The following is a general procedure to rapidly remove various cell lines from the substratum while maintaining cellular integrity. This procedure is not meant to be universally applicable for all cell lines. The optimal conditions and concentrations employed for individual systems should be determined empirically. Cell viability should be routinely monitored at the time of subculturing. Cell viability should be greater than 90%.
1. Remove and discard spent cell culture media.
2. Wash cells using a balanced salt solution without calcium and magnesium or wash with EDTA. Add wash solution to the side of the flask opposite the cells. Rinse the cell sheet by rocking the flask for 1 to 2 min and discard wash solution.
3. Add the dissociation solution of choice at 2 to 3 ml/25 cm2 to the side of the flask opposite the cells. Be certain that the dissociation solution covers the cell sheet. Incubate the flasks at 37°C. Rock the flasks gently. Generally, cells are dissociated in 5 to 15 min. The actual time needed to dissociate cells will vary according to cell line. Monitor the process carefully to avoid cell damage. In addition to rocking gently, flasks of cell lines that are characteristically difficult to remove from the substratum may be tapped to expedite removal.
4. When the cells are completely detached, stand the flask in the upright position to allow the cells to drain to the bottom of the flask. Add complete media to the flask. Disperse the cells by pipetting repeatedly over the surface of the monolayer. Count and subculture the cells.
- Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.