How To Make Baculovirus High-Titer Stocks

single use bioreactor
Baculovirus stocks have infectious titers that usually range between 6×106 and 3×108 pfu/mL. At Kempbio we have found that the best way to ensure that your working baculovirus titers are in the range of 1×108 pfu/mL is to infect Sf9 cells using a low MOI, (we use 0.02) and to incubate at 27°C for 3 days. It is also important to split your Sf9 cells to 5×105 cells per mL 18 – 24 hours before adding virus to make stock. Baculovirus stocks with good titer levels can be made in static flasks, shake flasks or stirred-tank bioreactors. We normally produce P0 stocks in T-25 flasks with 6 mL of serum-free medium and we produce most of our high-titer virus stocks in shake-flask culture unless we are running BacMam expressions at the 100-liter scale where we may be adding 10-liters of very fresh virus stock which we produce using a stirred-tank bioreactor. We titer our virus stocks using the ViroCyt Virus Counter but titers may also be determined by sending samples to Expression Systems (http://expressionsystems.com/product/baculovirus-titering-service/), running some of the commercially available immunoassay kits or performing a standard plaque assay (protocol on our website).

If you do not know the titer of the virus stock that you are trying to passage to make a high-titer virus stock you might want to try a series of small amplifications using a range of volumetric virus additions. For example, you could set up four, 250 mL shake flasks containing 50 mL of medium with Sf9 cells at 5×105 cells per mL. Incubate the flasks at 27°C on a shaker running at 80-100 rpm and add virus after 18-24 hours of incubation. The volumes of virus that we would recommend that you add separately to the four flasks is 0.01, 0.1, 0.5 and 1 mL. Incubate the flasks for 72h. at 27°C and obtain viable cell counts before you harvest. You are looking for a cell count of between 2 x106 and 4 x106 cells per mL and a viability of less than 70%. That should give you a virus stock with a titer somewhere around 1×108 pfu/mL. If the viability is high and the count is above 5×106 cells per mL the titer may be low. The titer will also be low if the cells growth arrested at 1×106 cells per mL and the viability is low (<50%). For test expressions you can use the same 50 mL in a 250 mL flask setup using Sf9 cells at a density of 1.5×106 cells per mL. If we know the titer of the virus that we are using we test express using an MOI of 0.1 and 1 and take samples at 48 and 72h. post-infection for western blot analysis. If you do not know the virus titer try running your test expressions using virus volumes of 0.2 and 2% V/V.