| Umbilical cord blood(UCB)is an alternative source to bone marrow(BM)and mobilized peripheral blood(MPB)of hematopoietic stem cells. Many studies have demonstrated that UCB has been used successfully for hematopoietic reconstitution in pediatric patients and low weight adults who are in bone marrow failure. It has been shown that UCB contains abundant CD34+ hematopoietlc stem cells, another characteristic of UCB transplantation is the lower sever severity and accident of acute graft-versus-host(GVHD)than BM and MPB. However, due to the relative volumes of placental blood and the limited number of stem cells collected routinely, cell dose presently a potential for the widespread use of UCB for transplantation in adults. The study of ex vivo expansion of UCB hcmatopoietlc stem cells is an interesting projection in basic and clinical research of the transplantation field.Density gradient centrifugation (Ficoll), gelatin sedimentation and natural sedimentation were compared. The result showed that the recovery of nuclear cells were high using gelatin sedimentation and natural sedimentation (79.76±2.74% and 83.26±3.84% respective), whereas the recovery of nuclear cells were relative low using Ficoll. Semisolid colony assays showed that the recovery of CFU using Ficoll and natural sedimentation were relatively low(59.26±4.38% and 68.22±3.21% respective), whereas the recovery of gelatin sedimentation was high(83.51±3.83%).Immunomagnetic bead separation was used to enrich CD34+ stem cells, next fluorescence activated cell sorter (FACS) was employed to determine the percentage of the enriched CD34+ cells after the purification. Experimental results showed that the purity of CD34+ cells amounted 70.98% after purification, which were enriched with 44.1 folds, recovery was 35.27%.The single effect of various hematopoietic cell growth factors on the expansion of cells was investigated. The results showed that the effect of G3 was most evident, the expansion folds of colony was most high(6.93±1.72); secondly were SCF(5.84±1.84)and IL-6(1.63±0.17);G-CSF was lowest. The expansion folds were generally low when hematopoietic cell growth factor was individually used.Combinational effects of three hematopoietic cell growth factors and their concentration on the expansion of stem cells were investigated employing uniform design. The optimal concentration of cell growth factors were: SCF 60ng/ml,G-3 30ng/ml,IL-6 10ng/ml. The amounts of CFU were expanded (10.39±0.17) folds when cultured after fourteen days under the optimal conditions, which was better than results under experimented conditions. Effects of various initial cells density and media exchange frequency on the expansion of stem cells were investigated. The results showed that when seeding density was low(103cells/ml), frequently media exchange(every three days) could reduce low expansion folds(MNC:84.15±4.26；CFU:27.24±1.97)；whereas seeding density was high(105cells/ml), increaseing media exchange frequency (every three days) could improve the expansion folds(MNC:137.10±4.04；CFU:43.21±2.22).Comprehensively , the most optimal conditions were seeding density 1～5×104cells/ml, exchange media every week.The expansion of cells in long-term cultivation with the optimal conditions was investigated. The results showed that after cultured in four weeks, total cells expansion folds could reach 1200 and still increasing, whereas CFU expansion reacheed peak during the second and third weeks(40～50folds), and then cuted down gradually.
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