Lentivirus-Production-CGMP

293T cells

1. Thaw a vial of 293T cells and place them in a Nunc T-75 flask.

2. Passage cells every three to four days into Nunc T-500 flasks

3. Transfer cells to 10-layer Nunc Cell Factory trays

Lentivirus production and Purification

1, Seed 1.25 × 10^5 cells/cm2 in 10-layer Corning CELLbind CellStack trays
or at 1 × 10^5cells/cm2 in T-75 flasks.

2. Two days later, transfect cells with three helper plasmids (pCgp, pCMV-Rev2, and pCMV-G) and a lentiviral construct expressing target gene in a ratio of 20:13:5:20 respectively with 0.8 μg DNA/cm2.

3. Remove transfection reagents (CaPO3) after a three-hour incubation

4. Add fresh media containing 2% serum and 6 μM sodium butyrate.

5. Harvest crude supernatant 72 hours later

6. Add Benzonase nuclease at 50 U/mL in the presence of 2 mM Mg for six hours at 37 °C.

7. Clarify culture supernatant by depth filtration using either a 0.45-μm capsule filter (Sartorius) or a 0.5-μm capsule filter (Millipore) with 3,000 cm2 of surface area.

8. Use a hollow-fiber cartridge with a 500-kDa NMWC (GE Healthcare) to concentrate supernatant (from 10 L down to 2 L).

9. diafilter viral supernatant to ~500 mL using 8 L chilled DF buffer (4 g/100 mL lactose from JT Baker in DPBS from MediaTech).

10. Diafilter with an additional 2 L of DF buffer.

11. Centrifuge viral suspension for 16–20 hours at 6,000g.

12. Resuspend pellet and spin at 1,000 rpm for two minutes to pellet and remove insoluble material.

13. Store viral vector at –80 °C

14. p24 Titer Assay using HIV-1 p24 ELISA kit from PerkinElmer (catalog #NEK050).