Hi Shantanu,

have you tried collagen coating of your flasks? That could help.

Also, confirm with the literature that you apply the correct culture conditions for these cells. They will not adhere nicely if the media, supplements etc. don’t fit their needs. Also, as you are cultivating from oral swabs, be very alert about possible contamination.

Good luck with your experiments!

Marvin

Hello Shantanu Saraf

There could be two possibilities:

1. Contamination could be a cause. Did you check the culture for mycoplasma contamination? Which antibiotic are you using?

2. Also, the serum used in the culture medium. FBS contains adherent proteins that facilitate adherence of cells to the substratum. Try using another FBS make.

Best.

Hi shantanu,

Are you sure that they have adherent characteristic? If they don't, you can grow them in growth medium. However, if they do, you should check medium and also incubator for contamination.

I hope you will solve it soon.

Probably your epithelial cells need a coated plastic to grow. Many epithelial cells without the proper ECM die for apoptosis. You can also try to add an anti-apoptotic to your culture media (y-27632) that can help to prevent the anoikis (cell detachment induced apoptosis).

Hi Shantanu,

I have been isolating and growing oral epithelial cells for years and I normally do not use a feeder layer or ECM coating (only tissue-culture plates for sure). My question for you is, how do you know that they are epithelial cells if they are not attached to the plate? I normally locate my epithelial cells from their morphology. Unless I misunderstood your question, you have suspended round cells that can be anything. However, if you already had a way to first isolate them from other cells in your swab sample and then showed that they were epithelial cells, then try to subculture them at a very high density (20-50 000/cm2). Use 24 or 48 well culture plates at first. You should use defined serum-free media with added keratinocyte growth kit. I buy from ATCC. If non of the above works, you can coat the plates with laminin5 (500-200ng/cm2 ) which works better than collagen.

Hope it helps!