Here is a general protocol for subculturing CHO cells:
- Assemble all necessary materials and reagents, including culture flasks, sterile pipettes, sterile tips, cell culture media, and trypsin-EDTA solution.
- Pre-warm the cell culture media and trypsin-EDTA solution to 37°C in a water bath or incubator.
- Remove the old culture media from the CHO cell culture flask and add enough pre-warmed trypsin-EDTA solution to cover the surface of the cells.
- Incubate the culture flask at 37°C for 3-5 minutes, or until the cells have detached from the surface.
- Tap the flask gently to help dislodge the cells, and add enough pre-warmed culture media to neutralize the trypsin-EDTA solution.
- Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 150 x g for 5 minutes.
- Aspirate the supernatant and resuspend the cell pellet in fresh pre-warmed culture media.
- Count the cells using a hemocytometer or an automated cell counter, and adjust the cell concentration to the desired density for subculturing.
- Plate the cells in a new culture flask, using a ratio of 1:3 to 1:6 (i.e., seed 1 part of cells into 3-6 parts of fresh media).
- Incubate the culture flask at 37°C with 5% CO2 and 95% humidity.
It is important to maintain proper sterile techniques and to avoid over-confluent cultures to ensure optimal growth and health of the CHO cells. The frequency of subculturing will depend on the growth rate of the cells and the desired cell density, but generally subculturing should be performed every 2-3 days for optimal cell growth and productivity.