Cryopreservation Of Neural Stem Cells

Neural stem cells (NSCs) hold tremendous potential in the newly emerging field of regenerative medicine, especially for diseased central nervous system, and to be a useful tool for basic scientific research. However, an essential prerequisite for the future widespread applications of NSC is the development of efficient cryopreservation protocols to facilitate their storage and transportation. At present, slow cooling as a conventional method is the adopted method for long-term storage for NSCs. Vitrification is a promising, novel and simple procedure that requires less time and is likely to become safer and more effective than slow cooling. Vitrification solution(VS) needs a combination of high concentrations of cryoprotectant agents, which have very high toxic and osmotic injuries when the VS is loaded and diluted. This paper focuses on the slow cooling and vitrification protocols, respectively.At first, this paper is attempted to cryopreserve the neural stem cell (NSC) spheres with different diameters and bio-states, aiming to improve the survival rate and explore the underlying mechanism of the cryopreserving process. After the growth curve of the NSC was measured by CCK-8 kit, three types of NSCs in logarithmic growth phase, i.e. the single cell suspension, NSC spheres with the diameters of 30~50μm and 80~100μm, respectively, were cryopreserved at the freezing rate of 1 ℃ /min and seven concentrations of DMSO (3%,5%,7%,8%,10%,1.5%,20%). For the NSC sphere with the diameter of 80~100μm and the DMSO concentration of 8%, the survival rate is 82.9%. The subtle "coordination" of NSC sphere and its diameter together affect the survival rate.Secondly, this paper aiming to select vitrification solution(VS) and loading procedure adapting to NSCs. Through observing on the cryomicroscope system and the survival rate comparision of NSCs, the combination with 20%DMSO+20% EG+10%Acetamide+0.3M Sucrose, is suitableto NSCs. After examing five loading time(30s 60s 90s 120s and 150s) and two loading temperatures(20~26℃ and 0~4℃), It is found that 20~26℃ and 60s is optimal for the good loading procedure of NSCs.