High-Pressure Freezing Improves Cell Cryopreservation

Cryopreservation is an established technique that currently uses slow-freezing techniques, which promote ice formation, cell dehydration and higher levels of cryoprotective agents (CPAs) to shield against freezing damage. These factors often compromise the quality of preserved cells. Now, a team from the University of Tokyo has a new approach, high-pressure freezing, which rapidly cools samples into a noncrystalline solid under high pressure. This approach, described in PNAS Nexus, avoids ice crystals and works well even for hard-to-freeze biological materials. 

Vitrification applies about 2,000 times atmospheric pressure to quickly freeze cells while blocking ice formation. It is not without its own issues though as it still relies on CPAs at a certain volume concentration for protection, but CPA cytotoxicity—damage from these agents—poses a challenge. “In vitrification, a trade-off lies between the CPA’s cytotoxicity and its ice-inhibiting ability,” said Masaki Nishikawa, 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.”

By incorporating high pressure, the researchers cut CPA needs to 20-30% volume, compared to 30-50% in standard methods. Tests showed superior cell viability and metabolic activity with high-pressure freezing (HPF) over normal-pressure freezing (NPF). HPF also reduced ice crystals significantly. The method succeeded with complex formats like spheroids and monolayers, yielding samples with transparent, fracture-free morphology. High pressure generates high-density amorphous ice, which resists recrystallization during thawing, potentially mimicking CPA effects—though more confirmation is required.

Further gains may come from pairing HPF with advanced thawing like joule warming, which converts electrical energy to heat, or nanowarming via iron-oxide nanoparticles for even internal heating. Complementary strategies could enable low-CPA or CPA-free vitrification.