As recommended by ATCC I freezed my Calu-3 cell line down(-- 80 °C ) in Complete growth medium supplemented with 5% DMSO but I got low cell viability AFTER just two weeks from freezing them down!..Any help and suggestions I would appreciated..
Hi Doaa, so I see a few red flags in the method. First make sure that immediately after the addition of cryopreserving medium ( to prvent ice crystal formation) you transfer the cells in a controlled cooling rate container (-1oC/min) and transfer at -80oC overnight (freezing to quickly causes crystals to form and burst cells). the day after transfer the vials in liquid nitrogen vapour phase for long term storage. Storage at -80 can affect viability. For thawing cells hold the vial in a 37oC waterbath ( thawing must be quick to reduce exposure time to dmso)while swirling the vial (make sure to keep the cap out of the water). Once only a tiny crystal is left go to the hood, transfer the now thawed suspension into a 50 ml tube, and add 10 mL of complete culture medium dropwise ( this is to prevent osmotic shock from thawing), then spin down and remove medium. Resuspend in fresh medium and plate cells.
To be able to help, there are a lot of details missing here. Do you freeze cells immediately after the addition of DMSO containing media? Do you use a controlled cooling rate container? What process do you use on thawing? Do you transfer in liquid nitrogen or keep at -80?
Thanks Dario for your response
1- YES I freezed them immediately at -80,I read also to insure high cell viability cells should be stored in liquid nitrogen not - 70C so how about - 80C!
2- Thawing
* When I brought them out I opened tube cape for its quarter then I closed it again warming it up in the normal Room temperature
*Transferred the vial contents to a centrifuge tube containing 9mL complete culture medium. and spun at approximately 125 x g for 5 to 7 minutes.
* Re-suspend cell pellet with the recommended complete medium (5ml) into a 25 cm2 culture flask.
3- a minus 80 freezer model similar to the attached photo
Hi Doaa, so I see a few red flags in the method. First make sure that immediately after the addition of cryopreserving medium ( to prvent ice crystal formation) you transfer the cells in a controlled cooling rate container (-1oC/min) and transfer at -80oC overnight (freezing to quickly causes crystals to form and burst cells). the day after transfer the vials in liquid nitrogen vapour phase for long term storage. Storage at -80 can affect viability. For thawing cells hold the vial in a 37oC waterbath ( thawing must be quick to reduce exposure time to dmso)while swirling the vial (make sure to keep the cap out of the water). Once only a tiny crystal is left go to the hood, transfer the now thawed suspension into a 50 ml tube, and add 10 mL of complete culture medium dropwise ( this is to prevent osmotic shock from thawing), then spin down and remove medium. Resuspend in fresh medium and plate cells.
Im not sure about the controlled cooling rate container!! I have never heard about it in my lab... so could we let tubes be frozen in Mr frosty in -80 overnight!
Yes indeed, Mr frosty is a controlled cooling container so it is perfectly fine. Let me know how it goes and good luck!
In our lab, what we use for freezing is this:
70% complete growth media
20% FBS
10% DMSO
Make sure you freeze when cells are healthy or when they are in exponential growth phase.
We freeze overnight in a cell freezing container in -80 and store in liquid nitrogen or -180 for long term storage.
For thawing, I heat up 10 ml medium to 37°C and rather than putting the cryo vial in a 37°C waterbath until sample is almost completely thawn, I take 500µl to 1 ml of the heated medium into the vial, pipet up and down for 4-5 times and immediately put then every dissolved cells into the 10 ml 37°C medium - I repeat this a few times till medium with cells is completely thawn. Advantage is that DMSO has less time to kill the cells during the thawing process.
Long term storage of the cryo vials is also possible at -150°C in a deep freezer, a colleage of mine is storing them there as he does not have a nitrogen tank. But -80°C is definitely too less for long term storage...
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