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

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive,low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics. The combination of BD FACS Aria Cell Sorter,CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation,production host cells are first transfected with an expression vector carrying the gene of interest (1),followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody,and are subsequently subject to flow cytometry analysis (2-4). Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed,these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup. This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes,the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure,this automated platform demonstrated advantages of significantly increased capacity,ensured clonality,traceability in cell line history with electronic documentation and much reduced opportunity in operator error. View Publication

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy,although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore,in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells,cetuximab concentration,and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets,respectively,in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore,supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level,cetuximab concentration,and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P textless 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a,and secreted significantly (P textless 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients. View Publication

Interleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin(-)Sca-1(+) hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6(-/-)) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6(-/-) BM progenitors is also observed in IL-6(-/-) long-term BM cultures,which show defective hematopoietic support as measured by production of total cells,granulocyte macrophage-colony-forming units (CFU-GMs),and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6(-/-) BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6(-/-) BM,stromal mesenchymal precursors,fibroblast CFUs (CFU-Fs),and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover,IL-6(-/-) stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1),Sca-1,CD49f,and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors,which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6(-/-) mice. View Publication