Bead to Bead Transfer, an Efficient Strategy to Scale-up the Mesenchymal Stromal Cells-Derived Secretome...

Secretome derived from mesenchymal stromal cells (MSCs) holds strong therapeutic potential for regenerative medicine. However, the large-scale production of MSC secretome still presents scientific and technical challenges. This study presents a novel approach using bead-to-bead (BTB) transfer in stirred-tank bioreactors (STR) to scale up the secretome production by turbulence stimulation. By applying three successive microcarrier additions, we could achieve 428 million MSCs in 16 days, corresponding to 5.3 cumulative population doublings (CPD). The BTB transfer significantly outperformed conventional batch culture by extending cell culture duration without aggregate formation. Indeed, a 2.4-fold increase in particle/cell yield, an 88-fold increase in total particle production, and a 113-fold enhancement in particle productivity were obtained in comparison with production in static flasks, demonstrating substantial improvements while reducing the initial number of cells required. The produced EVs display the presence of CD9, CD81 and CD63 tetraspanins and positive MSC markers, validating the identity of the produced EVs. This scalable and enzyme-free BTB transfer method thus offers an efficient and reproducible strategy for high-yield secretome production while maintaining its main characteristics.