Remove and discard culture medium. Briefly rinse the cell layer with DPBS solution to remove all traces of serum that contains trypsin inhibitor. Add 1.0 to 2.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 1~2min). Cells that are difficult to detach may be placed at 37℃ to facilitate dispersal. Add 4.0 to 6.0 mL of complete growth medium and aspirate cells by gently pipetting. Add appropriate aliquots of the cell suspension to new culture   vessels.