搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system. View Publication

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene,a codon humanized,red-shifted variant of the green fluorescent protein (GFP) gene,which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6,up to 16% of the cells expressed EGFP,as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers. View Publication

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines,serum-free media,and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS,Gibco; X-Vivo-10,BioWhittaker; QBSF-60,Quality Biological; and StemSpan SFEM,Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO],SCF,Flt-3 ligand [FL] with and without G-CSF,and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition,cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow,spleen,and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells,textlessor= 64.8-fold; CD34+CD38- cells,330-fold; CFU-granulocyte erythroid macrophage megakaryocyte [GEMM],248-fold) and CFUs of the myeloid (CFU-GM,407-fold) and erythroid (BFU/CFU-E,144-fold) lineages. The expansion of the megakaryocytic lineage was consistently higher in X-Vivo-10 (CFU-megakaryocyte,684-fold). Autologous plasma promoted colony formation but reduced CD34+ cells and CFU-GEMM. The addition of G-CSF or IL-6 improved cell yields; G-CSF was more effective for committed progenitors. Expansion products from cultures in QBSF-60 with the cytokines engrafted and differentiated into the myeloid and lymphoid lineages in NOD/SCID mice. CONCLUSION: The data supported the strategy of expansion. The optimized condition may be applicable to clinical expansion for the abrogation or reduction of posttransplant cytopenia. View Publication

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However,scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard,suspension cultures are a viable alternative,because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However,the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here,we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production. ?? 2014 The Authors. View Publication

The accurate measurement of biologically active erythropoietin (Ep) in human serum and plasma using present in vivo and in vitro bioassays is difficult because of the presence of both inhibitors and non-Ep stimulators of erythropoiesis. We have developed a simple procedure to quantitatively purify Ep from serum and plasma for subsequent testing in the phenylhydrazine-treated mouse spleen cell assay. The method involves absorption of Ep to an immobilized high-affinity anti-Ep monoclonal antibody and acid elution of the antibody-bound material. After neutralization,the eluted EP is then tested directly in the in vitro bioassay without interference by other serum proteins. By using magnetic beads as a solid support for the antibody,washing and elution steps can be performed rapidly and efficiently. Recoveries of Ep after this procedure show very little sample-to-sample variation and are consistently between 45% and 55%,which is close to the maximum binding expected for the anti-Ep antibody. Coupled with the 7.4-fold concentration that this procedure affords,there is an overall increase in sensitivity of three- to fourfold,which makes this assay suitable for accurately measuring Ep levels in patients with below-average titers. Results with this magnetic bead assay indicate that accurate and reproducible estimates for Ep levels in the serum and plasma from healthy donors as well as from patients with hematologic disorders can be obtained. Titers of biologically active Ep in the sera from a group of patients with either leukemia or lymphoma were found to be elevated,and the values correlated well with titers of immunoreactive Ep measured in the Ep radioimmunoassay. Because of its specificity and high sensitivity,the magnetic bead assay is a valuable alternative to immunoassays for the measurement of elevated,normal,and even subnormal Ep levels in human serum and plasma. View Publication