Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.
Animals ; Bone Marrow ; Cell Differentiation ; Cell Line ; Cytokines ; Erythrocytes ; cytology ; Erythroid Cells ; Leukocytes, Mononuclear ; cytology ; Mice

OBJECTIVE: To determine the biological traits and optimal condition for the induction and differentiation of endothelial progenitor cells from peripheral blood in healthy adults. METHODS: Mononuclear cells isolated from peripheral blood of healthy adults were cultured in M199 medium supplemented with VEGF, bFGF, IGF-1, and EGF. The appearing time of cell clusters or spindle-shaped cells was recorded respectively. Attached spindle-shaped cells were detached and labeled with a series of antibodies against blood vessel endothelial-specific markers. RESULTS: Attached spindle-like cells appeared 4 days after the culture, cell clusters were observed at 5 to 8 days, and cord-like structure was formed by 10th day. These cells expressed endothelial-specific markers. CONCLUSION Endothelial progenitors cells were derived from mononuclear cells of peripheral blood, which can be induced into endothelial cells at specific conditions.
Cell Differentiation ; Cell Separation ; Endothelial Cells ; cytology ; Endothelium, Vascular ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Stem Cells ; cytology

Cell Differentiation ; drug effects ; Cell Separation ; Endothelial Cells ; cytology ; Fibronectins ; pharmacology ; Humans ; Leukocytes, Mononuclear ; cytology ; Stem Cells ; cytology

To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.
CD4-Positive T-Lymphocytes ; cytology ; CD8-Positive T-Lymphocytes ; cytology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Leukocytes, Mononuclear ; cytology ; immunology

The purpose of study was to investigate the in vitro proliferation ability of PHA-induced CIK cells and traditionally prepared CIK cells, the effector cell level and its influence on killing activity to K562 cells, and to analyze the difference between them. The peripheral blood mononuclear cells(PBMNC) of healthy persons were isolated and divided into A and B group. The CIK cells in A group were obtained by using traditional culture method, the CIK cells in B group were prepared by PHA induction. During the cultivation, the cell survival rate and cell absolute value in the cell culture system were counted every 3 days. On day 15 of culture, the cell immunophenotype of 2 groups were detected by flow cytometry, and the ratios of CD3(+)CD56(+), CD3(+)CD8(+) and CD3(+)CD4(+) cells in total cell amount of culture system were accounted. Meantime, the killing activity to K562 cells in different effector-target ratios was detected by using CCK-8 kit between the 2 groups. The results showed that the method of preparing CIK by PHA induction promoted the cell proliferation more than that of the traditional method (P < 0.05), moreover, both the survival rate of cells in 2 groups was more than 90%. The CD3(+)CD8(+), CD3(+)CD56(+) cell ratio in 2 groups obviously increased. As compared with traditional method, the CD3(+)CD8(+) cell level in B group was enhanced (P < 0.05); but there were no statistical differences in increase of CD3(+)CD56(+) cell level and decrease of CD3(+)CD4(+) cell level between 2 groups. while the effector-target ratio is 5:1, 10:1, 20:1 and 40:1, the killing activity of PHA-induced CIK cells to K562 cells was more stronger than traditionally-prepared CIK cells (P < 0.05), moreover, along with increase of effector-target ratio, the difference of killing activity to K562 cells in 2 groups significantly increased. It is concluded that compared with traditional method for preparing CIK cells, the new way by PHA induction can increase the proliferation of CIK cells obviously, enhance the ratio of CD3(+)CD8(+) cells and strengthen the killing activity to the K562 cells. This new way provides a new source of CIK cells and reliable evidence for cyto-immune therapy of leukemia and other tumors.
Cell Proliferation ; Cytokine-Induced Killer Cells ; cytology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; Phytohemagglutinins ; pharmacology

The influencing factors on cord blood storage after collection and mononuclear cell separation as well as cryopreservation were studied. The mononuclear cell are separated from blood after blood collection, then cryopreserved and washed after thawed. Results showed that the cord blood kept at 4 degrees C or room temperature less than 24 hours after blood collection, mononuclear cell separated by hydroxyethylstarch and 2 centrifugations, mononuclear cell cryopreserved with 50% DMSO and autoplasma from cord blood as protectives and washing the cells after thawing. In conclusion, the optimal project in this study can effectively preserve cord blood mononuclear cells.
Blood Preservation ; Cell Separation ; methods ; Cryopreservation ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; physiology

For in vitro studies, both CD34+ selected cell and mononuclear cell (MNC) can be used to expand hematopoietic stem/progenitor cells. To investigate the expansion characteristics of mononuclear cells (MNC) and CD34+ selected cells the two cell fractions were cultured in the medium containing cytokine cocktails of SCF + IL-3 + IL-6 + FL + Tpo. It was found that the CD34+ selected cells had presented a high proliferation potential. The expansion of CD34+ selected cells could be maintained for 8 weeks while that of MNCs declined after 4 weeks. During the culture period, the maximum expansion of total cells in CD34+ selected cell culture achieved 31,270.9 +/- 8640.5 times, while that of MNC reached 50.9 +/- 8.2 times only. In the culture of MNCs, the colony density and the proportion of CD34+ cells increased from day 0 to day 7. However, in the culture of CD34+ selected cells, both the colony density and the proportion of CD34+ cells declined continuously during the whole culture period. During the ex vivo culture of CD34+ selected cells, the maximum expansion of CFU-GM and CD34+ cells achieved 185.7 +/- 14.1 fold and 191.7 +/- 188.8 fold, respectively. They are much higher than that of MNC, which were 12.4 +/- 3.2 fold and 50.6 +/- 33.2 fold only. While the BFU-E of both cell fractions only expanded by few times, which were 7.2 +/- 5.2 and 10.1 +/- 3.4 times, respectively. The results showed that the CD34+ selected cells culture could obtain more CFU-GM cells and CD34+ cells during the whole culture period.
Antigens, CD34 ; analysis ; Cell Count ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; physiology ; Leukocytes, Mononuclear ; cytology

The aim of this study was to establish the method of isolating and culturing endothelial progenitor cells (EPCs) from human umbilical cord blood. Mononuclear cells (MNCs) from human umbilical cord blood were cultured by using culture system supplemented with endothelial cell-conditioned medium. The obtained two types of cells were purified by picking up colonies, identified by uptake of acetylated low-density lipoprotein (Ac-LDL) and binding to lectin [Ulex European Agglatinin (UEA-1)], and were analyzed for the expression of markers by flow cytometry. The results showed that there were significant differences between two types of cells in proliferation, so they were referred as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs), respectively. They were in accordance with the standards of EPCs, could uptake DiI-Ac-LDL and bind to UEA-1, and expressed the markers of endothelial cells, such as CD31, CD144 and vWF detected by immunocytochemistry. The transcription of CD31, KDR, CD144 and ENOS in both of them could be detected by RT-PCR, but FACS analysis showed significant differences of surface marker expression between them. In conclusion, two types of EPCs are successfully obtained by culturing MNCs isolated from human umbilical cord blood using endothelial cell-conditioned medium.
Cell Separation ; Cells, Cultured ; Culture Media, Conditioned ; metabolism ; Endothelial Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Neovascularization, Physiologic ; physiology ; Stem Cells ; cytology

Colony-Forming Units Assay ; Cryopreservation ; methods ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunotoxins ; immunology ; Leukocytes, Mononuclear ; cytology ; Ricin ; immunology ; T-Lymphocytes ; cytology ; immunology ; Time Factors

OBJECTIVETo discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).

METHODSUCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.

RESULTSWith the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.

CONCLUSIONSThis non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Erythroblasts ; cytology ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Umbilical Cord