Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography

<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 10⁸transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm²tissue 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 × 10¹⁰TU 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

Lipase induction in Yarrowia lipolytica for castor oil hydrolysis and its effect on γ-decalactone production

γ-Decalactone is an aromatic compound of industrial interest, resulting from the biotransformation of ricinoleic acid, the major constituent of castor oil. In order to increase the availability of the substrate to the cells for the aroma production, castor oil previously hydrolyzed can be used. This hydrolysis may be promoted by enzymatic action, more specifically by lipases. In this work, the influence upon the aroma production of the lipase produced by Yarrowia lipolytica, a microorganism able to carry out the biotransformation, was studied. In a first approach, lipase induction conditions were analyzed using different Y. lipolytica strains and culture conditions, such as the inoculation mode of the lipase production medium. Lipase production was not affected by the cells centrifugation, so this step was eliminated, reducing the time and phases of the process. Moreover, Y. lipolytica W29 was shown to be the most adequate strain for lipase production. To investigate the importance of castor oil hydrolysis, the pre-addition of an inducer of lipase production (olive oil) to the biotransformation medium was tested. Results showed that the highest aroma production (1,600 mg L−1) was obtained without a lipase inducer. However, the pre-induction of lipase decreased the lag phase for γ-decalactone secretion.The authors acknowledge Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided (SFRH/BD/28039/2006 and SFRH/BD/63701/2009). They also acknowledge the kind supply of Yarrowia lipolytica IMUFRJ 50862 by Professor Alice Coelho from UFRJ, Brazil, and the supply of Y. lipolytica W29 by Prof. Yves Wache, ENSBANA, Dijon, France