Cryopreservation Process For The Human Umbilical Cord Blood

Human umbilical cord blood (UCB) has proven to be a feasible alternative source of hematopoietic stem cells (HSC) from bone marrow for pediatric and some adult patients with major malignant and nonmalignant hematological disorders. In order to make use of UCB more effectively, the cryopreservation of UCB has become a very important topic in the biomedical engineering area. At present, slow cooling is the adopted method for long-term storage for UCB 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 agents (CPAs) which has very high toxic and osmotic injury when the CPA is loaded and diluted. This thesis focuses on the cooling procedures, the loading and diluting of CPAs and CPA solution properties for the cryopreservation of UCB with slow cooling and vitrification protocols respectively.Firstly, during the process of the slow cooling of HSC, the optimum DMSO concentration and the appropriate cooling rate are investigated experimentally and at the same time, the cell viabilities are compared among seven CPAs for slow cooling method. The results indicate that the cooling rate of 1℃/min and 10% DMSO are the most satisfying for the slow cooling of HSC and XX3 CPA shows the best result among the seven CPAs selected in this paper.Secondly, CPA for vitrification is studied to find one suitable for HSC. The ability of the ice formation is compared among several chemicals, and then three permeable CPAs are chosen and compared in many aspects and finally the mixed vitrification CPA is made: 10% propane-1,2-diol, 25% DMSO, 17% formamide and 6% PEG (w/v) in IMDM media.Finally, the effect of operating temperature on the cell viability is studied when stepwise addition of low and high concentrations of CPAs to HSC suspension was performed at room temperature (17-19℃), on ice (0- 4℃) or at the lower temperature (-9-11℃). The effects of the slow cooling and vitrification protocols, one-step dilution, step-wise dilution and step-wise dilution with addition of different concentrations of sucrose solutions are compared and evaluated as well. The results indicate that lowering the operating temperature at high concentration CPA prior to cryopreservation results in theremarkable improvement of HSC viability and there is opposite result for the low concentration CPA. The slow cooling and vitrification of HSC can contribute a close viability before dilution and the lower operating temperature shows a negative effect for CPA dilution. One-step dilution is significantly more detrimental to viability of HSC compared with step-wise dilution and addition of 0.75mol/l sucrose solution has a positive effect on the dilution process.