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``#
Thaw AAV293 cells in 37°C waterbath.
Add to 5 mL of 293T culture medium.
Spin down for 5 min at 1000 rpm
Resuspend the cells with 10 mL 293T culture medium.
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.
Once the cells are 90% confluent, split them 1 to 4 using TrypLE express:
Rince with DPBS–
Add 5 ml of TrypLE express and incubator for 5-7 min
Spin down for 5 min at 1000 rpm, then aspirate supernatant
Resuspend in 8 mL medium per flask.
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#
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.
Collect the cells in a centrifuge tube from the flask.
Centrifuge at 1,700 - 2,000g for 10 min at 4℃ and discard the supernatant.
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.
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.
Add 2 ml of AAV Extraction Solution A plus.
Resuspend by vortexing for 15 sec.
Note
Continue to vortex until there are no remaining cell clumps.
Incubate for 5 min at room temperature and then vortex again for 15 sec
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.
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.
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.
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.
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.
Filter the supernatant using Millex-HV 0.45 μm.
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.
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.
Repeat step VI-2-6 4 times (total 5 times) to obtain an appropriate volume of solution.
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.