Self-pressurised rapid freezing at arbitrary cryoprotectant concentrations.

Area of Science:

• Cryo-EM and cryopreservation
• Materials science
• Biophysics

Background:

• Self-pressurised rapid freezing (SPRF) is an alternative to high-pressure freezing (HPF) for biological sample vitrification.
• Both methods leverage pressure to lower the freezing point of water, preventing damaging ice crystal formation.
• The effect of cryoprotective agents (CPAs) on pressure build-up in SPRF is not well understood, yet CPAs are crucial for vitrifying larger samples.

Purpose of the Study:

• To investigate how CPA concentration affects internal pressure during SPRF.
• To develop strategies for optimizing SPRF vitrification with CPAs.
• To assess the efficacy of a novel two-chamber capillary design for SPRF.

Main Methods:

• Raman spectroscopy was used to measure pressure inside sealed capillaries after SPRF, utilizing the pressure sensitivity of hexagonal ice's Raman shift.
• Dimethyl sulfoxide (DMSO) was used as a model CPA to study pressure changes at varying concentrations.
• A two-chamber capillary design was developed to independently control pressure and CPA concentration.

Main Results:

• SPRF pressure significantly decreases with increasing DMSO concentration, dropping to zero above 15 wt%.
• The two-chamber capillary design allows independent tuning of pressure and CPA concentration in different chambers.
• Differential scanning calorimetry (DSC) data confirmed improved vitrification performance with the two-chamber design.

Conclusions:

• CPA presence reduces the self-pressurization effect crucial for vitrification in SPRF.
• The novel two-chamber capillary architecture effectively decouples pressure and CPA concentration, enabling better control over vitrification.
• This design offers a promising solution for vitrifying larger biological samples using SPRF.