My cells don't seem to be growing and getting infected. The first batch I tried look like they were frozen with infection. I then got a supply from another lab and it has been a week and they don't seem to grow/double. I noticed that our unopened and sealed bottles of sf9 II medium had stuff growing in it. I think this media was quite old. So discarded this media and got a fresh bottle (which was just as old but clear).
I have thoroughly cleaned everything twice but still the cells get infected. The fumehood and incubator are in a side room. However, there are aktas in there where people have preps from E.coli.
All media had pen/strep added to it and I supplemented the media with 5%FBS. They are in suspension and spin at 130rpm. Someone suggested a lower speed which i tried but it made no difference (in terms of growth)
Yesterday the cells hadn't grown but they still looked good, round, no debris etc. Today they were completely infected.
I am getting a new batch of cells next week. Really wanted other people experience and any advice, as I don't want the next new batch of cells to be wasted.
Dear Chitra,
1. First of all, let me point out that the cells take some time in getting revived from the frozen stock. It also matters how you are thawing them. Could you tell me how do you thaw them? If water is used for thawing, make sure to just let them at 37 degrees for 10 seconds and water should not touch the cap of the frozen vial. Also clean it thoroughly with ethanol before opening it.
2. Also, the medium in which they were growing before being frozen also matters. Make sure that they were growing in SF900 II before they were frozen. Many people also use Graces medium which gives better results. Also, SF900 II is serum free medium which means it does not require FBS. We are growing SF9 in SF900 II without FBS.
3. It seems that you are growing SF9 in suspension culture. I hope that you are aware that SF9 are also quite adherent which means that you can also grow them in T25 or T75 bottles. Also, once you put the cells back into the culture from frozen stock, change the medium daily if you are scared of getting contamination. Also normally 85 rpm is sufficient for cells in suspension if you are growing them in spinner flasks. In case, you are growing them in shaker flasks, 130 rpm seems fine.
4. You mentioned that people make preps from E.coli in the room where the laminar hood is there. Make sure that they are not using the same laminar hood for purpose of E.coli.
I hope this helps.
Best Regards,
Thanks for your answer Archit. I thaw the cells at 37 C for about 30 seconds and spray the vial before transferring into media. I have also tried to start cells in a T75 flask but the same thing happened.
The hood is only used for insect cells. Will try all your suggestions and see if it works. I have new media now, so hopefully it may work. Thanks again.
I will recommend not to use the water for thawing if you are facing the problem of contamination frequently. What you can do is just thaw the vial in your hand for a minute and dip it into the beaker containing ethanol, then wipe it and transfer it into media.
Hi Chitra,
I would prefer to recheck the pH of media since insect cells prefer slightly acidic pH (e.g Graces media has pH of 6.2). Also consider the passage effect, aging of SF-9 at passage 30 begins, so try limiting the use cells at passage 28 or so. Better use low passage vials.
Caution: Be careful if you working with baculovirus, wipe out the platform well before and after use and avoid cross contamination of growth media.
Thanks Nikhil. I will have a look at what passage number the cells are as they were kindly given to me.
Oh yes thanks Nikhil. All doubling now and onto virus amplification. No infections. Started with a new batch of cells, new media, 100 rpm, pen/strep and it all seems fine.
Actually I spoke to soon! I just did a cell count and they look l ike they have not grown ver the weekend. No infection, they just haven't grown.
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