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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.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:4 is recommended

Owens RB, et al. Epithelial cell cultures from normal and cancerous human tissues. J. Natl. Cancer Inst. 56: 843-849, 1976. PubMed: 176412

Smith HS. In vitro properties of epithelial cell lines established from human carcinomas and nonmalignant tissue. J. Natl. Cancer Inst. 62: 225-230, 1979. PubMed: 283258

Smith HS, et al. Nuclear ultrastructure of epithelial cell lines derived from human carcinomas and nonmalignant tissues. Cancer Res. 39: 332-334, 1979. PubMed: 761205