FIG. 1.
Suspension bioreactor expansion of bone marrow cells. (A) The Bellco® bioreactor system used for the whole bone marrow cultures in suspension in the presence of fibronectin-coated microbead carriers (Cultispher®). Throughout the culture period, the bioreactors contained both a microbead-adherent as well as a nonadherent cell population. On scanning electron microscopy examination scarce number of cells were observed adherent to the microbeads after 2 weeks of culture, while after 5 weeks the microbeads were coated with cells. Occasional microbead clusters were also observed. Scale bar, 20 μm. (B) The cell expansion curves of parallel cultures of whole bone marrow aspirates in bioreactors (square) and monolayer (circle) conditions in 20% fetal bovine serum (FBS) medium. A significant difference in the number of cells/million cells of whole bone marrow aspirate input between the two culture conditions was observed after 35 days of culture (p<0.05). (C) The growth curve for the microbead adherent (open symbols) and nonadherent (closed symbols) cell fractions in regular mesenchymal stem cells (MSCs) media (20% FBS minimum essential medium [MEM], squares) or media supplemented with cytokines (interleukin-3 [IL3], interleukin-6 [IL6], and stem cell factor [SCF], circles); no statistical differences were noted between the two culture conditions. (D) The colony forming unit assay (CFU) forming capacity (per 100 cells) of the nonadherent and the adherent cells fractions, indicating similar proportion of MSCs in both culture conditions. The presence of MSCs in the nonadherent fraction allowed for culture growth simply by adding new beads and media. (E) The expansion of microbead-adherent MSCs (defined as CD45−/CD90+/CD73+ by multicolor flow cytometry) over time in bioreactors in regular MSCs media (20% FBS MEM, squares) or media supplemented with cytokines (IL3, IL6, and SCF, circles); no statistical differences were noted between the two culture conditions. Color images available online at www.liebertpub.com/tec