1. Remove and discard culture medium.

2. Briefly rinse the cell layer with 0.25% （w/v） Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.

3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed （usually within 5 to 15 minutes）.

   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.

5. Add appropriate aliquots of the cell suspension to new culture vessels.

6. Incubate cultures at 37°C.




1. Subc*tion Ratio: A subc*tion ratio of 1:3 to 1:10 is recommended
   Medium Renewal: 2 to 3 times per week

recommended serum:ATCC 30-2020

irradiated to be used as feeder cells:ATCC 56-X

21874: . Teratomas and differentiation. New York: Academic Press; 1975.

22387: . . Cell 6: 467-474, 1975.

22701: Martin GR, et al. The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev. Biol. 61: 230-244, 1977. PubMed: 590624