AAV-Production-293T and Purification using aqueous two phases partitioning

Cell culture

On day one HEK293 cell are maintained in DMEM  with 10% FBS (Cellgro, Mediatech. Manassas, VA).

On day 2, at 80% confluent, change  culture medium with 2% FBS.

Transfection

Mix plasmids: pAAV-GFP, pHELP and prep-Cap AAV8 or AAV2 in the ratio of 2:1:1.

Add PEI (DNA:PEI=1:3 or 1:2)

Incubate for 15 min at room temperature

Add dropwise to plates.

Harvest

Collect the culture media from  transfected HEK293 cells.

Spin at 3,000x g for 10 min to pellet the cells and cell debris.

~ 320 ml of culture medium from twenty 15cm dishes

Collect cells, pool into one 50 ml conical tube with 5 ml of lysis buffer:

50 mM TrisCl, 150 mM NaCl,  2 mM MgCl2, pH 8.0

Freeze/thaw 3 cycles using dry ice-ethanol and 37 oC water bath.

Add 50 U/ml benzonase and 10 U/ml RNase I

Add 0.5% sodium deoxycholate

Incubate for additional 30 min.

Spin at 2500x g for 10 minutes

Collect supernatant.

PEG Precipitation

Add 5X stock solution of 40% PEG 8000, 2.5 M NaCl to combined clarified cell lysate and culture supernatant to a final concentration of 8% PEG, 0.5 M NaCl respectively.

Incubate on ice for 1 h

Spin at 2000x g for 30 min.

Collect pellet.

Resuspend virus in a minimal volume of HEPES buffer (10 ml per 300 ml clarified cell lysate input from twenty 15cm dishes).

Aqueous two phases partitioning

Add an equal volume of chloroform to  crude AAV solution  at room temperature.

Votex vigorously for 2 minutes.

You will see milk-like solution.

Spin at 370x g using a desktop centrifuge for 5 min

Let supernatant stand for 30 min to evaporate the chloroform.

The phase system is prepared from stock solutions of PEG (50%, w/w) and (NH₄)₂SO₄ (20%, w/w)

10% PEG-1450-10%(NH₄)₂SO₄ (pH6.5), buffer with HEPES to pH 8.0

or use PEG-4000, or 10%PEG8000-13.2%(NH₄)₂SO₄

The best purity is obtained with 10%PEG8000/13.2% (NH₄)₂SO₄

The pH is measured after addition of the rough AAV.

Stock solutions can be stored at 4°C.

Before use, the temperature of the stock solution should be equilibrated to room temperature.

Add PEG/NaCl or PEG/sulfate or PEG/Na2CO3 two phase mixture to the supernatant

Vortex vigorously for two minutes at room temperature

Leave 15 to 30 minutes at room temperature.

Spin at 3000x g for 15 min at room temperature.

Collect the clear bottom phase containing virus carefully with a needle.

Transfer the bottom phase to a dialysis cassette (Slide-A-Lyser Dialysis Cassette) or dialysis spin tube (50K MWCO, Amicon) for removal of salt.

Store virus in PBS or MEM medium with 0.001% pluronic F68.

Usually, high concentration of stock virus should be kept in PBS or MEM medium with pluronic. Pluronic F68 can prevent AAV aggregation during storage in freezer.