Purification of Recombinant Virus Particles from Transfected Human Kidney Cells

Begin with a cell lysate containing cellular DNA aggregated with cellular proteins and viral particles.

Add magnesium ions, followed by a nuclease, to activate enzymatic degradation of cellular DNA and disrupt DNA–protein aggregates.

Centrifuge to separate debris and collect the supernatant containing viral particles.

Layer the supernatant onto a discontinuous iodixanol gradient with concentrations increasing from top to bottom, then fill the tube with buffer.

Ultracentrifuge to separate the components based on density. Virus particles accumulate at the interface between the high and medium-density layers.

Insert a needle at this interface and collect the viral particles into a tube.

Add a buffer containing stabilizing components, which prevents viral particle aggregation.

Then transfer the solution to a tube with a membrane filter.

Centrifuge to concentrate the viral particles above the filter.

Add the stabilizing agent-supplemented buffer and centrifuge again to obtain purified viral particles.

To harvest the transfected cells, dislodge and suspend the cells by using the culture medium to pipette up and down. Then transfer the cell suspensions to sterile 50-milliliter tubes. Centrifuge the cells at 500 times g for five minutes.

Then add five milliliters of PBS to each tube and resuspend the cell pellets before combining all the suspensions in one 50-milliliter tube. After spinning the cells at 500 times g for five minutes, discard the supernatant. To purify the AAV, resuspend the pellet in 10 milliliters of lysis buffer.

Freeze the lysate at minus 80 degrees Celsius or in a dry ice–ethanol bath. Then thaw it at 37 degrees Celsius. Vortex the lysate for one minute before repeating the freeze and thaw two more times.

Next, add one millimolar magnesium chloride to the thawed lysate. Then add nuclease to a final concentration of 250 units per milliliter. Incubate the solution at 37 degrees Celsius for 15 minutes to dissolve the DNA protein aggregation.

Centrifuge the sample at 4,800 times g and four degrees Celsius for 20 minutes. Then collect the supernatant. Meanwhile, to prepare a 17%iodixanol gradient solution, mix five milliliters of 10X PBS with 0.05 milliliters of one molar magnesium chloride, 0.125 milliliters of one molar potassium chloride, 10 milliliters of five molar sodium chloride, and 12.5 milliliters of density gradient medium.

Use water to adjust the total volume to 50 milliliters. After preparing 25%, 40%, and 60% gradient solutions according to the text protocol, use a needle and syringe to load five milliliters of 17% gradient solution into a polypropylene tube. Follow this with five milliliters each of 25%, 40%, and 60% gradient solutions.

Layer the lysate on top of the gradient. Then fill the tube with lysis buffer and cover it with a cork. Centrifuge the gradient at 185,000 times g and 16 degree Celsius for 90 minutes.

Following the spin, use a syringe to harvest the 40% viral fraction by inserting a 21-gauge needle into the intersection between the 40 and 60% fractions, completely avoiding the 25% layer. Mix the viral fraction with sterilized polyoxyethylene polyoxypropylene, or PEG-PPG block copolymer PBS solution, up to a total volume of 15 milliliters. Load the mixture into a filter tube with a molecular weight cutoff of 100 kilodaltons.

Then centrifuge the sample at 2,000 times g and four degrees Celsius for 30 minutes. Decant the solution from the bottom of the tube. Then use PEG-PPG to refill the filter tube to 15 milliliters.

Centrifuge the tube at 2,000 times g and four degrees Celsius for 20 minutes. After repeating the refilling and spin two more times, collect the purified rAAV9 virus fraction still above the filter.