Traditionally, in order to release intracellular retained rAAV vectors, producer cells are lysed by 3 to 4 freeze-thaw-cycles (liquid nitrogen-37 °C water), producing a crude cell lysate that is the starting material for a variety of subsequent purification and concentration methods. Therefore, efficient cell lysis is of crucial importance towards high-titer rAAV vector preparations. This application note investigates the use of the Minilys as an alternative solution to traditional freeze-thaw methods.
The images show that the Minilys does not impair rAAV vector infectivity (Figure 1) and efficiently releases rAAV vectors from HEK 293T producer cells (Figure 2), and thus shows similar results compared to the freeze-thaw method.
Figure 1: Minilys homogenization (A) vs. 3 freeze-thaw cycles (B) on untransfected HEK 293T cells resuspended with known concentrations of rAAV vector titers. 700 μl of the crude cell lysate obtained from each method was added to separate dishes of cultured HEK 293T cells. Images were taken 2 days later (20x objective).
Figure 2: Transduction of HEK 293T cells by rAAV vectors produced by transient transfection of HEK 293T cells and released thereafter by Minilys homogenization (A) vs. 4 freeze-thaw cycles (B). 10 μl of the crude cell lysate obtained from each method was added to separate dishes of cultured HEK 293T cells. Images were taken 6 days later (20x objective).