Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 217

the cells. Divide the remaining suspension equally between the two
centrifuge tubes. Spin at 1,000 rpm for 10 min at room tempera-
ture. The residual cell pellet may break easily and be diffi cult to
isolate. Carefully remove about 200 ml media from each centrifuge
tube, resuspend the cell pellet in the remaining media, and transfer
to a 50-ml Falcon tube. Spin this further for 10 min at 1,000 rpm
at room temperature. Aspirate the supernatant without disturbing
the cell pellet.
Add 15-ml lysis buffer (4.38 g NaCl (0.15 M), 25 ml 1 M Tris-
Cl pH 8.5 (50 mM) in 500 ml dH 2 O; autoclaved) to each 50-ml
Falcon tube, and resuspend the cell pellet. Add 500 l of pre-dis-

solved EDTA-free protease inhibitor cocktail (Roche) to each 15 ml
of resuspended pellet (one pellet in 1 ml lysis buffer). Carry out
four freeze-thaw cycles (freeze for 30-60 min in a −80 °C freezer;
thaw at 37 °C for approximately 15 min and shake well after the
thaw). Store the lysate at −80 °C until ready for purifi cation.

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Defrost the lysate for approximately 15 min at 37 °C. Add
Benzonase (purity grade I for Biotechnology, VWR) to give a fi nal
concentration of 50 U/ml, and incubate for 30-45 min at 37 °C,
shaking by hand every 10 min. Centrifuge at 3,700 × g for 20 min
at room temperature.
Meanwhile, prepare the iodixanol gradient in a cell culture
hood in Beckman Quick-Seal Ultra-Clear 25 × 89 mm centrifuge
tubes (Beckman). Make up the iodixanol fractions (Table 2 ) in
50-ml Falcon tubes prior to loading in the Beckman tubes to form
the gradient using iodixanol OptiPrep Density Gradient Medium
(60 %) (Sigma), phenol red solution (Sigma), and 5× PBS-MK
(250 ml PBS 10× (Gibco), 2.5 ml 1 M MgCl 2 , 6.25 ml 1 M KCl,
in 500 ml H 2 O).
The following amounts of each fraction should be layered in
the Beckman centrifuge tube using a Pasteur pipette:

4.5 Virus Purifi cation
by Iodixanol Gradient

7.2 ml of iodixanol 15 %
4.8 ml of iodixanol 25 %
4 ml of iodixanol 40 %
4 ml of iodixanol 60 %

Table 2
Preparation of iodixanol fractions per 4 AAV ultracentrifuge tubes

Fraction (%) Iodixanol (ml) 5 M NaCl 5× PBS-MK H 2 O Phenol red

15 8 6.4 ml 6.4 ml 11.2 ml -

25 10 - 4.8 ml 9.2 ml 40 ʼ l

40 13.4 - 4.0 ml 2.6 ml -

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60 20 - - - 40 l