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Complete Growth Medium
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A 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate and supplemented with 400 ng/ml hydrocortisone and 10% fetal bovine serum
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Subculturing
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Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
- 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 1:3 to 1:10 is recommended
Medium Renewal: Every 2 to 3 days
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Cryopreservation
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Freeze Medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage Temperature: Liquid nitrogen vapor phase
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Culture Conditions
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Atmosphere: Air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Growth Conditions: Cells are disaggregated to single cells when the culture is preconfluent. They do not require a feeder layer.
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