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Very high confluence after thawing cells– good or bad? - (May/19/2008 )

Dear All,

I am trying to thaw some frozen aliquots of a transformed cell line that were frozen down in an extremely high confluence.

Therefore, after thawing them, I have these problems:

1. Even after diluting to a more adequate confluence, it still fells like overcroweded, and I am not sure how that can affect the attachment of the cells.

2. I believe their viability is low, since not many cells attach.

3. Sometimes after 3h there are many cells trying to attach, but after 6h, it seems like their number decreased and there is less cells attached.
In parallell to that, there is a huge amount of dead or dying cells floating around, and I wonder if they release toxins or consume more of the medium (while alive), hindering the ones that were ok.


What would be the best approach to solve it?

1. Would it be too soon to change their medium 8h after thawing them? Could it disturb their attachment process, either mechanically or by removing growth factors?

2. If I increase the volume of the medium in each well, would it be a way of avoiding too many dead cells in the culture releasing toxins? (that is, they would be more diluted) and there would be more nutrients to the viable cells?

3. Many ppl suggest to increase the amount of cells in each well when we know their viability is low. To which point that can be beneficial, could it also become a problem?

Any other suggestions/ideas? This is one of my last aliquots and I am getting very worried...

Thank you very much!

Julianne.

-Julianne W-

JW,

I don't know why you are concerned aobut viable cell numbers.

Primary purpose of thawing is to get your cell-line going. It should never be to get them in confluent order, or for doing expts right away.

Ordinarily, I would change the medium after 24 hours, and once they reach near confluence, I would split the cells. Only on this passaged cells I would do any experiments.

Most of the cells that die, were anyway destined to die of DMSO, freezing and thawing process. Nothing to do with their confluence or your change in media after certain hours.

..

-cellcounter-

Sometimes I even change the medium 3-4 hours after thawing...

-Franz K.-

1 ) yes - increase the volume of medium in each well/flask - it will dilute any problems of toxins/dmso (more nutrients is pretty irrelevant - its concentation not volume)
2 ) no - its not too early to change the medium after 8 hrs if you are happy you are able to continue the cell line with the cells that are attached - your decision
3 ) if you're getting cells to grow why add more (seems like an overcomplication)

cellcounter is right - if you're growing these cells in wells directly for an experiment that could be your mistake - grow some up in flasks first, let them chill for a bit (metaphorically not literally), have the odd flask warming party etc, then when they are settled, calmly watching telly eating liquid pizza, chuck in your disruptive chemicals and redistribute to the wells - they'll thank you for it by acting like normal cells (think of it as doing an exam the first day you move into a new flat - you'd never show your true potential)

good luck

dom

-Dominic-

Hi, guys, thank you so much for replying and for your help!

The strange things with these cells is that ca. 3h after thawing, there are, lets say, 20% of attached cells (and loads of floating ones). After 6h, there are less cells attached, around 10%, and in the next morning, only 5% left attached and all the rest dead (loads of dead cells!).

I dont know if they are dying because they were not healthy anyway in the first place (even if they were able to attach), or if it's this huge amount of dead cells that is somehow affecting their medium or their growth conditions. But from my (short) experience growing these cells, once they attach, they tend to keep growing ok, except if they are in a very low confluence. So I don't understand why the ones that attached after 3-6h would die in the next morning...

But maybe it's the stock itself, each 1ml aliquot of frozen cells (medium + 10% DMSO) came from a 90% confluent flask, so they were extremely confluent when frozen down.

- Can a very high confluence affect the quality of the freezing down process? Or is it a good thing that there were so many cells in the aliquot, for this way at least in theory I would have more cells left after a huge amount of them dies in the thawing process?

And a very stupid question, sorry, I am rather new to all this…

– how can I recognize, after thawing, which cells are dead and which ones are rounded up just because they didn't attach yet? My cells take at least 3h to start to attach, so in the meantime, I am very confused about how many will make it or not, making it hard to decide the right confluence to seed them.

And that's why I was asking about viability:

- Would it be useful to do a viability test (eg, Trypan bluet) to know how good my cells are when I just thaw them, to decide the right confluence to seed them? They are a bit fussy about the right confluence to make them grow ok.

(By no means I intend to do experiments with them right away, even in the next month! First I want to prepare a whole new stock of frozen aliquots, but they just don't seem to get going….But thanks for the advice, it's good to know!)

Thank you so much again!

All the best,
Julianne.

-Julianne W-

are you putting the thawed cells directly in a culture dish?

because first you have to remove the freezing medium which contains toxic DMSO. eg. i take 1 vial of frozen cells (~1ml) from the liquid nitrogen directly to the 37°C water bath. when they are thawed i put them in prepared 15ml falcons containing ~6ml fresh medium and centrifuge for 3min/200g. then i remove the medium (containing the DMSO), resuspend the pellet in fresh medium eg. 4ml and put it in a new culture flask.

-Ned Land-

I would agree with Ned land. That's what we do also and we have no problems with our cells. As for your questions :

Can a very high confluence affect the quality of the freezing down process? Or is it a good thing that there were so many cells in the aliquot, for this way at least in theory I would have more cells left after a huge amount of them dies in the thawing process?

The confluence will most certainly affect the future growth of your cells (but you're working with cancerous cells, so who knows!). But you have to take something more in account. The confluence of the cells when they were frozen do not always equals the number of cells in your vial. Take for example two flasks grown a 50% confluency and pooled in the same vial. You could have the same amount of cells that in a vial with one flask grown to 100% confluency. So the in the last case, more cells would not mean more healty, viable cells, rather than in the first case. Let's hope i'm clear tongue.gif

And as for the two last questions, i would ask this one : why wouldn't you try to let your cells attach for 24h, and then go and see them? Maybe your cells act in such a way that number of cells are on the edge of diying, and attach at first, but finally die from exposure to DMSO..

-Madrius-

QUOTE (Julianne W @ May 19 2008, 03:08 PM)
Dear All,

I am trying to thaw some frozen aliquots of a transformed cell line that were frozen down in an extremely high confluence.

Therefore, after thawing them, I have these problems:

1. Even after diluting to a more adequate confluence, it still fells like overcroweded, and I am not sure how that can affect the attachment of the cells.

2. I believe their viability is low, since not many cells attach.

3. Sometimes after 3h there are many cells trying to attach, but after 6h, it seems like their number decreased and there is less cells attached.
In parallell to that, there is a huge amount of dead or dying cells floating around, and I wonder if they release toxins or consume more of the medium (while alive), hindering the ones that were ok.


What would be the best approach to solve it?

1. Would it be too soon to change their medium 8h after thawing them? Could it disturb their attachment process, either mechanically or by removing growth factors?

2. If I increase the volume of the medium in each well, would it be a way of avoiding too many dead cells in the culture releasing toxins? (that is, they would be more diluted) and there would be more nutrients to the viable cells?

3. Many ppl suggest to increase the amount of cells in each well when we know their viability is low. To which point that can be beneficial, could it also become a problem?

Any other suggestions/ideas? This is one of my last aliquots and I am getting very worried...

Thank you very much!

Julianne.



Hi Julianne,

no-one appeared to answer one of the questions that you asked- will dead cells affect the attached cells i.e give off signal that may in turn cause the death of the attached cells. I am wondering if you ever found this out, as I would also like to know the answer to this!

Thanks!

-hrobson-

For a near confluent medium flask (T-175) I would freeze a minimum of 4 tubes and thaw each one into another T-175, depending a bit on the cell pellet from thawing.

Perhaps you could try thawing into a larger volume/surface area that you are currently using.

-bob1-