The following section describes the common problems encountered during the cell culture and infection of insect cells and discusses potential solutions. If these solutions do not adequately address the issues, its best to contact the manufacturer of the insect cells, plasmid vectors, or baculoviruses in use.
Insect cells are particular about the type of serum they are grown in. Many different types and lots of serum may need to be tested in order to achieve optimal growth, infection, and protein production. To test the serum, grow small batches of insect cells in 75 cm2 flasks for one to two weeks and record the growth and death rates. Once a lot of serum has been shown to support the growth of the insect cells, it must also be tested for infection and protein production (see Basic Protocol 2). Finally, the serum needs to tested in a transfection reaction (see Basic Protocol 2). Make Transfection Buffer A containing a titration of serum starting from 1% to 10%, and following the transfection protocol in Basic Protocol 2. The buffer should turn cloudy white after the addition of Transfection Buffer B, but should not be opaque and there should not be any visible white precipitates. Continue following Basic Protocol 2 through step 10. Check the viability of the cells after 7 days in culture. Since no baculovirus or plasmid DNA was used in this titration, the cells should be healthy and not Trypan Blue positive. Choose the concentration of serum for Transfection Buffer A in which the buffer turned white, but the cells were still viable.
Since baculovirus is a lytic virus and kills infected cells, all glassware used to grow insect cells should be acid-washed and autoclaved (see Supporting Protocol 3). In addition, we recommend using plastic disposable pipets and filter pipet tips for transferring insect cells, media, or baculovirus. If the insect cells die while being maintained in culture, it is likely they are contaminated with a virus or the glassware they maintained in is contaminated with detergents. Overgrowth can also be an issue, and it is difficult for the cells to be recovered once they have grown above 3×106 cells/ml. Starting a fresh culture from a frozen stock and maintaining the cells between 2×105 and 2×106 cells/ml is recommended (see Supporting Protocol 2). If baculoviruses is found to have a low titer (less than 1×108 Units/ml), a new stock of baculovirus should be made (see Basic Protocol 3). For highest titers, infected Sf9s should be grown until most or all of the cells stain with Trypan Blue.
Careful titration of the baculoviruses is recommended before beginning large-scale infections for vaccines (see Basic Protocol 3). Adding too much virus to the cells can be detrimental, as the cells may die before achieving the peak of protein production. Adding too little virus may result in a poor infection and low protein yield. If the insect cells do not produce the protein of interest, there is likely a problem with the original plasmid construction. We recommend confirming the sequence of the plasmid construction prior to making baculovirus (see Basic Protocol 1) and confirming the sequencing of the final baculovirus clone prior to beginning large-scale infections (see Supporting Protocol 1). There are commercially available positive controls for baculovirus production if further trouble-shooting is necessary.