I am using insect cells for almost 3 years now. I usually thaw and grow them as a suspension culture. But sometimes for some back-up cells, I plate them on 75 cm plates. They are not easy to detach. What I do is to remove the media and bang them (yes, bang them on the bench of the Laminar real hard) and then use high force from the pipettor and repeatedly flush it on the cells. This is the only way out I found to detach uninfected cells. When I used a cell scrapper, most of them died. So, I usually stick to suspension cultures, and they work best whether it is growing or infecting. But the infected Sf9 cells detach really well from the plates.

Suspension cultures are so easy to split and maintain. I have been hearing this problem of Sf9 cells from Invitrogen not being revived the second time. I guess they have some issue that they have to look into. Why don't you borrow a vial of cells from someone and then make your own stocks ! It is much easier that way.

I have cultured SF21 cell line for which I used sterile plastic cell scrapper. The SF9 should be similar, I think!

With SF21, when culture is young, removing medium and just leaving around 3-4 ml in T-75, and gently tapping proves sufficient. When culture is old and cells are 100% confluent, cell scrapper is needed as cells tend to be more strongly attached.

With cell scrapper cells do die but that shouldn't kill that many cells!

Try this: Harvest cells when they are 80% confluent. Remove medium and leave only 3 ml in T75 flask. First, gently tap on sides with your hand two or three times. Some cells will detach. Scrap the rest with a scrapper. Avoid excessive tapping and pipetting. Cell death should not be so high. I hope this works with SF9 as well.

Good luck!

I tried all that Zaheer. Nothing did work for me. They were really attached strongly. I ended to use a scrapper but it resulted in a lot of dead cells. I will try suspension cultures instead. Hopefully I will get better luck.

Thanks for you time :)

For deattaching the adherent cells You can try Trypsin-EDTA solutio (0.05% trypsin and 0.05mM EDTA). Add 2 to 3 ml of this solution to your 75 cm2 flask with adherent cells and immediately observe the lifting of the cells from the surface of the flask in microscope. once all the cells deattach, immediately add 5 ml of 10% serum which will stop the trypsinization.

Then wash the cells as usual

Dear Delphine,

You grow them in stirrer culture vessels IN SUSPENSION. In this way you never need to trypsinise them as they are never adherent. Scrapping them off flasks in order to grow them is highly inefficient and causing damage to the cells. We only use adherent Sf9/Sf21 when it comes to infecting then cells with baculovirus. Then after 24/48 hours post infection the cells are scrapped off and the protein isolated. Please refer to our section in the book below:

http://www.ncbi.nlm.nih.gov/pubmed/10906992

The suspension system is so much easier and in the long run a lot cheaper.

I hope this is useful

Kindest regards

Neale

Thanks Neale!

That's what we tried after realizing adherent cultures of insect cells were a pain to work with.

Can I ask where you buy your sf9 cells? We bought them from invitrogen (The serum free adapted ones) but we have a hard time to get them growing. They look great....but they don't grow. I am waiting for the third vial of cells (that Invitrogen always replace for free....plus free media). I feel like I am working hard to find out the proper conditions to grow their line of cells...

Thanks for your time. This is appreciated.

Cheers,

Delphine

Insect cells grown in serum free medium will stick like glue on TC treated polystyrene, and will basically never come off. You have two options: (1) use untreated (hydrophobic) polystyrene (bacterial petri dishes OR untreated T flasks), or (2) use a medium like C-TNMFH. Cells grown in C-TNMFH can be detached by smacking the flask against your wrist with minimal cell death. Cells grown in untreated polystyrene won't stick at all and just float on the surface, and can be removed just by swirling.

I am using insect cells for almost 3 years now. I usually thaw and grow them as a suspension culture. But sometimes for some back-up cells, I plate them on 75 cm plates. They are not easy to detach. What I do is to remove the media and bang them (yes, bang them on the bench of the Laminar real hard) and then use high force from the pipettor and repeatedly flush it on the cells. This is the only way out I found to detach uninfected cells. When I used a cell scrapper, most of them died. So, I usually stick to suspension cultures, and they work best whether it is growing or infecting. But the infected Sf9 cells detach really well from the plates.

Suspension cultures are so easy to split and maintain. I have been hearing this problem of Sf9 cells from Invitrogen not being revived the second time. I guess they have some issue that they have to look into. Why don't you borrow a vial of cells from someone and then make your own stocks ! It is much easier that way.

I am also getting the same problem. I had seen these cells attach strongly to even untreated flask. I had faced a same problem recently from Invitrogen Sf9 suspension adapted. I will be getting replacement vial. Has anyone used ATCC or some other repository.  

very clear, nobody has good answer to this question.