Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T-flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex-1 (R), by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50 x 10(6) cell g(-1) Cytodex-1 (R)). Then, the MSC aggregates were put into contact with additional available surfaces (T-flask, fresh and used Cytodex-1 (R)) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T-flasks or fresh Cytodex-1 (R) carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex-1 (R) carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex-1 (R) microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number.