Sf9 Cells in Grace's - FAQs

What is the procedure to thaw frozen insect cells?

The following protocol describes a general procedure for thawing cryopreserved cells. For detailed protocols, always refer to the cell-specific product insert.

1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath.
2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial.
3. Transfer the vial into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol.
4. Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line dropwise into the centrifuge tube containing the thawed cells.
5. Centrifuge the cell suspension at approximately 200 x g for 5-10 minutes. The actual centrifugation speed and duration varies depending on the cell type.
6. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet.
7. Gently resuspend the cells in complete growth medium, and transfer them into the appropriate culture vessel and into the recommended culture environment.

Note: The appropriate flask size depends on the number of cells frozen in the cryovial, and the culture environment varies based on the cell and media type.

Why does the Insect cell line manual state: "Cells should be maintained at 27 degrees C in a non-humidified environment."

Insect cells do not require CO2 or high humidity to grow, they can grow in a lab drawer at room temperature. We recommend this so people don't waste CO2 and other resources necessary for maintaining a tissue culture incubator. It should be noted, however, that the cells will grow in a humidified incubator.

When growing Sf9 cells in a bioreactor, can I use a glass vessel that has been cleaned and autoclaved and then reused or do I need to use a disposable vessel?

Yes, you can grow Sf9 cells in glass vessels. The only concern would be if your glass vessels are not clean enough and there may be residual detergent left which will hurt your cells.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Are spindle-shaped Sf9 cells bad? How do I get rid of them?

These cells appear after cultures have been grown for several weeks. These do not seem to be detrimental to plating of high titer stocks or expression. However, if they are in high numbers, it may indicate that the cells are becoming old and that the culture should be re-started with a new stock of frozen cells.

What are small particles that appear after a few days when growing Sf9 cells? I am not using Gibco Fungizone reagent, but I am using gentamicin. Are they yeast?

Yellow particles could be cell organelles, aggregates, or debris. We see this when we first thaw frozen cells. To avoid this, you can let the shaker culture sit for 5 minutes, then transfer top 1/3 to a new flask, making sure to count cells first. You can also use heparin at up to 200 U/mL to decrease aggregation. Pluronic solution at a final concentration of 0.2% can also be used to decrease shearing, and increase shake speed to 100-120 rpm. Recently thawed cells seem to be breaking up and releasing small vesicles, as observed under high magnification. To reduce the amount of those small particles, cells need to be rapidly but completely defrosted for successful thawing to take place. Also:

1. Place vial on ice during transfer from water bath to sterile hood.
2. Pipette as gently as possible because cells shear easily due to larger surface area.
3. Cells may not have been placed in cold media after removal from defrosted vial into flask.
4. Media may not have been changed after 30-45 minutes once a majority of cells had attached. Media change should be with pre-warmed media (27degrees C). 10% DMSO in freezing medium will kill the cells if left on them for long periods of time (1 hour seems to be a maximum).
5. Lastly, cells should be checked for contamination. To do so, plate a small portion of culture in a T-25 flask and incubate for 3 days, checking for cloudiness.

I split my Sf9 cells last week to 1 x 10E5 cells/mL. However, three days later, they are just 1 x 10E6 cells/mL. They look fine, but double very slowly. What happened?

These cells were oversplit. Typically, we recommend the lowest density for the Sf9 cells to be 0.5 million cells/mL.

I have Sf9 cells in suspension culture. The cells look really bad: 50% dead three days post-thaw. Are they contaminated?

No, but the cells are still stressed. Determine the cell density and spin down the cells at 130 x g for 3 minutes and resuspend the cell pellet with fresh medium. If the cell density is at least 2 x 10E6 cells/mL, proceed by seeding cells at 0.4 x 10E6 cells/mL and transfer into a new flask (125 mL shake flask; cell culture volume: 30-50 mL). Let the cells grow for 2 days. If the cell viability is not at least 80% and the cell density is not at least 2 x 10E6 cells/mL, discard the culture and start thawing a new vial.

I want to split my Sf9 cells but don't know how to do this. What do you recommend?

We typically recommend dislodging Sf9 cells mechanically rather than enzymatically (i.e., with trypsin/EDTA). To do so, wrap your flask with a towel, and bang the narrow end along the hood. This should cause the cells to dislodge from the flask.

How many passages can I do with Sf9 cells before a frozen stock should be revived? Does the infectability of the cells drop off as the cells are passaged more?

Whenever the cells are counted, a trypan blue assay should be performed to check for cell viability. If the cells are maintaining viability above approximately 95% and are doubling every 25 hours or so, then they are still okay to use. If the viability drops and the doubling time increases, the infectability will be affected. Cells older than 30 passages may start to show signs of aging with delayed protein production.

What methods can be used to detach tightly adherent Sf9 and Sf21 cells cultured in Grace's media supplemented with 10% heat-inactivated FBS, Sf-900 II SFM, or Sf-900 III SFM?

Sf9 and Sf21 cells should be lightly adherent cells. However, there are some Sf9 and Sf21 cells that attach to culture vessels very tightly. The use of enzymes such as trypsin, collagenase, hyaluronidase, TrypLE Express, and TrypLE Select have been tried without success for passaging cells. The main problem is that the cells do not attach well after having dissociated with the enzymes.

The best method to use is to culture cells in a T-flask. Close cap tightly and hold flask with cap pointing towards the ceiling. Hit the bottom of the flask over a counter 2-3 times with medium force. Cell detachment may be 60-80% and not 100%. This will allow for detachment of enough cells for passaging. If tapping the flask over the counter is performed with too harsh of a force or too many times, cell viability will be greatly affected.

If possible, we recommend that you culture cells in suspension conditions. Cells in suspension cultures can be passaged directly into adherent conditions when needed. The culture of cells in suspension conditions will allow for higher cell densities as cell growth is not limited to the surface area.

How can I concentrate my insect cells to increase the cell density?

If the cell density is too low and the cells have been in culture for 4-5 days, we recommend concentrating the cells by centrifuging them at 100 X g for 5 minutes and resuspending them in fresh medium. Cells should not be left in the same medium for more than 4-5 days as nutrients in the medium will have been used up by the cells in that period, and the medium itself degraded due to prolonged exposure to warm temperatures. Cells should also be centrifuged and concentrated if a lot of cell debris is observed in culture.

What are the main differences between insect cell culture and mammalian cell culture?

Insect cells are much more fragile than a lot of mammalian cell lines. They suffer much more damage than mammalian cells from overgrowth and over-splitting. Never let cells go above 8 x 10E6 cells/mL or grow at densities less than 0.5 x 10E6 cells/mL in suspension. Insect cells require a little more osmotic pressure than mammalian cells (340 µOsM). Insect cells use a lot of O2, especially during protein expression. Insect cell culture media is more acidic than mammalian media (pH 6.0-6.4). The insect cell culture media is phosphate buffer based. Therefore, no CO² is needed to maintain the pH.

Which insect cell line is best to use?

We recommend Sf9 or Sf21 cells for transfection, purification, and amplification of recombinant virus. Sf9 cells are regular in size, easy to manipulate, and form good monolayers for plaque assays. Sf9 and Sf21 cells can also be used for expression of recombinant proteins, but the High Five cell line may produce higher levels. We recommend the High Five cell line for expression of secreted recombinant proteins. They are grown in serum-free medium, adaptable to suspension culture, and produce high levels of recombinant protein (Davis et al., 1992; see http://www.ncbi.nlm.nih.gov/pubmed/1368794).

Note: Generally it is easier to use one cell line for procedures up to optimization of protein expression. Once you have confirmed expression of your recombinant protein, other cell lines can be tried for optimization of expression levels.

Can I grow insect cells for short periods at 37 degrees C?

No, this is not recommended. Prolonged exposure to temperature higher than 29 degrees C will cause cell death. It is better to grow the cells at 27 degrees C or room temperature.

Is CO2 required for insect cell culture?

No, CO² exchange is not required for insect cell culture.

What is the best way to plate insect cells?

Prior to performing transfections and plaque assays, cells need to be evenly distributed over the surface of a tissue culture plate. This ensures that:
- Cells do not distribute unevenly, leading to asymmetric monolayers.
- Maximum cell surface area is available for infection.

To disperse cells:
1. Rock the flask or plate slowly by hand forward and backward, then side-to-side.
2. Repeat this four times, watching carefully to ensure that the liquid reaches all areas of the growth surface.
3. Do not use a circular motion to disperse the cells, because this causes a concentration of cells around the edges of the plate rather than an even distribution.

What are the doubling times for robust growth and maintenance of different insect cell lines?

Please see below for information related to the type of cells, media, and doubling time in hours:

D.mel-2; Schneider's Drosophila + 10% FBS heat-inactivated; 18 to 24
High Five cells; Express Five SFM; approximately 24
Sf9 cells; Sf-900 II SFM; 24 to 30
Sf9 cells; Sf-900 III SFM; 24 to 30
Sf21 cells; Sf-900 II SFM; 24 to 30
Sf21 cells Sf-900 III SFM; 24 to 30

What are the sizes of Sf9, Sf21, High Five cells, and D.mel-2 cells?

Please see below for information related to type of cells, media, and cell size in microns:

D.mel-2; Schneider's Drosophila + 10% FBS heat-inactivated; 10 to 12
High Five cells; Express Five SFM; 17.5 to 19.5
Sf9 cell; Sf-900 II SFM; 15 to 17.5
Sf9 cells; Sf-900 III SFM; 15 to 17.5
Sf21 cell;s Sf-900 II SFM; 15 to 17.5
Sf21 cells; Sf-900 III SFM; 15 to 17.5