Generation of High-Titer Baculovirus in Insect Cells

摘要

Source: Nasimuzzaman, M., et al. Production and Purification of Baculovirus for Gene Therapy Application. J. Vis. Exp. (2018)

This video demonstrates the scalable amplification of high-titer baculovirus in insect suspension culture by sequential infection, incubation, and clarification of viral supernatant, enabling downstream purification for gene delivery and other therapeutic applications.

方案

1. Production of Baculovirus

1. Culture Spodoptera frugiperda9 (Sf9) cells in an orbital shaker incubator at 135 rpm and 28 °C in a polycarbonate Erlenmeyer flask containing serum-free insect culture medium.
   NOTE: If Sf9 cells are thawed from frozen stock, allow at least two weeks for the cells to recover and enter the exponential phase of growth.
2. Count the Sf9 cells harvested at the exponential phase of growth with a hemocytometer after staining with Trypan blue. Seed 1 x 10⁶ viable Sf9 cells per well in a 6-well tissue culture-treated plate in 2 mL of medium. Allow the cell to attach for 1 h at 28 °C in an incubator.
3. Replace the growth medium with 1 mL of serum-free, unsupplemented Grace's insect culture medium.
4. Transfect the bacmid DNA into the Sf9 cells using an insect cell transfection reagent.
   1. Mix 8 µL insect cell transfection reagent with 100 µL of Grace's Insect medium.
   2. Dilute 2 µg of bacmid DNA into 100 µL of unsupplemented insect medium, and mix gently by vortexing.
   3. Combine the diluted DNA with the diluted insect cell transfection reagent, and mix gently. Incubate for 15 to 30 min at room temperature.
   4. Add the DNA-lipid mixture dropwise onto the cells. Incubate at 28 °C in an incubator for approximately 5 h.
5. Remove the transfection mixture from the cells and wash the cells with 2 mL of PBS (phosphate-buffered saline).
6. Add 2 mL of insect culture medium and continue to culture at 28 °C in an incubator without changing the medium.
   NOTE: If the bacmid contains a fluorescent protein, its expression can be detected in cells 48 h post-transfection. Baculovirus is produced by transfected Sf9 cells and secreted into the culture medium, which subsequently infects the untransfected cells. The entire cell population becomes infected with baculovirus in 5 days and shows signs of viral infection, also called cytopathic effect. These include increased cell diameter, vacuoles/granules in the cytoplasm, cessation of cell growth, and dead or lysed cells present in the cell culture. However, if the transfection or infection efficiency is not optimal, the culture may not show an obvious cytopathic effect. The cytopathic effect can be visualized with an inverted phase microscope at 200 - 400X magnification. Baculovirus-infected cells can be detected by staining with anti-gp64 antibody.
7. Harvest baculovirus supernatant 5 days after transfection and label it as the P1 virus stock.
8. Store the baculovirus supernatant, which is light-sensitive, in the dark with 0.5% bovine serum albumin (BSA) in PBS. Store at 4 °C for the short term (up to 90 days) or at -80 °C for the long term.
9. Seed 6 × 10⁶ Sf9 cells in a 10-cm tissue culture-treated plate in 10 mL of insect culture medium. Add 1 mL of initial baculovirus supernatant (P1) to the cells.
10. Harvest baculovirus supernatant (P2) on the 5th day post-infection.
11. Seed 50 mL of Sf9 cells (3 x 10⁶ cells/mL) in suspension culture in insect culture medium in a 250-mL polycarbonate Erlenmeyer flask and place the flask in an orbital shaker incubator. Add 2 mL of P2 stock to the cells and shake at 135 rpm while maintaining a constant temperature of 28 °C.
    NOTE: Three or four days after infection, the entire Sf9 cell population will show cytopathic effect, which is an indication of high-titer baculovirus production.
12. Sequentially amplify the baculovirus supernatants until the desired volume is obtained (P3, P4, ….) by increasing the 10- to 50-fold volume of cell culture in each subsequent infection.
    NOTE: P3 is the 3rd round of infection in which 500 mL to 1 L of Sf9 cell culture can be infected with 10 to 20 mL of P2 baculovirus supernatant; P4 is the 4th round of infection in which 5 to 10 L of Sf9 cell culture can be infected with 100 to 200 mL of P3 baculovirus supernatant, and so on. Typically, Sf9 cells are seeded at 3 × 10⁶ cells/mL in serum-free insect culture media.
13. Centrifuge the baculovirus supernatant at 1,000 x g for 30 min at 4 °C to remove cellular debris and treat the baculovirus supernatant with 50 units/mL nuclease (250 units/µL of stock) for 2 h at 37 °C to digest the genomic DNA and RNA. Filter the supernatants through a 0.45 μm filtration unit.

材料

本文使用的材料清单

姓名	公司	目录编号	评论
MaxQ 8000 orbital shaker incubator	ThermoFisher Scientific	Non-catalog item	Shaker for suspension culture
250 mL Erlenmeyer flasks	ThermoFisher Scientific	238071	Flask for suspension culture
1 L Erlenmeyer flasks	ThermoFisher Scientific	238072	Flask for suspension culture
Microscope (Olympus CKX41)	Olympus	Non-catalog item	Cell monitoring and counting
Table top centrifuge	ThermoFisher Scientific	75253839/433607	For clarification of Baculovirus supernatant
50 ml conical tube	ThermoFisher Scientific	14-959-49A	For collection of Baculovirus supernatant
Sf9 cells	ThermoFisher Scientific	11496-015	Insect cells
Grace’s insect cell culture medium	ThermoFisher Scientific	11605-094	Transfection medium
PBS	ThermoFisher Scientific	20012227	Washing cells, diluting samples
HyClone SFX-Insect cell media	GE Healthcare	SH30278.02	Serum-free insect cell growth medium
Benzonase nuclease	Sigma-Aldrich	E1014	Enzyme to degrade DNA and RNA
Bovine serum albumin (BSA)	Sigma-Aldrich	A4737	Stabilizes Baculovirus