Purification of Recombinant Virus Particles from Transfected Human Kidney Cells

Abstract

Source: Ding, J. et. al., Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)

This video shows the purification of recombinant virus particles from transfected human kidney cells. The virus is released by freeze–thaw lysis and purified using iodixanol gradients with block copolymer-assisted filtration.

Protocol

1. Transfection of HEK293 Cells with recombinant adeno-associated virus 9 (rAAV9) Plasmids
   1. Prepare 1 µg/µl of linear polyethylenimine (PEI) solution. Dissolve PEI powder in endotoxin-free dH₂O that has been heated to 70-80 °C. After cooling down to room temperature (RT), neutralize the solution to pH 7.0 with 1 M hydrochloric acid (HCl). Filter sterilize (0.22 µm) the solution. Aliquot the 1 µg/µl PEI stock solution (1,400 µl/tube) and store the solution at -20 °C.
   2. Culture HEK293 cells in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Culture the cells in a 37 °C incubator with 5 ± 0.5% carbon dioxide (CO₂).
   3. At day 0, plate HEK293 cells in ten 150-mm dishes 18-20 hr before transfection by splitting > 90% confluent cells in a 1:2 dilution.
      NOTE: At day 1, the cells should reach 90% confluence.
   4. At day 1, transfect HEK293 cells with the rAAV9 plasmid (e.g., rAAV9.cTNT::GFP or rAAV6.U6::shRNA constructs), Ad-Helper plasmid, and AAV-Rep/Cap plasmid using PEI.
      1. For 10 dishes of cells at 90% confluence, mix 70 µg of AAV-Rep/Cap plasmid, 70 µg pfrAAV9 plasmid, and 200 µg of Ad-helper plasmid in a 50-ml centrifuge tube.
      2. If the cells are less confluent, adjust the DNA amount proportionally. For instance, if the cells are at 75% confluent, reduce the DNA amount proportionally (75/90 of the amount shown in step 2.4.1): mix 70 x 75/90 = 58.3 µg of AAV-Rep/Cap plasmid, 70 x 75/90 = 58.3 µg of rAAV9 plasmid, and 200 x 75/90 = 166.7 µg of Ad-Helper plasmid in a 50-ml centrifuge tube.
      3. Add 49 ml of RT DMEM (without FBS) to the 50-ml tube and mix well.
      4. Add 1,360 µl of PEI solution to make the PEI: DNA ratio (v/w) be 4:1. Mix well. Incubate at RT for 15 - 30 min.
      5. Add 5 ml of the mixture prepared in step 1.4.4 to each 150-mm dish (50 ml of the mixture for ten 150-mm dishes).
   5. Culture the cells in a 37 °C incubator with 5 ±0.5% CO₂ for 60-72 hr.
2. Harvest of Transfected HEK293 Cells and Purification of rAAV9 Vectors
   1. Harvest the cells 60-72 hr after transfection. Dislodge and suspend the cells in the dishes by pipetting up and down with the culture medium. Transfer all the cell suspensions to sterile 50-ml tubes.
   2. Centrifuge the cells at 500 x g for 5 min. Resuspend the cell pellet with 5 ml of phosphate-buffered saline (PBS) in each tube and combine all the cell suspensions into one 50 ml tube.
   3. Centrifuge the cells at 500 x g for 5 min. Discard the supernatant. At this step, store the cell pellet at -80 °C or immediately purify the AAV from the pellet, as described in steps 2.4-2.15.
   4. Prepare the lysis buffer: 150 mM sodium chloride (NaCl) and 20 mM Tris(hydroxymethyl)aminomethane Hydrochloride (Tris-HCl, pH 8.0). Filter sterilize (0.22 µM). Store the buffer at 4 °C.
   5. Resuspend the pellet with 10 ml of lysis buffer.
   6. Freeze the lysate at -80 °C or in the dry ice/ethanol bath, then thaw it at 37 °C. Vortex for 1 min. Freeze and thaw the lysate 3 times.
   7. Add magnesium chloride (MgCl₂) solution to the thawed lysate (make the final concentration of MgCl₂ in the lysate be 1 mM). Add the nuclease to a final concentration of 250 U/ml. Incubate at 37 °C for 15 min to dissolve the DNA/protein aggregation.
      NOTE: If the DNA/protein aggregation does not get dissolved after nuclease or endonuclease treatment, dounce homogenize the lysates 20 times.
   8. Centrifuge the sample at 4,800 x g for 20 min at 4 °C. Collect the supernatant.
   9. Meanwhile, prepare the Iodixanol gradient solution:
      1. Prepare the 17% of the gradient solution by mixing 5 ml of 10x PBS, 0.05 ml of 1 M MgCl₂, 0.125 ml of 1 M potassium chloride (KCl), 10 ml of 5 M NaCl, and 12.5 ml of density gradient medium. Adjust the total volume to 50 ml using H₂O.
      2. Prepare the 25% solution by mixing 5 ml of 10x PBS, 0.05 ml of 1 M MgCl₂, 0.125 ml of 1 M KCl, 20 ml of density gradient medium, and 0.2 ml of 0.5% (w/v) Phenol Red. Adjust the total volume to 50 ml using H₂O.
      3. Prepare the 40% solution by mixing 5 ml of 10x PBS, 0.05 ml of 1 M MgCl₂, 0.125 ml of 1 M KCl, and 33.3 ml of density gradient medium. Adjust the total volume to 50 ml using H₂O.
      4. Prepare the 60% solution by mixing 0.05 ml of 1 M MgCl₂, 0.125 ml of 1 M KCl, 50 ml of density gradient medium, and 0.1 ml of 0.5% (w/v) Phenol Red.
   10. With a needle and syringe, load the Iodixanol gradient solution into the polypropylene tube in the order of 5 ml of 17%, 5 ml of 25%, 5 ml of 40% and 5 ml of 60%, starting from the bottom. Load all the lysate obtained from step 3.8 (14-16 ml) on top of the gradient. The gradient, listed from the bottom to top, is 60%, 40%, 25%, 17%, and the lysate layer. Fill the tube with lysis buffer and cover it with the cork.
   11. Centrifuge at 185,000 x g for 90 min at 16 °C.
   12. Harvest the viral fraction (40% layer) with a syringe. Insert the needle (21 gauge) into the intersection between the 40% and 60% fractions, only aspirating the 40% layer.
       NOTE: Avoid aspirating the 25% layer.
   13. Mix the viral fraction with sterilized polyoxyethylene-polyoxypropylene block copolymer PBS solution (10% polyoxyethylene-polyoxypropylene block copolymer stock 1:10,000 diluted in PBS) up to a total volume of 15 ml. Load the mixture into the filter tube (cut-off MW = 100 kD). Centrifuge at 2,000 x g for 30 min at 4 °C.
   14. Discard the solution at the bottom. Refill the filter tube with polyoxyethylene-polyoxypropylene block copolymer PBS solution to a total volume of 15 ml. Centrifuge at 2,000 x g for 20 min at 4 °C. Repeat this step two more times. Collect the purified rAAV9 virus (the fraction above the filter).
   15. Transfer the purified rAAV9 in the filter tube to 1.7-mL tubes. Aliquot the purified rAAV9 (100 - 400 µl/tube, depending on the volume and titer of the AAV) and store the virus at -80 °C.
       NOTE: Avoid repeated freeze-thaws.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyethylenimine, linear (MW 25,000)	Polysciences, Inc.	#23966-2
Tube, polypropylene, 36.2 ml, 25 x 87 mm, (qty. 56)	Beckman Coulter, Inc	# 362183
Nuclease, ultrapure	SIGMA	#E8263-25KU
Density gradient medium(Iodixanol)	SIGMA	#D1556-250ML
Centrifugal filter unit with Ultracel-100 membrane	EMD Millipore Corporation	#UFC910008
Laboratory pipetting needle with 90° blunt ends,gauge 14, L 6 in., nickel plated hub	SIGMA	#CAD7942-12EA
Poloxamer 188 solution (Pluronic® F-68 solution)	SIGMA	P5556-100ML
Proteinase K	SIGMA	3115828001
DNase I	Roche	10104159001
Centrifuge machine	Thermo Scientific	75004260
Centrifuge System	Beckman Coulter	363118
Ultracentrifuge	Beckman Coulter
DMEM medium	Fisher Scientific	SH30243FS
Fetal bovine serum	Atlanta Biologicals	S11150
rAAV9 vector	Penn Vector Core	P1967