Hello HEK-cellers.
I am having issues with my TG HEK293 cells when seeding from frozen stock; they are remaining in suspension and do not adhere even after 4 days in media (high glucose DMEM,10% FBS 1% genetecin). Those that do adhere do not have a typical HEK morphology (large rounded cell bodies with multiple processes). I have used this method for a while and have not seen this issue before; neither in these particular cells nor other TG HEK293 cells, even if seeded at the same time using the same media batches. The cells in question were cryostored back in 2012. All the cells I have used until now simple adhere oversight after seeding from frozen. Can anyone suggest why my cells are behaving this way, and whether I can reverse them back to adherent cells?
Dear Jan,
First make sure that your cells those don't adhere "Are alive or not ?"
and possibly change the proportion of the FBS/Medium for defreezing cells from freezed stock.
make sure when you added cells to the flask, all used material had been wormed upto 37 C; this might helps you.
I am having the same problem with Hek293-TRex cells.
I've tried two thawing methods: Directly adding the cells to the flask and changing the medium after 2 hours in the incubator, or spinning the cells to remove all DMSO and ressuspending in media without p/s.
I've also tried seeding them in smaller dishes, but nothing seems to be working.
Today I'll try increasing [FBS] to 20%.
Best of luck!
Hi Jan,
sounds like your cells are in bad shape which means you have to treat them carefully. As Matheus Schultz recommended, get the DMSO out before you put them in a flask and use 20 % FCS in your media. Other than that you could try and coat the flasks with fibronectin to help adherence until they recover. In my experience HEK cells are not very adherent to begin with but they grow like crazy. As long as you have even very few cells attached to the flask they will eventually take over. Just carefully decant the loose cells and add new media every few days and wait for 1 week or 2. I once recovered a culture like this, maybe it will work for you too.
Thank you every one for your suggestions. i'll give them a go.
best of luck to you all.
Regards
Hello
I agree with Todor Tschongov, using 20 % FCS in media after removing DMSO. This may help you
Good Luck
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