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store my cells in incubator

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store my cells in incubator

Postby summer » Tue Mar 15, 2005 8:37 am

i wonder how long can i store my cells in incubator if i don't need the cells right away, but in coming days? should i change the medium regularly when i store the cells in the incubator instead of freezing? or do i have to freeze down the cells immediately and thaw them when i need them?

appreciate for all help.
summer
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Postby DevGrp » Tue Mar 15, 2005 1:34 pm

are we talking about mamalian cells here? human?

It all depends on the type of cells


In my experence of adherent human cells growing in a monolayer its a good idea to change the media every few days, this removes dead cells and refreshes any antibiotics / growth factors. I also passage the cells before they become confluent as once this has occured they start undergoing apoptosis.
the problem then is that after multiple passages some cells start to change in charatcter. The only solution to this is to have frozen stocks and go back to the stock after 10-20 passages. This advice seems to hold for cells like T47D, MCF7, Eahy 926.

Primary cells on the other hand don't always like freezing ( a "Mr Frosty" seems to help when freezing) and often don't last beyond passage 10. (eg Primary breast culture / HUVECS / primary endometrium)


Hope this helps

Ian
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Postby summer » Tue Mar 15, 2005 5:28 pm

DevGrp wrote:the problem then is that after multiple passages some cells start to change in charatcter. The only solution to this is to have frozen stocks and go back to the stock after 10-20 passages.

so you mean we make freezing stock as a security and cells that have undergone 10-20 passages are no longer passaged hence we have to passage the freezing stock instead?
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Postby DevGrp » Wed Mar 16, 2005 10:50 am

Yes I tned to make 5-10 aliqouts of any new stock as early passage as I can and then go back to that whenever the cells starting behaving strangely. For the cells I used this was often passage 20ish.
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passages

Postby 2810712 » Wed Mar 16, 2005 1:22 pm

Hey what are those passages, please explain,
And why do th confluents cells become apoptotic[ I mean start apoptosis ? ? ?

please.......

thanx

hrushikesh
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Postby DevGrp » Wed Mar 16, 2005 2:08 pm

Passaging cells:

Posh word for splitting. You detach your cells from the flask they where growing on (using EDTA and or Trypsin) spin them down and add 10-25% of the cells in fresh media into a new flask

So cells that have been passaged 10 times have been split 10 times. Based on the split ratio -say 25% and the fact that you tend to passage at just below 100% then 10 passages means very approx 40 generations


Now I have only ever worked with adherent cells which grow in a monolayer.
When they reach confluence they stop growing due to a phenomen called contact inhibition. According to my last grad students thesis

"This is due to contact inhibition, where intercellular junction associated phosphatase activity blocks proliferative pathways"

Basically the cells detect that they are bumping into other cells and stop growing.

With all the cells I have worked with they start to die at this point.

As to why?

Well I'm not quite sure.
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Postby summer » Wed Mar 16, 2005 6:26 pm

So cells that have been passaged 10 times have been split 10 times. Based on the split ratio -say 25% and the fact that you tend to passage at just below 100% then 10 passages means very approx 40 generations

How can you calculate that it is 40 generations of 10 passages? :roll: Hope for inputs.
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yeah

Postby 2810712 » Thu Mar 17, 2005 6:28 am

Thanks Ian,
But, I also want to know how did U calculate that 40% ? ? ?

hrushikesh
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Postby DevGrp » Thu Mar 17, 2005 9:38 am

very rough

if you take 25% of the cells and allow them to grow to 100% then each cell will have to divide-------oops

once to give 50% then a second time to give 100%=2 generations

(for some reason I was thinking 25x4=100)

If this happens 10 times you get 20 generations (NOT 40).


I am more than welcome to be corrected on this, just a rough guess and maths was never my strong point.
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Postby summer » Thu Mar 17, 2005 11:39 am

I see. :)
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Postby 2810712 » Fri Mar 18, 2005 6:01 pm

But , why is this a rough calculation?
I think, every cell doesn't devide and some cells devide more than twice a passage.
What's the reason.

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