Insect Cell Culture Support—Getting Started

Please see the table below of cells, media, and cell size:

There are three main factors that are critical in maintaining robust cell growth and good viability. These are incubation temperatures, aeration, and cell seeding densities. The table below gives temperatures, seeding densities, and aeration for the different insect cells to maintain robust cell growth and good viability.

Suspension culture parameters for a 3-to-4–day subculture schedule

Adherent culture parameters for a 3-to-4–day subculture schedule

Recommended aeration conditions for adherent and suspension cultures

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.

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

Please see the phase contrast images of healthy Sf21 insect cells grown in suspension. The culture was started in a shake flask at a seeding density of 3 × 10E5 viable cells/mL in Sf-900™ II SFM medium and it was maintained in a 28°C, non-humidified, ambient air-regulated incubator. The images were obtained using 10X and 20X objectives (panels A and B, respectively) 3 days after seeding. 

The following phase contrast images are of Sf21 insect cells grown as an adherent monolayer in Sf-900™ II SFM medium. The cells were plated at a seeding density of 5 × 10E4 viable cells/cm² in a T-25 flask and grown as monolayers in a 28°C, non-humidified, ambient air-regulated incubator. The images were obtained using 10X and 20X objectives (panels A and B, respectively) 7 days after seeding, when the culture had reached confluency.

Please see the image below of High-Five™ Cells preadapted to Express Five™ SFM (Day 3 in culture).

Please see the image below of D. Mel 2 (Schneider S2) cells preadapted to suspension growth in Drosophila-SFM (Day 2 in culture).

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

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)

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.

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.

*Note: The cells are loosely adherent, and are confluent when they cover ~80% of a given surface are Therefore, it is suggested to seed flask plates at a lower percent confluence.

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 × 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.

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. Cells may detach for a few passages, but they don’t work after this. 

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 passage 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.

Whenever the cells are counted, a trypan blue assay should be performed to check for cell viability. If the cells are maintaining viability above ~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.

High Five™ cells are difficult to transfect and we typically get efficiencies of 5–13%. Other cell types are 40–50%. Also, High Five™ cells at confluence typically do not smoothly cover the whole surface of a flask. They still look a little patchy. They are confluent when they start to overlap and pile on each other.

We recommend that both High Five™ cells and Sf9 cells be tried in small-scale infections for protein production. Sf9 cells often produce just as much protein per milliliter of culture because they will be infected at 3–4 x 10E6 cells/mL. High Five™ cells are definitely going into log phase at >2 x 10E6 cells /mL; they should not be infected at a cell density higher than 2 x 10E6 cells/mL.

Heat inactivation is not necessary. Our team has routinely used serum that has not been heat-inactivated, and we have not observed any effect on cell growth or morphology.

Many antibiotics are suitable for use with insect cells. The following antibiotics are commonly used:

Penicillin/Streptomycine: 50–100 U/mL; 50–100 µg/mL

Amphotericin B (Fungizone™ antimycotic): 0.25 µg/mL

Gentamicin: 0.5 mL of 10 mg/mL solution in 500 mL media (final concentration: 10 µg/mL)

We would recommend using our Grace’s Supplemented Insect Medium (Cat. No. 11667037) or SF-900™ Medium (1.3X) (Cat. No. 10967032). 

Grace’s Supplemented Insect Media is a 2X formulation intended for use in the agarose overlay when performing the plaque assay. The media has been modified with: 

• Lactalbumin hydrolysate
• Yeastolate
• L-glutamine
• Sodium bicarbonate

Click here for the complete formulation.

SF-900™ Medium (1.3X) is a complete serum-free low protein insect cell culture medium for Sf9 and Sf21 cells, which can be used for 1% agarose overlays used for plaque assays. 

24 µg/L Tween-80 

910 µg/L Pluronic Poly-all

Our Express Five SFM is protein-free and sterile filtered but does not contain L-glutamine.

Heparin solution can be stored at room temperature. Heparin is usually added at 10 µg/mL although you can use it at 20 times higher concentration.

There is no added cholesterol, however, there are yeastolates and hydrosylates which are cholesterol precursors. This is common for all insect serum-free media.

Yes, we offer our methionine-free Grace’s Insect Medium (Cat. No. 11595030). 

The supplement medium is referred to as TNM-FH. It consists of the following components (per 500 mL):

TC Yeastolate: 1.66 g, Lactalbumin hydrolysate: 1.66 g, L-gluatmine: 0.3 g

These supplements aid in increasing the stability and shelf life of the medium. Please note the supplements are not sterile and must be filter sterilized (through a 0.2 µm filter) after addition.

TNM-FH is not considered to be a complete medium without the addition of 10% FBS, heat-inactivated. Serum provides additional nutrients and also protects the cells from hydrodynamic stresses in suspension culture conditions (i.e., spinner and shaker flasks).