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  • EX-VIVO EXPANSION  (2)
  • 1
    Keywords: THERAPY ; TRANSPLANTATION ; T-CELLS ; NK cells ; CANCER-PATIENTS ; GENE-EXPRESSION ANALYSIS ; PHASE-II ; ACUTE MYELOID-LEUKEMIA ; EX-VIVO EXPANSION ; LARGE-SCALE
    Abstract: BACKGROUND AIMS: Ex vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell-based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process. METHODS: Enriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus-transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression. RESULTS: Automated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (+/-0.9) x 10(9) activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-gamma and tumor necrosis factor-alpha and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell-activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential. CONCLUSIONS: We established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.
    Type of Publication: Journal article published
    PubMed ID: 25881519
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  • 2
    Keywords: IN-VITRO ; GENE-EXPRESSION ; DIFFERENTIATION ; bone marrow ; ADHESION ; STEM-CELLS ; cord blood ; BONE-MARROW-CELLS ; adipose tissue ; EX-VIVO EXPANSION ; mesenchymal stromal cells ; OSTEOBLASTIC DIFFERENTIATION ; OSTEOGENIC DIFFERENTIATION ; impedance monitoring ; MATRIX-MEDIATED RETENTION
    Abstract: Background aims. For their wide mesodermal differentiation potential, mesenchymal stromal/stem cells (MSC) are attractive candidates for tissue engineering. However, standardized quality control assays monitoring differentiation that are non-invasive and continuous over time are lacking. Methods. We employed a non-invasive assay, using two different systems, to discriminate osteogenic and adipogenic differentiation of MSC by monitoring impedance. Fibroblasts and keratinocytes served as nonspecific controls. Impedance profiles were recorded comparing MSC from bone marrow and adipose tissue, either non-induced or induced for osteogenesis or adipogenesis, for 5-14 days, and correlated with differentiation markers assessed by reverse transcription-quantitative polymerase chain reaction and Western blot. Additionally, differentiation modulating effects of extracellular matrix components were analyzed. Results. Adhesion and growth-related impedance profiles of non-induced MSC roughly resembled those of fibroblasts, whereas keratinocytes differed significantly. Distinct from that, osteogenic induction of MSC revealed initially rapid and continuously rising impedance, corresponding to mineralized calcium matrix formation. Conversely, adipogenic induction caused shallower initial slopes and eventually declining profiles, corresponding to more compact, adipocyte-like cells with numerous lipid vacuoles. Pre-coating with either collagen type I or IV apparently favored osteogenesis and fibronectin adipogenesis. Impedance recordings correlated well with the extent of differentiation evaluated by histochemical staining and protein and gene expression. Conclusions. Overall, our data demonstrate that impedance profiling offers a basis for standardized real-time, non-invasive high-throughput screening of MSC properties. It enables further testing of the influence of diffusible factors or extracellular matrix composites on MSC differentiation or maintenance of stemness, thus substantiating therapeutic application
    Type of Publication: Journal article published
    PubMed ID: 21619493
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