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

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

Cardiac malformations and disease are the leading causes of death in the United States in live-born infants and adults,respectively. In both of these cases,a decrease in the number of functional cardiomyocytes often results in improper growth of heart tissue,wound healing complications,and poor tissue repair. The field of cardiac tissue engineering seeks to address these concerns by developing cardiac patches created from a variety of biomaterial scaffolds to be used in surgical repair of the heart. These scaffolds should be fully degradable biomaterial systems with tunable properties such that the materials can be altered to meet the needs of both in vitro culture (e.g. disease modeling) and in vivo application (e.g. cardiac patch). Current platforms do not utilize both structural anisotropy and proper cell-matrix contacts to promote functional cardiac phenotypes and thus there is still a need for critically sized scaffolds that mimic both the structural and adhesive properties of native tissue. To address this need,we have developed a silk-based scaffold platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM composite scaffolds have tunable architectures,degradation rates,and mechanical properties. Subcutaneous implantation in rats demonstrated that addition of the cECM to aligned silk scaffold led to 99% endogenous cell infiltration and promoted vascularization of a critically sized scaffold (10 × 5 × 2.5 mm) after 4 weeks in vivo. In vitro,silk-cECM scaffolds maintained the HL-1 atrial cardiomyocytes and human embryonic stem cell-derived cardiomyocytes and promoted a more functional phenotype in both cell types. This class of hybrid silk-cECM anisotropic scaffolds offers new opportunities for developing more physiologically relevant tissues for cardiac repair and disease modeling. View Publication

Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures,as media containing expensive factors/cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline,an anti-bacterial agent,had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study,we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions,hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change,while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus,hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties,indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs. View Publication

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such,hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However,for their full potential to be realized,both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however,they suffer from some major limitations including lack of definition,xenobiotic nature,batch-to-batch variation,and labor-intensive production. Therefore,hESC culture definition is essential if hESC lines,and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state,for over 30 passages using MT and SP. Additionally,we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture,contributing to the standardization of hESC in vitro models and ultimately their application. View Publication

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy,we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters,the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters,suggesting a relative resistance to silencing. Furthermore,an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector,largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile. View Publication