i use 0.05% EDTA-trypsin to detach Hep G2 cells 6min then neutralize the trypsin- EDTA with DMEM(HG) with 10% FBS, 1% p/s, and 1(NEAA) and centrifuge gently then resuspend cells in fresh medium,but it still clump,how to solve this promble ?
Hello Chung wei Chang,
You should trypsinize the cells when the culture is 80% confluent. DNA gets released from lysis of dead cells when overconfluent culture is trypsinized. DNA being sticky in nature will form clumps of cells and debris.
Maybe the formation of clumps is due to overconfluent culture. Also, try to reduce the time of trypsin exposure. Keep it at 3-4 mins instead of 6 mins. Within 4 mins the cells should loosely adhere to the substratum so that by tapping the culture vessel from the sides, the cells should detach.
Hope this helps!
Best.
Hello,
If you want to avoid cell clusters, use a 40μm or 20μm pluriStrainer (sieve) before adding cells to the flask. It's a simple step that ensures a more uniform cell distribution.
I hope this helps!
Hello Chung wei Chang ,
I would say that the clumps come from the fact that your cells are not well dissociated from one another. Otherwise, your cells seem in good health on your pictures : I don't think passaging them before confluence, as suggested by Malcolm, would change anything to your problem. Your islets are dense (maybe too dense on the last picture), but still spaced out, so the cells still have space to grow.
When you dilute your cells, I would suggest 3-5 minutes of trypsin ; where I work, for HepG2 cells we use trypsin 0.25% but without EDTA, 5 minutes at room temperature is enough to detach them, and the secret is to thoroughly separate them from one another , first by tapping lightly on the sides of your culture surface and then by doing some up-and-downs when you inactivate the trypsin with fresh medium. I don't know what surface/volumes you work with, I work with T75 flasks so it is easier to tap then with a cell culture plate ...
If you really need to perform your centrifuge step (if you prefer working with trypsin-EDTA it is the case), maybe you don't resuspend the pellet enough after the centrifuge step and that might also explain the clumps ?
Hope this helps a bit, good luck with your cultures !
Hi Chung wei Chang,
To improve the separation of clumps without affecting HepG2 viability, what worked for me was using a sterile P200 tip on the serological pipette and doing ~20 ups-and-downs during trypsin inactivation with fresh medium. This technique adds more pressure to the stream and ensures a good separation. When working with T75 or T175 flasks, I typically use 2 or 6 mL of 0.05% trypsin-EDTA and incubate them at 37 °C for 10 minutes. I hope this helps a bit.
By the way, do you happen to know the doubling time of your culture?
Hello Chung wei Chang,
Do you give PBS wash to the cells before trypsinization? You can avoid cell clumps by giving PBS wash twice and then adding trypsin to the cells.
Regards
Richa
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