I am trying to thaw S2R+ cells from the frozen culture. The cells (1 ml of cell culture) were frozen at passage 9 and stored at -80. I followed the steps below to thaw the cells from the frozen stock:
1. Take the vial with the cells and thaw it at 30 degrees in a water bath.
2. As soon as the culture is thawed and liquid, remove the vial from the water bath, and clean it using 70% ethanol. Take the 1 ml cell culture from the vial and add it to a 10 ml falcon containing 4 ml of complete Schneiders media (Complete S2 Media: 10% FBS, 1% Pen-Strep, 89% Schnieders Medium). Mix them nicely with pipetting.
3. Place the falcon, now with 5 ml components (1 ml of culture from stock and 4 ml fresh media) in a centrifuge at 100g for 10 min.
4. Remove the supernatant, which would contain DMSO, and then add fresh 5 ml of media (2.5 Fresh S2 Complete Media + 2.5 Conditioned S2 Media) and add this culture to the T-25 flask and let it incubate for 3-5 days before starting passaging.
I have attached the images (phase contrast images), which were taken after P10 (one passage after the above procedure of thawing and reculturing the stock). I see a lot of dead cells (Counted using Luna Cell Counter--> Viability is approximately 40%).
Am I doing something wrong while I reculture the frozen stock?? Or is it alright and I should just clear out the dead cells using low centrifugation speed for 10 minutes or so?
Shubhanshu Pandey According to expressionsystems.com website, S2 cells are best stored below -125 degrees C. They suggest vapor phase nitrogen. Eukaryotic cells in general need to be frozen long-term below -130 degrees C, because metabolism comes to a stop below that temperature. In my experience with mammalian cells, they begin to die off after a few days to 2 weeks in -80 degrees C conditions, and by about 6 months of -80 storage they are all dead. We use a -150 degree Panasonic freezer that works well. In addition, you must "freeze slowly, thaw quickly"; using a Mr. Frosty that is at room temperature when you start, and has a 100% isopropanol sleeve, will freeze your cells (placed in freezing media at 4 to 25 degrees C) at 1 degree C per minute. Only leave the cells in the -80 for 1 to 3 days before transferring to the colder long-term storage site. When thawing, ensure the media has been in the incubator (whatever incubator fly cells require) for at least 30 minutes, then rapidly thaw the cells (90 seconds) and transfer to the equilibrated plate/dish/flask (note that this last part does not centrifuge the DMSO out; we exchange media as soon as the cells attach; your cells may require immediate DMSO removal though). Good luck.
Hey Melville B Vaughan That is a good insight. Although, I did not freeze the cell and I only had to use that frozen culture. But I will keep your points in mind when I make frozen stock!
A previous labmate suggested that DMSO removal is not required for s2 cells and hence I am letting them grow. Let's see how the culture proceeds. If it is successful, I will update it here on the thread!
As other mentioned, cryopreserved cells should be stored in vapor phase of liquid nitrogen or below -125°C. It may be too late to response to OP, but the cells look fine. The S2 cells can be grown as adherent or suspension in S2 medium containing 10% heat-inactivated FBS. Do not use antibiotics for the first 24-48 hours as it would affect the recovery of the cells after thawing. They love high cell density in culture. Make sure you keep the conditioned medium for cryostorage and future use. For passaging, I usually just dilute the cells a few folds with fresh medium and keep ~20% conditioned medium.
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