Self-Pressurized Rapid Freezing as Cryo-Fixation Method for Electron Microscopy and Cryopreservation of Living Cells

Abstract

Reduction or complete prevention of ice crystal formation during freezing of biological specimens is mandatory for two important biological applications: (1) cryopreservation of living cells or tissues for long-term storage, and (2) cryo-fixation for ultrastructural investigations by electron microscopy. Here, a protocol that is fast, easy-to-use, and suitable for both cryo-fixation and cryopreservation is described. Samples are rapidly cooled in tightly sealed metal tubes of high thermal diffusivity and then plunged into a liquid cryogen. Due to the fast cooling speed and high-pressure buildup internally in the confined volume of the metal tubes, ice crystal formation is reduced or completely prevented, resulting in vitrification of the sample. For cryopreservation, however, a similar principle applies to prevent ice crystal formation during re-warming. A detailed description of procedures for cooling (and re-warming) of biological samples using this technique is provided. © 2018 by John Wiley & Sons, Inc.