High Five™ Cells in Express Five™ Medium - 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.

How does one get High Five cells that have been adapted for suspension culture to adhere to flasks? This can be important when generating stable cell lines with High Five cells.

To get High Five cells that have been adapted for suspension culture to adhere to a flask for selection of stable clones, it is recommended to check for heparin in the media (heparin helps keep the cells in suspension) and add 0.1% FBS to the culture for 10-20 minutes, then change the media. Keep in mind that cells which have adapted to suspension culture often take time to revert to adherent culture. Maintain the cells in the same flask over a few passages under selection, each time passaging at 1:4 or 1:5, without spinning the cells down. This should select for the adherent cells.

At what density should High Five cells be frozen?

We recommend a density of 3.0x10E6 cells/vial for adherent cultures and 10-15x10E6 cells/vial for suspension cultures.

Why do I need to add 18 mM L-Glutamine to prepare High Five Cells in Express Five Medium (Cat. No. B85502) and Express Five SFM (Cat. No. 10486025)?

L-Glutamine is an essential nutrient in cell cultures for energy production as well as protein and nucleic acid synthesis. In general, insect cell media contain higher L-Glutamine levels than serum-free media for mammalian cells, primary cells, stem cells, etc. This is to support the fast metabolism of insect cells, especially when used for virus production and protein expression applications.

Note that removal of L-Glutamine from the media formulation aids in product shelf-life by preventing glutamine degradation during storage.

What is the biosafety level (BSL) for High Five Cells in Express Five Medium (Cat. No. B85502)?

The biosafety level (BSL) for High Five Cells in Express Five Medium is typically biosafety level 2. Please note that this can vary from country to country.

My High Five cells are in log-phase growth in Grace's Insect Medium plus serum. Why are the High Five cells forming large, free-floating clumps with very few cells attached?

The cells are most likely getting too confluent. High Five cells grow extremely fast in serum. Split the cells as soon as there is no more room on the flask for the cells. We suggest doing a 1:15 split and observing cell growth each day. Add heparin at 10 µg/mL, although it can be used at 20 times higher concentration. Try adding some FBS (0.1%) for about 10-20 minutes then change the media.

When using heparin for High Five cells, at what temperature do you store the heparin solution?

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.

Should I first try High Five cells for protein expression since they normally give higher expression than Sf9?

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 greater than 2 x 10E6 cells /mL; they should not be infected at a cell density higher than 2 x 10E6 cells/mL.

Are High Five cells more difficult to transfect than Sf21 or Sf9 cells?

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.

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