Are these cells stirred or in T- flasks? They look at too low a density in the T- flasks.

There are few things to consider. What is the stock concentration of Sf9 cells you are starting with. I generally start at 1:3 dilutions for both adherent culture or suspension culture. At first you would expect some cells to die but the growth should pick up after 2-3days (when I change media) and let them grow till confluency(about 4days).

The problem here might be because your cell culture can be either too dilute ( its not very clear really the picture as cells seems to dead or dying) or there is some problem with the medium. You have to consider which medium you are using SF900 (II) or SF900(III). The former media is much better for Sf9 cells in adherent cultures for recovering them from frozen stocks.

Room temperature incubators are good but I generally prefer to grow them at 27C plus minus 1. Sf9 cells are very prone to sensitive to sudden temperature changes.

It is preferable not to use Serum for Sf9 cells. Also, if cells have not reached confluency in four days try to change the media. This replenishes the stock of everything the cells need from the media.

Hopefully this will give you some idea to optimize your cells.

I am fullya agree with mr. Sandeep talapatra....

Dr. Talapatra have already mentioned some important points to take care of for Sf-9 culture. Sudden temp change is problematic for Sf-9 culture indeed. Initial seed density is might be also an issue. May be you can do one more thing, you may change your media and sterilize your all tools including pipettes. Some time you never know from where those viral infections come form. Also I would wash out the DMSO while putting the cells in culture form a frozen stock.

I agree with the point about cell density comments. It looks looks like you need at least 50-fold more cells per cm2. About the medium. TC100 will not work if not supplemented with at least 5% FBS, 10% is good. You might check to make sure that any serum that you are using is good with the Sf-9 cells. but most lots are fine. If you add it to any serum free media, 2.5% is fine. Also, stick with the same medium that the cells were in before you got them. Also, check that you are using the same culture surface as you previously used.

I would start with at least 2 or 3 x 10e5 cells per cm2, i.e., 100% of a monolayer or a little more. Carefully, decant and replace with room temp fresh medium every few days. Make sure that the cells are growing before you subculture.

Good luck.

Which is the source of your cells? They come from other labs? They seems to be sub-infected or contaminated, so I agree with Dr. Imran Khan.

After you ensure that your cell line is not contaminated, pay close attention to the media. It is best to use the same media that they were originally cultured in. Find out if they were in Sf-900 II, Sf-900 III, TC100, or another one? If all fails, call the technical services group of the media manufacturer (GIBCO or other) who can usually walk you through troubleshooting. Make sure that you have the lot number and catalog number of the media you are using anfd the same info for the media that the cells were previously cultured in. Sf9 cells are pretty finicky with adaptation from one medium to another..

Without seeing the growth kinetics, I'd say it could simply be that neither of these two media was rich enough to support extended batch growth of Sf9 insect cells, or that the cells would die anyway within certain period of time due to nutrient limitation/toxic metabolite build-up. Unlike CHO cells, some types of cells, such as Sp2/0 (and Sf9?) do not live long in in vitro culture no matter what.

I do not think it can be an issue with contamination as we received 3 vials of cells direct from Invitrogen -so these definitely should not be contaminated. This makes me think that maybe it is an issue with the serum as this has been the only constant within all cultures - but I just don't know if a dodgy batch of serum could cause such catastrophic effects to cells so quickly - these cells went from growing happily to being like this in about 12 hours.

What happens if you use trypan blue after they've started growing- how many viable cells do you get? above 90%? You could be spinning to fast or not fast enough. I would try adhering the cells to t flasks get a large batch frozen down etc. How are you spinning them with a flea or in an orbital shaker?

Can anyone else in the lab grow Sf9 cells without a problem?

A problem in 12 hrs rules out a baculovirus contamination "as long as the cells started out uninfected" since it would take ~70-80 hrs to see a problem with a low level baculovirus contamination say in your media.

And I would not rule out FBS as a problem, was it insect cell culture tested by the manufacturer? Geminibio sells insect cell tested FBS. Invitrogen sells Sf9 cells in serum free media, did you try growing those cells in serum free media and did you still have a problem? I stopped using FBS after a lot of strange issues with my cells.

And I agree with Imran, if those cells are going over 30oC then they will look bad. Do you have a temp. probe in the incubator and does it record the Hi and Low temps?

And again as Imran pointed out, media is a potential problem. Invitrogen either uses L-glutamine in their media (or recommend it be added), L-glutamine breaks down within a month and forms ammonia which is toxic to the cells (that's if you treat it right, but if the bottle is put in a warm water bath a lot L-gluamine will break down a lot faster), Glutamax does not. BD sells Max-XP media which must have Glutamax in it since is has a shelf life of a year.

And I have gotten a bad batch of Sf9 cells once, but not from Invitrogen, and they looked like they had a baculovirus infection after 3 days.

Sir Gary Laco ...you posted a very valuavle information....it is very banificial for us.....

The cells in the picture look contaminated to me. When we start a new Sf9 culture they are adherent in a 75cm2 flask in Grace's with 1x lipids, gentamicin, and 5% FBS. Then after they reach about 90% confluency, we switch them over to suspension in SF900 media w/ 1x lipids and gentamicin, (w/o serum). Once they are in suspension, they need to be at least 1.2x10^6 cells/mL. Low density could make them look grainy like that as well, but the speckles around the cells makes me think they're contaminated. When we had this issue, I cleaned everything in the tissue culture room, particularly the hood, and restocked it with fresh tips and everything. I also checked all of the media components for contamination under the microscope.

try to get cells from different sources other than invitrogen.

1] prewarm the media to 27C, open a fresh bottle of media and then add antimycotic antibiotic. [we use SFX insect from Hyclone]

2] then thaw the cells at 27C.

3] wash the cells by diluting in fresh media [20 ml] and centrifuging at 1K rpm for 5 min at 4C.

4] throw the supernatant and resuspend in fresh media [ 3ml]. and plate in a T25 flask.

5] incubate at 27 C. Have a stainlesss steel water tray filled with DI water.

6]wait for 4-5 days for the cells to recover before subculturing.

"I have tried 8 different stocks of cells from 4 different sources. I have tried 2 different types of media - Sf900 and TC100 both with and without serum and also different incubators and growing at room temp. Each time the cells grow fine for a few days and then suddenly disintegrate (see attached pics) - does anyone have any ideas? "

It seems like cells are infected by baculovirus. A wrong batch of serum, lower/higher (+-2 C degrees) temperature in the incubator or low cell density, don´t make cells to look like that after several days of culture. If I´m right, you must clean everything and start again.

If you have contamination by bacteria, medium turns cloudy very fast, usually next day after plating cells. The photo was taken after a few days. At this time, you should see nothing but a soup of bacteria. If there is yeast contamination, just leave a plate with medium and no cells in the incubator and check it everyday under the microscope. I have seen sometimes small round particles, that could be yeast by their size, but they grow slower than bacteria and don´t make the cells to burst. Of course, those cells went directly to waste. If you say cells grow fine for a few days and then disintegrate, those are the symptoms of baculovirus infection.

Here it is an interesting discussion about insect cell contamination:

http://www.structuralbiology.eu/support/forums/networks/p4eu/insect-cell-contamination

If you are working with wild-type baculovirus: wt baculovirus expresses the polyhedron protein which makes occluded viral particles which are visible with the microscope and your cells have those particles inside them, those particles are also very stable to UV. Maybe your hood has been contaminated with these particles. It takes ~72 hr to see cell lysis after a baculovirus infection.

Phyllobacterium myrsinacearum: Incubation at 28°C in carrot juice agar containing yeast extract (lysing bacteria would have similar nutrients) for morphological characterization.  Observed in phase-contrast microscopy are star clusters.  Seems your pictures show star clusters and the incubation temperature is correct.  They will grow more slowly in Sf9 media and thus why it occurs late.  These microbes exert broad antimicrobial activity (bacterial and fungal), likely another problem for you culture.

This is a thought for exploration, not an absolute as your picture is either disrupted or unformed clusters or just refractive components.  Others have noted contamination with this organism in Sf9 cells.

There is also the possibility of Mycoplasma sp. contamination which wouldn't be seen by microscopy, but can reek havoc on cultures and is hard to remove from contaminated lab incubators.  Hoescht stain is a simple method of evaluating or one can send out for PCR identification.

Dear James Embree,

I'm interested in your comment. How dou you mean exactly hard to remove from contaminated lab incubators"? The incubator can be infected as well, not only the culture? Sorry if I missunderstood :)

Best regards,

Do not scrape the cells and do not centrifuge. In order to change the flask remove the medium, add a new medium and hit the flask hardly on the table until you detached them. I had exactly the same problem as you