Pre-made viruses have become increasingly accessible and are useful for saving time and avoiding potentially costly set-ups. However, there are many cases where the specific viral particles you need are not available, or the cost of custom viruses are too high for your budget. In those cases, you’ll need to produce your viruses in-house.
Introducing the systems
There are many ways that viruses can be produced in the lab. The first decision you must make is which system you will use, or more specifically, the type of cells that will be used to make the virus.
Viruses can be produced in many different cell types, spanning both prokaryotic and eukaryotic organisms. Typically, what system is used is dictated by the specific type of virus you are trying to make. For example, bacteriophages will need to be produced in bacteria cells. However, some viruses can be produced in multiple different cell types, including plants, mammals, and insects.
In this post, we will compare two of the most widely used systems, mammalian and insect cells. Whether you’re new to in-house virus production or looking to improve your current methods, understanding the differences can help you make an informed decision.
Mammalian cells
Mammalian cells are a great option for viral production, particularly for labs that already do mammalian cell culture. HEK-293T cells are the most common cells used in viral production, partially because they contain some of the necessary machinery to make these viruses. The cells are also easy to cultivate, fast-growing, and widely available.
The viruses
Lentiviruses, AAV, and adenoviruses can all be produced in mammalian cells. However, it is much more common to make lentiviruses and AAV in-house. High-titer adenoviruses are difficult and time consuming to produce, so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies.
Production of both AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus two packaging plasmids (three packaging plasmids for 3rd generation lentivirus production). The packaging plasmids are widely available, so you’ll only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities depending on the virus. AAV has a packaging capacity of ~4.7 kb, while lentivirus has a capacity of 8–10 kb.
Time
Viral production in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within 24 to 48 hours. Harvesting timepoints will depend on your specific virus, so this is something you may have to optimize for your specific application.
With mammalian systems, it is standard to do only one passage (transfection). This differs from typical insect cell preparations, which we talk about below.
Processing
The viral supernatant is typically collected over the course of a couple days to increase yield. The collection process is a cycle of collecting the media and adding fresh media every 12 to 24 hours. Processing the virus-containing supernatant will depend on the virus type. AAV is typically purified using methods like ultracentrifugation, while lentivirus supernatants are centrifuged and filtered to eliminate any HEK-293T cells. If you titrate your viral preps, lentiviral titration requires an additional couple of days, as it requires transduction of more cells, while AAV can be titrated following purification using qPCR or digital droplet PCR.
Insect cells
Insect cells are a less common option for the generation of viruses, though they are a popular choice for protein production. The most commonly used cells in insect viral production are Sf9 cells, which are derived from the pupa ovarian tissue of fall armyworms (Spodoptera frugiperda). Much like HEK-293T cells, they are easy to cultivate, fast-growing, and widely available. Unlike mammalian cells, they do not require serum or an incubator to grow.
The virus
Insect cell systems are used to produce baculovirus, a family of viruses that specifically infect insects. This is what makes insect cells a popular choice for protein production. AAV and lentiviruses can be produced in insect cells, but they require cloning specific AAV or lentiviral machinery into a baculovirus plasmid. Production of baculovirus requires only two plasmids — your transfer plasmid and the baculovirus construct, called a bacmid. Like AAV and lentiviral packaging plasmids, bacmids are readily available, so the amount of cloning required is about the same between insect and mammalian systems. Baculovirus transfer plasmids have a much larger packaging capacity — up to 38 kb!
Generally, insect cell production methods produce lower titers of virus than mammalian systems. This is why many insect production methods add an additional viral passage. This involves taking your first round of virus, called P1 (or sometimes P0), infecting new Sf9 cells, and getting an amplified P2 virus. This P2 virus can be produced in a greater volume and will have a higher titer than a P1 virus.
Pro-tip! Centrifuge the viral supernatant in between passages to eliminate dead cells and cellular debris.
While adding time (about three to four days), this second round provides the added benefit of producing a stock P1 virus, so that if you ever need to remake the same virus, you can skip the first round of production, saving about a week of work. An important note is that this is only possible because baculoviruses are slightly less prone to recombination than other viruses, like lentivirus. You can even produce a P3 virus!
Time
Sf9 cells will start producing virus within a day or two, but unlike mammalian systems, the supernatant isn’t collected sequentially. This is because of the second passage and because you wait until the majority of cells have died to collect your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in cell lysis, releasing the virus to the supernatant. The virus will reach peak production when roughly 60–70% of cells have died, usually after four to five days. This means that viral production takes a little bit longer in insect cells.
Processing
Processing of your baculovirus is straightforward — simply spin down the supernatant in a centrifuge to remove any unwanted cellular debris, as you would when collecting a lentivirus. From there, you can either move into the next viral passage, titer the virus, or go straight into your viral application, like protein production.
Titration of baculovirus can vary depending on the protocol used. Plaque assays are standard but can take seven to 10 days. Commercially available kits provide titers in just two days, but add additional costs and may need to be optimized for your purpose.
Making your decision
The final decision of which system to use will come down to which differences are most important to you — virus type, cost, time, or reagents and equipment. Hopefully this post has provided some insight on these differences in viral production and will help you decide which is right for you.
Table 1: Mammalian versus insect cells for viral production
| Consideration | Mammalian cells | Insect cells |
| Viruses | AAV and lentivirus | Baculovirus; can produce AAV and lentivirus if needed |
| # of plasmids | Three or four | Two |
| Packaging limit | ~4.7 kb (AAV); 8–10 kb (lenti) | 38 kb |
| Cells | HEK-293T | Sf9 |
| Time to collection | 2–4 days | 5–10 days |
| Purification | Ultracentrifugation (AAV); centrifugation (lenti) | Centrifugation |
| Titration time | ~1 day (AAV); ~1 week (lenti) | 2–10 days |
| Initial titers | Higher | Lower |
Resources and references
More resources on the Addgene blog
Viral Vectors 101: Producing Your rAAV
AAV Titers: Where Do They Come From and What Do They Mean?
Overcoming the Challenges of Lentivirus Production
Tips for Titering Your Lentivirus Preps
More resources on Addgene.org
References
Shaw, G., Morse, S., Ararat, M., & Graham, F. L. (2002). Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. The FASEB Journal, 16(8), 869–871. https://doi.org/10.1096/fj.01-0995fje.
Tan, E., Chin, C. S. H., Lim, Z. F. S., & Ng, S. K. (2021). HEK293 cell line as a platform to produce recombinant proteins and viral vectors. Frontiers in Bioengineering and Biotechnology, 9. https://doi.org/10.3389/fbioe.2021.796991.
Urabe, M., Ding, C., & Kotin, R.M. (2002). Insect cells as a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https://doi.org/10.1089/10430340260355347.
Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., & McCawley, P. (1977). The establishment of two cell lines from the insectspodoptera frugiperda (lepidoptera; noctuidae). In Vitro, 13(4), 213–217. https://doi.org/10.1007/bf02615077.
Topics: Viral Vectors, Viral Vectors 101, AAV, lentivirus
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