Continuous Loading Of CPA At Low Temperature And Cryopreservation Of NSCs As Single Cell Suspension

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.At the present,slow cooling is the widely adopted method for storage for NSCs.But as a conventional method,it has many disadvantages.Vitrification is a promising,novel and simple procedure that requires less time and is likely to become safer and more effective than slow cooling,however,vitrification need a combination of high concentrations of cryoprotectant agent(CPA) which has very high toxic and osmotic injury when the CPA is loaded and removed.This thesis focuses on the axamination of the key units of computer-controlled cooler with automatic sample seeding and continuous loading of NSCs CPA at low temperature.Debugging the computer-controlled programmed cooler,the low temperature thermo couples,thermal insulation of the freezing vessel,and the automatic sample loading unit were tested.The radial temperature distribution within the freezing vessel was checked and modified to make sure the computer-controlled programmed cooler could satisfy the requirement of temperature controlling precision.The continuous loading and stepwise elution process of CPA is optimized.To inspect the process of continuous loading at low temperature,the exposure time,cooling rate,the optimized loading rate should be considersd.The experiments show that at initial temperature -5℃;5min contact time,the optimized cooling rate is 5℃/min;the optimized loading rate is 0.53ml/min,and finally the optimized loading program was ontained,5℃/min cooling rate, 0.53ml/min loading rate,every stepwise adds CPA 62μl,83μl,122μl,190μl,343μl; every stepwise exposer is 62s,52s,42s,32s,22s and get 90.5%NSCs viabilities.