We propose three-dimensional (3D) spheroid formation involving perfusion and a lab-on-a-chip containing spheroid-forming chamber arrays. Cells are collected forming a spheroid in the chamber in microrotation. We previously reported a single chamber form hepatic spheroids 130 to 430 µm in diameter, controlling size by varying chamber diameter and cell density. Here, we scaled the system up by a factor of 10 while maintaining size control of 180±30 µm in diameter. Results were comparable to those using a single-chamber device. Long-term culture confirmed that cells in the spheroid maintained viability and diameters did not change after 24 hours. The system is readily applicable for creating size-controlled spheroids ensuring reliable, predictable in vitro data for drug screening and biological research.