Perfusion Enhances Functions of Bone Marrow Stromal Cells in Three-Dimensional Culture

Perfusion of medium through three-dimensional (3D) collagen sponges enhanced viability and function of cocultivated marrow stromal and hematopoietic cell lines. Cells of the murine bone marrow stromal cell line GPIa were cultured in novel 3D collagen sponges, made from pepsin-digested bovine skin. Static cultures of sponges were maintained in dishes with media changes every other day. Perfused sponges were contained in a glass column with medium flow set at 1.3 mL/min. In some sponges, the 32D cl3 c-fmsm (CRX-1) hematopoietic progenitor cell line was added 7 days after GPIa cells. At 7 and 16 days, light microscopic evaluation showed poor viability of cells in static sponge cultures. In perfused sponge cultures, there was greater cellularity throughout the sponge and abundant accumulation of metachromatic extracellular matrix surrounding GPIa cells. Chondroitin 6-sulfate and heparan sulfate were identified as components of the matrix by immunohistochemical methods. DNA synthesis was evaluated by 15-h exposure of cultures to bromodeoxyuridine (BrdU), with subsequent immunohistochemical localization with monoclonal anti-BrdU antibody. Cells positive for BrdU were identified at the outer surfaces of both static and perfused sponges; however, positive cells were also seen throughout the internal areas of the sponges that were perfused. These results suggest that better nutrient exchange occurred in perfused sponges. In static cocultures of GPIa and CRX-1 cells, there was no detectable viability of the IL-3–dependent CRX-1 cells; however, under perfused conditions, CRX-1 cells flourished within the sponges as documented by BrdU incorporation. Thus, medium perfusion enhanced GPIa stromal cell line viability and function in 3D collagen sponge cultures, as demonstrated by BrdU incorporation, matrix production, and support of CRX-1 cells. This novel culture system may be useful for examining the interactions of bone marrow stromal cells with extracellular matrix molecules, soluble and matrix-bound factors, and with other cell types.