Concentration and Purification of Viral Particles

Abstract

Source: Fricano-Kugler, C. J., et al. Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection. J. Vis. Exp. (2016).

This video demonstrates the step-by-step process for concentrating and purifying lentiviral particles from a mixed suspension containing host cell debris. The procedure utilizes centrifugation, filtration, and PEG-salt precipitation to isolate viral particles while removing soluble impurities. The final product is resuspended in a buffer and stored at ultra-low temperatures to preserve viral infectivity and structural integrity.

Protocol

1. Concentration and Purification of the Virus

1. Make a 5x polyethylene glycol 6000 solution by adding 40% polyethylene glycol 6000 and 1.5 M NaCl to ddH₂O. Autoclave the solution on the liquid cycle for 45 min at 121 °C. Slowly mix the solution when cooling.
   NOTE: The solution will start cloudy and then become clear as it cools.
2. Centrifuge the 50 ml conical tube containing both collections of viral supernatant at 2,000 x g for 10 min in order to pellet insoluble material. Purify the viral media by filtering through a 0.45 µm low protein binding syringe filter (PES or PVDF).
3. Add 5x polyethylene glycol 6000 solution to media (The final concentration should be 8% polyethylene glycol 6000 and 0.3 M NaCl). Mix by inverting the tube several times (do not vortex). Incubate the viral containing polyethylene glycol solution at 4 °C for 12 or more hr, remixing occasionally.
4. (Day 9) Centrifuge the viral containing polyethylene glycol solution at 2,500 x g for 45 min. Remove and discard the supernatant and spin again for 2 min. Again, remove and discard the supernatant.
5. Resuspend the pellet by adding 320 µl of sterile phosphate-buffered saline (the 320 µl is 1/100th of the original volume of viral containing media collected) and incubate overnight at 4 °C. Optionally, resuspend the pellet at room temperature on a rocker for 30 min.
6. After re-suspending the pellet, aliquot the virus (5-10 µl per 0.5 ml tube) and freeze aliquots at -80 °C (freeze thawing or storage at 4 °C will dramatically reduce the titer).
7. Titer the viruses using standard protocols, i.e., perform a dilution series on a 6-well plate of HEK293T cells and manually count fluorescent colonies 48 hr later.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
List of Transfection Reagents
5 ml polystyrene tubes	Fisher Scientific	352054
polyethylene glycol 6000 (PEG 6000)	Millipore	528877
(10x) Phosphate Buffered Saline (PBS)	National Diagnostics	CL-253
0.5 ml microcentrifuge tubes	USA Scientific	1605-0000
Matrigel	Fisher Scientific	CB-40230A