Reversible Cryopreservation of Living Cells Using an Electron Microscopy Cryo-Fixation Method

Abstract

Rapid cooling of aqueous solutions is a useful approach for two important biological applications: (I) cryopreservation of cells and tissues for long-term storage, and (II) cryofixation for ultrastructural investigations by electron and cryo-electron microscopy. Usually, both approaches are very different in methodology. Here we show that a novel, fast and easy to use cryofixation technique called self-pressurized rapid freezing (SPRF) is–after some adaptations–also a useful and versatile technique for cryopreservation. Sealed metal tubes with high thermal diffusivity containing the samples are plunged into liquid cryogen. Internal pressure builds up reducing ice crystal formation and therefore supporting reversible cryopreservation through vitrification of cells. After rapid rewarming of pressurized samples, viability rates of > 90% can be reached, comparable to best-performing of the established rapid cooling devices tested. In addition, the small SPRF tubes allow for space-saving sample storage and the sealed containers prevent contamination from or into the cryogen during freezing, storage, or thawing.

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

“…In both approaches, it is necessary to cool far below the freezing point of water to achieve complete arrest. However, crystallization of (intracellular) water must be avoided or at least minimized, since ice crystals may deform cellular structure and can be lethal (Jacques Dubochet, ; Huebinger et al., ; Pegg, ).…”

“…However, the warming step ending the duration of cryostorage must be considered more vulnerable to ice crystal formation than the initial cooling step. Technically, the very fast re‐warming is even more demanding than fast cooling (Huebinger et al., ), and is therefore most challenging for cell survival (Seki & Mazur, , ). Possibly because of these technological limitations, most of the developments of cooling and warming devices during the last decades have focused on the quality of cooling devices such as plastic straws, or simply using smaller sample volumes which freeze faster (He et al., ; Kuwayama, Vajta, Kato, & Leibo, ; Schiewe et al., ; Vajta et al., , ).…”

“…Self‐pressurized rapid freezing (SPRF) is a novel technique to arrest biological samples like living cells, tissues, or small organisms very rapidly either for microscopic investigation (Han, Huebinger, & Grabenbauer, ; Leunissen & Yi, ) or for long‐term storage in a living but arrested state, i.e., cryopreservation (Huebinger, Han, & Grabenbauer, ). Samples are rapidly cooled (and re‐warmed) in tightly closed metal tubes of high thermal diffusivity.…”

“…From this stage, samples can either be processed by freeze substitution for conventional electron microscopy at room temperature (Leunissen & Yi, ) or directly imaged at close‐to‐native state by cryo‐electron microscopy (Han et al., ). Alternatively, they can be thawed again and further investigated live after a certain period of cryopreservation (Huebinger et al., ).…”

“…If cryo‐fixation for (electron‐) microscopy is the main purpose of using SPRF, it is recommended to use a plunge freezer and a secondary coolant such as ethane or propane to achieve the highest possible cooling rates. To use SPRF for cryopreservation and long‐term storage, cooling in liquid nitrogen by hand may be sufficient, since the re‐warming procedure is the limiting step for the survival of cells (Huebinger et al., ). The cryoprotective medium containing the samples should be well adapted for each organism, cell type, and specific application.…”