Intracellular ice formation (IIF) during freeze–thaw repetitions

Intracellular ice formation (IIF) plays a crucial role in cryobiology. The cell membrane is believed to play important roles in IIF initiation, however the underlying biophysical mechanisms are still not entirely understood. In this work we use a unidirectional freezing design for observations of the freezing process across the cell membrane, which then allow cell membrane integrity detection via dual fluorescent staining. Important IIF information including the location of the initiation site, the growth rate, and the ice dendrite morphology was well obtained. It is found that IIF always initiates at the cell membrane in contact with extracellular ice. Cell membranes are found to be disrupted after IIF in the high freezing rate region (>20 °C/min), however they are observed to keep their integrity after IIF in the low freezing rate region (<20 °C/min). The extracellular to intracellular ice penetration seems to be easier and the ice dendrites become larger when IIF is applied again to the intact cells in subsequent freezing cycles. The results give insight into the IIF mechanisms as well as the relationship between IIF and the cell membrane.