A proof-of-concept study on high-pressure freezing for cryopreservation

Cryopreservation by vitrification typically requires 30-50 v/v% of cytotoxic penetrable cryoprotective agents (CPAs) to prevent ice crystal formation during freezing and thawing, limiting its broader application. Since pressure suppresses ice crystallization, applying high pressure during vitrification may enable reducing CPA concentrations while maintaining cell viability. In this study, we used a high-pressure freezing (HPF) device, commonly used for cryofixation, to successfully cryopreserve 2D cell monolayers and 3D cell spheroids with 20-30 v/v% penetrable CPA. Compared with commonly used plunge freezing, HPF cell monolayers exhibited higher postthaw viability and better retention on the substrate, allowing for subsequent proliferation. HPF cell spheroids showed improved cell viability, metabolic activity and maintained cell-cell adhesion. Developing HPF devices specifically for cryopreservation, in combination with advanced warming techniques, holds promise for achieving vitrification with low or even no CPA.