Lentivirus Concentration by Ultracentrifugation

Protocol 1

Concentrate Lentivirus by ultracentrifugation.
Ultracentrifuge: Beckman LE 80K
Rotor: SW28
Tubes: #326823 autoclaved (polyallomer)

1. Pool all viral media into and filter through a 0.45ÎŒm HV Durapore membrane (Millipore #SCHVU01RE).
2. Place tubes into rotor holders and place on a scale. Slowly add ~37ml of virus/media to each tube and balance. (One 15cm plate should correspond to one ultracentrifuge tube).

Make sure the tubes are filled to about 1-3mm from the tube, or the tube will collapse while spinning.

3. Cap holders with a dab of grease and connect to the rotor.

4. Spin at 25,000 rpm (~82,000 × g) for 1 hour 45 min at 4°C. (It is not necessary to prechill the rotor.)

5. Carefully pour off supernatant and invert tube on kimwipes to dry (5-10′ at RT).
There should be a visible pellet.

6. Resuspend each pellet in 125ÎŒl of MEC growth media (or growth media for the cells you will be transducing).

Incubate at 4°C O/N to allow pellets to fully resuspend.

7. Combine all virus into 1 tube.

Dispense into 25ÎŒl aliquots and freeze at -80°C.

Protocol 2

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

2. Transfer ~ 32-ml aliquots of filtered lentivirus-containing cell culture supernatant into each of the six UV-sterilized Ultra-clear SW28 tubes.

3. Fill a 10-ml pipette all the way up to the 12-ml mark with a 20% sucrose solution. Insert the pipette all the way to the bottom of the SW28 tube filled with the filtered vector-containing supernatant and gently underlay it by slowly expelling 4 ml of the sucrose solution. Repeat this step for two more of the SW28 tubes using the remaining 8 ml of the 20% sucrose solution from the same 10-ml pipette. For the remaining three tubes, use a new 10-ml pipette and draw up 12 ml of the 20% sucrose solution and proceed in the same way as for the first three tubes.

4. Adjust the weight of each tube by adding PBS until they are within 0.1 g of each other.

5. Place all the six tubes into a Beckman SW28 ultracentrifuge rotor.

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

7. 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.

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

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

10. Incubate the tubes at 4 1C for 2 h. Vortex very gently every 20 min.

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

12. 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.

If the pooled viral vector-containing suspension is to be subjected to titration, it can be aliquoted in screw-cap microfuge tubes in 25- to 100-ul portions, snap-frozen in crushed dry ice and stored at -80 oC. Alternatively, the pooled viral vector suspension can be further purified by anion exchange chromatography using Acrodisk units with Mustang Q membranes.

20% sucrose solution

Dissolve 20 g of UltraPure sucrose, 100 mM NaCl, 20 mM HEPES (pH 7.4) and 1 mM EDTA. Adjust the volume to 100 ml by adding H2O and filter-sterilize. This solution can be stored at 4 1C for at least 6 months.

Protocol 3

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 4

Before Starting:
required equipments and consumbles:

1. SW-28 rotor with buckets and caps (Kept in 4C Refrigerator in Centrifuge Room)

2. SW-28 thin walled ultra clear centrifuge tubes (Beckman # 344058)

3. 0.45um filters (Corning # 431220)

4. 60mL syringes with luer lok cap (Fisher # 13-689-8)

5. UV irradiate SW-28 thin walled tubes, SW-28 rotor buckets and SW-28 bucket caps in the hood overnight.

Purification Steps:

1. Filter the 40mL of collected viral supernatant through a 0.45um filter using a 60mL syringe into a fresh 50mL centrifuge tube to remove any 293T packaging cells.

2. Place the sterile SW-28 thin walled centrifuge tubes into the SW-28 rotor buckets using sterile forceps. Do not force the SW-28 tubes down into the rotor buckets, let them drop down slowly by themselves.

3. Add 37mL of filtered virus to the sterile SW-28 thin walled centrifuge tubes.

Note: Two plates of virus production yield 40mL. Add 37mL of filtered virus to the SW-28 centrifuge tubes. If necessary, bring the volume up to 37mL with
DMEM media.

Note: It is extremely important that every tube have exactly 37mL. No more and no less.

4. Carefully screw on the caps to the SW-28 rotor buckets, matching the proper caps to the proper tubes. The numbers on the tubes and caps should be aligned.

5. Take the SW-28 capped buckets into the centrifuge room and carefully attach the buckets onto the SW-28 rotor. Be sure the buckets are securely fastened onto the rotor. If they are not securely fastened, they will detach from the rotor and cause the centrifuge to become unbalanced and at high speeds, this could be disastrous.

Note: SW-28 buckets are specific to certain SW-28 rotors. Be sure that the rotor and buckets numbers match.

Note: The tubes inside of the buckets are full. Keep the buckets upright at all times and take care not to tilt them.

6. Place the rotor with attached buckets into the centrifuge, being careful to not tilt anything even slightly. Be sure the rotor is in proper position in the centrifuge. Spin at 16,600 RPM for 90 minutes at 4oC.

7. When the spin is complete, remove the buckets carefully from the rotor, and bring back into the virus room. Place the rotor back in the 4 oC fridge.

8. Unscrew the caps, and aspirate the first 10mL of media in the tubes (to avoid spilling).

9. Using sterile forceps, remove the tubes from the rotor buckets and place into a tube rack.

10. Carefully aspirate off the media by tilting the tube so that the media flows towards the top and gets aspirated.

Note: The pellet will be invisible, but will be stuck in the bottom and should not move with the supernatant as you tilt.

Note: When tilting to aspirate, be sure you never tilt back. Tilting back can cause the supernatant to splash back into the bottom of the tube and disrupt the pellet.

11. Add 500ul of plain OptiMEM media (no serum, no antibiotic) to the pellet. Cover the entire tube holder rack with saran wrap and place in the cold room overnight.

12. The next morning, resuspend the virus carefully by pipet. Pipet up and down about 20 times, being careful to avoid making too many bubbles.

13. Combine all the tubes together and mix gently. Aliquot the pooled virus into 1.5mL microfuge tubes. Each tube should contain 200-500ul.

14. Remove 20ul of resuspended, combined virus from one of the tubes for titering.

15. Freeze the tubes of concentrated virus at -80 oC. Be sure to label tubes with clone number and lot number.

16. Titer the virus using a p24 HIV ELISA kit.