Lentiviral concentration by anion-exchange chromatography

Protocol 1

Lentiviral concentration by anion-exchange chromatography and ultracentrifugation

1. Set up two Acrodisk units with Mustang Q membranes. Assemble the two Acrodisk units in a tandem manner.

2. Add 27 ml of PBS without Ca/Mg to lentivirus sample.

3. Add 3 ml of buffer C.

4. Pass the sample through the tandem Acrodisk units. Avoid the formation of bubbles.

5. To wash the tandem Acrodisk units, attach a 5-ml syringe containing 3 ml of buffer A. Push the buffer through the two columns. Avoid the formation of bubbles.

6. To elute the vectors, attach a 10-ml syringe containing 10 ml of buffer B and push the buffer through. Collect the eluate in an Ultra-clear SW28 centrifuge tube containing 25 ml of PBS.

7. Centrifuge for 2 h at 25,000 rpm. (82,700g) and 4 oC using an ultracentrifuge.

8. Carefully remove tubes from the rotor, pour off supernatant and leave tubes on a paper towel in an inverted position for 10 min to allow the residual liquid to drip away from the pellet. Aspirate the remaining medium droplets. There should be a miniscule pellet at the bottom of the tube.

9. Add 100 ml of PBS without Ca/Mg to each pellet.

10. Place the SW28 ultracentrifuge tubes into a 50-ml conical tube and close tube with the lid.

11. Incubate the tubes at 4 oC for 2 h. Vortex very gently every 20 min.

12. Spin the tubes at 500g for 1 min at 25 oC to collect the vector-containing liquid.

13. Using a 200-ul pipette, resuspend the pellet by gently pipetting the liquid up and down. Avoid the formation of bubbles. Combine liquid from all resuspended pellets in a single SW28 ultracentrifuge tube.

14. Transfer resuspended vector sample into screw-cap microfuge tubes in 25- to 100-ul portions.

15. Snap-freeze the tubes in crushed dry ice and store at -80 oC.

Buffer A—0.15 M NaCl, 25 mM Tris-HCl, pH 8.0;
Buffer B—1.5 M NaCl, 25 mM Tris-HCl, pH 8.0;
Buffer C—1.5 M NaCl, 250 mM Tris-HCl, pH 8.0.
These buffers can be stored at 4 1C for up to 1 month.

PES filter units, 0.45 mm (Corning, cat. no. 430768)
PES filter units, 0.22 mm (Corning, cat. no. 431096)
Ultra-clear SW28 centrifuge tubes (Beckman, cat. no. 344058)

Protocol 2

Lentiviral concentration by anion exchange chromatography using Mustang Q anion exchange membrane cartridges

1. Sterilize six Ultra-clear SW28 centrifuge tubes by spraying them down with a 70% ethanol solution. Place tubes into a laminar flow hood with the UV light on for 30 min.

2. Set up two Acrodisk units with Mustang Q membranes. Assemble the two Acrodisk units in a tandem manner.

3. Pipette 25 ml of PBS without Ca/Mg into each of the six sterilized Ultra-clear SW28 centrifuge tubes.

4. Add 20 ml of buffer C to 204 ml of the vector-containing cell culture supernatant after passing it through a 250-ml 0.45-uM PES filter flask.

5. To load the lentivirus-containing sample onto the tandem Acrodisk units, draw up about 32 ml of the sample into a 30-ml syringe and pass the sample through the tandem Acrodisk units. Avoid the formation of bubbles during loading of the sample.

6. To wash the sample adsorbed to the Acrodisk units, attach a 5-ml syringe containing 3 ml of buffer A. Push the buffer through the two Acrodisk units. Avoid the formation of bubbles.

7. To elute the vectors, attach a 10-ml syringe containing 10 ml of buffer B and push the buffer through. Collect the eluate in an Ultra-clear SW28 centrifuge tube containing 25 ml of PBS.

8. Regenerate the Acrodisks by passing 5 ml of a 1 N NaOH solution through followed by 5 ml of buffer A.

9. Load another 32 ml of the vector-containing supernatant, repeat this cycle four more times until all of the vector-containing supernatant has been processed. The six Ultra-clear SW28 centrifuge tubes will each contain 10 ml of vector-containing eluate and 25 ml of PBS.

10. Centrifuge the samples using ultracentrifuge.

11. Resuspend the pellets.

12. Aliquot resuspended sample in screw-cap microfuge tubes in 25- to 100-ul portions.

13. Snap-freeze tubes in crushed dry ice and store at -80 oC.

Buffer A—0.15 M NaCl, 25 mM Tris-HCl, pH 8.0;
Buffer B—1.5 M NaCl, 25 mM Tris-HCl, pH 8.0;
Buffer C—1.5 M NaCl, 250 mM Tris-HCl, pH 8.0.
These buffers can be stored at 4 1C for up to 1 month.

PES filter units, 0.45 mm (Corning, cat. no. 430768)
PES filter units, 0.22 mm (Corning, cat. no. 431096)
Ultra-clear SW28 centrifuge tubes (Beckman, cat. no. 344058)
Acrodisk units with Mustang Q membrane, 0.8 mm, 25 mm (Pall, cat. no. MSTG25Q6)