We present an approach based on acoustic levitation to grow the cells without wall interactions in order to promote 3D cell architecture (spheroïd, organoïd). The 3D microenvironment is indeed closer to In Vivo physiological behavior. The suspended cells are moved toward the acoustic pressure nodes where they are trapped and maintained in acoustic levitation in perfectly straight monolayers reaching very quickly the classical confluency of the cells in 2D culture. Interestingly, by maintaining the monolayers of MSCs in culture over a 24-h period, the MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. This approach of 3D cell culture is based on the use of the acoustic wave coupled with microfluidics. We designed a standing wave cavity to generate a large acoustic radiation force (ARF) with an optical access, which allows the characterization of the self-organization dynamics. This 3D cell culture method has been validated on MSCs over 24h experiments. The MSCs viability has been checked. Moreover, they show a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.