| Summary: | <p>Abstract</p> <p>Background</p> <p>During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells <it>in vitro </it>and <it>in vivo</it>. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.</p> <p>Methods</p> <p>Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography.</p> <p>Results</p> <p>Our results show that unconcentrated LV vector stocks with titers in excess of 10<sup>8 </sup>transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm<sup>2 </sup>tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 × 10<sup>10 </sup>TU were recovered from a single HYPERFlask vessel.</p> <p>Conclusion</p> <p>The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.</p>
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