Influence Of Trehalose On The Vitrification Cryopreservation Process Of Neural Stem Cells

It is necessary to achieve the long-term cryopreservation of Neural stem cells (NSCs) forits offshelf availability. This paper mainly investigate the influence of trehalose to the stemcell culture process and its vitrification ability, and then the vitrification cryopreservation ofNSCs was preliminarily conducted by adding the trehalose in CPA.Firstly, the influence of trehalose to the stem cell culture process has been investigated.Adding trehalose with concentration of 0mol/L,0.3mol/L,0.15 mol/L,0.075mol/L,0.05mol/L,0.025 mol/L resepectively during the NSCs culture process, and being testedcontinuously by cck-8 agent kits for six days, it was found that the addition of trehaloseduring the NSCs culture process could not affect the proliferation of cell, but kept thecharacteristic of the stem cell, differentiating into neurons, oligodendrocytes and astrocytesStar. Therefore, it can be ensured the safe application of trehalose to NSCs vitrificationcryopreservation.Secondly, through the experiments using cryo-stage with microscope and DifferentialThermal Scanning, the vitrification ability between trehalose and sucrose has been comparedwith respect to the instance of vitrification transition temperature and devitrification. With thefreezing rate of 100℃/min,both 60% trehalose and 60% sucrose could achieve vitrification.But devitrification phenomena also occurr during the rewarming process at the same thawingrate, the devitrification capability of 60% trehalose was less than that of 60% sucrose.Moreover, the minimum concentration of trehalose to be vitrified at such a condition was55%, but that of sucrose was 58%. The vitrification transition temperatures measured by thedifferential thermal scanning technology for the sample solution of 60% trehalose, 55%trehalose, 60% sucrose and 58% sucrose, respectively, are-29.7℃,-30.8℃,-39.4℃and-45.6℃.Finally, trehalose combined with DMSO, glycol and acetamide as the vitrificationcryopreservation agent (CPA) is applied to the vitrification cryopreservation of NSCs, beingobserved dynamicly and directly on the cryomicroscope system and compared the cellviability of NSCs, the proper cryopreservation agent has been preferentially selected, whichincludes 20%(v/v) DMSO+20%(v/v)EG+10%(w/v) Acetamide +0.5M Trehalose.After that,Using the obtained vitrification CPAs, two NSCs samples, one without trehalose addition during the culture process,and another with 50mmmol/L trehalose addition are tested. As areault, it is found that the addition of trehalose during the culture process of NSCs leads to anobvious increase of the cell viability after the vitrification cryopreservation. Moreover,compared the cell viability of vitrified NSCs in this paper with that in literature with the CPAof 20%(v/v) DMSO+20%(v/v) EG+10%(w/v) Acetamide+0.3M Sucrose, it is concluded thatthe cell viability for the present CPA and culture ptotocal is 71.7% higher than that reported inliterature.