10 replies to this topic

[skiier]

I thawed some HEK293 cells that I froze down before, but only a few attached and seeded at a very low density lower then 5% of a 100mm plates. The HEK293 cells got 100% confluent after 2 weeks or so, but some giant cells appeared. and other cells look more flatten out. They look very differently from some healthy HEK293 I culture before. I pass this cell lines twice, the giant cells still present. some of other cell lines appear flatten a bit, when they were seeded at low density two.

I googled, but couldnt find any helpful info for my case.
What exactly cause these happen? are they reversible after more passing or should I discard them? any idea,  experts? Thank you
-skiier

[Samantha]

is it the giant cells look like clumps?i have the similar problem lately

samantha

skiier, on Jul 11 2010, 07:30 PM, said:

I thawed some HEK293 cells that I froze down before, but only a few attached and seeded at a very low density lower then 5% of a 100mm plates. The HEK293 cells got 100% confluent after 2 weeks or so, but some giant cells appeared. and other cells look more flatten out. They look very differently from some healthy HEK293 I culture before. I pass this cell lines twice, the giant cells still present. some of other cell lines appear flatten a bit, when they were seeded at low density two.

I googled, but couldnt find any helpful info for my case.
What exactly cause these happen? are they reversible after more passing or should I discard them? any idea,  experts? Thank you
-skiier

[Rsm]

Big round cells with multiple nuclei? Your cells are maybe senescent. Look at the Wikipedia article, they've got pictures there. If yes, no need to panic, you'll never get rid of them. Well, if you do, tell me the secret

Cheers,

Minna

[skiier]

Sammatha, they do look more clumpy than before.
Minna, yep they have multiple nuclei, and vacuoles in the cytoplasm. thank you for the wikipedia tips. I looked at it.

I googled more about it too.  it seems that the cells are not in "well-being", which could be due to aging, or overusing trypsin. I supposed HEK293 is immortal. and I usedthe same procedure and amount of trypsin on other hek293 cell lines and they are fine.

I will use HEK293 for stable transfection, so I will avoid use this cell line before I know exactly what's happening to it.
Skiier

[bob1]

One big secret to cell culture is that if your cells look abnormal compared to the other times you have used them, then you really shouldn't be using them for your experiments as they MAY give atypical results which you then don't know if that is the result of the cells being odd or if it is a normal result.

Long story short - throw out your senescent cells and get a fresh batch up.

[Samantha]

do your cells take a longer time to attach to the flask than usual?? mine are in clumps and floating
dont know whats wrong

[skiier]

bob1, thanks for revealing the big secret. I think you are right, one simple solution does it all.
Samantha, the senescent cells do look clumpy, but only took a long time to attach when they were thawed at the first time.

[skiier]

i finally took a pic of it. you can see one giant cell at the center, other cells are in normal size but weird morphology. anything look similar?

Attached Thumbnails

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[bob1]

Definitely a senescent cell.  They will not come out of sensecence, eventually they just die.

[skiier]

That's what i think. but my question is, are there just any healthier cells that will stay alive and while the senescent ones gradually die out after a couple more pass? thanks

[bob1]

The smaller cells around the senescent cell looked healthy enough.  In any population of cells you can expect to see a few sensecent cells, so don't worry about them too much, unless you see a lot of them.