Improve Lentiviral Production Using Lipofectamine 3000 Reagent

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Invitrogen™ Lipofectamine™ 3000 Transfection Reagent is a highly efficient, cost-effective tool for lentiviral production (Figure 1). This versatile reagent enables high viral titers even with genes that are large or difficult to package. Here, we demonstrate the effectiveness of Lipofectamine 3000 reagent for the production of high-quality, high-titer lentivirus while minimizing time and reagent usage. A workflow is provided in Figure 2, followed by a more detailed protocol.

Advantages of lentiviral production using Lipofectamine 3000 reagent:

• Superior performance—higher unconcentrated viral titers than leading reagents (Figure 3).
• Larger gene sizes—high viral titers even with genes that are large or difficult to package.
• Simpler—no additional reagents required for boosting lentiviral production (e.g., chloroquine phosphate, sodium butyrate, caffeine). 
• Faster—time to virus collection is 24 hours less than other reagents, and reverse transfection time is almost 2 days less than other reagents with comparable viral titers. 
• Easier—eliminates the need for coating of plates or flasks.
   

Figure 1. Lentiviral production using Lipofectamine 3000 reagent. A lentiviral construct containing the gene of interest along with lentiviral packaging mix is cotransfected into 293T or 293FT cells using Lipofectamine 3000 reagent. Following incubation of cells, supernatant containing lentivirus is harvested and cellular debris is removed by centrifugation. Depending on the lentiviral construct, viral titer is determined either by blasticidin selection or analyzing the percentage of GFP-positive cells.
 

Figure 2. Lentiviral production workflow using Lipofectamine 3000 reagent.
 

Figure 3. Lipofectamine 3000 reagent performs significantly better than leading competitor transfection reagents for lentiviral production. (A) Titers obtained using Lipofectamine 3000 reagent compared to commonly used reagents in 293FT cells using a 3 kb insert gene. (B) Summary of fold increases in titer levels using two different cell lines and insert sizes. (C) Serial dilution and Blasticidin selection of concentrated virus demonstrates the high titer levels achieved using Lipofectamine 3000 reagent.
 

Materials

Instructions below are tailored to the 6-well plate format. For scaling up transfections, see Table 1.

Day 1 (afternoon): seeding of cells

1. Seed 293T or 293FT cells onto 6-well culture plates at 70–80% confluence. For a 6-well culture plate, plate cells at ~1.2 x 10⁶ cells per well in 2 mL of lentivirus packaging medium.
2. Incubate cells overnight at 37°C, 5% CO₂.

Day 2 (morning): transfection

At time of transfection, cell density should be 95–99% confluent.

1. Bring Opti-MEM I Reduced Serum Medium to room temperature and prepare Tube A and Tube B as described in the following table:

Component	Volume
Tube A
Opti-MEM I Reduced-Serum Medium	250 µL
Lipofecatmine 3000 Transfection Reagent	7 µL
Tube B
Opti-MEM I Reduced-Serum Medium	250 µL
P3000 Enhancer reagent	6 µL
ViraPower Lentiviral Packaging Mix (1 μg/µL)	2.25 µL
pLenti expression vector	0.75 µL

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.

2. To prepare lipid-DNA complexes, transfer contents of Tube A to Tube B and mix well.
3. Incubate the complexes for 10–20 minutes at room temperature.
4. Prior to adding complex, remove 1 mL of medium from each well, leaving a total of 1 mL in each well.
5. Add 500 μL of lipid–DNA complex to each well, taking care to dispense liquid against the well wall to avoid disrupting cells. Gently agitate plate to evenly distribute.
6. Incubate plate for 6 hours at 37°C, 5% CO₂.
7. At 6 hours posttransfection, change plate medium from each well. Carefully remove medium that contains lipid-DNA complexes from each well and treat with 10% bleach prior to disposal. Replace with 2 mL of pre-warmed lentivirus packaging medium.



Note: Because removed medium contains a small amount of lentivirus, it should be appropriately treated with bleach and discarded as per institutional guidelines.

8. Return plate to incubator, and incubate overnight at 37°C, 5% CO₂.

Day 3 (morning): harvest of first batch of virus

1. At 24 hours posttransfection, collect the 2 mL cell supernatant from each well and store in 15 mL conical tubes at 4°C.
2. Replace the collected medium with 2 mL of pre-warmed lentivirus packaging medium.
3.  Incubate the plate overnight at 37°C, 5% CO₂.

Day 4 (mid-afternoon): harvest of second batch of virus

1. Approximately 52 hours posttransfection, collect 2 mL of cell supernatant from each well and combine with the first collection for a total of 4 mL of collected supernatant.



Note: Prior to disposal, treat all cell culture vessels and tips with 10% bleach.

2. Centrifuge the 4 mL of collected supernatant at 2,000 rpm for 10 minutes at room temperature to remove cellular debris. Collect and transfer supernatant, discarding cell pellet.
3. Filter the clarified lentiviral supernatant using a 0.45 μm pore filter to remove any remaining cellular debris.
4. Aliquot virus into cryovials and store at –80°C. Virus can also be concentrated prior to freezing. Limit freezing and thawing of virus aliquots to maintain titer.

Day 1 (morning): complex formation and seeding of cells

1. Prepare cell suspension in pre-warmed lentivirus packaging medium at the appropriate cell concentration (see Table 2).
2. Bring Opti-MEM I Reduced Serum Medium to room temperature and prepare Tube A and Tube B as described in the following table:

Component	Volume
Tube A
Opti-MEM I Reduced-Serum Medium	250 µL
Lipofecatmine 3000 Transfection Reagent	7 µL
Tube B
Opti-MEM I Reduced-Serum Medium	250 µL
P3000 Enhancer reagent	6 µL
ViraPower Lentiviral Packaging Mix (1 μg/µL)	2.25 µL
pLenti expression vector	0.75 µL

3. Add contents of Tube A to Tube B, and incubate for 10–20 minutes at room temperature.
4. Add 500 µL of lipid–DNA complex directly to the plate well.
5. Seed 293T or 293FT cells onto 6-well culture plates at ~3.6 x 10⁶ cells per well in 1 mL of lentivirus packaging medium.



Note: For reverse transfection, seed cells at 3x higher density than for forward transfection.

6. Incubate plate for 6 hours at 37°C, 5% CO2.

Day 1 (afternoon): media change

7. At 6 hours posttransfection, change plate medium from each well. Carefully remove complex-containing medium from each well and treat with 10% bleach prior to disposal. Replace with 2 mL of pre-warmed packaging medium.
8. Return plate to incubator, and incubate overnight at 37°C, 5% CO₂.

Day 2 (morning): harvest of first batch of virus

1. At 24 hours posttransfection, collect the 2mL cell supernatant from each well and store in 15 mL conical tubes at 4°C.
2. Replace the collected medium with 2 mL of pre-warmed packaging medium.
3. Incubate plate overnight at 37°C, 5% CO₂.

Day 3 (mid-afternoon): harvest of second batch of virus

1. Approximately 52 hours posttransfection, collect 2 mL of cell supernatant from each well and combine with first collection for a total of 4 mL of collected supernatant.
2. Centrifuge the 4mL of collected supernatant at 2,000 rpm for 10 minutes at room temperature to remove cellular debris. Collect and transfer supernatant, discarding cell pellet.
3. Filter the clarified lentiviral supernatant using a 0.45 μm pore filter to remove any remaining cellular debris.
4. Aliquot virus into cryovials and store at –80°C. Virus can also be concentrated prior to freezing. Limit freezing and thawing of virus aliquots to maintain titer. Titers achieved using forward and reverse transfection are compared in Figure 4.

Figure 4. Comparative titers achieved using forward vs. reverse transfection. Titers were compared in 293FT and 293T cells using forward and reverse transfection protocols and 2 different gene sizes. Titer levels were comparable between the two methods.
 

Titering by GFP selection

Materials

• Lentivirus
• HT1080 cell line
• Culture medium:
  • Gibco DMEM, high glucose
  • GlutaMAX Supplement,  pyruvate with 10% FBS
• Polybrene™ reagent at 10 mg/mL (Fisher Scientific™ Cat. No. NCO663391)
• Corning™ 96-well round-bottom plate for dilutions (Fisher Scientific™ Cat. No. 05539200)

Day 1 (morning)

This protocol is tailored for a 96-well plate format for high-throughput flow cytometery analysis.

1. Seed a 96-well plate with HT1080 cells at a density of 7,000 cells/well in 100 μL of culture medium.
2. Store in an incubator for 4–5 hours until transduction.
3. Prior to transduction, freshly prepare virus dilutions as follows:
   1. Combine 15 mL of fresh culture medium and 12 μL of 10 mg/mL Polybrene reagent (final concentration of 8 mg/mL). Vortex well to combine.
   
   

   

   2. Per vial sample, add 135 µL of medium prepared in the previous step to 16 wells of a 96-well round-bottom plate in a 4-well by 4-well pattern (see illustration above).
   
   

   Note: n = 4 is recommended for titering.

   3. Add 15 μL of lentiviral supernatant to each well in row 1 (total volume 150 μL; 1:10 dilution).
   4. Mix well using a pipette.
   5. Perform a serial dilution of row 1 (use a multichannel pipette, if available):
      1. Transfer 15 μL from row 1 to row 2, mix well (1:100)
      2. Transfer 15 μL from row 2 to row 3, mix well (1:1,000)
      3. Transfer 15 μL from row 3 to row 4, mix well (1:10,000)
4. To transduce cells, remove plating medium from HT1080 cells and transfer 100µL of the prepared dilutions to each corresponding well (use a multichannel pipette, if available).
5. Centrifuge the plate at 2,000 rpm for 30 minutes at room temperature.
6. Incubate the cell plate overnight.

Day 2

1. Remove medium containing viral supernatant, and replace with fresh HT1080 culture medium (without Polybrene reagent).
2. Incubate cells for an additional 3 days. Analyze the percentage of GFP-positive cells (flow cytometry analysis is recommended).

Calculation of titer

To calculate the titer in transducing units (TU) per mL, determine the appropriate dilution factor to use based on the percentage of GFP-positive cells. The desired transduction range is 1–20%.

Use the following formula:
Titer = (F × C/V) × D

where:
F = frequency of GFP-positive cells (percent GFP-positive cells/100)
C = cell number per well at the time of transduction (7,000 cells)
V = volume of inoculum in mL (0.1 mL)
D = lentivirus dilution factor

Titering by Blasticidin selection

Materials

• Lentivirus
• HT1080 cell line
• Culture medium:
  • Gibco DMEM, high glucose
  • GlutaMAX Supplement,  pyruvate with 10% FBS
• Polybrene™ reagent at 10 mg/mL (Fisher Scientific™ Cat. No. NCO663391)
• Crystal violet (Fisher Scientific™ Cat. No. ICN15251150)
• Selection medium:
  • Culture medium with Gibco™ Blasticidin S HCl (Cat. No. A1113903, 10 µg/mL final concentration)

Day 1 (morning)

This protocol is tailored for a 24-well plate format.

1. Seed 24-well plate with HT1080 cells at a density of 42,000 cells per well (30–50% confluent) in 500 µL of culture medium.
2. Store in incubator for 4–5 hours until transduction.



Note: Duplicate titering is recommended.

3. Prior to transduction, freshly prepare virus dilutions as follows:
   1. Combine 15 mL of fresh culture medium with 12 μL of 10 mg/mL Polybrene reagent (final concentration of 8 mg/mL). Vortex well to combine.
   2. Prepare 5 aliquots of 135 μL of medium from step a, and label tubes as 1–5.
   3. Add 15 μL of lentivirus to tube 1 and pipette to mix well.
   
   Perform a serial dilution:
   i. Transfer 15 µL from tube 1 to tube 2, mix well (1:100)
   ii. Transfer 15 µL from tube 2 to tube 3, mix well (1:1,000)
   iii. Transfer 15 µL from tube 3 to tube 4, mix well (1:10,000)
   iv. Transfer 15 µL from tube 4 to tube 5, mix well (1:100,000)
   
   
   
4. At 4–5 hours post-seeding, aspirate medium from 24-well plate and replace with 450 μL of medium prepared in step a. Number wells 1–5 and a control.
5. Transduce HT1080 cells by transferring 50 μL of dilution 1 to well 1 and mix well by pipetting.



Note: This step further dilutes your virus by 10-fold, thus well 1 now contains a 10² dilution.

6. Repeat Step 5 for remaining prepared dilutions 2–5, transferring dilutions to corresponding numbered wells.
7. For the control, add 50 μL of culture medium with Polybrene reagent (from step a) to control well.
8. Gently swirl the plate to mix.
9. Spin plate at 2,000 rpm at room temperature for 30 minutes.
10. Incubate cell plate overnight.

Day 2

1. Change medium in each well and replace with 500 μL of culture medium (without polybrene).
2. Incubate cells for an additional 24 hours.

Blasticidin selection

1. 48 hours posttransduction, remove medium and replace with selection medium (culture medium with Blasticidin S HCl at 10 μg/mL final concentration)
2. Change and replace the selection medium every 2 days for 10 days.

Note: The control well should have no surviving cells prior to staining.

Day 12

1. Remove medium and wash each well with 1 mL of PBS.
2. Prepare a working solution of 1% crystal violet in a 10% solution of ethanol in water.
3. Stain wells by adding 250 μL diluted crystal violet.
4. Incubate plate for 20 minutes at room temperature.
5. Remove and collect staining solution (staining solution can be reused, or appropriately discarded).
6. Wash plate with water several times to reduce background.

Calculation of titer

To calculate the viral titer, count the number of colonies stained with crystal violet per well. Use the following formula to calculate the titer (TU/mL) of the viral stock:

TU/mL = (No. of discrete colonies x dilution factor)/volume of inoculum