Journal: Stem Cell Research & Therapy
Article Title: Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell
doi: 10.1186/s13287-021-02190-3
Figure Lengend Snippet: Assessment of hBM-MSC characteristics after CellSTACK cell culture expansion confirms cell identify. a Representative microscopic images of undifferentiated hBM-MSCs (i) or chemically stained for detection of adipocytes (ii), osteocytes (iii), and chondrocytes (iv) following induction of differentiation of the cells into the three-mesoderm lineages. All images were taken using a × 20 objective (FOV of 0.43) and the Zeiss Axio Observer 5 microscope. Adipocytes were stained with Oil Red O, osteocytes stained with Alizarin S, and chondrocytes stained with Alcian Blue. Each hBM-MSC donor ( N = 4 donors; hBM-MSC-48RB/81RB/55RB/85RB) was assessed for differentiation potential. b Flow cytometric analysis of established positive and negative MSC surface markers. Red indicates the cell population stained with the respective antibodies for the detection of positive and negative antigens. Blue indicates the cells stained with an IgG isotype control. Negative antibody (ab) cocktail indicates cells stained with a mixed cocktail of antibodies for the detection of MSC negative markers: HLA-DR, CD11b, CD19, CD34, and CD45 (i). Quantification of percentage of positive cells analyzed by flow cytometry (ii). c Viability analysis (i) of each hBM-MSC donor ( N = 4 donors; hBM-MSC-48RB/81RB/55RB/85RB) recovered from the CellSTACK culture expansion system prior to inoculation into the bioreactor system. Percentage of viable cells were generated by automated counting of dye negative over dye positive cell populations stained using Trypan Blue ( n = 2 technical replicates per donor). Population doublings (ii) and doubling time (iii) of each hBM-MSC donor ( N = 4 donors; hBM-MSC-48RB/81RB/55RB/85RB) calculated over 4 days of expansion in the CellSTACK culture expansion system ( n = 2 technical replicates per donor)
Article Snippet: For large-scale expansion in CellSTACK culture chambers (10-stack), 20 million hBM-MSCs from the working cell bank described in the “ ” section were seeded in the chambers at a seeding density of 3145 cells/cm 2 (Corning, cat.#3271).
Techniques: Cell Culture, Staining, Microscopy, Control, Flow Cytometry, Generated
