Effects On Viability And Bio-characteristics Of Vitrified Hematopoietic Stem/Progenitor Cells Derived From Cord Blood And The Design Of Equilibrium Time In Loading Process

Umbilical cord blood is one of the most important sources of hematopoietic stem/progenitor cells(HS/PCs) which are used in clinical transplantation for restoring hematopoietic system.Until putting into practice,HS/PCs have been required professional cryopreservation skills Currently,vitrification gradually becomes promising besides of the conventional slow-cooling method.An effective cryopreservation skill should to keep the viability and bio-characteristics of stem cells.First,this thesis focused on the evaluation of a newly designed vitrification protocol for HS/PCs.Assays included Tryptan Blue dying,flow cyto-metry of CD34~+ cells,cell culture and growth curve within 72 hours after cryopreservation,and colony forming assay in 14 days. Second,the equilibrium time in loading process was designed.Microscope and CCD software were used to record the loading process and we analyzed volume change of cells at different time.Then we study the viability of cells after each step loading,and finally determined the equilibrium time.The results showed that the recovery of mononuclear cells of vitrification was 81.5±1.7%,higher than that of the slow-cooling sample,is 74.7±2.6%.The recovery of CD34~+ cells for both methods were similar,49.7%and 52.3%for vitrification and slow-cooling,respectively.The cells of untreated control,vitrified sample and slow-cooling sample all grew well and after 72 hours expanded 2.7,2.3 and 1.9 times,respectively.After 14 days colony culture,all the samples formed BFU-E,CFU-GM and CFU-GEMM,and the CFU recovery of vitrified sample was above 90%.The non-optimized equilibrium time between each loading step was the same,90s,and the total loading time was 450s.At that time,the volume of cells shrunk below 35%of the original size without recovery.The cell viability was 88.1±3.0%.Comparably,the optimized equilibrium time was uneven among different loading steps,that is 180s,180s,90s,90s and 60s.After loading,the volume of cells recovered above 63%of the original size and never below 60%during the whole process.The cell viability apparently increased to 95.4±0.4%.Therefore,the newly vitrification method effectively increased the viability of mononuclear cells,achieved comparable CD34+ cells,and the vitrified stem cells also maintained good expansion and colony forming ability.Moreover,the optimized equilibrium time was design uneven,longer between the former steps and shorter for the latter ones, which effectively reduced the loss of cells in loading process.