Hello,
I recently started a cell culture, but I have problem with the cell aggregation problem when detaching cell from a 100mm cell culture dish to another dish.
After treating trypsin, I usually pipette cells several times to completely detach cells loosely attached on the culture dish. After ultracentrifugation, I add 2~3ml of medium to disperse cells, and pipette the suspension around 30 to 40 times to make sure there are no cell clumps.
I do not pipette the medium strongly to avoid the bubble formation. However, after passaging, when I check cells through optical microscope, there are always dark and packed cell clumps attached on a culture dish.
Are there any other ways to remove the cell aggregations?
Should I re-start the cell culture with new cells?
Try rinsing, then icubsting your flasks with calcium/magnesium-free buffered salts solution first. Then Trypsinize. Use a gentle centrifugation (tabletop/clinical) centrifuge. Don’t be afraid to pipette(triturate) the cells. To avoid bubbling, don’t draw up the full volume when pipettting..
What is the reason for using ultracentrifuge? At what speed do you centrifuge cells? If you are using high speed, cell membrane may get damaged.
For subculturing adherent cells, centrifugation is not normally required. The procedure we follow is, the monolayer washed with prewarmed trypsin (0.25%, containing EDTA) twice, cells are incubated for about 3 to 5 min at 37 C after removing trypsin. Required volume of medium is added to the flask and cells are dislodged from petridish/flask by pipetting gently. Try using fresh trypsin.
If trypsin does not work for your culture, you can try using collagenase.
Try rinsing, then icubsting your flasks with calcium/magnesium-free buffered salts solution first. Then Trypsinize. Use a gentle centrifugation (tabletop/clinical) centrifuge. Don’t be afraid to pipette(triturate) the cells. To avoid bubbling, don’t draw up the full volume when pipettting..
For subculturing NIH 3T3 you should wash the flask/dish with an appropriate amount of pre-warmed PBS, remove the PBS and add 1x Trypsin (approx. 1ml for a T75 flask). Incubate (without removing the trypsin) for 5-7 minutes at 37°C. Check cell detachment using a light microscopy (if a portion of the cells remain on the surface gently tap the flask/dish a few times). Use cell media (containing FBS) to stop the trypsin reaction and centrifuge at 1500rpm for 5min. Resuspend your cells using a 1000µl pipet. After resuspending 5-10 times you should receive a single-cell suspension. Check for cell aggregation after seeding your culture vessels using a light microscope. If a lot of cell aggregates are visible return the flask/dish to the bench and use a serological pipet (5 or 10ml) to resuspend the cell suspension once more (5-10 times).
- Badges
- Science topic