Nov 23, 2023
Lentivirus production & concentration
This protocol is a draft, published without a DOI.
- 1Cancer Research UK, Cambridge Institute
Protocol Citation: Allan JW Lui 2023. Lentivirus production & concentration. protocols.io https://protocols.io/view/lentivirus-production-amp-concentration-byphpvj6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 30, 2021
Last Modified: November 23, 2023
Protocol Integer ID: 53705
Keywords: Lentivirus production, lentivirus concentration, lentivirus
Abstract
Lentivirus production protocol based on official protocol for Lipofectamine 3000 and TransIT-Lenti
Attachments
Protocol materials
LentiFugeCellectaCatalog #LFVC1
Step 17
Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985047
Step 7
GlutaMAX™ SupplementThermo FisherCatalog #35050038
Step 7
Sodium Pyruvate (100 mM)Thermo FisherCatalog #11360070
Step 7
DMEM, low glucose, GlutaMAX™ Supplement, pyruvateThermo FisherCatalog #21885025
Before starting
Fetal Bovine Serum, qualified, One Shot™ format, BrazilThermo FisherCatalog #A3160801
Before starting, Step 7
Before start
Prepare culture media for HEK293T:
DMEM, low glucose, GlutaMAX™ Supplement, pyruvateThermo FisherCatalog #21885025 supplemented with 10% Fetal Bovine Serum, qualified, One Shot™ format, BrazilThermo FisherCatalog #A3160801
HEK293T seeding density titration
HEK293T seeding density titration
1d
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
Note
>28 x 10E6 cells required in total; recommend detaching cells from 1x T175 at 70-80% confluence.
1h
Incubate plates at 37 °C incubator (5% CO2, humidified) for up to 24:00:00
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.
1d
Lentivirus production (Lipofectamine 3000)
Lentivirus production (Lipofectamine 3000)
Detach, count & seed HEK293T cells at a density that yields 80-90% confluence 18 - 24 hours after plating
Incubate at 37 °C incubator (5% CO2, humidified) for up to 24:00:00
Note
Lentivirus packaging medium (step 7 below) can be used when plating cells & during transfection the next day, but DMEM+10% FBS works well too
1d
When HEK293T cells are at 80-90% confluence, transfect cells with lentiviral transfer and packaging plasmids using lipofectamine 3000
Table 1.
Note
If transfecting multple vessels, make a master mix of 1.1x the total volume required
Source document:
Lipofectamine3000-LentiVirus-AppNote-Global-FHR.pdf
Lipofectamine3000-LentiVirus-AppNote-Global-FHR.pdf Calculate the amount of each plasmid required
Example using lentiCas9-Blast, psPAX2 and pMD2.G:
| A | B | C | D | E | F | G | H | |
| Mass of plasmids per vessel (ug) | ||||||||
| Plasmid | Size (bp) | Equimolar ratio (ug) | 6-well | 10cm | T75 | 150mm | T175 | |
| lentiCas9-Blast | 12859 | 2.209 | 1.313 | 7.88 | 10.50 | 21.00 | 23.63 | |
| psPAX2 | 10709 | 1.839 | 1.093 | 6.56 | 8.75 | 17.49 | 19.68 | |
| pMD2.G | 5822 | 1.000 | 0.594 | 3.57 | 4.75 | 9.51 | 10.70 | |
| Total | 3 | 18 | 24 | 48 | 54 | |||
Note
Optimisation of plasmid ratios may be required for each transfer plasmid; an equimolar ratio is a good starting point. Zhang Lab uses a 4:3:2 transfer : psPAX2 : pMD2.G ratio that approximates an equimolar ratio for lentiCas9-blast, lentiGuide-Puro & lentciCRISPR v2 plasmids. With lentiCas9-blast, 4:2:1, 9:8:1, 7.5:1.5:4 ratios all result in similar titres (+/- 20% of 4:3:2), around 1-2e6 TU/ml.
Make up DNA mix by diluting transfer and packaging plasmids into Opti-MEM, then adding P3000 reagent (2ul / 1ug DNA)
Mix well by vortexing 00:00:03
3s
Make up lipofectamine mix by diluting Lipofectamine 3000 (7ul / 3ug DNA) into Opti-MEM (volume specified in Table 1)
Mix well by vortexing 00:00:03
Note
Lipofectamine 3000 reagent diluted in Opti-MEM medium should be used within 15 minutes of dilution. Longer times can result in a loss of transfection efficiency
3s
Combine both lipofectamine and DNA mixes and incubate for 00:20:00 Room temperature to allow DNA-lipid complexes to form
Remove 50% volume of media from HEK293T culture vessels intended for transfection
| A | B | C | D | E | |
| Volume of media to remove per vessel (ml) | |||||
| 6-well | 100mm dish | T75 | 150mm dish | T175 | |
| 1 | 5 | 7.5 | 15 | 17.5 | |
20m
Gently add DNA-lipid complexes to cells
| A | B | C | D | E | |
| Volume of DNA-lipid complex to add per vessel (ml) | |||||
| 6-well | 10cm dish | T75 | 150mm dish | T175 | |
| 0.5 | 3 | 4 | 8 | 9 | |
Gently rock culture vessel back-and-forth and from side-to-side to evenly distribute
Incubate at 37 °C incubator (5% CO2, humidified) for 06:00:00
6h
Prepare Lentivirus packaging medium (Opti-MEM + 1x GlutaMAX + 1mM sodium pyruvate + 5% FBS)
Supplement 500 mL Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985047 with:
- 5 mL GlutaMAX™ SupplementThermo FisherCatalog #35050038
- 5 mL Sodium Pyruvate (100 mM)Thermo FisherCatalog #11360070
- 25 mL Fetal Bovine Serum, qualified, One Shot™ format, BrazilThermo FisherCatalog #A3160801
5-6 hours after transfection, replace media with pre-warmed packaging medium
Incubate plates at 37 °C incubator (5% CO2, humidified)
Note
Consider supplementation with 1 millimolar (mM) sodium butyrate or 2 millimolar (mM) caffeine to improve titer.
Supplements will be present in viral supernatant if not concentrated
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 incubator (5% CO2, humidified) for up to 28:00:00
48-52 hours post-transfection, collect supernatant from culture vessels and combine with supernatant harvested the day before.
Centrifuge at 300 x g, Room temperature, 00:05:00 to separate any detached cells, and collect the supernatant.
5m
Filter the supernatant through a 45μm PES filter to remove any remaining cellular debris
Note
Smaller filters (22μm) improve purity but can lower the viral titer.
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.
Note
It is recommended to avoid subjecting lentiviral preparations to multiple freeze-thaw cycles, since each cycle can result in a 10%–20% loss in functional titers.
Lentivirus production (TransIT-Lenti)
Lentivirus production (TransIT-Lenti)
Detach, count & seed HEK293T cells at a density that yields 80-90% confluence 18 - 24 hours after plating
Incubate at 37 °C incubator (5% CO2, humidified) for up to 24:00:00
1d
Change media for HEK293T cells
When HEK293T cells are at 80-90% confluence, transfect cells with lentiviral transfer and packaging plasmids using TransIT-Lenti
Note
If transfecting multple vessels, make a master mix of 1.1x the total volume required
Calculate the amount of each plasmid required
Example using lentiCas9-Blast, psPAX2 and pMD2.G:
| A | B | C | D | E | F | G | H | |
| Mass of plasmids per vessel (ug) | ||||||||
| Plasmid | Size (bp) | Equimolar ratio (ug) | 6-well | 10cm | T75 | 150mm | T175 | |
| lentiCas9-Blast | 12859 | 2.209 | 0.875 | 5.25 | 6.56 | 13.13 | 15.31 | |
| psPAX2 | 10709 | 1.839 | 0.729 | 4.37 | 5.47 | 10.93 | 12.75 | |
| pMD2.G | 5822 | 1.000 | 0.396 | 2.38 | 2.97 | 5.94 | 6.93 | |
| Total | 2 | 12 | 15 | 30 | 35 | |||
Note
Optimisation of plasmid ratios is required for each transfer plasmid but an equimolar ratio is a good starting point, Zhang Lab uses a 4:3:2 ratio that approximates an equimolar ratio for lentiCas9-blast, lentiGuide-Puro & lentciCRISPR v2 plasmids.
Warm TransIT-Lenti reagent to room temperature and vortex 00:00:03
3s
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 00:10:00 to allow transfection complexes to form
Note
Precipitation may be observed when excess DNA is used during complex formation. This may
negatively impact transfection efficiency.
10m
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 incubator (5% CO2, humidified) for 48:00:00
2d
Harvest virus 48 hours post-transfection by following steps 10 - 12.
Lentivirus concentration
Lentivirus concentration
1h
Add 1 µL LentiFugeCellectaCatalog #LFVC1 per 1 mL lentiviral supernatant
Incubate at 4 °C 01:00:00
1h
Centrifuge at 12000 rpm, 4°C, 01:00:00
Note
Pellets generated from small supernatant volumes may be difficult to see. It is advised to mark the centrifuge tube or bottle with a marker at the site where you expect the virus pellet to be.
1h
Resuspend the pelleted lentiviral preparation in 1/100 of original volume using sterile phosphate buffered saline (PBS).
Prepare single-use aliquots and snap freeze virus in dry ice, then store at -80°C.