Process development for expansion of human mesenchymal stromal cells in a 50 L single-use stirred tank bioreactor

Abstract

The application of cell-based therapeutics requires development of refined and scalable culture processes. Stirred tank bioreactors facilitate growth of human mesenchymal stromal cells (hMSC) while meeting these needs. A process for expansion of hMSCs in a 50L bioreactor was developed. Parameters evaluated include agitation rate, pH and dissolved oxygen (DO) control set-points, and media formulation. The pH and DO levels were determined empirically in 3L bioreactors prior to implementation at the 50L scale. The agitation operating range for microcarrier cultures in the 50L bioreactor was calculated based on theoretical and empirical analyses of solid suspension and shear limitations. Additionally, small-scale experiments demonstrated that hMSC growth was improved in αMEM supplemented with human platelet lysate in comparison to DMEM supplemented with FBS. A 43-fold expansion of harvested hMSCs was achieved in 11days in a 50L bioreactor incorporating these process improvements. Cells expanded in the bioreactor exhibited the expected surface marker expression, trilineage differentiation potential, T cell growth inhibition and indoleamine 2,3-dioxygenase inducibility. The results highlight that identification of optimal pH, DO, agitation rates and culture medium for microcarrier-based bioreactor expansion of adherent cells is paramount to developing a platform to support cell-based therapies.