Hey All,
Our team wants to complete cytotoxicity per USP , using the L929 cell line and I've been tasked with this. I've never worked with this cell line (or any animal cell lines for that matter) before, but I figured most of it out. We got all the right equipment, a liquid nitrogen dewar for long-term storage and I am capable of passaging cells well with high viability.
However, recently, I've noticed contamination in my cell line - mostly bacterial. The media changes its pH and turns pink, while some flasks turn visibly turbid as well.
For contaminated flasks, is there a way to de-contaminate cells? There are vialble L929 cells present, but adding 1% Antibiotic-Antimycotic doesn't seem to work too well.
Also, would anyone have any advice on preserving high viability of cells post-cryopreservation? I get my L929 from ATCC (they come at >80% viability), but my post-freezing viability average at ~58%.
Any help would be appreciated. Thank you!
-Tim M Seyidov
Hello,
I would suggest you discard the contaminated flasks. De-contaminating the flask with antibiotic treatment is only wastage of time and the quality of cells will also be compromised. Such cells cannot be used for any cell-based assays. You should thaw a new vial of L929.
Thawing of cells from liquid nitrogen must be done as quick as possible. Place the freezing vial at 37 degree C to thaw and add cell growth medium to dilute the contents. Centrifuge and plate cells in smaller culture flask like T-25. The next day replace the culture medium with fresh medium to get rid of traces of DMSO which is used in the freezing medium as a cryoprotectant.
The viability of cells after thawing will depend on how well the process of thawing has been done as well as on the freezing protocol (gradual freezing of cells).
I hope this helps.
Best.
Our lab rarely have encounter cell contamination, for what I remember, Normocin from InvivoGen have been advised by lab co-worker for prevention. In case of contamination, after disperse L929 with trypsin-EDTA (ATCC recommended 0.25%, but I think 0.05% should work), we will first use same amount of growth medium to attenuate trypsin's activity, then add PBS to wash off or dilute any bacteria in medium (up to 3 times), then provide a fresh prepared antibiotic (you may also switch antibiotic to deal with resistant strain) (1% penicillin/streptomycin or 0.2% Normocin) containing growth medium and observe for another 2 days. The reason is maybe the medium been through too many freeze-thawing cycle, the antibiotic can loose its inhibition ability, there will be a chance to inhibit bacterial growth by adding a fresh one.
For L929 cryopreservation, ATCC recommends 5% DMSO, and a single cryo. vial we do not store more then 1x106 cells. After subculture, remove the sup. then apply 1ml medium-DMSO mixture and freeze the cell as soon as possible (we use cell freezing container and directly into -80oC).
To thaw a cryopreserved cell, prepare a 15c.c. tube filled with 9c.c. growth media, after take out the cryo-vial from -196oC or -80oC, vial should directly go into a prewarmed 37oC water-bath, do not wait until the ice melt completely, but left a small piece of ice floating, just before adding into media containing tube it should be totally melted, this can slightly increase cell viability.
Same procedure have always bring >85% viability in our cell lines.
Best of luck.
Hello Tim,
Toss the contaminated flask. Efforts to decontaminate the cell lines seldom work. Take a new cryovial of your cell line, thaw and bring it to body temperature (37 C). Centrifuge the vial to get the cell pallet. Re-suspend the pallet in complete media (compatible with your cell line) having antibiotics- antimycotics (generally 1%) and supplemented with FBS (10%). Seed the suspended cells in a fresh culture flask (size depending on cell count). Your cells should grow normally.
To get maximum viability, try to preserve cells in 90% FBS and 10% DMSO (as cryoprotectant). Also, the freezing of your cells should be gradual. Directly transferring cells to very low temperatures (-80 C or -196 C) can put them into shock. After making your vial, keep cells in 20 C for a couple of hrs, followed by freezing in 80 C for 8 hrs and then finally transferring it to liquid nitrogen (-196 C). Similar strategy should also be followed while thawing (gradual thawing).
Abu, Mu-Shen, and Malcolm - thank you all for your advice.
I ended up thawing one of the stored vials. I had the same ~58% viability, but I can now passage and grow some more. Hopefully, the antibiotic/antimycotic will prevent further contamination.
My cryopreservation procedure is very close to what was described. The only think I can think of to be a problem is my medium I use for cryo. I don't make my own - instead I get CellBank II (serum supplemented). Interesting to know that cryopreservation medium is almost entirely serum!
Thank you all again for your advice.
-Tim
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