Respected people,
I have recently started working with mammalian cell lines, especially HepG2 cells for which I'm culturing cells in Nunc coated T25 flasks(Cat No. 156397) with 89% DMEM, 10%FBS & 1% Pen Strep.
The media is changed every 3rd day and cells are split once reached 70-80% confluency at split ratio 1:3.
Before, trypsinization, I washed cells twice with 1X PBS.
I earlier used 0.05% Trypsin EDTA for detaching cells by keeping it at 37C for 5 minutes but a maximum of 70% of cells did not detach.
I then used 0.25% Trypsin-EDTA solution and kept it at 37C for 15 minutes and tapped the flask vigorously, still, around 50% cells did not detach.
Kindly help me with this problem.
Thanks in advance.
This is a common problem for some cell lines. There are a few things you can try:
1. Wash cells after removing the media with PBS-EDTA instead of just PBS
2. Pre-warm your trypsin to 37C before using
3. Once you've added your trypsin, place the flask directly on the metal shelf in your incubator, don't pile your flasks on top of each other - the warm metal will also help
4. I know you said you split the cells 1:3, but you could also try for example 1:2. The longer you leave cells without trypsinisation, the more they attach to the plastic, making it harder to remove them. Try splitting them 2-3 times a week.
I hope that helps. If you still have trouble, there are alternatives to trypsin.
@Diana Baxter Thank you for your suggestion.
I'll try washing with PBS-EDTA instead of only PBS.
I do not generally stack my flasks in incubator.
These are how my cells look under the microscope.
Could you tell if they are growing fine and are really HepG2 cells or not?
The cells look ok to me, although they seem a bit sparse, have you just split them? It's difficult to see, but it looks like there is a lot of debris in your culture, I'm not sure if this is just your pictures, or how your flasks look under the microscope, or if it really is debris.
Diana Baxter Thank you for your reply. These cells have been split 2 days back. It is debris due to dead cells. The cells looked at 20X are shown below.
Kindly help what can I do to maintain a healthy stock of HepG2 cells.
How many days do you normally leave the cells to grow before splitting them and how confluent are they? If this is what your cells look like after 2 days, I would split them less. Also try washing away the cell debris with PBS and give them fresh media. Finally, when you trypsinise them, then add media to inhibit the enzyme activity, do you centrifuge the cells? You could also try this to remove all trypsin from the cells, then resuspend in fresh media - some cells prefer this.
- Badges
- Science topic
- Similar topics
- Physics
- Condensed Matter Physics
- Solid State Physics
- PEAKS