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    Insect cells—first week in culture

    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.

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

    This is normal for Sf9 cells. Typically, we recommend leaving the cells for 1 week after recovery, then checking cell viability. There should be approximately 1–2 million cells/mL, and can be split to a lower density.

    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.

    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. 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 (27°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).

    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. 

    Insect cells—after the first week in culture
    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.

    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.

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