AAVpro Concentrator allows easy and efficient concentration of AAV vectors using a standard centrifuge. In contrast with other concentration methods that involve only precipitation of AAV particles, AAVpro Concentrator incorporates filtration steps to yield AAV preps of higher purity. The scalable mix-and-spin protocol can accommodate a range of input volumes and requires less than one hour of hands-on time. Figure 1 illustrates the workflow for preparing and purifying AAV vectors using AAVpro Concentrator.
Rapid and efficient AAV concentration
AAV vectors can be used to transduce genes into both proliferating and nonproliferating cells and can impart long-term transgene expression in nondividing cells. In addition, AAV has little immunogenicity and is suitable for the transduction of genes into whole organisms (as an in vivo transduction tool). However, it is necessary to prepare highly concentrated AAV vectors for transduction, since the amount of AAV vector injected into an individual animal depends on the administration method and target species, and the injection volume for commonly used animals (e.g., mice) is limited due to their small body size. AAVpro Concentrator provides a simple, highly efficient method for concentrating AAV particles of any serotype from cell culture supernatants using a standard centrifuge. This kit can be used for various applications, such as the concentration of AAV vectors in culture supernatant with or without serum, and buffer exchange of AAV vector solutions.
- Efficient concentration of AAV particles from cell culture supernatant
- Simple mix-and-spin protocol requires less than one hour of hands-on time and avoids ultracentrifugation
- Filtration steps included for removal of impurities
- Scalable method enables processing of various input volumes
Concentration of AAV vectors from serum-free culture supernatant
Culture supernatant containing AAV1 vectors expressing ZsGreen1 was concentrated from a starting input of 40 ml (one T225 flask) down to a final volume of 200 μl using AAVpro Concentrator. The viral titer was measured before and after concentration to determine the viral recovery rate using the AAVpro Titration Kit (for Real Time PCR) Ver.2.
Biological titer of concentrated AAV vectors
A biological assay commonly used to test the infectivity of an AAV preparation involves infecting permissive cells with AAV and measuring transgene expression to determine a titer in transducing units (TUs). To confirm the infectivity of AAV prepared using AAVpro Concentrator, flow cytometric analysis was used to compare ZsGreen expression in HT1080 cells infected with AAV1-ZsGreen1 vectors before or after concentration. HT1080 cells were infected with AAV at 5,000 or 500 vg/cell (MOI) and analyzed by FACS after three days. Concentrated AAV particles administered at MOIs of 5,000 or 500 were able to infect cells at rates of 44.7% or 9.2%, respectively.
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