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 pipetting gently.
5. Add appropriate aliquots of cell suspension to new culture vessels.
6. Incubate culture vessels at 37°C.

Protocol for Control Conditioned Medium:

1. Split the cells 1:10 in 10 mL culture medium in 10 cm petri dishes and let the cells grow for 4 days (approximately to confluency).
2. Take off the medium and sterile filter. This is the first batch of medium.
3. Add 10 mL fresh culture medium and culture for another 3 days.
4. Take off the medium and sterile filter. This is the second batch of medium. Discard the cells, because they will be overgrown.
5. Mix the first batch and second batch of medium 1:1. This is the L Cell conditioned medium. It is stable at 4°C and can be frozen.

Willert K, et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448-452, 2003. PubMed: 12717451