The freezing point temperature of intracellular water is lower than extracellular water due to the intracellular presence of osmotically active molecules. In addition, ice has a lower potential energy than liquid water. Therefore, as extracellular water freezes, water moves from the intracellular to the extracellular space leading to cellular dehydration, resulting in damage to membranes, DNA, RNA, and proteins with precipitation of various intracellular molecules. The membrane change is especially severe and is considered the primary site for freezing-related damage.
At the present time, non-naturally occurring molecules such as dimethyl sulfoxide (DMSO) are used as osmotically active cryopreservative agents. However, DMSO has known toxicity on cells, tissue, and whole organisms, including humans. It would be extremely useful if non-toxic, endogenous molecules could be found to serve as effective cryopreservation agents.
To combat freeze-induced cellular damage, we propose to prevent the physicochemical-induced cellular damage by extracellularly adding naturally occurring non-toxic osmotically active molecules which can replace intracellular water and thereby prevent intracellular ice formation and cellular shrinkage.
An additional way to prevent freeze-induced cellular damage is by using naturally occurring molecules to enhance membrane stabilization and repair; providing naturally-occurring antioxidants; and maintaining optimal intracellular ATP levels.
In this patent application we describe endogenous molecules which act as osmotically active cryopreservative agents as well as naturally-occurring molecules which lead to membrane repair, maintain normal cellular levels of energy metabolites, and act as antioxidants. None of the endogenous molecules described have demonstrated toxicity in the concentrations proposed.