PURPOSE: Cryopreservation has been the standard method of storing hematopoietic cells for the past 20 years, but this prdegrees Cedure is laborious and expensive. So, we evaluated the hematopoietic recovery of stored PBSCs at 4degrees C for a variable storage period MATERIALS AND METHODS: Eight leukapheresis products were kept unprdegrees Cessed at 4degrees C for 96 hours. To evaluate the effect of storage period on the hematopoietic recovery of PBSCs, assays for viability of mononuclear cells (MNCs), CFU-GM colony counts and CD34 cell counts were performed every 24 hours after PBSC collection. We tried to compare hematopoetic recovery of stored PBSCs at 4degrees C with that of cryopreserved PBSCs by using repeated measures ANOVA. RESULTS: Viability of MNCs, CFU-GM colony counts and CD34 cell counts were monitored at 24 hour, 48 hour, 72 hour and 96 hour after PBSC collection. Data are expressed as percentage of baseline value and shown as mean s.d.; MNCs viability (96+/-2%, 94+/-2%, 92+/-2%, 88+/- 3%), CFU-GM colony counts (87+/-10%, 79+/-11%, 65+/-13%, 56+/-15%), and CD34 cell counts (93+/-13%, 93+/-12%, 88+/-14%, 85+/-19%). After storing PBSCs at 4degrees C for 96 hours, viability of MNCs and CFU-GM colony counts were significantly reduced (p<0.05) except CD34 cell concentration (p>0.05). Prdegrees Cedures of controlled-rate freezing and thawing resulted in a notable loss of viability (77+/-9%) and CFU-GM colony count (71+/-29%). CFU-GM colony counts of 72 hour-stored PBSCs at 4degrees C was similar to those of cryopreserved PBSCs. CONCLUSION: If G-CSF mobilized PBSCs are stored at 4degrees C in less than 72 hours after collection, those hematopoietic recovery would be comparable to that of cryopreserved stem cells which are achieved by the rate-control freezer.
Bezafibrate ; Cell Count ; Cryopreservation ; Freezing ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Progenitor Cells ; Leukapheresis ; Stem Cells*

PURPOSE: Many studies for hematopoietic stem cell have investigated CD133, instead of CD34, as a new surrogate stem cell marker. Counterflow centrifugal elutriation (CCE) is a physical separation of a homogeneous cell population through cell sedimentation characteristics. We evaluated the stem cell distribution and hematopoietic function from cord blood (CB) and bone marrow (BM) through CCE. METHODS: We obtained total nucleated cells from CB and BM, and separated the cell fractions according to media infusion flow rates (17 mL/min (FR 17), 24 mL/min (FR 24), 29 mL/min (FR 29), and rotor off (R/O) ) by CCE. We analyzed the proportion of CD34+ and CD133+ cells in each fraction, and performed methylcellulose-based colony assay. RESULTS: In CB, the cell recovery rates after CCE were 5.9+/-4.3% in FR 17, 4.2+/-2.1% in FR 24, 19.4+/-11.9% in FR 29, and 61.9+/-11.7% in R/O. In BM, they were 14.9+/-8.2% in FR 17, 17.4+/-13.4% in FR 24, 23.6+/-6.11% in FR 29, and 27.1+/-8.9% in R/O. The distributions of CD133+ and CD34+ cells in CB were more abundant in R/O (2.91%, 1.85%) than in other fractions. In BM, CD133+ and CD34+ cell rates in R/O (5.40%, 2.75%) were similar with those in unmanipulated BM (5.48%, 2.78%). In both CB and BM, there was more CFU-GM and BFU-E in R/O than in other fractions. CONCLUSION: We suggested that the distribution of CD34+ and CD133+ cells might be different between CB and BM. However, the R/O containing relatively large cells could have an effective clonogenicity compared with the unmanipulated sample in both CB and BM.
Bone Marrow* ; Erythroid Precursor Cells ; Fetal Blood* ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cells ; Stem Cells

BACKGROUND: An essential prerequisite for successful procurement of sufficient peripheral blood stem cells (PBSC) for engraftment is the optimal timing of collection. The Sysmex SE-9000 automated hematology analyzer provides the immature information (IMI) channel for the identification and counting PBSC. In this study, The optimal timing of PBSC collection was studied using IMI channel. METHODS: 193 peripheral blood stem cell collections were performed from 52 patients with hematologic disorders or solid tumors and 15 donors. Pre-harvest peripheral blood WBC, mononuclear cells (MNC) and IMI were tested and compared with CD34+ cell count and CFU-GM count of harvested products. RESULTS: Peripheral blood WBC and MNC count showed a weak correlation with CD34+ cell yield (r=0.38, P<0.0001 and r=0.38, P<0.0001) and peripheral blood IMI had a stronger correlation (r=0.58, P<0.0001) with collected CD34+cells than did WBC and MNC count. A receiver operating characteristic (ROC) curve was drawn for cutoff value of IMI and predictive values of the chosen cutoff value of IMI for different target CD34+ cell collections were calculated. The ROC curve showed that the best cutoff value of IMI was 465/microliter for the target CD34+ cells >1x10(6)/kg with sensitivity of 88.7%. Positive and negative predictive values of IMI >465/microliter for CD34+ cell >1x10(6)/kg were 65.5% and 87.5%, respectively. CONCLUSIONS: The automated IMI might be used as a simple and efficient indicator of PBSC mobilization and applying variable cutoff values of IMI would be a useful tool to predict the optimal timing of PBSC collection.
Cell Count ; Granulocyte-Macrophage Progenitor Cells ; Hematology* ; Humans ; ROC Curve ; Stem Cells* ; Tissue Donors

BACKGROUND: To standardize the separation method of cord blood and establish the Korean Cord Blood Bank, we analysed the data regarding cord blood separation from 8 different hospitals which involved in cord blood banking project. METHODS: We analysed 306 samples which collected with heparinized syringes and separated within 24 hours by Ficoll-Hypaque density gradient separation method and RBC depletion method with 3% gelatin. We performed the mononuclear cell (MNC) count, viability, CD34+ cell count and CFU-GM count following separation of cord blood. We assessed the yield of hematopoietic stem cells by different separation methods in 9 hospitals and the variability among hospitals which performed cord blood separation by same method. RESULTS: 1) In 204 samples of cord blood separated with Ficoll-Hypaque, there were significant differences in MNC count, viability, CD34+ cell count and CFU-GM count among all engaged hospitals, but not in each individual hospital. 2) In 102 samples of cord blood separated with 3% gelatin, there were significant differences in viability, CFU-GM count and CD34+ cell count among all engaged hospitals, but not in MNC count. 3) The yield of MNCs, CD34+ cells and CFU-GM was higher in the cord blood separated with 3% gelatin than Ficoll-Hypaque. CONCLUSION: We suggest that RBC depletion method with 3% gelatin would be better than Ficoll-Hypaque method in order to increase the yield of hematopoietic stem cells and standardize the method of cord blood separation.
Cell Count ; Fetal Blood* ; Gelatin ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cells ; Heparin ; Syringes

PURPOSE: For separation of RBC from cord blood, it is important to minimize RBC contamination without significant loss of nucleated cells using sedimentation agent that is safe for human use. This study was performed to investigate the possibility of replacing 6% hydroxyethylstarch (HES) with 10% pentastarch (PS) which is a lower molecular weight hetastarch-analog that is cleared from the circulation rapidly. METHODS: After dilution of cord blood till hematocrit 25%, PS or HES were added by the ratio of 7:1 and 5:1 respectively. Sedimentation was performed for 2 hours by gravity. RESULTS: PS was used in 14 cases with volume of 72.4+/-22.3 mL (45~126 mL) and HES in 8 cases with volume of 58.4+/-8.0 mL (50~70 mL). Sedimentation rate has reached at plateau by 90 minutes in PS group and it was slightly faster than in HES group. Recovery rate of nucleated cells and residual RBC were 82.9+/-10.7%, 7.6+/-5.4% in PS group, and 84.0+/-4.7%, 10.7+/-2.3% in HES group. There were no significant differences between the two groups (P=0.657, 0.219). Cell viabilities were high in both groups; 92+/-3% before separation and 97+/-2% in PS group and 98+/-3% in HES group. CD34+ cells were 0.75+/-0.28% before separation and 0.64+/-0.21% in PS group and 0.60+/-0.30% in HES group (P=0.690). CFU-GM after 2 week culture were 27.4+/-20.0 per 1 105 mononuclear cells in PS group and 22.9+/-8.6 in HES group (P=0.856). CONCLUSION: These results demonstrated that PS has similar efficacy to HES for separation of RBC from umbilical cord blood. Considering its rapid clearance and faster sedimentation rate, PS can replace HES for RBC separation in cord blood banking.
Cell Survival ; Fetal Blood* ; Granulocyte-Macrophage Progenitor Cells ; Gravitation ; Hematocrit ; Humans ; Hydroxyethyl Starch Derivatives* ; Molecular Weight

BACKGROUND: Umbilical cord blood (UCB) is generally stored overnight and it undergoes a CD34 positive selection process the next day for reducing the cost and due to the convenience. We intended to determine whether overnight storage of cord blood cells affects the short and long-term repopulating capacity. METHODS: Five individuals' UCB samples were analyzed by colony assay, apoptotic cell counts and long term bone marrow culture. All the samples were divided to four groups, which were the fresh group (immediate use of harvest), the overnight storage group (overnight storage at room temperature after harvest), the immediate cryopreservation group (immediate cryopreservation after harvest) and the overnight cryo group (cryopreservaton after overnight storage at room temperature after harvest). RESULTS: The number of colony forming units-granulocyte macrophage (CFU-GM) was 116.2+/-20.1 in the fresh group and 90.8+/-15.8 in the overnight storage group (P=0.07). The number of CFUs-GM was similar between the immediate and overnight cryo groups (P=0.79). The immediate cryo group showed a significantly lower number of CFUs-GM as compared to that of the fresh group (P=0.03). The apoptotic cells were detected at 21+/-6.8% in the fresh group and this was 24.2+/-2.4% in the overnight storage group (P=0.32), and this was similar between immediate and overnight cryo groups (P=0.80). The fresh group had a significantly lower number of apoptotic cells compared to that of the immediate cryo group (P=0.02). After long term stromal-based culture, the mean production of CFU-GM colonies was similar between all the groups (P>0.05). CONCLUSION: These results support the continue use of overnight storage of UCB before cryopreservation as a convenient, cost reducing measure.
Bone Marrow ; Cell Count ; Cryopreservation ; Fetal Blood ; Granulocyte-Macrophage Progenitor Cells ; Macrophages

BACKGROUND: Umbilical cord blood (UCB) cells are an alternative source of hemopoietic stem cells for transplantation. However, the use of UCB is limited by the lack of number of hemopoietic stem cells to reconstitute bone marrow of adults or higher weight recipients. The aim of this study was to evaluate the ex vivo expansion effect of various cytokines, especially thrombopoietin (TPO) in isolated CD34 positive UCB cells. METHODS: CD34 positive UCB cells were isolated by biotin-avidin immunoaffinity method. Isolated CD34 positive cells were cultured with various combination of cytokine including TPO, SCF, IL-3, & GM-CSF. CFU-GM, CFU-Mix, and CFU-Meg colony assays were performed. RESULTS: After 3 weeks of culture, the maximal expansion of total nucleated cells was seen in the combination of TPO+SCF+IL-3. After 1 week of culture, the number of CFU-GM was significantly increased in the combination of TPO+IL-3 (P<0.05) or SCF (P<0.05). In the presence of TPO+IL-3, significantly increased number of CFU-Mix was observed (P<0.05). TPO alone enhanced colony formation of CFU-Meg. In the combination of TPO+SCF, the number of CFU-Meg was increased significantly (P<0.05). CONCLUSIONS: TPO expanded the number of CFU-Meg as well as CFU-GM and CFU-Mix. These results suggest TPO acts not only in the megakaryocytopoiesis but also in the early stage of erythropoiesis and granulopoiesis.
Adult ; Bone Marrow ; Cytokines ; Erythropoiesis ; Fetal Blood* ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocyte-Macrophage Progenitor Cells ; Humans* ; Interleukin-3 ; Stem Cells ; Thrombopoiesis ; Thrombopoietin* ; Umbilical Cord*

BACKGROUND: During ex vivo expansion of cord blood (CB) CD34+ cells, differentiation of the expanded cells happened and hematopoietic potential of the progenitor cells decreased. In this study, we evaluate the effect of the expression of Fas antigen, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during ex vivo expansion. METHODS: CD34+ and AC133+ cells isolated from human CB were cultured in serum free medium supplemented with several cytokines for 7 days. After expansion culture, we re isolated CD34+ and AC133+ cells and compared the numbers of granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, and macrophage colony-forming units (CFU-GEMM), and expression of Fas antigen, Bcl-2, and Bax with unexpanded cells. RESULTS: CFU-GM was expanded 23.94 fold in CD34+ cells and 15.22 fold in AC133+ cells at day 7 of culture but CFU-GEMM was not expanded. The expression of Fas antigen and Bax was 7.44% and 2.75%, respectively, in fresh isolated CD34+ cells and increased to 19.71 % and 33.67%, respectively, in expanded CD34+ cells at day 7 culture, but Bcl-2 was not changed. In case of AC133+ cells, the expression of Fas antigen and Bax were also increased from 5.87% and 6.19% to 24.85% and 22.83%, respectively, and Bcl-2 was slightly decreased. Apoptosis was not changed in CD34+ cells and AC133+ cells during ex vivo expansion. CONCLUSION: These results indicate that the nature of expansion was similar between CD34+ and AC133+ cells, and expression of Fas antigen and Bax increased on CD34+ and AC133+ cells during ex vivo expansion. Selection of the expanded progenitor cells without apoptosis may be useful for the hematopoietic stem cell transplantation.
Antigens, CD95* ; Apoptosis* ; Cytokines ; Erythrocytes ; Fetal Blood ; Granulocyte-Macrophage Progenitor Cells ; Granulocytes ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells* ; Humans ; Macrophages ; Monocytes ; Myeloid Progenitor Cells ; Stem Cells

PURPOSE: Retrovirs-mediated transduction of target genes into bone marrow progenitor cells or peripheral lymphocytes has been less optimal due to low efficiency and minimal expression on long-term analysis. This study aims to establish an efficient 4-day culture condition for the increased transduction efficacy into CD34+ cells selected on umbilical cord blood by comparing combination of various cytokines. METHODS: CD34+ cells from umbilical cord blood selected by Isolex-50R were incubated with supernatant containing XM5/PA317 vector for 96 hours. Cytokine combinations were used including IL-6+SCF, IL-6+IL-3+SCF, and IL-6+IL-3+SCF+TPO. Methylcellulose colony assay was done after culture. The data were expressed as mean+/-SD with 3 experiments. The efficiency of gene transfer was assessed by the ability of transduced CFU-GM to grow in the presence of G418 and PCR analysis of individual CFU-GM. RESULTS: The mean recovery rate of CD34+ cells after purification was 22%, and the purity of the final CD34+-enriched fraction was 82+/-13% (mean+/-SD). After a 4-day culture, the cell number increased 5~10 fold in each culture condition. The transduction efficiency evaluated by both G418-screened CFU-GM and PCR-positive CFU-GM with the above cytokine combinations was 46% and 64%, 41% and 57%, and 28% and 45%. However, there were no significant differences of colony counts between the cytokine combinations. CONCLUSION: We were unable to establish the best recipe of cytokine combination as the number of experiments was small and we tried only a fixed concentration of cytokines. For the future, the study of developing a novel vector, a better condition of transduction, and better combination of cytokines is warranted to attain the goal of highly effective, long-lasting method of gene transfer.
Bone Marrow ; Cell Count ; Cytokines ; Fetal Blood* ; Granulocyte-Macrophage Progenitor Cells ; Lymphocytes ; Methylcellulose ; Polymerase Chain Reaction ; Stem Cells ; Umbilical Cord*

PURPOSE: We tried to evaluate the clinical usefullness of fractionated low-dose infusions of peripheral blood stem cells (PBSCs) as a supportive care. MATERIALS AND METHODS: Four patients were entered onto this study who were diagnosed to have gastric lymphoma (n=1) and advanced ovarian carcinomas (n=3). To overcome the hematologic toxicities, G-CSF-mobilized PBSCs were collected early in disease course. Harvested products were cryopreserved in aliquotes and then infused after each cycle. Planned therapeutic schedules should be performed without changes of dose and interval regardless of hematologic toxicities. RESULTS: 20 cycles of chemotherapies were performed and data of infused cell doses were as follows: median number of PBSCs infusions, 4.5 (3~5); median MNCs, CFU-GM colony counts per infusion of low-dose PBSCs, 1.7 108/kg (1.0~2.4), 3.2 104/kg (2.1~11.8). Among 20 cycles, delayed recovery of thrombocytopenia was shown on 10 cycles. Leukopenia (III/IV) and thrombocytopenia (III/IV) were shown on 8/6 cycles and 8/2 cycles. In spite of myelosuppression, they were successfully treated with planned dose-intensity. Especially incomplete platelet recovery was successfully rescuced by using fractionated infusions of low-dose PBSCs. CONCLUSION: These data warrant further clinical trials to evaluate the potentials of fractionated low-dose infusions of PBSCs collected early in disease course for overcoming accumulated hematologic toxicities, especially thrombocytopenia complicated by repeated chemotherapies.
Appointments and Schedules ; Blood Platelets ; Drug Therapy ; Drug Therapy, Combination* ; Granulocyte-Macrophage Progenitor Cells ; Humans ; Leukopenia ; Lymphoma ; Stem Cells* ; Thrombocytopenia