I started working in a U937 line. On the day I thawed the cryopreserved vial of cells they were nicely viable. When I checked them the next day, they were at 30 % viability. and shriveled. Has anyone else observed this large drop-off in the first 24 hours post-thaw? Thanks in advance.
Hello Alaa Al-Emerieen
The viability of U937 cells should not drop after cryopreservation if you have carried out the cryopreservation process in an appropriate manner.
Slow rate of freezing at an optimum cooling rate of -1 degree celcius per minute is needed for the cells. The rate of cooling controls the size of the ice crystals and the rate at which they are formed, both of which may affect cell recovery. Thus, a slow uniform cooling rate of -1°C per minute from ambient is effective.
To achieve this you may store the cryogenic vials in an isopropanol-containing cryo-freezing container such as Mr. Frosty and place them overnight in a -80°C freezer. This will allow the cells to freeze at approximately -1°C/minute, which is ideal for freezing most cell types. You may then transfer the cryogenic vials to a liquid nitrogen tank the next day.
The viability of U937 cells should also not drop after thawing unless and until you have not thawed the cells in a proper manner, namely quick thawing. It is commonly assumed that cryopreserved cells must be thawed rapidly otherwise ice recrystallization events occur during warming which are detrimental to cell recovery. So, quickly thaw the cells in a 37°C water bath by gently swirling the vial. Remove the vial when a small amount of ice remains. Do not vortex the cells.
After thawing the vial immediately transfer the cells to a large volume of pre-warmed cell growth medium (~10 mL/1-mL aliquot of cells). For the next step, you will have to decide which one works well for your cells.
There are two schools of thought on whether the cryoprotectant should be immediately removed from the cells:
1. In some labs, the cells are centrifuged, and the cell pellet is resuspended in fresh cell growth medium prior to placing cells in a culture dish.
2. However, some cells are extremely fragile after thawing and that centrifugation may increase cell death. So, it is recommended that the cells be plated, and that the growth medium be changed after the cells have attached (7-8 hours) or the next day.
Besides freezing and thawing, there could be other conditions that could have led to low viability in 24 hrs. post-thaw namely, environmental stress which could include:
1. Inadequate nutrients in growth media
2. Incubator temperature fluctuation
3. Inappropriate environmental gas mixture and
4. Contamination
Please check for these above conditions as well.
Also,
The culture medium for U937 is RPMI-1640 supplemented with 10% FBS and 2mM L-glutamine.
The cells should be seeded in T-75 flasks with the cell density between 1 х 10^5 and 2 х 10^6 viable cells/mL and cultured in 5 % carbon dioxide at 37 ℃. The medium should be renewed every 3 to 4 days depending on the cell density.
Finally, the cryopreservation medium for U937 is complete growth medium supplemented with 5 % (v/v) DMSO and storage should be in liquid nitrogen vapor phase.
Best.
The viability may be affected by the cryopreservation process and/or the revival process. Here are some considerations to check if your processing was correctly done: https://lab.plygenind.com/how-to-cryopreserve-mammalian-cell-lines
https://lab.plygenind.com/considerations-revive-cell-cryogenic-preservation
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