TITLE: Lentivirus production &amp; concentration
AUTHORS: Allan JW Lui

[DESCRIPTION]
Lentivirus production protocol based on official protocol for Lipofectamine 3000 and TransIT-Lenti

[BEFORE_START]
Prepare culture media for HEK293T:
  supplemented with 10%   


[STEPS]
SECTION: HEK293T seeding density titration
1. Detach, count & seed HEK293T cells into T75 flasks at:
4 x 10E6 cells
6 x 10E6 cells
8 x 10E6 cells
10 x 10E6 cells

 


SECTION: HEK293T seeding density titration
2. Incubate plates at 37 °C for up to 1440 min 
Observe confluence of cells under a microscope at 18 - 24 hours after plating.

The optimal plating density for transfection yield 80-90% confluence 18 - 24 hours after plating.

SECTION: Lentivirus production (Lipofectamine 3000)
3. Detach, count & seed HEK293T cells at a density that yields 80-90% confluence 18 - 24 hours after plating
Incubate at 37 °C for up to 1440 min 
 

SECTION: Lentivirus production (Lipofectamine 3000)
4. When HEK293T cells are at 80-90% confluence, transfect cells with lentiviral transfer and packaging plasmids using lipofectamine 3000 
 
     
 

Source document:  






SECTION: Lentivirus production (Lipofectamine 3000)
4.3. Make up lipofectamine mix by diluting Lipofectamine 3000 (7ul / 3ug DNA) into Opti-MEM (volume specified in Table 1)
Mix well by vortexing 3 s 


 

SECTION: Lentivirus production (Lipofectamine 3000)
5. Combine both lipofectamine and DNA mixes and incubate for 20 minRoom temperature to allow DNA-lipid complexes to form

Remove 50% volume of media from HEK293T culture vessels intended for transfection
 


SECTION: Lentivirus production (Lipofectamine 3000)
4.2. Make up DNA mix by diluting transfer and packaging plasmids into Opti-MEM, then adding P3000 reagent (2ul / 1ug DNA)
Mix well by vortexing 3 s 
SECTION: Lentivirus concentration
17. 
Add 1 µL   per 1 mL 
Incubate at 4 °C 60 min 

SECTION: Lentivirus production (TransIT-Lenti)
13. Detach, count & seed HEK293T cells at a density that yields 80-90% confluence 18 - 24 hours after plating
Incubate at 37 °C for up to 1440 min 
SECTION: Lentivirus production (Lipofectamine 3000)
6. Gently add DNA-lipid complexes to cells 
 

Gently rock culture vessel back-and-forth and from side-to-side to evenly distribute
Incubate at 37 °C for 360 min 
SECTION: Lentivirus production (Lipofectamine 3000)
8. 5-6 hours after transfection, replace media with pre-warmed packaging medium

Incubate plates at 37 °C


 

SECTION: Lentivirus production (Lipofectamine 3000)
7. Prepare Lentivirus packaging medium (Opti-MEM + 1x GlutaMAX + 1mM sodium pyruvate + 5% FBS) 

Supplement 500 mL     with:
5 mL  
5 mL  
25 mL   

SECTION: Lentivirus production (Lipofectamine 3000)
4.1. Calculate the amount of each plasmid required


Example using lentiCas9-Blast, psPAX2 and pMD2.G:  
 

 
 
 


SECTION: Lentivirus production (Lipofectamine 3000)
10. 48-52 hours post-transfection, collect supernatant from culture vessels and combine with supernatant harvested the day before.
Centrifuge at 300 x g, 5 min to separate any detached cells, and collect the supernatant.

SECTION: Lentivirus production (Lipofectamine 3000)
11. Filter the supernatant through a 45μm PES filter to remove any remaining cellular debris


 


SECTION: Lentivirus production (TransIT-Lenti)
15. When HEK293T cells are at 80-90% confluence, transfect cells with lentiviral transfer and packaging plasmids using TransIT-Lenti 

  

 

SECTION: Lentivirus production (TransIT-Lenti)
15.1. Calculate the amount of each plasmid required


Example using lentiCas9-Blast, psPAX2 and pMD2.G:   
 

 

SECTION: Lentivirus production (TransIT-Lenti)
15.3. Combine plasmids in quantities determined in step 14.1
Add Opti-MEM (Volume specified in Table 2), mix by pipetting
Add TransIT reagent (3ul / 1ug DNA), mix by pipetting

Incubate at Room temperature 10 min to allow transfection complexes to form

 

SECTION: Lentivirus production (TransIT-Lenti)
15.4. Add transfection copmlexes drop-wise to different areas of vessel
Gently agitate culture vessel back-and-forth and from side-to-side to evenly distribute

Incubate at 37 °C for 2880 min 

SECTION: Lentivirus production (Lipofectamine 3000)
9. 24 hours post-transfection, collect supernatant from culture vessels and store at 4 °C 
Replace media with pre-warmed packaging medium
Incubate plates at 37 °C for up to 1680 min 
SECTION: Lentivirus production (TransIT-Lenti)
16. Harvest virus 48 hours post-transfection by following steps 10 - 12.
SECTION: Lentivirus production (Lipofectamine 3000)
12. Prepare single-use aliquots in cryovials and snap freeze virus in a dry ice-ethanol bath, then store at -80°C.

Alternatively, proceed with virus concentration or infection of cells.


 

SECTION: Lentivirus production (TransIT-Lenti)
15.2. Warm TransIT-Lenti reagent to room temperature and vortex 3 s 

SECTION: Lentivirus concentration
18. Centrifuge at 12000 rpm, 60 min, 4 °C 

 

SECTION: Lentivirus concentration
19. Resuspend the pelleted lentiviral preparation in 1/100 of original volume using sterile phosphate buffered saline (PBS). 
SECTION: Lentivirus concentration
20. Prepare single-use aliquots and snap freeze virus in dry ice, then store at -80°C.
SECTION: Lentivirus production (TransIT-Lenti)
14. Change media for HEK293T cells