Optima X Series Ultracentrifuges and SW 32 TI Rotor for Lentiviral Vector Preparation

Viral vectors are commonly applied in the manipulation of mammalian genes, for both gene silencing and gene expression. Lentiviruses are frequently used vectors because they can infect both dividing cells and non-dividing cells, including stem cells. In cases where a large number of cells need to be infected or when the cell line is resistant to transduction, the virus particles often need to be concentrated to attain high titer. This concentration of viral particles can be achieved simply and effectively using centrifugation.

Optima XPN Centrifuge

Optima XPN Centrifuge

This article presents two simple techniques for concentrating these viral vectors.

These techniques can also be used to concentrate viral particles for other studies such as electron microscopy or nucleic acid sample prep.

Concentration of Lentiviral Particles using Polyethylene Glycol (PEG):

The steps followed for concentrating lentiviral particles using polyethylene glycol are detailed below:

  1. In order to eliminate any cell debris and loose cells, the viral supernatant is collected from transfected packaging cells and passed through a sterile 0.45µm filter.
  2. The supernatant is mixed with 40% PEG solution to achieve a final PEG concentration of 10%, after which the mixture is incubated on ice for 3 to 6 hours.
  3. The mixture is then spun in a centrifuge at 2000 x g for 30 minutes.
  4. The supernatant is then discarded and the viral particles pellet is dispersed by pipetting in 1/20 of the original harvest PBS (phosphate buffered saline) volume or another media of choice.
  5. The viral particles are then moved to pre-sterilized ultracentrifuge tubes for further concentration.
  6. The tubes are placed in buckets and then weighed and balanced.
  7. In a Beckman Optima X Series ultracentrifuge, the tubes are spun at 100,000 x g (24,500rpm) at 4°C in a SW 32 Ti rotor for 90 minutes.
  8. The tubes are then inverted or pipetted to remove the supernatant, with care taken to ensure that the viral pellet is not dislodged.
  9. The pellet is then resuspended in PBS or the media of choice.
  10. To completely dissolve the pellet, pipette up and down or shake for a few minutes if necessary.
  11. Next, aliquot and store at the preferred temperature. For long term storage, ultra-low temperature (ULT) storage is advised.

Concentration of Lentiviral Particles Using Sucrose Cushion

The method for concentrating Lentiviral particles using sucrose cushion is detailed below:

  1. To eliminate any cell debris and loose cells, the viral supernatant is collected from transfected packaging cells and passed through a sterile 0.45µm filter.
  2. 3-5mL of 20% sucrose is carefully added to the bottom of pre-sterilized ultracentrifuge tubes.
  3. The viral supernatant is carefully layered over the sucrose cushion.
  4. The tubes are then placed into buckets and weighed and balanced
  5. Using a Beckman Optima X Series ultracentrifuge, the supernatant is centrifuged at 125000 x g at 4°C in SW 32 Ti rotor for 90 minutes.
  6. Without dislodging the viral pellet, the tubes are inverted or pipetted to remove the supernatant.
  7. Next, the pellet is resuspended in PBS or the media of choice.
  8. To completely dissolve the pellet, pipette up and down or shake for a few minutes, if necessary.
  9. Next, aliquot and store at the preferred temperature. For long term storage, ULT storage is advised.

References:

  1. Lentiviral vector production—DS-12662A.
  2. Miest T, Saenz D, Meehan A, Llano M, Poeschla E; Intensive RNAi with lentiviral vectors in mammalian cells—Methods. 2009 April; 47(4): 298–303.
  3. Houzet L, Morichaud Z, Didierlaurent L, Muriaux D, Darlix JL, Mougel M; Nucleocapsid mutations turn HIV-1 into a DNA-containing virus—Nucleic Acids Res. 2008 April; 36: 2311–2319.

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Last updated: Mar 1, 2019 at 4:38 AM

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