Cryoprotectants are the basis of cryopreservation of various human cells, tissues and organs. Since the discovery of the cryoprotection of glycerol in 1960s, more than 50 drugs or reagents have been applied to cryoprotection. In recent years, with the development of vitrification cryopreservation technology, many researchers have paid more attention to the research of cryoprotectants.
There are two kinds of biological sample tissues which are usually stored at 2~10°C at sub-normal temperature and -80~196°C in deep low temperature. Cryopreservation at 2~10°C is generally applicable to tissues and organs which can be transplanted or transported in a short time. The commonly used protective solutions are UW solution developed by Wisconsin University, which has been used for nearly 20 years, IGL solution developed by Georges Lopez Research Institute in France, low-potassium, low-sodium, non-body fluid HTK preservation solution, combining the advantages of UW solution and HTK solution, and Celsior solution (CS solution) for donor heart preservation. SMO solution, Shanghai multi-organ preservation solution, has been developed in China. In addition, there are standard preserved EC solution for clinical kidney transplantation widely used in Europe, and HC-A solution for main irrigation and cryopreservation of organ transplantation in China.
With the development of science and technology and the increasing demand for long-term preservation of samples, cryopreservation has become the best choice for the preservation of cells, tissues and organs.
Cryoprotectants can be classified into two categories according to their role in cryopreservation: permeable and impermeable protective agents. The permeability protection agents are mainly polyols, including ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol, glycerol, formamide and acetamide. The role of osmotic protectants is to reduce or prevent the formation of intracellular ice crystals by lowering the freezing point and replacing the water surrounding the intracellular proteins, DNA and other components. The impermeable protective agent mainly includes low molecular weight monosaccharides (such as sugar, glucose, maltose, etc.), disaccharides (sucrose, trehalose, etc.), polysaccharides (raffinose, etc.) and high molecular compounds (polysucrose, dextran, polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, hydroxyethyl starch, etc.) having a molecular weight greater than 1000 Da. The impermeable protective agent is beneficial to improve the vitrification property of the protective solution, improve the cryoprotection effect, meanwhile, it can promote the cell dehydration, stabilize the cell membrane, reduce the dosage of cryoprotectant required for the vitrification itself, thereby reducing the toxicity of the vitrification solution.
New substances different from the above two categories, such as ice crystal blockers, antifreeze proteins, magnetic nanoparticles, and even some Chinese herbal ingredients such as ginsenosides, have also been documented to have good cryoprotection. According to the characteristics and requirements of different cryoprotected biological samples, the above-mentioned substances are analyzed systematically, and the appropriate combination of cryoprotectants is screened according to the properties and requirements of different cryoprotected biological samples, and the parameters are determined by advanced instruments and technical means, and the cooling and recovery procedures are screened to protect the biological samples from ice crystal damage, thus providing good biological samples for clinical and basic scientific research and becoming a new research direction.
In addition, on the basis of the research and development of existing protective agents, it is also an important research direction for us to actively pay attention to some other substances with potential application value, such as antioxidants (propyl orange, vitamin E, vitamin C, amino acid, lecithin, protein hydrolysate, sodium thiosulfate, sodium ascorbate) and so on.
On the basis of cryopreservation of many kinds of cells, more protective substances were selected and optimized, combined with new materials such as antifreeze protein and nano-magnetic particles, suitable temperature-lowering and rewarming procedures were screened to achieve high-quality preservation of clinical tissues and organs.