ABSTRACT
OBJECTIVE: To study the impact of anticoagulant to the quality of umbilical cord blood (UCB). METHODS: 6060 cord blood units (CBUs) were classified into five groups, such as 28 ml: (10-29) ml, 28 ml: (30-69) ml, 28 ml: (70-109) ml, 28 ml: (110-150) ml and 28 ml: (>150) ml according to volume ratio of anticoagulant and CBVs. The count of pre-cryopreservation total nucleated cell (pre-TNC), the viability of nucleated cell (VNC), the amount of CFU-GM and the ratio changes of CD34+ were evaluated and analyzed statistically. RESULTS: It was found that pre-TNC increased with the growth of volume of CBUs (r=0.9937) under the certain volume of antico-agulant, and the TNC in the minimum UCB volume group was (2.57±0.89)×108; the VNC grew up with the increasing count viability of volume (r=0.9897), and the average viability of the minimum volume group remained over 95%; the CFU-GM climbed up with the increasing of volume (r=0.9024), and the number of CFV-GM in minimum volume group reached to of 89/×105; CD34+% grew up with the increase of volume of CBUs (r=0.9641), and the ratio was (0.30±0.19)% for the minimum volume group. CONCLUSION: In certain volume of anticoagulant in collection-bag, pre-TNC, VNC, CFU-GM and CD34+% are all dropped with the decrease of CBUs volume , however, all above-mentioned indexes in the minimun random group still meet the requirement for clinical administration.
Subject(s)
Cryopreservation , Fetal Blood , HumansABSTRACT
The aim of the present study was to obtain the qualified hematopoietic stem/progenitor cells (HSC/HPC) and human umbilical cord-mesenchymal stem cells (MSC) in vitro in the co-culture system. Cord blood mononuclear cells were separated from umbilical cord blood by Ficoll lymphocyte separation medium, and then CD34+ HSC was collected by MACS immunomagnetic beads. The selected CD34+ HSC/HPC and MSC were transferred into culture flask. IMDM culture medium with 15% AB-type cord plasma supplemented with interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (Flt-3L) factors were used as the co-culture system for the amplification of HSC/HPC and MSC. The cellular growth status and proliferation on day 6 and 10 after co-culture were observed by using inverted microscope. The percentage of positive expression of CD34 in HSC/HPC, as well as the percentages of positive expressions of CD105, CD90, CD73, CD45, CD34 and HLA-DR in the 4th generation MSC, was tested by flow cytometry. Semisolid colony culture was used to test the HSC/HPC colony forming ability. The osteogenic, chondrogenesis and adipogenic ability of the 4th generation MSC were assessed. The karyotype analysis of MSC was conducted by colchicines. The results demonstrated that the HSC/HPC of co-culture group showed higher ability of amplification, CFU-GM and higher CD34+ percentage compared with the control group. The co-cultured MSC maintained the ability to differentiate into bone cells, fat cells and chondrocytes. And the karyotype stability of MSC remained normal. These results reveal that the appropriate co-culture system for MSC and HSC is developed, and via this co-culture system we could gain both two kinds of these cells. The MSCs under the co-culture system maintain the biological characteristics. The CFU-GM ability, cell counting and the flow cytometry results of HSC/HPC under the co-culture system are conform to the criterion, showing that the biological functions of HSC/HPC are maintained.