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The ability to efficiently isolate and expand various stem cell populations in vitro is crucial for successful translation of cell-based therapies to the clinical setting. One such heterogeneous population that possesses a remarkable potential for the development of cell-based treatments for a variety of degenerative diseases and disorders is called the Side Population (SP). For many years, investigators have isolated these primitive cells based upon their ability to efflux the fluorophore Hoechst 33342. This attribute enabled separation of SP cells derived from multiple tissue sources from other endogenous cell populations using fluorescence-activated cell sorting (FACS). While all tissue-specific SP fractions appear to contain cells with multi-potent stem cell activity, the therapeutic utility of these cells has yet to be fully realized because of the scarcity of this fraction in vivo. In view of that, we developed a method to expand adult murine bone marrow and skeletal muscle-derived SP cells in vitro. Here, we describe a spinner-flask culture system that supports the growth of SP cells in suspension when they are combined with feeder cells cultured on spherical microcarriers. In this way, their distinguishing biological characteristics can be maintained, attachment-stimulated differentiation is avoided, and therapeutically relevant quantities of SP cells are generated. Modification of the described procedure may permit expansion of the SP from other relevant tissue sources and our method is amenable to establishing compliance with current good manufacturing practices.

Growth of Bone Marrow and Skeletal Muscle Side Population Stem Cells in Suspension Culture