Presently the MDF's are maintained in RPMI media supplemented with 15% FBS, sodium pyruvate and NEAA in a tissue culture flask.
But I am finding the cells are undergoing apoptosis after passage 3 and the growth is slow.
Can anyone suggest plausible reasons for the same and methods to prevent it.
Should I go for the gelatin coating of the tissue culture flask??? Will it help??
Kindly reply.
Thanking You
To culture primary cells you need to take lot of precautions. I think you make check with collagen/gelatin coated plates although there might be other reasons also for this.
Most of the dermal fibroblasts you get on a primary culture are very differentiated cells, which allows you to achieve not many passages with them. In our experience, we have observed that most primary fibroblast cell cultures start to die out after passage 10. So, what you see is normal. Using collagen coated plates might help. We also have had great results using regular plates coated with a little layer of FBS just before seeding. For this, we apply a little bit of FBS to the plate to cover all seeding surface and then we let it dry. Then we seed. Also, you might want to avoid the use of trypsin for subculturing and using the scraper instead. Good luck!
Hi Tarun,
What you see is absolutely normal - cell senescence. (Irreversible arrest of normal cells after a limited number of divisions). Cells become big, flat, with pale nuclei and large nucleoli, also prominent actin cables in the case of fibroblasts. Just like yours. If you wanted to be sure you could stain for senescence markers like beta-galactosidase (at pH 6), p21, p16 etc, but the morphology is exactly right and around passage 4 sounds right too (for mouse). Senescent cells don't reattach very well after passage, so you may then see cell death just after subculture.
However mouse fibroblasts readily escape from senescence and become immortal - maybe 1 in 104 or 105 cells. So if you just keep the cultures and passage them regularly at between 1:3 and 1:1 [spin down to remove residual trypsin if 1:1], soon you will see proliferating (immortal) cells emerging, and they will take over the cultures and grow fine after that.
No special culture conditions needed, as the immortal cells will probably be selected more quickly under normal conditions.
PS, may I keep a copy of your photo, for teaching? I teach students about cell senescence and this image illustrates all the features so well!
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