Trypsinization has generally been used as a technique to detach adherent mesenchymal stromal cells (MSC). However, this technique involves chemical manipulation. This study was designed to identify whether detachment of MSC can be induced by cold without using trypsin. MSC isolated from bone marrow were detached via trypsin or exposed to -20C for 1, 5 or 10 min at all passages. Compared with trypsinization, exposing MSC to -20C for 10 min resulted in a significant decrease in MSC number and viability. In conclusion, although detachment of adhered MSC on culture dishes via exposure to cold may allow structurally and functionally intact detached cells, the technique requires improvement of the thermotolerance of MSC.