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Suspension cultures, in which human mesenchymal stem cells are cultivated on microcarriers in scalable single-use stirred bioreactor types, have been shown to be a promising alternative to planar flask cultures. However, stirred single-use bioreactors were originally developed for production processes with robust, permanent cell lines. Human mesenchymal stem cells are adherent primary cells and thus expanding them in such bioreactor systems imposes more stringent requirements on bioreactor systems. For low-serum conditions (5%) and different types of stirred single-use bioreactors, a suspension criteria-based approach for expanding human adipose tissue-derived mesenchymal stem cells (hASCs) from milliliter to pilot scale was successfully developed. For process scale-up, experimental and numerical investigations were performed to (i) predict optimum impeller speeds, (ii) determine the main engineering parameters (local shear stress, turbulent dissipation rate, Kolmogorov microscale), and (iii) verify suspension criteria NS1 and NS1u for rapid process transfer from 100 mL to 2 L and 35 L cultures. Using optimized medium-microcarrier combinations as well as NS1 and NS1u as scale-up factors, total hASC quantities between 3 × 107 (100 mL scale) and 1 × 1010 (35 L scale) were obtained. The cell quantities obtained are the highest reported to date for scalable single-use bioreactors under low-serum conditions. [ABSTRACT FROM AUTHOR]