Samples were aspirated from suspension cell cultures, cells were counted, and appropriate amounts of suspension cells were inoculated into fresh medium in new culture flasks to bring the cell concentration to the same level as at the start of the culture.
Operation method
Scheme 13.3 Suspension cell passaging
Principle
Samples were aspirated from suspension cell cultures, cells were counted, and appropriate amounts of suspension cells were inoculated into fresh medium in new culture flasks to bring the cell concentration to the same level as at the start of the culture.
Materials and Instruments
Starting Cultures Move 1. Prepare the ultra-clean table and place the reagents and materials on the ultra-clean table ready to start the experiment. For more product details, please visit Aladdin Scientific website.
Growth medium Suction tubes Centrifuge tubes Culture flasks Stirring flasks Pipettes Suction earballs Suction paper towels Suction buckets Anti-ethanol markers Hematocrit plates
2. Carefully examine the culture for signs of contamination or decline. This step is more difficult for suspension cultures than for monolayers of adherent cells. Poor cell condition is characterized by crumpling, irregular shape, and/or granulation.
Healthy cells are clean and clear, the nucleus is clearly visible under a phase contrast microscope, and small cell clusters are often visible when the culture is left to stand.
3. Cultures (starting cultures: HL60, L1210 or P388, inoculated at 2 x 104 ml in 25 cm2 flasks for 7 and 14 days) are placed in a sterile work area, from which samples are taken and cells counted.
4. Decide whether or not to passivate based on the suspension culture passaging criteria described above and knowledge of the behavioral characteristics of the culture. If passaging is required, proceed as follows:
5. mix the cell suspension and disperse the small cell clusters by blowing up and down.
6. Add 50 ml of medium (growth medium, e.g. MEM with Spinner's salt (S-MEM) or RPMI 1640 with 23 mmol/L NaHCO3, 5 % calf serum).
Add 50 ml of medium (growth medium such as MEM with Spinner Salt (SMEM) or RPMI 1640 with 23 mmol/L NaHCO3, 5 % calf serum) to 2 freshly stirred culture bottles.
7. Add 5 ml of medium to each of the 4 25 cm2 bottles.
8. Add enough cells to each bottle. Add enough cells to each bottle to reach a final concentration of 1 x 105 /ml for slow-growing cells (doubling time 36.48 h) and 2 x 104 /ml for fast-growing cells (doubling time 12-24 h). 1 x 104 ml for the cells mentioned in the "Materials".
9. Blow gas into the bottle with 5 % CO2.
10. Cover the flask and place it in the incubator. Place the flask flat as for monolayer cells.
11. Cover the stirring bottle and place it on a magnetic stirrer in an incubator or 37°C greenhouse and rotate it at 60-100 rpm. Be careful not to overheat the culture with the stirring motor. If necessary, place a polystyrene foam mat at the bottom of the bottle. Electromagnetic stirrers produce low heat and have no moving parts.