Study On Mice Brain Cells Cryopreserved By Vitrification

In present study, adult ICR mice were used as experimental material for brain cells, which were cryopreserved by vitrification method. The experiment mainly divides into two parts. On one hand, the effects of pre-equilibrium time and temperature to brain cell's survival rates were detected, and analysis the toxicity of vitrification solutions (VSi, i=1~6 and VSx, x=a~e) to cells to obtained the appropriate pre-equilibrium time and temperature. On the other hand the suitable vitrification solution for mice brain cells were selected due to recovery rates, survival rates, vitality of SDH in mitochondria and structure of cell membrane in brain cells.The selection of pre-equilibrium time and temperature: Survival rates of cells were detected after pre-equilibration and elution, and found that survival rates were reduced gradually along with the pre-equilibrium time. There was no significant differences (P>0.05) between 2 min and 4 min in survival rates, so choose 4 min as the suitable pre-equilibrium time. In temperature selected experiment, we found survival rate of 0℃was higher than 20℃, so 0℃was the suitable temperature.The selection of vitrification solution: According to the detection of recovery rates, survival rates, vitality of SDH, we found that the VS2 which contain 12 % ethylene glycol (EG), 16 % dimethyl sulfoxide (DMSO), 14 % acetamide, 6 % glycerol, 0.7 M sucrose and 6 % PEG, as well as VSb which contain 14 % dimethyl sulfoxide (DMSO), 16 % acetamide, 0.15 M trehalose and 2 % PEG, were showed better effects to brain cells.The effects of vitrification to cells: We have studied the recovery rates of cells, and found recovery rates were decreased by cryopreserved time. The vitality of SDH were descended remarkable in first 20 days, but 20day and 30day group were have no significant difference (P>0.05). The damage of membrane was increased with cryopreservation time.During the vitrification cryopreservation, cells were affected by the chemical toxicity of cryoprotectants, the process of freezing-thawing and the elution of vitrification solutions, but this technique still maintained the viability of cells. So it was applicable to cryopreserved the brain cells of for long times. These results proved that this technology could be used to clinic research.