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 targe...
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| Format: | Article |
| Language: | English |
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BMC
2009-02-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/9/10 |
| _version_ | 1818006709929508864 |
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| author | Marino Michael P Puthli Sharon Kutner Robert H Reiser Jakob |
| author_facet | Marino Michael P Puthli Sharon Kutner Robert H Reiser Jakob |
| author_sort | Marino Michael P |
| collection | DOAJ |
| description | <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> |
| first_indexed | 2024-04-14T05:05:03Z |
| format | Article |
| id | doaj.art-f408c1dae6d0480ea9a410a0e42af703 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-14T05:05:03Z |
| publishDate | 2009-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-f408c1dae6d0480ea9a410a0e42af7032022-12-22T02:10:44ZengBMCBMC Biotechnology1472-67502009-02-01911010.1186/1472-6750-9-10Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatographyMarino Michael PPuthli SharonKutner Robert HReiser Jakob<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>http://www.biomedcentral.com/1472-6750/9/10 |
| spellingShingle | Marino Michael P Puthli Sharon Kutner Robert H Reiser Jakob Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography BMC Biotechnology |
| title | Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography |
| title_full | Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography |
| title_fullStr | Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography |
| title_full_unstemmed | Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography |
| title_short | Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography |
| title_sort | simplified production and concentration of hiv 1 based lentiviral vectors using hyperflask vessels and anion exchange membrane chromatography |
| url | http://www.biomedcentral.com/1472-6750/9/10 |
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