“The colder something is stored without freezing the longer it remains viable.”

-Olga Kukal

This hypothesis begs the question: How cold can something be stored in stable liquid state without freezing? The freezing point of a substance depends on its water content. An orange will not freeze until its temperature is below -2.1°C. Mammalian tissue freezes below -0.55°C. The specific freezing point is based on the concentration of solutes in the type of tissue. Any dissolved solid (solute) in water (solvent) colligatively depresses the freezing point of the solution by 1.86°C per Mole. The freezing point depression of mammalian tissue at 0.55°C equating to a solute concentration of o.3 Moles (300 millimoles).

There are three methods of further lowering the freezing point of mammalian tissue:

The tissue can be suspended in an aqueous solution containing a specific type of solute (tonics). These compounds rapidly cross the membranes of the cells in the tissue equalizing osmotic pressure and preventing dehydration. The freezing point of the system (aqueous media and mammalian tissue) is depressed by 1.86°C per mole allowing mammalian tissue to be stored as cold as -6°C before the toxicity of the tonic causes cell mortality during extended storage intervals.

An alternative method of storing mammalian tissue below its colligatively freezing point is to add antifreeze proteins to an isosmotic solution (300 millimoles). Antifreeze proteins depress the freezing point of a solution without altering the melting point this property is termed: thermal hysteresis. Antifreeze proteins sequester (isolate) microscopic ice crystals immediately after nucleation as they form in a bulk liquid. The proteins are frozen into the growing ice front limiting the size of crystals. Highly active antifreeze proteins can lower the freezing point of a 300 millimole solution to -6°C.

The method that provides the lowest freezing point depression of aqueous media is pressure. Pressurizing water in a closed vessel to 210 megapascals (MPa) depresses its freezing point to -22°C. 210 MPa is not an insignificant amount of pressure being equivalent to twice the pressure of the Challenger Deep in the Marianas Trench.

CryoStasis Inc. has Patent Pending technologies employing all these technologies.