Interrupted cooling protocols in cryopreservation: A review of fundamentals, methods and experimental outcomes for...

Interrupted cooling protocols are effective techniques used in cryobiology to study how cells respond to the freeze/thaw processes. By interrupting either rapid or slow cooling processes within specific sub-zero temperature ranges, researchers can improve the viability of cells after cryopreservation. These methods also provide insights into the differences between slow and rapid cooling injuries. Interrupted cooling protocols help researchers optimize critical factors affecting post-thaw viability and function, such as cooling profiles, cryoprotectants, and plunge temperatures. Over more than 60 years, numerous studies have used interrupted cooling protocols to understand the nature of damage during cooling and warming, identify the sub-zero temperature ranges where most damage occurs, and select appropriate types and concentrations of cryoprotectants for specific cell types. This paper begins with a review of crucial cryobiology concepts that are important to understand interrupted cooling, such as slow cooling and rapid cooling injuries, different types of cryoprotectants and how each type protects cells during cooling and warming, and the damage caused by cryoprotectants. This paper continues with providing a comprehensive review of studies that used different interrupted cooling protocols and to highlight their important findings for a broad range of cell types.