Suspension Culture Infections: Low MOI

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Infection of suspension cultures is the most common way of producing protein in the baculovirus system. Suspension cultures are easily scaleable from about 10 mL to more that 100 L. Generally, the suspension cultures are maintained in serum free media. These media are available from several commercial sources, all of which work well, though there is variation in effectiveness dependent upon cell line and protein.

It is probably best to test your protein and cell line in a variety of media and pick the one that best produces your protein in your cells. Give your cells a chance to adapt to various media before testing expression or the expression test may not be representative of results you would get after doing so.

Media Sources (see vendor page)

NOTE: You must know the normal growth parameters of your cell line in order to optimize expression; however, I will present some common values in order to facilitate getting started. My favorite Sf9 cell line can be split easily to 2E5 and recovers almost without a lag. It will double in under 20 hours in log phase, and grow to about 5E6 without too much trouble. These are the growth parameters that I will be assuming in my protocol. If your line behaves differently, you should take this into consideration and make appropriate adjustments.

Introduction to Low MOI Infections

Low MOI infections (Multiplicity Of Infection = ratio of infectious virus particles to cells) are the best strategy for expanding or amplifying virus stocks. This is for two reasons. Expanding at low MOI provides the quickest and easiest way to rapidly expand the volume of virus that you have. In addition, low MOI amplification is the best strategy for preserving the genetic integrity of your virus and preventing the buildup of DIPs (Defective Interfereing Particles that represent partial genomes packaged by complementation from intact genomes coinfected in the same cell).

Low MOI infections can also be used to produce protein, but they are more difficult to control and less reliable for producing maximal amounts of protein. They do have the advantage, however, of requiring much less virus stock. This can be a great advantage for large scale production, provided they can be reliably reproduced. This seems to be dependent upon the particular recombinant virus. I prefer a high MOI strategy for routine small-scale protein production.

In general, the strategy with Low MOI infections is to use the first day or so to produce the virus necessary to infect the rest of the culture. In other words, you infect a small number of cells with the virus, these cells replicate the virus and produce sufficient budded virus to infect the remaining cells in the culture, and these secondarily infected cells are the main producers of virus or protein.

It is important that the cells not overgrow the culture or there won't be sufficient medium resources to dedicate to virus or protein production. On the other hand, it is important that there not be so much virus present that the cells are killed before they can expand into the culture and utilize all its resources. Ideally, the virus will stop the cell number between about one third and two thirds the maximum density obtained with your cell line in the absence of the virus. I usually target about one half that density.

Execution of this strategy requires that you infect the cells with enough virus to stop growth and infect all cells in the first 24-48 hours. Then, depending upon growth arrest as an indicator of full infection, the culture is allowed to continue for approximately 48 additional hours beyond full infection. If protein production is your goal, it may be desirable to proceed longer.


Split your cells to a density of 2-4E5 cells/mL in a shake flask or spinner with a total volume between 20% and 40% of the flask or spinner capacity. I personally prefer shake flasks for most of my small scale (less than 500 mL) work. I culture them at 27±2°C with shaking between 100 and 130 rpm, usually 120 rpm.

Day 0 Incubate the cells until they reach a density of appoximately 4-8E5 cells/mL, that is until entered into log phase growth, but still at a quite low density, and add a small amount of virus. The amount depends on the titer of your virus stock, but you would prefer to have an MOI between 0.1 and 0.5. If your cells are at 6E5 cells/mL and your virus stock is at 3E7 pfu/mL, an MOI of 0.1-0.5 would be 0.2%-1% by volume (e.g. add between 100 µL and 500 µL of virus stock to a 50 mL culture in 250 mL flask). Return the flask to a 27°C shaker for 24 hours.

1 DPI (Day Post Infection): Count your cells and estimate their size. If the titer of the virus stock is higher than you estimate, that is, your MOI is more than 0.5, you will see clear signs of growth arrest and cell swelling. If the MOI is closer to 0.3 or below, you will see very little effect on the cells; they will have doubled to near 1.2E6 cells/mL and they will look healthy. At this time, infected cells should be releasing budded virus into the medium to infect other cells.

2 DPI: Again, count your cells and estimate their size. At this point most, if not all, of the cells should be infected. The cell number should be substantially below the 2.4E6 cells/mL that would be expected if they had continued doubling every 24 hrs., and they should show very substantial swelling. If the cell number did not increase between Day 0 and 1 dpi, then all the cells were infected immediately and the media can be harvested at 2 dpi, otherwise, return the flask to a 27°C shaker for 24 hours.

3 DPI: Again, count your cells and estimate their size. If the infection proceeded as expected, there will be no more cells on this day than at 2 dpi. If there was no increase in cell number between 1 dpi and 2 dpi, then all the cells were infected by 1 dpi and they can now be harvested as they've been infected for about 48 hours. This is typically the case. IF there was still some cell number increase between 1 dpi and 2 dpi AND the cell number has not yet reached a level indicating that you've reached stationary phase for your culture conditions, then return the flask to a 27°C shaker for 24 hours.

4 DPI: Again, count your cells and estimate their size. By 4 dpi the cells had better be thoroughly infected, swollen and growth arrested, or there were not sufficient virus in the infection to stop the culture before it reached stationary phase. Harvest the culture, spin down the cells, and store the medium at 4°C protected from light or frozen at -70°C.

Usually it is best to store the pellets frozen. My favorite method is to suspend the cells in a very small volume (ca. 1/4 vol) of PBS or TBS containing protease inhibitors at about 5x the usual concentration. After the cell pellet is uniformly resuspended, the slurry is dripped directly into a clean Dewar flask full of liquid nitrogen, usually from a pipet. The drops of suspension freeze instantly upon hitting the nitrogen and form little pellets. These pellets can be poured and weighed almost like granulated material as long as they're kept very cold. They can be aliquoted into 50 mL conical tubes, simply stored in Revco freezer boxes, or whatever. When it is time to purify protein from the cells, just add them gradually to 3 to 5 volumes of rapidly stirring lysis buffer at room temperature. They will thaw uniformly and very quickly and drop the buffer temperature to near 0°C. Individual pellets can be easily transferred with forceps to eppendorf tubes and analyzed in mini-scale experiments.

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