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

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

Human embryonic stem cells (hESCs) are pluripotent cells derived from the embryo at the blastocyst stage. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cell types. Based on these properties of self-renewal and pluripotency,hESCs represent a unique source to generate a large quantity of certain specialized cell types with clinical interest for transplantation-based therapy. However,hESCs are usually grown in culture conditions using fetal bovine serum and mouse embryonic fibroblasts,two components that are not compatible with clinical applications. Consequently,the possibility to expand hESCs in serum-free and in feeder-free culture conditions is becoming a major challenge to deliver the clinical promises of hESCs. Here,we describe the basic principles of growing hESCs in a chemically defined medium (CDM) devoid of serum and feeders. View Publication

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal,chondrogenic,and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However,there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study,we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently,a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media,MesenCult™-ACF (ACF),and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology,surface markers,senescence status,differentiation capacity,and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. View Publication

Human embryonic stem cells,because of their unique combination of long-term self-renewal properties and pluripotency,are providing new avenues of investigation of stem cell biology and human development and show promise in providing a new source of human cells for transplantation therapies and pharmaceutical testing. Current methods of propagating these cells using combinations of mouse fibroblast feeder cultures and bovine serum components are inexpensive and,in general,useful. However,the systematic investigation of the regulation of self-renewal and the production of safer sources of cells for transplantation depends on the elimination of animal products and the use of defined culture conditions. Both goals are served by the development of serum-free culture methods for human embryonic stem cells. View Publication

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br),and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations),to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD,n=30) nucleated cells and was enriched in cells expressing CD34,CD117,CD105,CD127,CD133 and CD166,and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells,and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes,osteoblasts and chondrocytes. DISCUSSION: Hematopoietic,endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications. View Publication