Complete Growth Medium
The base medium for this cell line is 400 mL DMEM (Lonza cat# 12-741) plus 100 mL F12 Medium (Lonza cat# 12-615F). To make the complete medium add the following components:
- 56 mL Fetal Bovine Serum (ATCC 30-2020; heat-inactivate before using)
- 5.6 mL T-medium supplement (See instructions for preparing T-medium suppplement below)
To prepare the T-medium supplement:
Prepare 100 mL of a 0.1% BSA (Sigma cat# A8022) in PBS (ATCC 30-2200) solution (0.1g of BSA in 100 mL PBS) and add the following components:
- 50 mg Insulin (Gibco cat# 12585-014)
- 136 ng Triiodo-L-thyronine (Sigma cat# T2877)
- 50 mg Transferrin (Sigma cat# T4382)
- 2.5 mg D-Biotin (Sigma cat# 47868)
- 250 mg Adenine (Sigma cat# A3159)
Combine all components, mix well, filter sterilize the T-medium supplement using a 0.22-micron filter. Aseptically dispense 5.7 mL into sterile tubes. Store at -20°C.
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
- Remove and discard culture medium.
Briefly rinse the cell layer with Dilute 1:5 - 0.05% Trypsin (ATCC catalog # PCS-999-003) in PBS (ATCC catalog # 30-2200)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.
Cultures can be established between 3x 104 and 6 x 104 viable cells/cm2.
- Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 3 X 104 and 1 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Atmosphere: air, 95%; carbon dioxide (CO2), 5%