AAV production with HEK293T#

Note

This page is still under active development

Cell lines#

AAV 293 or HEK293T

Materials & Reagents#

  • Cell

  • DMEM (high glucose without L-glutamine)

  • Fetal bovine serum (FBS)

  • TrypLE Express

  • Penicillin/Streptomycin (P/S)

  • AdDeltaF6 (Addgene, #112867)

  • AAV2/1 (Addgene, #112862)

  • AAV2/1 (Addgene, #104964)

  • AAV retrohelper (Addgene, #81070)

  • Transfer plasmid (ITR-ITR)

  • Lipofectamine 3000

  • Opti-MEM

  • AAVpro Purification Kit Midi (All Serotypes) Takara

  • 0.5 M EDTA (pH 8.0)

Culture medium : DMEM +10% FBS + 1%P/S

Reagent Volume
DMEM 445 ml
FBS 50 ml
P/S 5 ml
Total 500 ml

Protocols#

Thawing cells from freezing cells``#

  1. Thaw AAV293 cells in 37°C waterbath.

  2. Add to 5 mL of 293T culture medium.

  3. Spin down for 5 min at 1000 rpm

  4. Resuspend the cells with 10 mL 293T culture medium.

  5. Plate in 150 cm culture dish.

Important

Change medium every day or every two days. If you don’t and the medium gets yellow, the cells will detach and die.

  1. Once the cells are 90% confluent, split them 1 to 4 using TrypLE express:

  2. Rince with DPBS–

  3. Add 5 ml of TrypLE express and incubator for 5-7 min

  4. Spin down for 5 min at 1000 rpm, then aspirate supernatant

  5. Resuspend in 8 mL medium per flask.

  6. Plate 2 mL cell suspension + 8 mL of fresh medium per flask to split ¼.

Transfection#

Collection Option 1#

Important

The protocol below describes purification of AAV particles from producer cells in one T225 flask or four 10 cm dishes.

See also

https://takara.co.kr/file/manual/pdf/6675_6675S_e.v2001Da.pdf

VI-1 Preparation of AAV extract solution#

  1. Detach the AAV-producing cells by adding 1/80 volume of 0.5 M EDTA (pH 8.0) to the culture medium; incubate at room temperature for 10 min.

  2. Collect the cells in a centrifuge tube from the flask.

  3. Centrifuge at 1,700 - 2,000g for 10 min at 4℃ and discard the supernatant.

  4. Centrifuge again at 1,700 - 2,000g for 1 min at 4℃ and remove the supernatant completely.

Note

Be sure to completely remove the supernatant, because remaining supernatant could impair virus purification.

  1. Loosen the cell pellet well by tapping or vortexing.

Note

If the cell pellet is not loosened sufficiently, the purification efficiency can be decreased. Make sure there are no cell clumps before proceeding to the next step.

  1. Add 2 ml of AAV Extraction Solution A plus.

  2. Resuspend by vortexing for 15 sec.

Note

Continue to vortex until there are no remaining cell clumps.

  1. Incubate for 5 min at room temperature and then vortex again for 15 sec

  2. Centrifuge at 4,000 - 9,000g for 10 min at 4℃

Note

In some cases, the virus recovery can be improved by repeating steps 7 - 9.

  1. Transfer the supernatant in a new sterile centrifuge tube using a pipet to avoid contamination. Add 1/10 volume of AAV Extraction Solution B to the supernatant.

Note

The virus suspension can be stored at -80℃. Alternatively, promptly proceed to step VI-2-1. If the virus suspension is stored at -80℃, thaw in a 37℃ incubator before using. Be sure to use a tube that is resistant to freezing and centrifugation when a virus suspension is stored at -80℃.

Note

When AAV Extraction Solution B is added, the color of the solution may turn pink in some cases; this does not affect performance.

VI-2. Purification and concentration of AAV particles#

Purification

Note

Use swing bucket rotors for steps VI-2-5, VI-2-6, and VI-2-7.

  1. Add 1/100 volume of Cryonase Cold-active Nuclease to the virus suspension at step VI-1-10, and then incubate at 37℃ for 1 hr.

  2. Add 1/10 volume of Precipitator A, vortex for 10 sec, incubate at 37℃ for 30 min, and vortex again for 10 sec.

Note

Precipitator A may produce a white precipitate at low temperature, however this does not affect the quality or performance of this reagent. If a precipitate is present, dissolve it completely at 37℃ before use.

Note

Although a precipitate may form during the incubation, this is not a problem. Proceed to the next step.

  1. Add 1/20 volume of Precipitator B to the mixture at the step above, vortex quickly for 10 sec, and then centrifuge at 5,000 - 9,000g for 5 min at 4℃.

Note

A precipitate may be formed after adding Precipitator B, but proceed to centrifugation.

  1. Filter the supernatant using Millex-HV 0.45 μm.

  2. Transfer the filtrate containing AAV vector into a filter device of Amicon Ultra-4, 100 kDa. Centrifuge at 2,000g for 5 min at 15℃, and then confirm that the AAV solution in the filter device is <0.4 ml.

Note

If the volume of the solution is >0.4 ml, continue to centrifuge.

  1. After removing the filtrate, add 1 ml of Suspension Buffer in the filter device of the Amicon Ultra-4 and mix the solution uniformly by pipetting. Centrifuge at 2,000g for 5 min at 15℃, and then confirm that the AAV solution inside the filter device device is <0.4 ml.

Note

If the volume of the solution is >0.4 ml, continue to centrifuge.

  1. Repeat step VI-2-6 4 times (total 5 times) to obtain an appropriate volume of solution.

  2. After discarding the filtrate, resuspend the solution inside of the cup of the Amicon Ultra-4, 100 kDa filter device by pipetting or vortexing for 30 sec and transfer the virus suspension to a new tube.

Option 2#

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