| Recommended Culture Conditions:
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From MGH/CMT as specified by cell provider: Leibovitz's L-15 medium with 2 mM L-glutamine supplemented with 20 ng/ml human EGF and 20% fetal bovine serum.
Growth Conditions: The cells grow very slowly, and growth is enhanced by using 20% fetal bovine serum and adding epidermal growth factor (20 ng/ml) to the medium.
Protocol:
Remove and discard culture medium.
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.
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.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.
Subcultivation ratio: A subcultivation ratio of 1:2 to 1:3 is recommended
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