Adeno-associated virus (AAV) was discovered in 1965 and has since been used in a myriad of applications¹. AAVs have been applied in neuroscience research to study gene and neuronal function, map neurocircuits, or produce animal models for disease². Traditionally, this is done by injecting directly at the site of interest, as most natural serotypes do not cross the blood-brain barrier or need a high dose to do so¹^,²^,³.

With the discovery of AAV.PHP.B⁴ and next-generation capsids such as AAV.PHP.eB⁵ and AAV.CAP-B10⁶, it is possible to target the central nervous system (CNS) using a simple systemic injection. Spatial mapping reveals the cells targeted by AAV.PHP.eB at cellular level⁶^,⁷. In combination with specific promoters/enhancers, these capsids offer extensive opportunities for neuroscientists to study genes and brain function by non-invasive AAV delivery⁴^,⁸.

While a lower dose is needed for AAV.PHP.eB (typically 1 to 5 x 10¹¹ Genome Copies (GC)/mouse) compared to AAV9 (4 x 10¹² GC/mouse)⁷, still more vector needs to be produced compared to direct injection strategies (typically 1 x 10⁹ GC/µL injection). Most natural serotypes can be produced using the classical adherent cell culture system in combination with iodixanol purification⁹^,¹⁰^,¹¹^,¹². For AAV.PHP.eB this entails a labor-intensive process to culture and transfect cells to obtain sufficient vectors for one experiment⁸. Therefore, the production of AAV in suspension cell culture in conical tubes was developed. Conical tubes, with a capacity of up to 300 mL, are compact, saving both incubator space and plastics. Suspension cells are much easier to culture and handle in large amounts than adherent cells on 15 cm plates. The transfection components of the protocol remain the same. Therefore, plasmids previously used with the adherent system can easily be used in this protocol based on production in suspension cells. The protocol was successfully transferred to other researchers in the laboratory and successfully used for various capsids and constructs.

Iodixanol Gradient Purification of Transfected HEK293 Cell Lysates for Concentrated Adeno-Associated Virus Production