Vitrification Process Of HSCs Derived From Umbilical Cord Blood

Human umbilical cord blood (UCB) has plenty of hematopoietic stem cells (HSCs) and can be used in the cure of leukaemia, malignancy and thalassemia major. It has been shown to contain a similar or higher proportion of HSCs than adult bone marrow and has proven to be a feasible alternative source of HSCs from bone marrow and peripheral blood. At present, slow cooling is the adopted method for long-term storage for HSCs 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 characteristic of CPA, the loading and removing process of CPA and the vitrification and culture of HSCs.Firstly, the characteristic of CPA is researched. The composing of CPA is: 25% DMSO, 17% formamide, 10% propane-1.2-diol and 6% PEG (w/v). With the method of dilution, the osmolality of CPA of different concentration is measured. By observing the cryomicroscope system it is found that when the cooling rate is lower than 30°C/min CPA can't be vitrificated. Increasing the warming rate can decrease the devitrification time effectively.Secondly, the continuous addition and stepwise elution process of CPA is optimized. CPA is added by continuous addition with different time at 20°C and 1.5°C and then removed with five steps. The experiments show that the best result of the addition time of 60s is got at 1.5°C. In the process of elution, the damage is lower than which in the addition process.Finally, the vitrification process and the culture of HSCs is researched. The effect of the cooling and warming process on HSCs is evaluated and compared with the addition and elution process. After a 4-day-culture, we observe the growth status. The cryopreservation protocol is proved to be effective and can meet the requirements. By the culture, 60s is further proved to be the best addition time. And the addition temperature has no influence on the growth condition of the HSCs.