Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 33

Add 6 ml of trypsin-EDTA, rock flask slowly and incubate cells
at 37 °C until they have detached (1-2 min).
Resuspend cells in 24 ml of DMEM-10 % FBS. Gently pipette
up and down to dissociate cell clumps.
Add 15 ml of the cell suspension to a 250-ml conical tube
containing 85 ml of DMEM-10 % FBS.
Add 100 ml of the cell suspension to a triple flask. Incubate
cells at 37 °C.
(b) Transfection
To transfect one triple flask, prepare the DNA/PEI complex in
the following order:
- 50 μg of AAV cis plasmid
- 150 μg of pDG helper plasmid
- 20 ml of serum-free, antibiotics-free DMEM
- 700 μl of PEI
Vortex the DNA/PEI mixture and incubate for 45 min at
room temperature.
Add the DNA/PEI complex to a 250-ml conical tube contain-
ing 100 ml of prewarmed DMEM supplemented with 2 %
FBS and 25 mM HEPES.
Aspirate media and add the DNA/PEI/DMEM mixture into
the triple flask.
Incubate cells for 3 days at 37 °C.
(c) Cell harvesting
Gently tap the flask to remove cells from the surface. Transfer
cells and medium in a 250-ml conical tube.
Add 50 ml of PBS to the flask and shake to remove any remain-
ing cell from flask surface. Harvest cells and combine with
previously collected cells.
Spin down cells at 1,000 rpm for 10 min at 4 °C.
Discard supernatant. Wash cell pellet with 20 ml of PBS.
Repeat wash step.
Gently resuspend cell pellet in 10 ml of lysis buffer.
The cell pellet can be stored at −20 °C or processed immediately.

3.3 rAAV Virus
Purification

(a) Preparation of crude lysate by freeze/thaw.
Transfer the crude lysate to a 50-ml conical tube and vortex.
Freeze the crude lysate in dry ice until the pellet is completely
frozen (at least 30 min).
Thaw the crude lysate in a 37 °C water bath for at least 15 min.
Mix by swirling every 5 min.
Repeat the freeze-thaw steps two more times.