Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 222

Fig. 6 SH-SY5Y cells transduced with rAAV2/2. Image shows GFP fl uorescence
5 days after transfection with rAAV2/2 virus. Scale bar 50 ʼ m

5.2 Ex Vivo Retinal
Culture

Testing AAV vectors on cell lines is a useful screening tool; however,
the cultures lack the complex cellular architecture of retinal tissue,
which may affect transduction patterns and effi ciency. Therefore,
testing vectors on retinal explants and ideally on human tissue
would provide a better indication of cellular tropism and effi cacy of
transduction.
Retinal explants may be prepared from a rodent tissue by har-
vesting the eye and carefully dissecting the retina from the
RPE. We have also recently developed a system for culturing
human retinal tissue taken with consent from patients having a
retinectomy surgery for complex retinal detachment (REC refer-
ence no. 10/H0505). Following a routine 23-gauge pars plana
vitrectomy for retinal detachment surgery, disks of scarred retina
are cut with a vitrectomy system (Ocutome; Alcon Surgical),
refl uxed into the eye, aspirated with a fl ute needle, and placed in
balanced salt solution.
The retinal explants (rodent or human) can be cultured as fol-
lows. Transfer the samples using a cutoff 5 ml pipette to minimize
crush injury to the tissue into organotypic culture inserts (BD
Falcon) and place in a 24-well plate. Add 700-

l culture media per
well consisting of Neurobasal A, 0.08 M L -glutamine, 100 U/ml
penicillin, 100

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g/ml streptomycin, 2 % B27 supplement, and 1 %
N2 supplement (Invitrogen). Maintain explants at 34 °C in a 5 %
CO 2 environment.
After 24 h, change the media and add virus (10

ʼ

l of titer
1 × 10 12 vg/ml was used in our experiments) to each well, ideally
testing at least two wells per virus. Change the media every 48 h.
Due to the transparent nature of the culture inserts, explants can

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