Vitrification Of HSCs Derived From Umbilical Cord Blood

Human umbilical cord blood (UCB) has proven to be a feasible alternative source ofhematopoietic stem cells (HSCs) from bone marrow and peripheral blood. In order to makeuse of HSCs derived from umbilical cord blood more effectively, the cryopreservation ofHSCs has become a very important topic in the biomedical engineering area. At present, slowcooling is the adopted method for cryopreservation of HSCs, but it has many disadvantages.Vitrification is a promising novel and simple procedure that requires less time and is likely tobecome safer and more effective than slow cooling, however, vitrification need a combinationof high concentrations of cryoprotectant agent (CPA) which has very high toxic and osmoticinjury 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 of HSCs.Firstly, the characteristic of CPA is investigated. The composing of CPA is: 25% DMSO,17% formamide, 10% propane-1, 2-diol and 6% PEG (w/v). With the method of dilutedsolution, the osmolality of CPA of different concentration is measured. Through observing onthe cryomicroscope system, it can be found that when the cooling rate is lower than 30℃/minCPA solution can't be vitrificated. Increasing warming rate can decrease the ice crystalnumber and restrain the growth of ice crystal of devitrificstion effectively. Meanwhile, theCPA solution shows very high toxic and osmotic injury when the CPA is loaded in one stepmethod.Secondly, the loading and removing process of CPA is optimized by using stepwise anddifferent equilibrium time method. The effect of adding different concentrations of trehalosein the removing process on the removing result is investigated also. The experimental resultsshow that five steps addition, equilibration time at each intermediate step is 90s and five stepsdilution, equilibration time at each intermediate step is 60s get the best HSC viability. Adding0.5M trehalose in the dilution process can increase the HSC viability effectively.Finally, the vitrification process of HSCs is studied. The effects of cooling and warmingrates on HSC viability after vitrification are examined. The effects of slow cooling andvitrification protocol are compared and discussed as well. The experimental results indicatethat increasing the cooling and warming rates can increase the HSC viability effectively. TheHSC viability after vitrification is higher than that after slow cooling.