Adeno-Associated Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 13

11. Two days after transfection, gently add 10 ml of fresh serum-
free medium to each dish.
( NB : Step 11 is optional ; it helps to maintain cells in better con-
dition and may therefore improve virus yield , but the increase in
volume will lengthen the purification steps .)
12. Five days after transfection, add Benzonase nuclease to each
dish to the final concentration 25 U/ml. Incubate at 37 °C for
a further 2 h.
( NB : Instead of step 12 , Benzonase may be added directly to the
pooled supernatant in step 14. Add Benzonase to a final concen-
tration of 25 U / ml , incubate at 37 °C for 2 h, and then proceed
to clarify the lysate by centrifugation as described in step 14 .)
13. To each dish, add NaCl solution (iltered through 0.22 ʼm
Nalgene Thermo Scientific Rapid-Flow filter) to a final con-
centration of 500 mM and incubate for 2 h.
14. Collected medium from all dishes is pooled to a final volume
of approximately 1 l. Since it contains cell debris, it must be
clarified by centrifugation (3,850 × g for 5 min, Sigma 3-16PK,
rotor 11180), followed by iltering through a 0.22 ʼm vac-
uum filter (Thermo Scientific Nalgene Rapid-Flow filter unit).
Filtered supernatant may be used immediately or stored at
4 °C overnight.
15. Finally, concentrate the supernatant by approximately 75 times.
In the absence of tangential flow filtration equipment, Amicon
Ultra-15 Centrifugal 100K Filters (Millipore) can be applied.
Supernatant is centrifuged at 3,800 × g at 4 °C until the volume
is reduced to 14 ml. While not laborious, this step can take
several hours. Keep the supernatant refrigerated at all times.

For the iodixanol gradient ultracentrifugation use Quick-Seal
39 ml tubes (Beckman Coulter #344326) by underlaying and
displacing the less dense supernatant. The solutions must be added
slowly in order to prevent mixing of the layers and bubbles intro-
duction by inserting a 100 mm, 18 gauge blunt-end needle
(Hamilton #7750-09) and filling the tube from the bottom
upwards.

3.8 AAV Purification
by Iodixanol Density
Gradient

1. Preparation of iodixanol solutions:

Percentage
iodixanol

5 × PBS-MK
( see below) H 2 O Iodixanol 5 M NaCl Phenol Red

15 % 12.5 ml 10 ml 10 ml 17.5 ml -

25 % 20.8 ml - 10 ml 19.2 ml 100 ʼl

40 % 33.3 ml - 10 ml 6.7 ml -

54 % 45 ml - - 5 ml 100 ʼl