Fig. 3
Post-bioreactor analysis of hBM-MSCs confirms retention of MSC characteristics. 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. b Flow cytometric analysis of established MSC surface markers following immediate recovery of cells from the hollow-fiber bioreactor system (N = 4 donors; hBM-MSC-48RB/81RB/55RB/85RB). Red indicates the population stained with the respective antibodies for the detection of positive and negative markers. 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 for the expression of markers from hBM-MSCs post-bioreactor recovery (ii). c Flow cytometric analysis of established MSC surface markers was performed following 7 days of recovery of cells in 2D flask-based culture system using normal RoosterBio culture medium. Representative flow cytometric plots (i) and quantification of the flow cytometric results (ii) of hBM-MSC #55RB and #81RB donors. Red indicates the population stained with the respective antibodies for the detection of positive and negative markers. 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