Cryoprotection trehalose used

Trehalose is a relatively new bulk sweetener with potential for use in soft drinks. It is a di-glucose sugar and it occurs in nature in shellfish and mushrooms, where it confers a degree of protection to plant and animal cells in conditions causing dehydration. This led to its use as a cryoprotectant in freeze-drying systems in the pharmaceutical industry. In food markets, its potential use is as a bulk sweetener. It is manufactured using the Hayashibara patented process using starch as a raw material. The process involves enzymatic conversion and crystallisation to the trehalose dehydrate crystal (LFRA, 2001). 

Cryoprotectants are added to the culture medium in order to protect cells. Glycerine, the first cryoprotectant to be used, was discovered in the 1940s, and it was used for the cryopreservation of bovine sperm. More recently, it has been discovered that other substances such as dimethyl sulfoxide (DMSO), saccharides," and proteins also function as cryoprotectants. However, the mechanism of the cryopreservation process in each case remains unclear. In the present study, we compared DMSO and trehalose, which are cryoprotectants of different molecular sizes that may differ in their ability to pass through the cell membrane. 

Three types of cryoprotectants at various concentrations were selected DMSO (5-20 wt%), trehalose (3-17 wt%), and a mixture of trehalose (10 wt%) and methanol (8 wt%). A mixture of methanol and trehalose has been reported to be effective for the cryopreservation of salmon sperm.Therefore, this mixture was also used to check its effectiveness in the cryopreservation of neurons. 

Some neurons recovered after cryopreservation in 17 wt% trehalose at a cooling rate of 10 K/min as determined by microscopic observations and immunofluorescence staining using MAP2 however, neurons did not recover at lower concentrations of trehalose. These results indicated that trehalose functions as a cryoprotectant only at high concentrations. 

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