Passage of Cells
What is cell passaging?
Cell subculture (subculture), when the primary culture is successful, with the extension of the culture time and the continuous division of the cells, on the one hand, the cells contact each other and the contact inhibition occurs, and the growth rate slows down or even stops; Unfavorable growth or poisoning due to insufficient nutrients and accumulation of metabolites.
The process of dividing the culture into small parts, re-seeding them into another culture vessel (flask), and re-culturing is called cell subculture. For monolayer cultures, cells that are 80% confluent or just confluent are the ideal passaging stage.
Consumables preparation
Before the experiment starts, the experimental supplies need to be prepared.
Alcohol, pre-warmed complete medium, PBS buffer, trypsin, culture flasks or petri dishes, pipettes, pipettes, centrifuge tubes, etc.
The ultra-clean workbench used in the experiment needs to be irradiated with ultraviolet light half an hour in advance.
Experimental operation
Take adherent cell culture as an example
1. Wear a lab coat for the cell room, sterilized gloves and a mask.
2. Take out the cell culture flask or petri dish from the incubator (Tip1: generally choose cells in log phase), sterilize the outer package with 75% alcohol, and transfer it to the ultra-clean bench.
3. Open the lid (Tip2: if it is a petri dish, just open a gap to avoid contamination), gently suck out the old culture medium, add an appropriate amount of PBS buffer to wash 1-2 times (Tip3: pay attention to uncultured cells side walls to avoid washing out cells), discard the PBS buffer.
4. Use a pipette to add an appropriate amount of trypsin (Tip4: the specific amount is determined according to the experimental needs, preheating the trypsin at 37°C in advance, the effect is better), cover the lid, and shake "cross" to make the trypsin fully contact the cells.
5. Transfer the cell culture flask with trypsin to the inverted microscope stage and observe the cell digestion under the microscope. When the cells become round and fall off a lot, prepare to terminate the digestion (Tip5: If the initial cell density is relatively high, add pancreatic After the enzyme, the cells can also be observed to fall off with the naked eye. When about half of the cells fall off, the digestion can be terminated. Avoid repeatedly taking the culture dish out of the ultra-clean bench to avoid increasing the risk of contamination), spray the culture bottle with 75% alcohol surface and transfer to a clean bench.
6. Add an equal volume of serum-containing medium with a pipette to terminate the digestion. Pipette well and gently to avoid air bubbles to completely detach the cells.
7. Transfer the mixed liquid in the culture flask or petri dish to a centrifuge tube (the size of the centrifuge tube is determined according to the needs of the experiment), and centrifuge for about 3-5 minutes after balancing (the speed of the centrifuge depends on the type of cells, the common one is 1000-3000rpm /min).
8. After centrifugation, spray the surface of the centrifuge tube with an alcohol watering can, transfer it into the ultra-clean bench, open the lid, and pour the supernatant into the waste liquid tank.
9. Add an appropriate volume of serum-containing medium, and gently pipette with a pipette or Pasteur pipette to prepare a cell suspension.
10. Divide the cell suspension into 2 (or more) clean and sterilized culture flasks or petri dishes, add an appropriate amount of culture medium, and close the lid.
11. Place the subpackaged culture flask on the stage of an inverted microscope to observe the cells. The cells should be in a certain number. Too little number will affect the growth.
12. Mark the culture flask and indicate the passage time, cell type, operator and other information. Before putting it into the incubator, you should spray the surface of the culture bottle with alcohol again, and then remember to tidy up the operating table, wipe the ultra-clean table with 75% alcohol, and clean up waste liquid and garbage.
——
