storage in incubator? - (Mar/14/2005 )

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.

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It depends. How fast are they growing? If you're going to need them in a few days, then keep them in the incubator. If you'll need them in a few weeks, you may want to freeze some down and passage a plate or two so that they can have room to grow. Just remember, it all depends on what kind of cells you're working with...some cells are limited by the number of passages they can withstand. If passage number doesn't matter, you may want to keep a few plates in the incubator between experiments and freeze down extra cells. If it does matter, then freeze them down and thaw them about a week or two before you need them. For your last question, ALWAYS keep feeding cells in your incubator! Otherwise, waste products will build up, nutrients will become scarce, and your cells will die. Hope this has been helpful and not too wordy.

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so fast growing cells are frozen down and slow growing cells are stored in incubator?

you mean passage in culture flasks or wells or dishes and keep them in incubator? does it matter which one i choose?
what does it mean with passage number? is that the number of times the cells are frozen and thawed ?

i thought i should thaw the cells just days before work. so if the cells don't like passage numbers then i have to freeze and thaw them instead storing them in incubator? Besides i thought too much freeze and thaw cycles will damage the cells and this will get worse for cells which don't like passage numbers.
how do i know that i should change my medium? how many times a day?

thank you very much. you have helped alot. do you have any links to say about storage of mammalian cells?

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hi
slow or fast cells are treated differntly due to the fact fast-growing cells need to be divided more. but if you don't need your cells for more than two weeks i would recommend you to freeze them.
Passage in flasks or plates should be choosen regarding the relative surface needed by your cells. A flask (as i know) has a bigger area than a plate of diameter 10cm, but smaller than a 15cm plate. But in all cases cells need o be divided at 90confluency max. so take a look at your cells every day and see when it's tilme to divide them.

passage number: it means number of divisions of cells. (as i know)
some cells that are not immortal (for example HUVEC) do not support more than 5passages. they start a de differenciation and loose markers of endothelial cells. for an other example, IMR 90 cells do not support more than 30passages. After that they start to die lot and dedifferenciate too.
But for immortal cells, Hek293, HeLa cells... they can be passaged as many as you want / need.

Thawing of cells : usuall, i thaw cells two days before my experiment (my cells are dividing well so two days are ok but in case of slower dividing cells, i thaw them one week before needed).

I change my medium every two days at least. Some elements are unstable at 37° so cells need fresh medium regulary.

for storage of mammalian cells, you can read this topic :

http://www.protocol-online.org/forums/inde...wtopic=3689&hl=

I hope i've answered your questions. Ask if you need more.
Fred

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so we have to split the cells into new flasks if the cells are crowded enough?


does it mean the number you seed the cell and split it when it is cofluent? or does it mean the number the cell divide (generation time)? because some cells dvide every 2 hours, so the passage numbers can be very high.

thanks.

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Hi

Passage number is the number of times you have split the cells. Generation number is the number of population doublings your cells have undergone. These two numbers will generally be very different, as most people split their flasks/plates at ratios between 1:3 and 1:20 depending on cell line and growth rate. If you split at 1:20 then the population will be able to undergo many more doublings before requiring passage, than if you split at 1:3. However some cell lines can't tolerate being split at high ratios, as then need a certain level of extra cellular proteins to function/grow properly, that can only be achieved by close proximity to other cells.

In the lab I am currently at, we work with a large number of immortal cell lines (293, HeLa, A549 etc) that we use all the time, therefore people maintain one medium (80cm2) flask of the cell lines they need and split when the flask is at 80-90% confluent, discarding the remainder of the cells that they don't need. This cuts down on number of flask each person uses and saves freeze/thawing cells. However if we are not using a particular cell line for a while (month or more) then we will freeze some down and thaw them when we need them.

Cell lines that are not immortal such as most primary cell lines need to have some vials frozen at each passage as they are more and more likely to die as the passage number goes up.

I suggest you read a good text book such as Freshney (1994) "Cell Culture" that will explain all or most of your questions.

Sorry for the long post.
Bob

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hi
bob1 you are a master for me. You've clarified very good the notion of passage.
Thanks.

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Thank you for the help.

But what does it mean when most people split their flasks/plates at ratios between 1:3 and 1:20? i don't get the ratios 1/3 and 1/20. what does 1 specify for? what does 3 or 20 specify for? thank you again.

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hi
bob1 meant that when you get a plate at 80% confluency (or till 100%), then you trypsinise all cells, and divide the quantity in 3 new plates, than you have done a 1:3 (from 1plate, seeded 3 plates)

it's the sdame 1:20 from 1 plate you seed 20 plates

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thanks. i got it.

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