Complete Growth Medium
The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium:
- 0.5 mg/mL G-4180.5 mg/mL hygromycin B
- fetal bovine serum to a final concentration of 10%
Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Protocol for Wnt-3A, R-spondin and Noggin Conditioned Medium:
- Remove and discard culture medium. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC 30-2200) or 0.25% Trypsin – 0.53mM EDTA (ATCC 30-2101) solution to remove all traces of serum which 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 1:4 to 1:10 is recommended.
- Split the cells 1:10 in culture medium (without G418 and Hygromycin B to prevent carryover of drugs in the conditioned medium) and seed 25 mL cell suspension into T-150 flasks
- Incubate the flasks for 3 or 4 days or until the cells become over-confluent and a number of cell aggregates come off.
- Remove medium and rinse flasks with 10 mL medium and discard rinse.
- Add 25 mL fresh medium and incubate flasks for 24 hours.
- Remove the medium to a centrifuge tube. Add new medium to the flasks. Centrifuge the conditioned medium at 2000 x g for 5 minutes and decant supernatant into a 1 L bottle. Store the conditioned medium at 4°C. This is the first batch of medium.
- Every 24 hours, collect 2nd, 3rd and 4th conditioned medium. Centrifuge and add to same same bottle.
- After the 4th collection, add an equal volume of fresh medium to the bottle (final concentration: 50%), mix well and aliquot media into 50 mL centrifuge tubes and store at -20°C.
- Collect 5th to 8th and 9th to 12th conditioned media, if desired.
Medium Renewal: Every 2 to 3 days.
Freeze Medium: complete growth medium, 70%; fetal bovine serum, 20%; DMSO, 10%
Storage Temperature: liquid nitrogen vapor phase
Temperature: 37°C; Atmosphere: air, 95%; carbon dioxide (CO2), 5%