Optimization Of Separation And Cryopreservation Methods Of Umbilical Cord Blood And Study On Related Factors Affecting Quality

Objective:Hematopoietic stem cell(HSC)has many advantages,such as abundant sources,great potential for differentiation,low immunogenicity,many immature T cells,easy to preservation,not easy to cause tumor,no damage and side effect to the donor when collected,so it is getting more and more popular.The purpose of this study is to recycle more umbilical cord blood hematopoietic stem cell(UCB-HSC),look for more suitable cryoprotectant for UCB-HSC preserved,and analyze factors affecting the quality of UCB-HSC to provide reference for the quality control of UCB.Methods:It optimizes the isolation technology with Hydroxyethyl Starch(Hydroxyethyl Starch,HES)of UCB-HSC in two ways:(1)Compares the results centrifuging UCB for 3 min or 5 min after precipitating erythrocyte for 15 min,30 min,45 min and 60 min with those by centrifugation for 7 min directly.Thus,the optimal time for HES to separate UCB is obtained.(2)Compares the results recycling nuclear cells twice with that recycling once traditionally.In addition,this study proposes to compare 10%DMSO+10%autologous serum with 10%DMSO and 10%DMSO+10%dextran 40 as cryoprotectant,to test the protective effect of 10%DMSO+10%autologous serum on UCB-HSC frozen for 3 months,6 months and 12 months.Finally,according to statistical analysis,this paper summarizes the factors affecting the quality of UCB-HSC,proposes targeted improvement measures and tracks the effect of practical test to determine the key factors which affect the quality of UCB.Results:(1)When UCBs are centrifuged for 3 min after precipitating erythrocyte by HES for15 min,30 min,45 min and 60 min,the recovery rate of nuclear cell and the nuclear cell viability and the percentage of CD34~+and CFUs yield all have no significantly difference comparing with the results of centrifugation for 7 min directly(P>0.05).When UCBs are centrifuged for 5 min after precipitating erythrocyte for 15 min and 30 min,the results are the same with the above,but when precipitating erythrocyte for 45 min and 60 min,the other results except the nuclear cell viability are all significantly higher than those of centrifugation for 7 min directly.Especially precipitating erythrocyte for 45 min,the percentages of CD34~+are the highest,the group has statistical differences comparing with the centrifugation for 7min directly(P<0.05),while the nuclear cell viabilities have no significantly difference(P>0.05).(2)When recycling the nuclear cells twice,the recovery rate of nuclear cell,the percentage of CD34~+,CFUs yield are all significantly higher than that of recycling once.There are significant statistical differences(P<0.05),but the nucleated cell viabilities have no statistical difference(P>0.05).(3)Taking 10%DMSO+10%autologous serum as cryoprotectant,HSCs are cryopreserved for 3 months,6 months,12 months,the recovery rate of nuclear cells,the recovery rate of CD34~+cells and the recovery rate of CFUs are all higher than that of 10%DMSO+10%dextran 40 than that of 10%DMSO.There are statistically significant differences(P<0.05).However,there is no significant difference in the nuclear cell viabilities no matter the cryopreservation time(P>0.05).(4)Factors which influence the quality of UCB are summarized after analysis and practical verification,they include the way of delivery and the way of collection,transport time,HES precipitation time,centrifugation,pollution and personnel practice in the isolating process,freezing time control,temperature change and pollution in the freezing process.Conclusions:(1)Precipitating erythrocyte for 45 min and then centrifuge for 5 min after HES is poured into UCB,the yield of HSC increases by 40%and the cell viability is not affected.(2)Recycling nuclear cells twice,the recovery rate of HSC increases by 60%,which is an effective way to improve the recovery rate of HES.(3)10%DMSO+10%autologous serum is an efficient,safe and economical cryoprotectant for UCB-HSC.(4)The most blood can be collected by selecting the appropriate collection way according to the way of delivery.The transport time should be shorten to ensure cell viability.Optimizing of separation process,preventing pollution and personnel training can improve the quality of UCB-HSC.Controlling operation time exactly,reducing temperature change and preventing pollution can effectively improve the number and vitality of cells cryopreserved.