this may act as a CPA, though further verification is needed.

The use of complementary techniques could provide a vitrification process with low-CPA or CPA-free conditions. Researchers hope to achieve even better results by coupling HPF with advanced warming techniques upon thaw, such as joule warming or nanowarming. Joule warming utilizes electrical energy to convert to heat, and nanowarming uses iron-oxide nanoparticles to heat uniformly, internally.

Cryopreservation and subsequent thawing could see most use in regenerative medicine research and could save time to culture cells, facilitate standardization, and reduce variability in batches for drug testing and cell transplantation.

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