Study On The Separation And Storage Process Of Cord Blood Hematopoietic Stem Cells

Hemopoietic stem cell(HSC)is a group of primitive hematopoietic cells found in hematopoietic tissue.It is not a tissue fixed cell and also can be found in hematopoietic tissue and blood.Hematopoietic stem cells appear in in the yolk sac of 2 weeks'human embryo and then shift to the embryo liver after 4 weeks.pregnancy later,the bone marrow hematopoietic begin to work after five months and function as the main site after birth.According to the sources of hematopoietic stem cells,the transplantation could be classified as bone marrow hematopoietic stem cell transplantation,cord blood hematopoietic stem cell transplantation and peripheral blood hematopoietic stem cell transplantation.Because of the advantage of autologous cord blood hematopoietic stem cell transplantation,such as no limited by donor,less graft versus host disease,low dose of immune inhibitor usage,less serious complications and low cost,the storage of autologous cord blood become many people's choice.Hematopoietic stem cells separation and storage process have been studied in this paper.In the first part,mainly clarified the relationship between umbilical cord blood amount and number of mononuclear cells were clarified.The umbilical cord blood was separated by the method of 6%hydroxyethyl starch density gradient centrifugation.By counting the number of mononuclear cells,we found there are linear correlation between umbilical cord blood amount and number of mononuclear cells(r=0.985,P<0.0001).That means,in the premise of having no effect on mother and baby,the more umbilical cord blood is collected,the more mononuclear cells will be separated,which further ensure Success rate of transplantation.The second part in this paper showed the effect of different methods on hematopoietic stem cells separation.Three methods(Ficoll stratification,6%hydroxyethyl starch natural sedimentation and 6%hydroxyethyl starch density gradient centrifugation)are used to separate hematopoietic stem cells.The result showed more mononuclear cells were collected though 6%hydroxyethyl starch density gradient centrifugation and least using Ficoll stratification.The following semisolid agar culture result pointed out CFU-GM colonies using 6%hydroxyethyl starch density gradient centrifugation are significantly higher than that of the other two methods.Meanwhile,we also compared the Cell phenotype of mononuclear cells collected from different methods and found more CD3~+?CD14~+and CD34~+were detected by the method of 6%hydroxyethyl starch density gradient centrifugation.Overall,6%hydroxyethyl starch density gradient centrifugation is an ideal separation method for mononuclear cells collecting.According to the published paper,many elements influence the mononuclear cells separation.In this paper,we chose three main factors,centrifugal temperature,centrifugal force and centrifugal time,and setup response surface method to find out the optimal factors co-ordinate.Finally,we find the combination of 15?,1000g and 17.8min could keep the cell viability reach up to95.7%In the last part,the effect of cell cryopreservation on the quality of mononuclear cells were analyzed.As we know,hematopoietic stem cell cryopreservation is the prime requirement for construction of umbilical cord blood bank and successful transplantation.Currently,two-steps slow cooling is the popular method which will influence cell viability remarkable with different cooling process.Three factors of cooling rate,cooling time and cooling terminal temperature were chosen to optimize the cooling process.According to the result of response surface method,under optimized conditions of 1.5?/min(cooling rate),40h(cooling time)and-80?(terminal temperature),cell viability after resuscitation could reach up to 98.7%.On the other hand,because of fast cooling,no ice crystal and less cell injury,vitrification has a good application prospect for preserving umbilical cord blood hematopoietic stem cells.According to the published paper,the types and dosage of antifreeze,the acceleration of freezing agents and the temperature of freezing agents are influence factors against cell activity.Though optimizing the three factors above,17.1%of human serum albumin,-8.32?of drop temperature and 2.84%/min of drop acceleration were found out as optical condition.The cell viability after resuscitation could reach up to99.1%.