Adherent mesenchymal cell populations derived from bone marrow (BM) have the capability of differentiating into multiple cell lineages in vitro and in vivo. The potential use of these populations, referred to as marrow stromal cells (MSC), in clinical applications to treat nonhematopoietic disorders is being intensely investigated. Ex vivo expansion is being used to increase the number of multipotent MSC available for cell and gene therapy applications. There are currently no standardized methodologies for the culture of these cells, however plating and culturing cell densities have been found to be critical in maintaining the multipotentiality of these cells. Aastrom has previously developed a proprietary single-pass perfusion (SPP) process that has successfully expands BM stem cells with demonstrated engraftment capability in humans. This process technology has been effectively implemented in an automated clinical culture system, the AastromReplicell (tm) Cell Production System (ARS), which enables cells to be produced in a single step process without pre-selection or step-cultures. The culture chamber of this system, along with the operating system software, can both be modified to apply for the growth of other cell types. Using this platform technology, the overall goal of the proposed studies is to develop a GMP compliant automated SPP culture process for the generation of large numbers of highly functional MSC suitable for clinical use. This process will be reliable and reproducible, and the unique combination of SPP and closed system automation should allow MSC production under GMP conditions with minimal manipulation. In Phase I, basic culture parameters known to significantly effect adherent cell proliferation and differentiation will be defined in small-scale cultures to optimize the number of functional MSC produced. MSC will be identified by flow cytometry, colony assays, and differentiation assays. Plastic surface characteristics, including roughness, patterning, and tissue-culture treatment, will be defined. Next, several serum-free media formulations and surface coatings will be examined. Initial studies on the effects of medium perfusion and oxygenation levels will also be performed. In Phase II, after further optimization, defined parameters will be used to modify the ARS components, and the expansion process will be validated to confirm feasibility in the automated SPP system