Thermally defined cryomicroscopy and some applications on human leucocytes

Area of Science:

• Cryobiology
• Cell Biology
• Biophysics

Background:

• Standard cryopreservation methods lack precise control over cooling rates, impacting cell viability.
• Observing cellular responses during freeze-thaw cycles is crucial for understanding cryoinjury mechanisms.

Purpose of the Study:

• To develop and validate a novel freezing stage for light microscopy enabling controlled freeze-thaw cycles.
• To investigate the osmotic and ice formation responses of biological cells during rapid cooling.

Main Methods:

• A specialized freezing stage for light microscopy was developed, offering linear cooling/warming rates from 0.1 to 10,000 K/min.
• Video-monitoring was employed to observe volume changes (osmotic water efflux) and intracellular ice crystallization in biological cells.
• Human lymphocytes and granulocytes were frozen at rates from 2 to 500 K/min to assess cellular responses.

Main Results:

• Cellular shrinkage up to 36% of initial volume was observed in human leucocytes at cooling rates up to 100 K/min.
• Intracellular ice formation was detected starting at cooling rates of 10 K/min.
• A sharp increase in intracellular ice formation probability occurred between 10 and 100 K/min, with nearly all leucocytes forming intracellular ice.

Conclusions:

• The developed cryomicroscope stage provides reproducible and precise control over freeze-thaw rates for cellular observation.
• Rapid cooling rates significantly induce cell shrinkage and intracellular ice formation in human leucocytes, contributing to cryoinjury.
• The findings offer insights into optimizing cryopreservation protocols by understanding critical cooling rate thresholds for cell survival.