10 replies to this topic

[spyderman]

I've just started my first SF9 culture. I'm using serum free medium from Novagen, in T-75 flasks and 100mm dishes. The cells are growing, but less than 10% are adhering. I'm on my 3rd passage. What can I try to get them to stick? Or is there another way to transfect than draining the media off so that I can use these suspended cells?

[gfischer]

spyderman, on Apr 8 2009, 02:16 PM, said:

I've just started my first SF9 culture. I'm using serum free medium from Novagen, in T-75 flasks and 100mm dishes. The cells are growing, but less than 10% are adhering. I'm on my 3rd passage. What can I try to get them to stick? Or is there another way to transfect than draining the media off so that I can use these suspended cells?

I don't know much about your specific cell line, but here are a few thoughts:

1.  Are you sure it's an adherent cell line (I know you probably sure since you're asking, but it can't hurt to check)?
2.  Are the flasks/plates made for adherent cells?  Several companies make plates with a special treatment of the polyethylene that promotes adhesion.
3.  have you tried coating the plates with anything?  I've had good success in other cell types with poly-L-lysine and/or laminin.

Hope this is of some help.

[spyderman]

Thanks for  the reply. The cell line is supposed to be adherent, although without contact inhibition so they won't stop growing at confluence. The plasticware is treated from the manufacturer. Interestingly, after the fourth passage I am now getting about 25% adherence in some (but only some) of my flasks. Should I check the lot # with the manufacturer, or just keep passaging till they stick better? I'm not currently equipped here to be coating my flasks.

[klinmed]

spyderman, on Apr 8 2009, 09:16 PM, said:

I've just started my first SF9 culture. I'm using serum free medium from Novagen, in T-75 flasks and 100mm dishes. The cells are growing, but less than 10% are adhering. I'm on my 3rd passage. What can I try to get them to stick? Or is there another way to transfect than draining the media off so that I can use these suspended cells?

I assume that you will be using the cells for protein expression using a baculovirus approach. If this is the case, the health of your cells is critical to success.
When you first work with Sf9 they can appear difficult to handle. However, after you get use to them you wonder what the problem was!

The cells are adherent in stationary flask cultures and do NOT need specially coated surfaces. Normal tissue culture flasks are fine. In flasks, adherence is a sign of healthy cells. They should contain <10% "floaters" and have a doubling time of ca 18 - 25  hours. Indeed, in serum-free cultures, getting cells of the surface is usually the problem...

Your problem may be that the cells are just recovering from liquid nitrogen storage and will be fine after a few passages. You mention that you use serum-free medium. I assume that your frozen stock was made from Sf9 adapted to serum-free conditions. Suggestions:

1) Do not over split your cells. They like reasonably high densities 2E5 - 1E6 and slow down if over split.

2)  Always transfer a little of the old culture medium when splitting. For example, take 1 ml of culture to 4 ml of fresh (room temperature) medium. The cells seem to secrete some form of  "growth promoter" which helps to maintain healthy cells.

3) With one of your flasks you could try the following:  Remove non-adherent cells, and GENTLY suspend the adherent ones by pipetting. Tranfer the cells to a smaller flask (or culture well) to get about 50% confluence. Incubate, then split no more than 1:5 (as above), when just confluent. Always aspirate all non-adherent cells at each split.

Good luck and hope this helps.

Edited by klinmed, 11 April 2009 - 12:58 AM.

[spyderman]

I am putting these suggestions into play: keeping the cell density higher is underway. As for removing the floaters and plating the adherent cells, I barely get enough adherent cells to line the bottom of a single well in a 96 well plate at the required densities.
I called the supplier of the cell line and they stated the problem was I wasn't using polycarbonate plates. Any opinion on that? I hate to think I have to dispose of all the tissue culture supplies I laid in and purchase more.
Thanks for all the help.

[klinmed]

spyderman, on Apr 17 2009, 06:27 PM, said:

I am putting these suggestions into play: keeping the cell density higher is underway. As for removing the floaters and plating the adherent cells, I barely get enough adherent cells to line the bottom of a single well in a 96 well plate at the required densities.
I called the supplier of the cell line and they stated the problem was I wasn't using polycarbonate plates. Any opinion on that? I hate to think I have to dispose of all the tissue culture supplies I laid in and purchase more.
Thanks for all the help.

I´m sorry to hear that you are still having problems. As I have mentioned before, healthy Sf9 are usually very adherent to tissue culture plastic. The representative you talked to does not know his insect cell culture. Sf9 stick well to "normal" tissue culture plastic (like Nunc or Costar cell flasks). These are made of charge-modified polystyrene not polycarbonate.  Indeed, polycarbonate flasks are used for suspension cultures when you don´t want too much adherence.

I have worked with this cell line for many years but have not used Novagen medium. With the serum-free medium from Gibco/Invitrogen (Sf900II) or Hyclone (Sfx-insect) the cells stick very well (sometimes too well!) to polystyrene tissue culture flasks. The same is true for the more traditional serum-containing media such as Graces+10% FBS.

Your plastic is fine. Therefore the problem must be with the cells themselves, or the culture medium.  Do the cells grow as adherent layers in the presence of 10% heat-inactivated FBS?

Regards

[see_mj]

I've just started SF9 cells as well. I thawed my cells, suspended them in 60mL serum free media and put them in a 250mL shaker flask. I let them incubate at room temp shaking at 200rpm for 2 weeks. I trasnferred the suspension, pelleted the cells, resuspened in fresh media and put in a clean shaker flask. I repeated this process every week and at passage 4 the cells began adhering to the wall of the shaker flask. Next week I will pass into T25s. My manufactures instructions recommended starting the cells in a 125mL shaker flask with 30mL of media, but I didn't have any available. My shaker flasks are sterile, disposable filter capped tissue culture flasks from BD. Best of Luck

[Majid]

I try to express protein in Sf9 cells.I'm using serum free medium from Novagen, in T-75 flasks. The cells are growing slowly and less than 10% are adhering. They accumulate in center of flask. What can I do to obtain a monolayer cell culture?
Thanks
Majid

[protolder]

Hola, it seems that your stock cells come from other medium or they had bad viability. The phenomenom that you describe occurs changing to a different medium withouth any adaptation. tell us more details. Buena suerte

[Majid]

Thanks for replay. I replaced about 20% of old medium by different medium after 48h.
I think cells viability is about 30%. I have not another cell stock. What can I do? please help me

Best
Majid

[protolder]

Hola I´m not sure, but I would return to the initial medium and once I have enought cells to freeze and to have a master culture or bottle to mantain, I would start to play with media changes, having a high confluence bottle and changing media sequentially. But Why do you want change the medium?. Have you any secreted protein to purify? does it have any tag?, if yes you dont need to adapt cells to a new medium you could  work with the medium with serum and change it by the same(grace´s or whatever) without serum for two days untill harvesting. Buena suerte