Lentivirus Concentration by Ultrafiltration

1. Grow low passage (<12) 293T cells below confluence in 500 cm2 TripleFlasks in 150 mL D10 medium

(DMEM with 10% fetal bovine serum, 50 U/mL penicillin,50 ug/mL streptomycin, and 2 mM l-glutamine).

Passage cells at 1:4–1:5 every 2 days, or 1:8–1:10 every 3 days.

2. Transfect cells with lentivirus plasmids mix

Add 1 mL GeneExpresso to 50 mL of DMEM

Add
150 ug of vector plasmid,
150 ug of pGagPol
30 ug of pCMV-VSVG
incubated for 20 min at room temperature.

3. Wash cellwith 15 mL 0.05% trypsin EDTA in HBSS to bind and remove trypsin inhibitors.
digest with 15 mL of 0.05% trypsin incubate for 3–5 min incubation at 37 oC.
Add 15 mL of D10 to inactivate the trypsin.
Transfer cells a sterile polystyrene bottle.
Rinse TripleFlask with 30 mL of D10 .
Combine cells.

4. Add transfection mix to 7 × 10^8 cells (from three TripleFlasks, NUNC 132867) in ∼150 mL D10.

5. Transfer cells/transfection mix to a HYPERFlask. Add D10 to dislodge air bubbles from the layers

6. Place the flask in a horizontal position at 37 oC overnight.

7. Change medium 18–20 h post-transfection: D10 containing 10 mM sodium butyrate and 20 mM HEPES.

8. After 6–8 h, rinse cells once with 500 mL DPBS and then add fresh harvesting medium, consisting of UltraCULTURE
with 20 mM HEPES, 50 U/mL penicillin, 50 ug/mL streptomycin, and 2 mM l-glutaminewas added to HYPERFlask (Corning 10010.

Use one finger to gently tap the side of the flask, while held in a vertical position to dislodge air without appreciably disrupting the cell monolayers.

9. About 40 h after addition of harvesting medium, harvest LV-containing medium from HYPERFlasks, store medium at 4 oC.

10. Add fresh medium.

11. Harvest medium 24 h later

12. Filter medium with 0.45 or 0.8 micron filter. Combine filtered medium

13. Concentrate filtered medium using KrosFlo Research II TFF System
KrosFlo Research II TFF System (Spectrum Labs, SYR2-U20-01 N).
Flow path 1 [FPI] (Spectrum Labs, 500 kDa, EZ-M1-500S-260-01N-I).
Flow path 2 [FPII] (Spectrum Labs, EZ-CHIL07-01-I).