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AAV Serotype Testing on Cultured Human Donor Retinal Explants

Publicatiejaar 2018
Gepubliceerd in Methods in Molecular Biology
Auteur(s) Thilo M Buck, Lucie P Pellissier, R.M. Vos, Elon H C van Dijk, Camiel J F Boon, J. Wijnholds

This protocol details on a screening method for infectivity and tropism of different serotypes of adeno-associated viruses (AAVs) on human retinal explants with cell-type specific or ubiquitous green fluorescent protein (GFP) expression vectors. Eyes from deceased adult human donors are enucleated and the retinas are isolated. Each retina is punched into eight to ten 6-mm equal pieces. Whatman™ paper punches are placed on the retinas and the stack is transferred onto 24-well culture inserts with the photoreceptors facing the membrane. AAVs are applied on the retinal explant punches to allow transduction for 48 h. Retinas are nourished by a serum-free Neurobasal®-A based medium composition that allows extended culturing of explants containing photoreceptor inner and outer segments. The protocols include quality control measurements and histological staining for retina cells. The cost and time effective procedure permits AAV transgene expression assays, RNAi knockdown, and pharmacological intervention on human retinas for 21 days ex vivo.

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