Cell detachment and reseeding are typical operations in cell culturing, often using trypsin exposure and pipetting, even though this process is known to damage the cells. Reducing the number of detachment and reseeding steps might consequently improve the overall quality of the culture, but to date this has not been an option. This study proposes the use of resonant vibration in the cell cultivation substrate to selectively release adherent calf chondrocyte cells: Some were released from the substrate and collected while others were left upon the substrate to grow to confluence as a subculture-without requiring reseeding. An out-of-plane vibration mode with a single nodal circle was used in the custom culture substrate. At a maximum vibration amplitude of 0.6 µm, 84.9% of the cells adhering to the substrate were released after 3 min exposure, leaving a sufficient number of cells for passage and long-term cell culture, with the greatest cell concentration along the nodal circle where the vibration was relatively quiescent. The 72-h proliferation of the unreleased cells was 20% greater in number than cells handled using the traditional method of trypsin-EDTA (0.050%) release, pipette collection, and reseeding. Due to the vibration, it was possible to reduce the trypsin-EDTA used for selective release to only 0.025%, and in doing so the cell number after 72 h of proliferation was 42% greater in number than the traditional technique.