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liquid nitrogen storage


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6 replies to this topic

#1 shs

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Posted 18 March 2009 - 05:37 PM

hi all
I am planing to start culturing Hep G2 cell line and keep them in liquid nitrogen.
My question is that can I store the cells in liquid nitrogen for more than 6 months and then start treat the cells with the chosen drugs or storage for long period like this will weaken the cells and then give a false results when I start the treatment.
Please any help will be apprecieted :lol:
shirri
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#2 CKtong

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Posted 18 March 2009 - 05:48 PM

you can keep the cell more than yrs in Liq. N2. after revive, culture for few day then check if the cell is healthy... it should not be any problem...

#3 cotchy

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Posted 19 March 2009 - 06:11 AM

Liquid nitrogen is the preffered way of long term storage of cells.

The - 80 C should really only be used for short term storage, so your cells should be perfectly fine when taken from liquid nitrogen

Thaw the cells from the liquid niotrogen very rapidly (in palm of your hand is best) as the DMSO is very toxic to the cells, so as soon as the last crystal of ice is gone place the cells in 1 ml of pre-warmed media and spin them down and remove the supernatant and resuspend the cells in fresh pre warmed media, this will remove all DMSO.

However,

You can also place them straight into the pre-warmed media (in a T25 or T75 for example) without centrifuging them and just place in the incubator to culture them as the DMSO will be diluted out, we work with HepG2 and never have any problems doing this.

Another tip is if you are losing many cells when you thaw them out (many float and do not survive you can increase the FBS to 10 % or even up to 20% to help them resucitate, just remeber to reduce this back to normal levels when the cells start growing well

After culturing for a few days you can then seed them for analysis

Cotchy.

Edited by cotchy, 19 March 2009 - 06:13 AM.


#4 bob1

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Posted 19 March 2009 - 03:59 PM

As Cotchy says, LN2 is best storage. Thawing quickly is good too, I thaw in a water bath, as it is quicker than in the hand.

Be aware that Hep G2 may shed live Hepatitis B virus!!!! You should get yourself checked for this before you use the cells, and preferably immunised against it if your aren't alreacy.

#5 shs

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Posted 19 March 2009 - 05:25 PM

Thank you soooooo much ^_^ :wacko: :lol: ;)
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#6 cotchy

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Posted 20 March 2009 - 08:05 AM

Im pretty sure Hep G2 will only shed hepatitis B virus from cells which were exposed to the virus previoulsy (for example somebody using the cells to study the virus)

However in theory the cells you have at the moment could have "once upon a time" been exposed to the virus or transfected with a plasmid for the virus, and thus possibly produce the virus without you knowing

Its not something i would worry about if your happy with the supply of your cells

I dont think production of the virus is an occurence that can happen in routine culture of your cells

If i'm wrong please let me know as myself and alot of opther post grads in my college use these cells.

#7 bob1

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Posted 22 March 2009 - 03:41 PM

Sorry, I was thinking of Hep 3B. The Hep G2 cell line is fine.




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