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cells not adhering to flask after thawing - (Jun/13/2006 )

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yesterday i thawed out CHO cells from liquid nitrogen and grew them up in a t-25 flask (for thawing i placed the vial in 37C water bath until cells were thawed and then added them to a flask containing 5 mL of warmed media (DMEM & 10%FBS). Today i checked them and almost all of the cells are floating...none of them adhered to the flask...is there something I can do??

-mmini-

it seems that they didn't like the freezing... check what was used for freezing.
Did you thaw quick and resuspend them quick in complete medium?

-fred_33-

Dear mmini,

Thaw cells in your hand until the media just starts to melt and then add your fresh media. Dilute your cell suspension at least 1:20 in a centrifuge tube. Spin at 100g for 5 minutes and then remove media leaving the cell pellet intact. Resuspend in fresh media and then add to flask. You should be getting 80-90% adherence to your T25 flask if you have frozen the cells properly. The reason why you have no viable cells is that you have not diluted out the DMSO which is cytotoxic to the cells at concentrations above 0.1%.

-Rhombus-

After thawing the cells and seeding them in a flask, i change media after 3 hrs to get rid of DMSO, as its toxic.

-scolix-

QUOTE (scolix @ Jun 13 2006, 02:04 PM)
After thawing the cells and seeding them in a flask, i change media after 3 hrs to get rid of DMSO, as its toxic.


Have you already done trypan exclusion to verify that your floating cells are alive and not adhering vs just dead??

-InsulateMe-

QUOTE (mmini @ Jun 13 2006, 09:41 AM)
yesterday i thawed out CHO cells from liquid nitrogen and grew them up in a t-25 flask (for thawing i placed the vial in 37C water bath until cells were thawed and then added them to a flask containing 5 mL of warmed media (DMEM & 10%FBS). Today i checked them and almost all of the cells are floating...none of them adhered to the flask...is there something I can do??



fred 33...the freezing medium was dmem, fbs, and dmso--so i just took them directly from the liquid nitrogen into the 37C water bath and then once they were thawed i put them directly into warmed media (so it was all pretty quick)--i just tried spinning them down and removing the supernatant and then resuspending them in media and transfering to a new flask to see if they will grow in there once the freezing media is gone....is there an alternative freezing medium that would be better for CHO cells

-mmini-

QUOTE (fred_33 @ Jun 13 2006, 09:48 AM)
it seems that they didn't like the freezing... check what was used for freezing.
Did you thaw quick and resuspend them quick in complete medium?


thanks!...i just tried that we'll see tomorrow if it worked...thanks again smile.gif

-mmini-

QUOTE (Rhombus @ Jun 13 2006, 10:46 AM)
Dear mmini,

Thaw cells in your hand until the media just starts to melt and then add your fresh media. Dilute your cell suspension at least 1:20 in a centrifuge tube. Spin at 100g for 5 minutes and then remove media leaving the cell pellet intact. Resuspend in fresh media and then add to flask. You should be getting 80-90% adherence to your T25 flask if you have frozen the cells properly. The reason why you have no viable cells is that you have not diluted out the DMSO which is cytotoxic to the cells at concentrations above 0.1%.


oh okay...i did it after 24 hrs so maybe that could be part of why they did not adhere....thanks!

-mmini-

QUOTE (scolix @ Jun 13 2006, 02:04 PM)
After thawing the cells and seeding them in a flask, i change media after 3 hrs to get rid of DMSO, as its toxic.


thats a good pt....i didnt do that yet...i just tried to put them into another flask..but they might be dead anyways so that would've been pointless...i'll try adding trypan...thanks!

-mmini-

as rhombus said, it's important to dilute cells in great volume of medium (to dilute DMSO basically), spin an remove supernatant wich contains DMSO. After that step, you can put your cells in the flask.

You can't directly put cells from DMSO stock to the flask (that's i understood regarding your post, please forgive me if i'm wrong)

-fred_33-

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