Abstract
BackgroundDuring 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 in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.MethodsHere 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.ResultsOur results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 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 × 1010 TU were recovered from a single HYPERFlask vessel.ConclusionThe 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.
Highlights
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 in vitro and in vivo
Simplified production of lentiviral vectors using HYPERFlasks With a view toward improving high-titer LV vector production for preclinical studies in animals, we tested the usefulness of HYPERFlask vessels that have a total growth area of 1720 cm2, corresponding to ten standard T175 flasks
To test the usefulness of HYPERFlasks for LV vector production involving calcium phosphate-mediated transfection [10], we compared the titers of LV vector stocks prepared side-by-side using either ten 150-cm2 dishes or a single HYPERFlask
Summary
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 in vitro and in vivo. Despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly. The production of LV vectors is typically carried out using transient transfection approaches involving tissue culture dishes or flasks [3], cell factories [4,5,6], or stirred-tank bioreactors [7]. These protocols are cumbersome to scale up (dishes, flasks) or technically challenging (cell factories, bioreactors), preventing their routine use in a standard laboratory setting
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