Cryoprotective agents

Abstract

Two forms of artificial cryoprotection are immediately available for experimental use. Foremost are the penetrating agents functioning on a colligative basis and used in concentrations of 2–4 m. These apparently function simply by preventing the concentration of extracellular solution and thus reducing cell dehydration to a tolerable degree. There are two immediate obstacles to the general use of such compounds. First, the penetration of cells must be uniform and achieved without the imposition of osmotic stresses which can in themselves be destructive. The particular virtues of DMSO, ethanol, and methanol are the speed and apparent universality of their penetration. The major defect of glycerol is its slow movement across those membranes which are permeable to it and the largo number of tissues which appear to be virtually impermeable. The second obstacle is the toxicity of penetrating agents in the high concentrations necessary to achieve protection. Ethanol and methanol can be ruled out on this basis, and studies of DMSO, dimethylformamide, and ethylene glycol have not been particularly encouraging. A second means of cryoprotection is through the use of nonpenetrating agents. We have presented evidence which suggests that the efficacy of nonpenetrating cryoprotective agents lies in their ability to enable the cell membrane to leak solute reversibly under osmotic stress. The success of such an approach presumably depends upon the achievement of a freezing rate which is rapid enough to limit solute leak to low-molecular-weight solutes, prevent loss of the larger and more essential protoplasmic elements and yet not be so fast as to produce intracellular freezing. It will also be essential that the cell have an opportunity to heal and restore its normal solute content. This hypothesis for the mechanism of extracellular cryoprotection also provides an explanation for the general failure of extracellular agents to afford significant cryoprotection for packed cells, tissues, or organs. If the cell avoids excessive dehydration through the influx of extracellular solute, it is clearly necessary that a substantial amount of extracellular solute be available which is only the case in a relatively dilute cell suspension. In a tissue, extracellular solute is present in such insignificant amounts as to be of little value in facilitating the leak mechanism. The provision of a suitable environment for cell healing ex vivo is also difficult to achieve. Ideally, the prevention of nucleation would provide the simplest and most effective means of avoiding freezing injury but whether compounds which will do this exist and whether they could be artificially introduced into animal cells can at the moment be no more than speculation. The most promising approach to the preservation of organized tissues and organs through artificial cryoprotection would still appear to be through the development of nontoxic, rapidly penetrating cryoprotective agents or means of preventing their toxicity or of reversing its effects.