Ice nucleation and freezing in undercooled cells.

Area of Science:

• Cryobiology
• Biophysics
• Materials Science

Background:

• Understanding cellular freezing is crucial for cryopreservation.
• Extracellular ice formation and chemical cryoprotectants are common considerations in cell cooling.
• Previous studies have not fully elucidated the intracellular freezing mechanisms.

Purpose of the Study:

• To investigate cellular freezing behavior using differential scanning calorimetry (DSC).
• To determine if cells freeze at temperatures above homogeneous nucleation of ice.
• To explore potential mechanisms of heterogeneous ice nucleation in various cell types.

Main Methods:

• Differential scanning calorimetry (DSC) was used to monitor cellular freezing.
• Experiments were conducted excluding extracellular ice and chemical cryoprotectants.
• Kinetic analysis of ice nucleation temperature dependence was performed.
• Freeze-fracture electron microscopy validated assumptions about ice growth rates.

Main Results:

• All studied cells, including human erythrocytes, yeast, and plant cells, froze above the homogeneous nucleation temperature of water.
• Plant cells and yeast showed a significant difference (approx. 9°C) compared to homogeneous nucleation, while human erythrocytes showed a smaller difference (0.5°C).
• Ice nucleation kinetics suggest heterogeneous mechanisms initiated within or on the cell, with plant cells/organelles as potential nucleators.

Conclusions:

• Cellular freezing is initiated by heterogeneous nucleation, either intracellularly or at the cell membrane.
• The kinetics of ice nucleation in human erythrocytes are highly temperature-sensitive and distinct from the suspension medium.
• Cell dimensions and internal structures likely play a role in heterogeneous ice nucleation during cooling.