Our lab uses viral factors as a platform to deliver therapeutic genes to the injured nervous system. Recently, numerous non-invasive viral factors which are able to cross the blood-brain barrier have been developed. The use of these factors requires high titers, and therefore an improved production process.
The implementation of our HEK293 suspension cell procedure makes this AAV protocol less laborious compared to other adherent cell-based systems, while at the same time gaining higher yields of AAV vectors. There are no treatments to repair the damage in the central nervous system. We aim to use non-invasive viral vectors to stimulate central nervous system repair.
The advantage of this protocol is that we can now produce viral vectors at high yield, which allows us to perform non-invasive therapeutic gene delivery to the injured central nervous system. Our current work will pave the way to develop non-invasive gene therapy for multiple neurodegenerative diseases, such as multiple sclerosis, Parkinson's, and Alzheimer's disease. To begin, place all materials required for culturing HEK293 cells in the clean biosafety hood.
After thawing, transfer HEK293 cell suspension from the vial to a 30 milliliter pre-warmed suspension cell medium in a 50 milliliter tube. Incubate the cells at 37 degrees Celsius with 80%humidity, 8%carbon dioxide, and 200 RPM for three to four days. Add 100 microliters of cell suspension to the 400 microliters of 0.4%trypan blue solution, and gently invert the tube to mix.
Place 50 microliters of the trypan blue-treated cell suspension on the hemocytometer slide. Count total viable cells, and calculate the required amount of cell suspension for transfection. Add the required amount of cell suspension to a pre-warmed suspension cell medium, and incubate at 37 degrees Celsius with 8%carbon dioxide.
Add 1 million cells per milliliter HEK293 cell suspension in 300 milliliters of suspension cell medium and incubate at 37 degrees Celsius with 8%carbon dioxide. On day two, warm the suspension cell medium, plasmids, and transfection reagent to room temperature. Add plasmids to the prepared medium and vortex briefly.
Then, add transfection reagent and vortex again briefly before incubating at room temperature for 30 minutes. During incubation of the transfection mix, count the cells cultured on day one, and if necessary, adjust the cell density between two and 2.5 million cells per milliliter. After 30 minutes, dropwise, add the transfection mixture to the cells while gently swirling the tube, and incubate for 72 to 96 hours.
On day five, add 33 milliliters of 10X cell lysis buffer to the cells, and mix by gentle shaking. Incubate the suspension at 37 degrees Celsius for 1.5 hours with shaking. Centrifuge the cell suspension at 3, 428G at four degrees Celsius for 60 minutes.
Filter the supernatant through a 0.45 micron PES vacuum filter, and collect the clarified lysate in the filter container. Add 90 milliliters of 40%PEG 8, 000 solution to 360 milliliters of filtered cell lysate. Add 70%ethanol-sterilized stir bar to the cell lysate and stir at 300 RPM on ice or in a cold room for one hour.
Then, incubate overnight at four degrees Celsius without stirring. To begin, transfer the entire PEG-precipitated adeno-associated virus HEK293 culture to a large conical tube. Centrifuge the culture at 2, 820G for 15 minutes at four degrees Celsius.
Discard the supernatant and resuspend the pellet in 15 milliliters of PBS with calcium and magnesium. Transfer the resuspended part to a 50 milliliter tube. Add 10 milliliters of PBS to collect the remaining PEG precipitate, and transfer all to a 50 milliliter tube.
Then, add 40 microliters of DNase 1 and incubate for one hour at 37 degrees Celsius. Using a glass Pasteur pipette, fill a 25 millimeter by 89 millimeter polyallomer tube with 15.5 milliliters of concentrated cell lysate. Using a new glass Pasteur pipette, gently infuse nine milliliters of the 15%iodixanol solution beneath the cell lysate.
Then, add five milliliters of the 25%and 40%iodixanol solutions. And lastly, add five milliliters of the 60%iodixanol solution. Use a syringe to top off the tube with PBS, ensuring most air bubbles are removed without disturbing the iodixanol layers.
Seal the tube using an electrical tube topper. Check that the different layers of the iodixanol all gradient are clearly visible. Centrifuge the tubes using a non-swing rotor at 490, 000G for one hour and 10 minutes at 16 degrees Celsius.
Upon centrifugation, assemble the tubes in a metal clasp. Attach a 19 gauge needle to the syringe. Next, create a puncture at the top of the tube using a 30 gauge needle, leaving the needle inserted.
Carefully puncture the tube just below the 40%60%iodixanol interface, as indicated by the phenol red indicator. Ensure the needle bevel faces the 40%layer. Remove the 30 gauge needle with the non-dominant hand while slowly extracting the virus iodixanol mixture.
About halfway, rotate the needle for the bevel to face down, and continue extraction, avoiding collection from the protein layer. After extraction, immediately transfer the ultracentrifuge tube to a 50 milliliter tube to discard it. Add collected a AAV iodixanol suspension to a 15 milliliter tube, and dilute it fivefold in PBS, filling up to 15 milliliters.
Transfer the suspension to a centrifugal filter tube and centrifuge at 3, 428G for 10 minutes at four degrees Celsius. After discarding the flow-through, add 15 milliliters of PBS 5%sucrose to the filtrate and resuspend. Collect the concentrated AAV from the centrifugal filter tube.
Finally, store the virus aliquot at four degrees Celsius for up to three months.
