Hi, I am using A549 cells. I have taken a cryovial from -80 to revive the cells. I have revived at 37 for around 2 min and then gave two media wash. then seeded all the cells in a T-25 flask (Nest). Cells did not get adhered even after 3 days and starts dying.
Please tell me the possible reasons and improvements.
Note: Cells were stored in 10% DMSO in FBS.
Hi Sandeep,
did you check the viability of the cells using a trypan blue exclusion test before seeding? This way you will know how many of your cells are viable and you will be able to exclude this possibility from your troubleshooting.
For A549, we freeze them in 20% FBS and 10% DMSO in DMEM or Ham's. After 24 hours in a minus 80 (in a cryo box filled with 2-propanol or inside a styrofoam), we transfer them to a liquid nitrogen tank and keep them in the vapor phase.
I generally make 5 vials from a confluent 10 cm plate. When I unfreeze it, I wash them once in DMEM to remove DMSO, plate them in a 10 cm plate in complete medium, let them recover overnight and change the medium the next day. Of note, when I unfreeze the vial, I just wait until it just starts unfreezing (not a fix amount of time) and gently transfer the cells to a tube containing 9ml of warm medium using a P1000.
I have attached the protocol for the exclusion assay.
Best,
Nicolas
https://www.thermofisher.com/ca/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/trypan-blue-exclusion.html
Hi Nicolas, I did not used the Trypan blue assay after thawing but from the look itself (under microscope), cells look viable.
I have pelleted down the cells and again washed the cells three times with chilled PBS and once with media to wash out any remains of DMSO (if left unknowingly). Then did trypan blue and found around 40% cells were viable.
Then seeded in new T-25 flask. But cells are still not getting adhered.
Thank you.
Hi Sandeep, it could be because you washed the cells three times which possibly means you spin it down three times? Preserve cells are fragile which some I have experienced before. Some cannot even be spin down when thawed, just transfer to T25 in 10ml warm media and left for a day before changing fresh media (probably a good thing to increase FBS %, normally I would use 10% but in case of slow growing or cells that are floaters more than 2-3 days, I would increase FBS to 15%). I usually just spin down once and resuspend in 10ml warm media. I have nutlin resistant cell lines which took almost a week to recover and attached (cryopreserved for almost 3 years, preserved in 90% FBS 10% DMSO). But not all thawed vials managed to survived though. If your cells are still 40% viable, then probably transfer to a smaller plate, ie 60mm (smaller surface area for the cells to find each other as we know cells do not like to be alone for too long when growing) and increase FBS %. That was what I did though. Good luck!
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