Background <p>The production of human mesenchymal stromal cells (hMSC) for therapeutic use requires scalable, efficient and standardized manufacturing processes that could further benefit from shortening manufacturing time, automate and simplify operations with closed systems. This study aimed to develop an industry-ready process for the expansion and integrated downstream processing of human adipose tissue-derived MSC (ASC) using xeno-free medium and microcarriers in stirred-tank bioreactors. The proposed workflow provides controlled culture conditions and is compatible with closed and large-scale cell production.</p> Methods <p>ASC were directly inoculated after thawing in stirred-tank bioreactors without the need for a seed train. Microcarrier type (Plastic vs. Synthemax II-coated) in combination with xeno-free medium and stirring profile during cell attachment (intermittent vs. continuous) were optimized in 0.2&#xa0;L bioreactors and subsequently scaled-up to 2&#xa0;L bioreactors using power input per volume as scale-up criteria. Focusing on further process scale-up, two strategies were evaluated: (1) bead-to-bead transfer, enabling cell migration from colonized to fresh microcarriers, and (2) enzymatic detachment followed by re-inoculation as single cells. A closed downstream process, using counterflow centrifugation was integrated as an alternative to conventional open centrifugation.</p> Results <p>Synthemax II-coated microcarriers supported efficient cell attachment without a seed train, even under continuous stirring. Two strategies were deemed feasible for process scale-up (cell migration via bead-to-bead transfer and cell detachment and re-attachment to microcarriers as single cells) in stirred-tank bioreactors. Counterflow centrifugation achieved cell recovery yields comparable to standard centrifugation (67% vs. 75%, respectively). High cell viability (&gt; 96%), expression of MSC characteristic surface markers (&gt; 95%) and immunomodulatory function were preserved.</p> Conclusions <p>This work presents a fully integrated and scalable ASC manufacturing platform from inoculation to harvest, without requiring seed train. The process supports an efficient (up to 4-5 × 10⁵ cell/mL) and robust cell expansion (regardless of donor variability, similar cells yields are obtained), enables closed downstream processing (integrating filtration and counterflow centrifugation), and shortens overall production timelines. This approach achieves a volumetric productivity, after microcarrier filtration and counterflow centrifugation, of approximately 3–4 × 10⁸ cell/ L, corresponding to cell numbers that could support early-phase clinical trials. Additionally, glucose and lactate concentrations directly correlate with cell density, allowing these metabolites to be used as predictive parameters for defining harvesting day. These findings contribute to the development of a clinically- and industrially-relevant bioprocess for large-scale MSC production.</p>