CHO Cell Culture
CHO cells can be maintained as a suspension or as adherent to a substrate. For suspension, maintain cells in culture by revolving cultures continuously at approximately 50 RPM. Cell lines should be kept in such 10 mL “roller cultures”. Maintenance roller cultures should be diluted every 3-4 days. CHO cells should be cultured in Ham’s F12K (ATCC suggestion) or DMEM modified with 10% FBS. If cells are not doubling every 14-17 hours, supplement the medium with 1-2% FCS.
CHO Subculture Protocol (adherent)
CHO cells grow quickly and easily and cell doubling time is 14-17 hours.
- Rinse cells with 0.25% Trypsin/0.53mM EDTA
- Add 3 mL of Trypsin-EDTA to flask and watch for cell layer detachment under an inverted microscope. This should occur within 5-15 minutes. Do not agitate cells during this type as agitation encourages clustering. If cells are not detaching, place in incubator for 5 minutes to facilitate dispersal.
- Centrifuge cells and remove supernatant to remove Trypsin-EDTA
- Add 6-8 mL of growth medium and aspirate cells with pipette, gently.
- Add aliquots of cells suspension to culture vessels.
- Incubate at 37°C in 8% CO2.
Split confluent culture 1:4 to 1:8 every 4-7 days with trypsin/EDTA. Make sure to renew medium 1-2 times between subculturing.
CHO Cell Storage
Freeze CHO cells in complete growth medium with 5-10% DMSO at a concentration of 0.5-1 x 107 cells/mL if growing in suspension. For adherent cells, freeze cells in modified medium containing 10% DMSO with a cell concentration of approximately 1 x 106 cells/vial. Cells should be frozen at a slow rate using a freezing vial (i.e. -1°C per minute in a -80°C freezer). Store the frozen cell vials in the liquid nitrogen vapor phase.
Subculture Troubleshooting Procedures
Low cell viability after passaging cells:
- Cells exposed to dissociation agent for too long; leave dissociation agent on cells until cells detach, not any longer
- Harsh pipetting procedures during cell collection; treat cells more gently
If cells are difficult to detach:
- Cells reached too high confluency, cell-to-cell junctions are tight and preventing dissociation agent from reaching cell interface; resolve by subculturing the cells before becoming confluent
- Dissociation agent is weak and must use a higher concentration; also, incubate flask containing the dissociation agent at 37°C while waiting for cell detachment to increase enzymatic activity.
- Ensure the flask was washed with sterile 1x PBS prior to addition of the dissociation agent; if necessary, two 1x PBS washes can be used.
If cells form clumps after detachment from flask:
- Cells centrifuged too hard during washing steps; spin cells no harder than 100 x g for 5 minutes to pellet
- If cells will not be immediately used after dissociation, place the flask or vial on ice to decrease cell aggregation