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SF9 Cells lose exponential growth, lack stationary phase

sf9 insect cell culture Baculovirus

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#1 Alopex

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Posted 27 January 2012 - 01:28 PM

Hey there,

I've been maintaining shaker flasks of SF9 insect cells for the past 2.5 years using a single cell line, but recently I've been having some problems.

When I subculture the cells to 2.5x10^5 cells/mL, they typically double in 24 hours. After those 24 hours have elapsed, however, they rarely double again, and seem to enter a phase of linear growth (i.e. day 2: 5x10^5 cells/mL, day 3: 7x10^5, day 4: 9x10^5). What is also disturbing is that they have a relatively low high density maximum around 1x10^6 (when my lab could typically get around 2x10^6 cells/mL easily), and also that they do not remain at their maximum for any period of time -- while it used to be the case that the cells could be left for up to a week in stationary phase, these cells are now reaching about 9x10^5 cells/mL on average before slowly decreasing.

Let me provide some more information:

-I have been using the same cells all along, and have retrieved cells from frozen stock to make sure the passage number is not too high.
-I use grace's insect medium +10% FBS +pluronic surfactant in a shaking incubator at 100 RPM at 27.0 celsius. These parameters have not changed. They are in shaker flasks with a relatively low volume, with sealed caps. I have tested vented caps and different medium (tc-100) but nothing has changed.
-There is no visible sign of contamination, at least nothing obvious.
-I work with baculovirus, and the stocks are negative for contamination with that.
-When viewing the cells using typan blue exclusion, they seem reasonably normal/happy, and I hardly ever see a dead cell

Anyone have any ideas what could be going wrong? The help would be greatly appreciated!

#2 klinmed

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Posted 31 January 2012 - 11:05 AM

Did the problems start after switching to a new batch of FBS? If you heat-inactivate FBS, has the protocol been altered? Was your primary frozen stock prepared some time before the problem started? You could (should) check for mycoplasma contamination.

#3 Alopex

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Posted 01 February 2012 - 09:27 AM

Thank you for the input -- to answer these questions:

-We don't/have never heat-inactivated FBS for this cell culture, and the protocol has been the same generally speaking

-We did in fact have some problems when switching to new FBS. Back in August of last year, we switched vendors and the cells started growing to about 70% of their typical high density maximum. We figured that it was due to a problem with the cells themselves (since they were at a very high passage number), because when we tested the lot of FBS for a couple of weeks, growth was fairly on par with what we had been using. It may be that problems with the FBS manifest very slowly over the period of the next couple of months, which is something that I've been thinking more recently. When we restarted the cells from a frozen lot, they were grown in this new FBS. Within the last month, the cells are growing alright but definitely not doubling every day for several days consecutively like I would expect.

-The frozen stock was prepared many years ago and I think it should be okay.

-I have not checked for mycoplasma contamination, so that is still a possibility, as well, and I will look into assays/kits for that.

On a side note, I think that poor and linear growth near the higher densities might be due to oxygen limitation in the culture. We used closed erlenmeyer flasks on an orbital shaker at 100 RPM, but after reading through several vendors' technical papers on their SF9 lines, it seems that shaking speeds of 125-150 RPM are recommended (or possibly more if the line is particularly oxygen dependent). I'm worried of destroying the cells with shear force but I will be testing this possibility in the near future.

Thanks again!

#4 rhombus

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Posted 02 February 2012 - 03:36 AM

Thank you for the input -- to answer these questions:

-We don't/have never heat-inactivated FBS for this cell culture, and the protocol has been the same generally speaking

-We did in fact have some problems when switching to new FBS. Back in August of last year, we switched vendors and the cells started growing to about 70% of their typical high density maximum. We figured that it was due to a problem with the cells themselves (since they were at a very high passage number), because when we tested the lot of FBS for a couple of weeks, growth was fairly on par with what we had been using. It may be that problems with the FBS manifest very slowly over the period of the next couple of months, which is something that I've been thinking more recently. When we restarted the cells from a frozen lot, they were grown in this new FBS. Within the last month, the cells are growing alright but definitely not doubling every day for several days consecutively like I would expect.

-The frozen stock was prepared many years ago and I think it should be okay.

-I have not checked for mycoplasma contamination, so that is still a possibility, as well, and I will look into assays/kits for that.

On a side note, I think that poor and linear growth near the higher densities might be due to oxygen limitation in the culture. We used closed erlenmeyer flasks on an orbital shaker at 100 RPM, but after reading through several vendors' technical papers on their SF9 lines, it seems that shaking speeds of 125-150 RPM are recommended (or possibly more if the line is particularly oxygen dependent). I'm worried of destroying the cells with shear force but I will be testing this possibility in the near future.

Thanks again!



Dear Alopex,

I have grown Sf9/Sf21 cells for many years. This is what I have observed in my lab:-

FCS/FBS is VERY IMPORTANT........We use exclusively New Zealand (NZ) Serum in our experiments.....the serum is batch tested because there can be huge batch variations...even with NZ sources serum.

The cells change dramatically over time and passage. That is why we grow the cells for 2 months and then get fresh cells up from our frozen master store

We grow the cells in Techne Biological stirrers bottles..... these are specifically manufactured to grow suspension cultures. We spin the cells at between 20-25 RPM.... and we do that because the cells are adversely affected by shear stresses at RPM's above that speed.

We use vented caps to allow oxygen penetration. I have never use closed caps so I cannot say with certainty that the lack of oxygen will affect the cells.

We grow our cells at 21oC.....we do this because the cells will grow too quickly at higher temperatures. When we do bacullovirus infections we do this at 28oC and on cells that have been adherent on plates for 24hours.

Frozen stocks of cells in my lab under our conditions have given us great results even though they may have been frozen down 25 years previously. Cells in Liqiud Nitrogen should in theory last forever. If you keep cell stocks in a -80 freezer then this will cause major problems....your stocks may perish after only a couple of years.


I hope some of this is useful.

Kindest regards

Uncle Rhombus





Also tagged with one or more of these keywords: sf9, insect cell culture, Baculovirus

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