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HACAT cells - very low cell viability after thawing cells (Apr/23/2009 )

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Hi all, a colleauge of mine has recently started to work with HACAT cells.

He bought them in a while ago from CLS (cell line company) at a low passage number and passaged them a few times and then froze them down in the - 80C (70 % DMEM, 20 % serum, 10 % DMSO) and then after a few days places them in liquid nitrogen

When he brings them up from the liquid nitrogen (in the proper recommended media - DMEM w/ 10 % serum ) for use the viabilty of the cells is always very low, around 2 - 5 % and thus it takes 2 to 3 weeks to get a T 25 confluent.

Does anybody have any suggestions as to what is causing the low viability of the cells or has anybody had the same problems and managed to overcome them?

-cotchy-

Hi,

the Hacat cells we use here are rather looking good after thawing. The have been stored at -80C for some vial.
Are you sure the DMSO is still fine and has not beeon oxidized or something?

We use only 5% DMSO for freezing (though I doubt that this makes the difference) in regular media.
What we use here is DMEM with lower bicarbonate content so that the media can be used in incubators with 5% CO2 and doesn't become alkaline (pink) in between.
Do you buffer the media with HEPES or anything?

-Bomber-

Bomber on Apr 23 2009, 10:34 AM said:

Hi,

the Hacat cells we use here are rather looking good after thawing. The have been stored at -80C for some vial.
Are you sure the DMSO is still fine and has not beeon oxidized or something?

We use only 5% DMSO for freezing (though I doubt that this makes the difference) in regular media.
What we use here is DMEM with lower bicarbonate content so that the media can be used in incubators with 5% CO2 and doesn't become alkaline (pink) in between.
Do you buffer the media with HEPES or anything?



Hi Bomber,

I dont get pink media so i pressume i am ok on this point.

I dont use HEPES as i use the 5 % CO2, should i use HEPES also?

Have you tried to use 10 % DMSO for freezing or do you always use 5 %?

Can you tell me your full recipe for the culturing media?

Mine is:

DMEM (no hepes or L-glut)
10 % FCS
0.5 % penn/strep
0.5 % amphotericin
1 % L- Glutamine

Also what is your freeze media?
is it 75 % DMEM
20 % FCS
and 5 % DMSO?

Many thanks for your help.

-cotchy-

Hi,

the media is DMEM (high glucose) containing L-glutamine but no Hepes.
We use 10% FBS - that is all.
For freezing them we use this media plus 5% DMSO.
Though I haven't tried it with 10% DMSO I doubt that this is a problem for these cells.
Are your sure the DMSO is fresh?

As an alternative you might try to culture/to adapt the cells to regular RPMI media.

Best regards

-Bomber-

Ok thanks for your advice bomber.

-cotchy-

Hy,

Can somebody tell we what the abreviation HACAT stands for. i know that its a keratinocyte cell line but I mean the exact long version of the abreviation.

Thanks!

-marie-

HaCaT is the abreviation of: Human adult low Calcium Temperature keratinocytes

-Chakchel-

We freeze them in 90% serum, 10% DMSO and directly from the room to the liquid nitrogen.... They are just fine like that when we thaw them.... like 80%... they will fill the dish in 2 days :P

-Neurite-

Hi,
I would like to seed and grow HaCaT cells to form monolayer. How long time does it take to them to be confluent? Does anybody have experiences with this issue?

-polarka-

polarka on Tue Jan 11 11:52:17 2011 said:


Hi,
I would like to seed and grow HaCaT cells to form monolayer. How long time does it take to them to be confluent? Does anybody have experiences with this issue?


Hi,

It all depends on how many you seed to start with, of course the more cells you put in the faster they'll get to confluency, but also HaCaT cells do not like being at low density. They take longer to double if you seed at 10% then if you seed at 50%. I usually seed 2 X 105 in a 9.5 cm2 plate and by the next day they are at 60% confluent.

P

-Penguin-
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