For Research Use Only. Not for use in diagnostic procedures. LV-MAX ™ Lentiviral Production System USER GUIDE For suspension format lentiviral production in a chemically defined, serum-free medium Catalog Number A35684 Publication Number MAN0017000 Revision D.0
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For Research Use Only. Not for use in diagnostic procedures.
LV-MAX™ Lentiviral Production SystemUSER GUIDE
For suspension format lentiviral production in a chemically defined,serum-free medium
Catalog Number A35684Publication Number MAN0017000
Revision D.0
Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0017000
Revision Date DescriptionD.0 22 March 2019 Overhaul of the user guide to bring it up to current style and standards.
C.0 24 August 2018 Remove a related product
B.0 13 June 2018 Add flask type
A.0 14 July 2017 New document
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
IMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.
Product description
The Gibco™ LV-MAX™ Lentiviral Production System is a high-yield lentiviralproduction system based on HEK293F cells adapted to a special chemically defined,serum-free and protein free LV-MAX™ Production Medium in suspension form.
The LV-MAX™ Lentiviral Production System provides cells, production medium,supplement, transfection reagent, and enhancer to produce high titer lentiviralvectors.
Contents and storage
Reagents provided in the kit are sufficient for 300 mL of lentiviral production volume.
Table 1 LV-MAX™Lentiviral Production System Starter Kit (Cat No. A35684)
Component Cat. No. Amount Storage
Viral Production Cells[1] (1 X 107 cells/mL) A35347 2 × 1 mL Liquid nitrogen[2]
LV-MAX™ Production Medium A3583401 1 L
• 2°C to 8°C
• Protectedfrom light
LV-MAX™ Transfection Kit
• LV-MAX™ Supplement
• LV-MAX™ Transfection Reagent
• LV-MAX™ Enhancer
A35346 1 Kit
• 15 mL
• 2 × 0.9 mL
• 12 mL
[1] In 90% LV-MAX™ Production Medium and 10% DMSO[2] Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at −80°C.
1
6 LV-MAX™ Lentiviral Production System User Guide
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
Table 2 Materials required for lentiviral vector production (all methods)
Item Source
CO2 resistant orbital shaker 88881101
125-mL shaker flasks for Viral Production Cells MLS
Adjustable micropipettors MLS
Laboratory mixer (Vortex mixer or equivalent) MLS
Equipment and reagents to determine cell density and viability MLS
LV-MAX™ Packaging Mix A43237
Opti-MEM™ I Reduced Serum Medium 31985088
For cryopreservation:
Sterile 0.2-μm filter MLS
Cryovials MLS
Table 3 Additional materials required for lentiviral vector production in a 96-deepwell block
Item Source
3 mm orbital shaker MLS
2-mL sterile 96-deep well block MLS
2-mL sterile 96-deep well block (V bottom) MLS
96-well round bottom plate MLS
Multi-channel micropipettors MLS
PureLink™ Air Porous Tape 12262010
Table 4 Additional materials required for lentiviral vector production in a 50-mLconical tube
Item Source
Nunc 50-mL conical tube 339653
Corning™ Mini Bioreactor Centrifuge Tube 07-202-150
50-mL conical tube holder MLS
Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterile
Chapter 1 Product informationRequired materials not supplied 1
Table 5 Additional materials required for lentiviral vector production in a shaker flask
Item Source
Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterile
125 mL 4115-0125
250 mL 4115-0250
1 L 4115-1000
Components of the Gibco™ LV-MAX™ Lentiviral Production System
The Gibco™ LV-MAX™ Lentiviral Production System is designed to produce high-titerlentiviral vectors using transient transfection of a high-density suspension cell systemcultured in chemically defined, serum-free medium.
The Gibco™ Viral Production Cells are a clonal derivative of the original HEK293F cellline, that have been adapted to suspension culture in LV-MAX™ Production Medium.These cells can be thawed directly into LV-MAX™ Production Medium.
Cell line characteristics:• Transformed via culture with sheared human adenovirus 5 DNA• Expresses E1A adenoviral gene• Lacks the SV40 large T antigen• Cell doubling time of ~26 hours• Achieves maximum cell densities of ~1 × 107 cells/mL in shaker flask cultures• High lentiviral production capabilities between cell passages 5–20
LV-MAX™ Production Medium is a complete, chemically defined, serum-free andprotein-free medium, developed for growth and transfection of Gibco™ ViralProduction Cells.
LV-MAX™ Supplement is a chemically defined, serum-free and protein-freeformulation designed to control cell growth during transfection and increase lentiviralvector production without compromising cell viability.
LV-MAX™ Transfection Reagent is uniquely designed for high efficiency co-transfection of multiple plasmids into high density Gibco™ Viral Production Cells,with low toxicity.
Viral productioncells
Growth medium
Supplementsolution
Transfectionreagent
Chapter 1 Product informationComponents of the Gibco™ LV-MAX™ Lentiviral Production System1
8 LV-MAX™ Lentiviral Production System User Guide
LV-MAX™ Enhancer is a chemically defined, serum-free, protein-free formulation thatis designed to boost lentiviral vector production in Gibco™ Viral Production Cells.
Enhancer solution
Chapter 1 Product informationComponents of the Gibco™ LV-MAX™ Lentiviral Production System 1
LV-MAX™ Lentiviral Production System User Guide 9
Thaw and establish Gibco™ ViralProduction Cells
Procedural guidelines
• All solutions and equipment that come in contact with cells must be sterile.• Thaw and transfer cells directly into a 125-mL shaker flask with 30 mL of pre-
warmed 37°C LV-MAX™ Production Medium. Centrifugation and mediumchange are not required when seeding the cells.
• Do not centrifuge the cells as this can lower cell viability.• Three days post-thaw, viable cell density should be approximately 1 × 106
cells/mL with ≥ 90% cell viability.• Subculture cells when they reach a density of ~3.5 to 5.5 x 106 viable cells/mL,
typically every 3–4 days.• Allow freshly thawed cells to recover in culture for 5 passages post-thaw before
using cells for virus production.• Inspect cells to ensure that round cell morphology is maintained over passages
for most of the cell population with minimal cell clumping. Proper cell phenotypeis critical for optimal transfection and virus production.
• Warm LV-MAX™ Production Medium to room temperature.For a faster warmup, place your medium at 37°C for no longer than 20 minutes.
• We recommend discarding cells after passage 20 from thaw.• The following table shows the recommended cell density for routine cell culture.
Subculture Seeding density
3 day 0.55 × 106 viable cells/mL
4 day 0.35 × 106 viable cells/mL
• The following table shows the recommended shaker speed and culture volumefor cultivation.
Production vessel Shaker speed(Orbit diameter) Vessel size
Cell culture volumerange (24%−40% of
vessel size)
Shaker flask(vented, notbaffled)
125 rpm (19 mm)120 rpm (25 mm)95 rpm (50 mm)
125 mL 30 mL − 50 mL
250 mL 60 mL − 100 mL
1 L 240 mL − 400 mL
2
10 LV-MAX™ Lentiviral Production System User Guide
Count suspension cells
Cell density is the critical driver of successful lentiviral production. The cell densitydepends on accurate cell counting. Follow these steps to ensure consistent andaccurate cell counts.
1. To minimize settling of cells after agitation, load the pipet aid in preparation.
2. Shake the flask in an orbital path,clockwise 3 times.
3. Shake the flask in an orbital path,counter clockwise 3 times.
4. Shake the flask back and forth threetimes, then left and right three times.
5. Pipet the cells up and down 3 times.
6. Remove 1 mL for cell counting, then count the cells.
7. Repeat cell counting at least 2 times to ensure accurate cell densities.Recount any cell counts with >10% variability to ensure a reliable average celldensity.
Chapter 2 Thaw and establish Gibco™ Viral Production CellsCount suspension cells 2
LV-MAX™ Lentiviral Production System User Guide 11
Workflow: Thaw and establish Gibco™ Viral Production Cells
Thaw cells
Cul
ture
cel
ls
Culture cells
Continue
P0
P1
P2
P3
P4
P5
P6
subculture
1. Day 1
3. Day 8
5. Day 15
7. Day 22
4. Day 12
6. Day 19
2. Day 4
Week 1
Week 2
Week 3
Week 4
Follow LV production protocol
8. Subculture and prepare cells for
next day LV production
Monday
Thursday
Monday
Friday
Monday
Friday
Monday
Chapter 2 Thaw and establish Gibco™ Viral Production CellsWorkflow: Thaw and establish Gibco™ Viral Production Cells2
12 LV-MAX™ Lentiviral Production System User Guide
Thaw and establish Gibco™ Viral Production Cells
Week 1: P0−P11. Day 1 Passage 0 (P0): Partially thaw a vial of Gibco™ Viral Production Cells in a
37°C water bath until only a small piece of ice remains (~2–3 minutes).
Note: We recommend starting on a Monday.
2. Transfer the contents of the vial into a 125-mL shaker flask containing 30 mL ofpre-warmed LV-MAX™ Production Medium.
Note: Centrifugation and medium change are not required for these cells.
3. Place the flask on an orbital shaker platform in an incubator set to 37°C, 8% CO2,and 80% relative humidity (for recommended shaker speeds, see Table 3).
4. Day 4 Passage 1 (P1): Seed cells at a density of 0.35 × 106 cells/mL in a totalculture volume of 40 mL in a new 125-mL shaker flask.
Week 2: P2−P35. Day 8 Passage 2 (P2): Seed cells at a density of 0.35 × 106 cells/mL in a total
culture volume of 40 mL in a new 125-mL shaker flask.
Note: By Passage 2, cell viability should be ≥90%.
6. Day 12 Passage 3 (P3): Seed cells at a density of 0.55 × 106 cells/mL in a totalculture volume of 40 mL in a new 125-mL shaker flask.
Note: Aliquot some cells for long-term storage, see “Cryopreserve Gibco™ ViralProduction Cells“ on page 14.
Week 3: P4−P57. Day 15 Passage 4 (P4): Seed cells at a density of 0.35 × 106 cells/mL in a total
culture volume of 75 mL in a new 250-mL shaker flask.
8. Day 19 Passage 5 (P5): Seed cells at a density of 0.55 × 106 cells/mL in 300 mL oftotal culture volume in a 1-L shaker flask to ensure appropriate cell numbers forlentivirus production.
Week 4: Cells are established and ready for lentivirus production9. Day 22 Continue to subculture cells when required until P20. For recommended
seeding densities for subculture, see List item. on page 10.
Proceed with lentiviral production.• For high throughput lentiviral production in a 2-mL 96-deep well block, proceed
to Chapter 3, “Produce lentiviral vector in a 2-mL 96-deep well block“.• For lentiviral production in a 50-mL conical tube, proceed to Chapter 4, “Produce
lentiviral vector in a 50-mL conical tube“.• For lentiviral production in a shaker flask, proceed to Chapter 5, “Produce
lentiviral vector in a shaker flask“.
Chapter 2 Thaw and establish Gibco™ Viral Production CellsThaw and establish Gibco™ Viral Production Cells 2
LV-MAX™ Lentiviral Production System User Guide 13
Cryopreserve Gibco™ Viral Production Cells
Use cells at passage 3 when cell density reaches 3.5−5.5 × 106 viable cells/mL and cellviability is >95%.
1. Prepare the appropriate amount of cryopreservation medium by combining LV-MAX™ Production Medium and DMSO according to the following table.
Reagent volume to add
LV-MAX™ Production Medium 9 mL
DMSO 1 mL
Total 10 mL
2. Mix well, then filter through a 0.2-µm filter.
3. Centrifuge the cells at 100 × g for 5 minutes to collect the contents at the bottom,then discard the supernatant.
4. Gently resuspend the cell pellet in cryopreservation medium.
5. Dilute the cells with cryopreservation medium to a final density of 1 × 107 viablecells/mL.
6. Aliquot 1 mL of cells per cryovial, then freeze the cells at −80°C for one day.
7. Transfer the frozen vials to liquid nitrogen for long-term storage.
Chapter 2 Thaw and establish Gibco™ Viral Production CellsCryopreserve Gibco™ Viral Production Cells2
14 LV-MAX™ Lentiviral Production System User Guide
Produce lentiviral vector in a 2-mL96-deep well block
Procedural guidelines
• Follow the guidelines in Chapter 2, “Thaw and establish Gibco™ Viral ProductionCells“ to culture cells.
• Warm LV-MAX™ Production Medium to room temperature before transfection.• The LV-MAX™ Supplement, LV-MAX™ Transfection Reagent, and Opti-MEM™ I
Reduced Serum Medium can be used at 2°C to 25°C.• LV-MAX™ Enhancer can be added 5–14 hours posttransfection.• Recommended long-term storage of lentiviral vectors is at −80˚C.• Avoid repeated freeze and thaw cycles as it can decrease lentiviral vector activity.• Each well of the 2-mL 96-deep well block is appropriate for 1 mL of lentivirus
production.• The lentiviral production volume for each 96-deep well block is 105 mL, which
includes 10% overage.• Triplicate runs are recommended for each individual lentiviral transfer vector.
Optimized transfection conditions
Condition Amount
Total Opti-MEM™ I Reduced Serum Medium 2 × 5% of lentivirus production volume
Total DNA per mL of lentivirus production 2.5 μg/mL (LPM + LTV)
Ratio of lentiviral packaging mix (LPM) to lentiviraltransfer vector (LTV) 3 to 2
LV-MAX™ Transfection Reagent 6 µL/mL of lentivirus production volume
LV-MAX™ Enhancer 4% of the lentivirus production volume
Gibco™ Viral Production Cells 4 × 106 cells/mL production volume
Total LV-MAX™ Supplement 5% of lentivirus production volume
3
LV-MAX™ Lentiviral Production System User Guide 15
Workflow: Lentiviral production in a 2-mL 96-deep well block
1. Prepare cells
2. Plate cells
3. Make complex
4. Transfect cells
5. Add enhancer
7. Measure Titer
Day 0Monday
Day 1Tuesday
Day 3Thursday
24 - 28hr
1min
10min
5-6 hr
48 -55 hr
TfxRDNA
AB
CD
EF
GH
12
34
56
78
910 11 12
AB
CD
EF
GH
12
34
56
78
910 11 12
21
6. Harvest lentiviral vector
Chapter 3 Produce lentiviral vector in a 2-mL 96-deep well blockWorkflow: Lentiviral production in a 2-mL 96-deep well block3
16 LV-MAX™ Lentiviral Production System User Guide
Transfect cells and produce the lentiviral vector
Note: This workflow guides you through the production of lentiviral vector from105 mL of production volume, which is enough for one 96-well block. For scaling uptables, see Appendix A, “Scaling up tables“.
1. Determine the cells/mL of the 3-day cultured high density cells.
2. Dilute the high density cells to 3.5 × 106 viable cells/mL, then subculture for afurther 24 hours.
1. Count the high density cells.
2. In a new sterile flask, add 4.20 × 108 cells to LV-MAX™ Production Medium tomake a final volume of 89.25 mL.
3. Add 5.25 mL of LV-MAX™ Supplement.
4. Mix the cells well by pipetting up and down, then transfer 900 µL to each well ofa 2-mL 96-deep well block.
5. Seal the 96-deep well block plate with PureLink™ Air Porous Tape, then place ona 3-mm orbital shaker at 1,250 rpm in an incubator.
1. Add 5.25 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 1: DNA.
2. Add 157.5 µg of packaging plasmid.
Note: The ratio of packaging plasmid to transfer plasmid is 3 to 2. For 2.5 µg/mLof total DNA, this translates to 1.5 µg of packaging plasmid to 1 µg of transferplasmid.
3. Pipet 50 µL into each well of a 96-well round bottom plate.
4. Add 1 µg of transfer plasmid to each well.
5. Add 5.25 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 2: TfxR.
6. Add 630 µL of LV-MAX™ Transfection Reagent (6 µL/mL).
7. Vortex briefly to mix, then incubate for 1 minute at room temperature.
8. Immediately pipet 50 µL into each well of the 96-well round bottom platecontaining the prepared DNA solution, then mix well by pipetting up and down.
9. Incubate the combined solution for 10 minutes at room temperature.
1. Add 100 µL of the DNA-lipid complex to each well of the previously prepared96-deep well block of cells.
Note: The DNA-lipid complex is stable for up to 1.5 hours.
Prepare cells
Dilute cells
Make complexes
Transfect cells
Chapter 3 Produce lentiviral vector in a 2-mL 96-deep well blockTransfect cells and produce the lentiviral vector 3
LV-MAX™ Lentiviral Production System User Guide 17
2. At 5–6 hours posttransfection, add 40 µL of LV-MAX™ Enhancer to each well ofthe 96-deep well block.
Note: The optimum time frame for LV-MAX™ Enhancer addition is anytimebetween 5–14 hours posttransfection.
1. At 48–55 hours posttransfection, centrifuge the 96-deep well block at 900 × g in aswinging bucket centrifuge for 15 minutes.
2. Harvest the lentiviral vector by transferring the supernatant to a fresh 96-wellround bottom plate.
Note: To avoid multiple freeze thaw cycles, aliquot the supernatant to multiple96-well round bottom plates.
3. Immediately following harvest, store the lentiviral vector at -80°C.
Thaw, then titer the lentiviral vector using your method of choice.
Note: Thaw the virus on ice before use.
For recommended protocols, see Chapter 6, “Titer lentiviral vector“.You can optimize your lentiviral vector yield by trying different starting cell densities,such as 2.5, 3, and 4 × 106 viable cells/mL.
Harvest thelentiviral vector
Titer the lentiviralvector
Chapter 3 Produce lentiviral vector in a 2-mL 96-deep well blockTransfect cells and produce the lentiviral vector3
18 LV-MAX™ Lentiviral Production System User Guide
Produce lentiviral vector in a 50-mLconical tube
Procedural guidelines
• Follow the guidelines in Chapter 2, “Thaw and establish Gibco™ Viral ProductionCells“ to culture cells.
• Warm LV-MAX™ Production Medium to room temperature before transfection.• The LV-MAX™ Supplement, LV-MAX™ Transfection Reagent, and Opti-MEM™ I
Reduced Serum Medium can be used at 2°C to 25°C.• LV-MAX™ Enhancer can be added 5–14 hours posttransfection.• Recommended long-term storage of lentiviral vectors is at −80˚C.• Avoid repeated freeze and thaw cycles as it can decrease lentiviral vector activity.• This protocol is appropriate for lentiviral production volumes of 5–30 mL.• Fix a 50-mL conical tube holder onto an orbital shaker in an incubator. Set the 19-
mm orbital shaker speed to 250 rpm.
Optimized transfection conditions
Condition Amount
Total Opti-MEM™ I Reduced Serum Medium 2 × 5% of lentivirus production volume
Total DNA per mL of lentivirus production 2.5 μg/mL (LPM + LTV)
Ratio of lentiviral packaging mix (LPM) to lentiviraltransfer vector (LTV) 3 to 2
LV-MAX™ Transfection Reagent 6 µL/mL of lentivirus production volume
LV-MAX™ Enhancer 4% of the lentivirus production volume
Gibco™ Viral Production Cells 4 × 106 cells/mL production volume
Total LV-MAX™ Supplement 5% of lentivirus production volume
4
LV-MAX™ Lentiviral Production System User Guide 19
Workflow: Lentiviral production in a 50-mL conical tube
2. Dilute cells
3. Make complex
4. Transfect cells
5. Add enhancer
7. Measure Titer
24-28hr
1min
10min
5-14 hr
48-55 hr
TfxR DNA
AB
CD
EF
GH
12
34
56
78
910 11 12
2 1
Day 0Monday
Day 1Tuesday
Day 3Thursday
1. Prepare cells
6. Harvest lentiviral vector
Chapter 4 Produce lentiviral vector in a 50-mL conical tubeWorkflow: Lentiviral production in a 50-mL conical tube4
20 LV-MAX™ Lentiviral Production System User Guide
Transfect cells and produce the lentiviral vector
Note: This workflow guides you through the production of lentiviral vector from10 mL of production volume, in one 50-mL conical tube. For scaling up tables, see Appendix A, “Scaling up tables“.
1. Determine the cells/mL of the 3-day cultured high density cells.
2. Dilute the high density cells to 3.5 × 106 viable cells/mL, then subculture for afurther 24 hours.
1. Count the high density cells.
2. In a new 50-mL conical tube, add 4 × 107 cells to LV-MAX™ Production Mediumto make a final volume of 8.5 mL.
3. Add 0.5 mL of LV-MAX™ Supplement.
4. Mix the cells well by pipetting up and down, then place on a 19-mm orbitalshaker at 250 rpm in an incubator.
5. Use a mini bioreactor cap to seal the top of the conical tube in place of theoriginal cap.
1. Add 0.5 mL of Opti-MEM™ I Reduced Serum Medium to a 1.5-mLmicrocentrifuge tube and label it Tube 1: DNA.
Note: The ratio of packaging plasmid to transfer plasmid is 3 to 2. For 2.5 µg/mLof total DNA, this translates to 1.5 µg of packaging plasmid to 1 µg of transferplasmid.
2. Add 15 µg of packaging plasmid.
3. Add 10 µg of transfer plasmid, then vortex briefly to mix.
4. Add 0.5 mL of Opti-MEM™ I Reduced Serum Medium to a 1.5-mLmicrocentrifuge tube and label it Tube 2: TfxR.
5. Add 60 µL of LV-MAX™ Transfection Reagent (6 µL/mL).
6. Vortex briefly to mix, then incubate for 1 minute at room temperature.
7. Combine Tube 1:DNA with Tube 2:TfxR, then mix well by pipetting up anddown.
8. Incubate the combined solution for 10 minutes at room temperature.
1. Add 1 mL of the DNA-lipid complex to the previously prepared tube of cells,then mix well by pipetting up and down gently.
Note: The DNA-lipid complex is stable for up to 1.5 hours.
Prepare cells
Dilute cells
Make complexes
Transfect cells
Chapter 4 Produce lentiviral vector in a 50-mL conical tubeTransfect cells and produce the lentiviral vector 4
LV-MAX™ Lentiviral Production System User Guide 21
2. At 5–6 hours posttransfection, add 0.4 mL of LV-MAX™ Enhancer.
Note: The optimum time frame for LV-MAX™ Enhancer addition is anytimebetween 5–14 hours posttransfection.
1. At 48–55 hours posttransfection, collect the culture medium, then centrifuge thecells at 1,300 × g in a swinging bucket centrifuge for 15 minutes.For smaller volumes, use a 1.5-mL microcentrifuge tube and centrifuge in abenchtop centrifuge at 13,000 rpm for 5 minutes.
2. Harvest the lentiviral vector by transferring the supernatant to a fresh tube.
3. Further remove cell debris by filtration through a 0.45 µm low protein bindingfilter.
4. Immediately following harvest, store the lentiviral vector at -80°C.
Note: To avoid multiple freeze thaw cycles, aliquot the supernatant to multipletubes.
Thaw, then titer the lentiviral vector using your method of choice.
Note: Thaw the virus on ice before use.
For recommended protocols, see Chapter 6, “Titer lentiviral vector“.You can optimize your lentiviral vector yield by trying different starting cell densities,such as 2.5, 3, and 4 × 106 viable cells/mL.
Harvest thelentiviral vector
Titer the lentiviralvector
Chapter 4 Produce lentiviral vector in a 50-mL conical tubeTransfect cells and produce the lentiviral vector4
22 LV-MAX™ Lentiviral Production System User Guide
Produce lentiviral vector in a shakerflask
Procedural guidelines
• Follow the guidelines in Chapter 2, “Thaw and establish Gibco™ Viral ProductionCells“ to culture cells.
• Warm LV-MAX™ Production Medium to room temperature before transfection.• The LV-MAX™ Supplement, LV-MAX™ Transfection Reagent, and Opti-MEM™ I
Reduced Serum Medium can be used at 2°C to 25°C.• LV-MAX™ Enhancer can be added 5–14 hours posttransfection.• Recommended long-term storage of lentiviral vector is at −80˚C.• Avoid repeated freeze and thaw cycles as it can decrease lentiviral vector activity.• This protocol is appropriate for lentiviral production volumes of 30–500 mL.
Production vessel Shake speed(orbital diameter) Vessel size
LV production volumerange (24%−50%[1] of
vessel size)
Shaker flask(vented, no baffle)
125 rpm (19 mm)
120 rpm (25 mm)
95 rpm (50 mm)
125 mL 30−62.5 mL
250 mL 60−125 mL
500 mL 120−250 mL
1,000 mL 240−500 mL
2,000 mL 480−1,000 mL
[1] LV production volume can be up to 54% of vessel size, including LV-MAX™ Enhancer. Do not exceed 60%.
Optimized transfection conditions
Condition Amount
Total Opti-MEM™ I Reduced Serum Medium 2 × 5% of lentivirus production volume
Total DNA per mL of lentivirus production 2.5 μg/mL (LPM + LTV)
Ratio of lentiviral packaging mix (LPM) to lentiviraltransfer vector (LTV) 3 to 2
LV-MAX™ Transfection Reagent 6 µL/mL of lentivirus production volume
5
Lentiviralproductionspecifications
LV-MAX™ Lentiviral Production System User Guide 23
Condition Amount
LV-MAX™ Enhancer 4% of the lentivirus production volume
Gibco™ Viral Production Cells 4 × 106 cells/mL production volume
Total LV-MAX™ Supplement 5% of lentivirus production volume
Chapter 5 Produce lentiviral vector in a shaker flaskOptimized transfection conditions5
24 LV-MAX™ Lentiviral Production System User Guide
Workflow: Lentiviral production in a shaker flask
1. Prepare cells
2. Dilute cells
3. Make complex
4. Transfect cells
5. Add enhancer
7. Measure Titer
24-28hr
1min
10min
5-14 hr
48-55hr
TfxR DNA
12
AB
CD
EF
GH
12
34
56
78
910 11 12
Day 0Monday
Day 1Tuesday
Day 3Thursday
6. Harvest lentiviral vector
Chapter 5 Produce lentiviral vector in a shaker flaskWorkflow: Lentiviral production in a shaker flask 5
LV-MAX™ Lentiviral Production System User Guide 25
Transfect cells and produce the lentiviral vector
Note: This workflow guides you through the production of lentiviral vector from30 mL of production volume, in one 125-mL shaker flask. For scaling up tables, see Appendix A, “Scaling up tables“.
1. Determine the cells/mL of the 3-day cultured high density cells.
2. Dilute the high density cells to 3.5 × 106 viable cells/mL, then subculture for afurther 24 hours.
1. Count the high density cells.
2. In a new 125-mL shaker flask, add 1.2 × 108 cells to LV-MAX™ ProductionMedium to make a final volume of 25.5 mL.
3. Add 1.5 mL of LV-MAX™ Supplement.
4. Mix the cells well by pipetting up and down, then place on a 19-mm orbitalshaker at 125 rpm in an incubator.
1. Add 1.5 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 1: DNA.
Note: The ratio of packaging plasmid to transfer plasmid is 3 to 2. For 2.5 µg/mLof total DNA, this translates to 1.5 µg of packaging plasmid to 1 µg of transferplasmid.
2. Add 45 µg of packaging plasmid.
3. Add 30 µg of transfer plasmid, then vortex briefly to mix.
4. Add 1.5 mL of Opti-MEM™ I Reduced Serum Medium to a 15-mL conical tubeand label it Tube 2:TfxR.
5. Add 180 µL of LV-MAX™ Transfection Reagent (6 µL/mL).
6. Vortex briefly to mix, then incubate for 1 minute at room temperature.
7. Combine Tube 1:DNA with Tube 2:TfxR, then mix well by pipetting up anddown.
8. Incubate the combined solution for 10 minutes at room temperature.
1. Add 3 mL of the DNA-lipid complex to the previously prepared flask of cells,then mix well shaking gently.
Note: The DNA-lipid complex is stable for up to 1.5 hours.
2. At 5–6 hours posttransfection, add 1.2 mL of LV-MAX™ Enhancer.
Note: The optimum time frame for LV-MAX™ Enhancer addition is anytimebetween 5–14 hours posttransfection.
Prepare cells
Dilute cells
Make complexes
Transfect cells
Chapter 5 Produce lentiviral vector in a shaker flaskTransfect cells and produce the lentiviral vector5
26 LV-MAX™ Lentiviral Production System User Guide
1. At 48–55 hours posttransfection, collect the culture medium, then centrifuge thecells at 1,300 × g in a swinging bucket centrifuge for 15 minutes.For smaller volumes, use a 1.5-mL microcentrifuge tube and centrifuge in abenchtop centrifuge at 13,000 rpm for 5 minutes.
2. Harvest the lentiviral vector by transferring the supernatant to a fresh tube.
3. Further remove cell debris by filtration through a 0.45 µm low protein bindingfilter.
4. Immediately following harvest, store the lentiviral vector at -80°C.
Note: To avoid multiple freeze thaw cycles, aliquot the supernatant to multipletubes.
Thaw, then titer the lentiviral vector using your method of choice.
Note: Thaw the virus on ice before use.
For recommended protocols, see Chapter 6, “Titer lentiviral vector“.You can optimize your lentiviral vector yield by trying different starting cell densities,such as 2.5, 3, and 4 × 106 viable cells/mL.
Harvest thelentiviral vector
Titer the lentiviralvector
Chapter 5 Produce lentiviral vector in a shaker flaskTransfect cells and produce the lentiviral vector 5
LV-MAX™ Lentiviral Production System User Guide 27
Titer lentiviral vector
IMPORTANT! Handling of lentivirus must be performed as per institutionalguidelines. All materials should be treated with a 10% bleach solution prior todisposal.
Titer using GFP expression
• Thaw the virus on ice before use. Do not use any acceleration method because itcan decrease the virus titer.
• Mix the virus by tapping or inverting the tube. Do not vortex and avoid mixingvigorously.
Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
Item Source
HT1080 cell line ATCC, CCL-121
DMEM, high glucose, GlutaMAX™ Supplement, pyruvate[1] 10569010
Polybrene [2] Fisher Scientific™,NC0663391
96-well culture plates MLS
96-well round bottom plates MLS
Large swinging bucket centrifuge MLS
TrypLE™ Express Enzyme (1X), no phenol red 12604013
DPBS, no calcium, no magnesium 14190250
[1] Add 10% FBS to make culture medium for HT1080 [2] Stock solution: 10 mg/mL in sterile H2O
Day 1 (morning) - infect HT1080 cells
1. Four hours before infection, seed a 96-well culture plate according to thefollowing table:
Cells Density Volume of media
HT1080 7,000 cells/well 100 µL/well
Note: Cells are ~30% confluent at time of infection.
6
Proceduralguidelines
Requiredmaterials notsupplied
Perform lentiviralvector titration
28 LV-MAX™ Lentiviral Production System User Guide
2. Two hours before infection, thaw the virus on ice.
Note: Do not accelerate the lentiviral vector thawing process because it candecrease the titer of the virus.
3. Prepare dilution medium by combining 15 mL of fresh culture medium with12 µL of 10 mg/mL Polybrene (final concentration of 8 µg/mL), then vortex tomix.
4. Prepare a 4-log serial dilution (10-1 to 10-4) by sequentially diluting 15 µL of virusinto 135 µL of dilution medium per well of a round bottom 96-well plate.
Note: We recommend performing each dilution in quadruplicate.
Note: If your virus was concentrated, you may need more dilutions.
5. Remove the culture media from the HT1080 cells, then infect by transferring100 µL of the diluted virus to each corresponding well.
6. Centrifuge the infected cell plate at 900 × g for 30 minutes at room temperature.
7. Incubate the infected cell plate overnight.
Day 2 (morning) – change medium, then incubate cells
1. Remove the medium from each well, then add 100 µL/well of fresh HT1080culture medium (without Polybrene).
Note: When replacing medium, always start from the Negative Control andwork backwards to minimize the amount of virus carried over.
2. Incubate the infected plate for 72 hours.
Day 5 - prepare cells, then run through a flow cytometer
1. Remove the medium from each well, then add 150 µL of trypsinization medium(75% TrypLE™ + 25% DPBS mixture) to each well.
2. Incubate the infected plate for 15 minutes in an incubator at 37°C.
3. Use a microscope to check the cells ands ensure that the cells are rounded up.
4. Detach the cells from the culture plate bottom by gently pipetting up and down.
5. Process the cells through a flow cytometer.
Chapter 6 Titer lentiviral vectorTiter using GFP expression 6
LV-MAX™ Lentiviral Production System User Guide 29
1. To calculate the titer (Transforming Units/mL), determine the appropriatedilution factor to use based on the % of GFP+ cells.The optimal infection range is 1−20% of GFP+ cells. Over 20% of GFP+ cellsresults in an underestimated lentiviral vector titer.
Lentivirus dilution EmGFP+ cells
102 96%
103 65%
104 18%
Using this table of results, the 104 dilution is selected for the calculation becausethe % of GFP+ cells falls in the desired 1−20% range.
2. Use the following formula to calculate the titer:Titer = (F × C/V) × D
• F = frequency of GFP+ cells (%GFP+ 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
Example:• F = 18/100 (using the table in Step 1)• C = 7,000 (cell number at the time of infection)• V = 0.1 (100 µL of medium)• D = 104
Chapter 6 Titer lentiviral vectorTiter using GFP expression6
30 LV-MAX™ Lentiviral Production System User Guide
Titer using antibiotic selection
• Thaw the virus on ice before use. Do not use any acceleration method because itcan decrease the virus titer.
• Mix the virus by tapping or inverting the tube. Do not vortex and avoid mixingvigorously.
Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
Item Source
HT1080 cell line ATCC, CCL-121
DMEM, high glucose, GlutaMAX™ Supplement, pyruvate[1] 10569010
Polybrene[2] Fisher Scientific™,NC0663391
Crystal violet Fisher Scientific™,ICN15251150
Blasticidin S HCl[3] A1113903
Puomycin[4] A1113802
6-well culture plates 140675
TrypLE™ Express Enzyme (1X), no phenol red 12604013
[1] Add 10% FBS to make culture medium for HT1080 [2] Stock solution: 10 mg/mL in sterile H2O and filter through 0.2 µm filter[3] Selection medium: culture medium with Blasticidin S HCl, 10 μg/mL final concentration (the selection
medium based on pLenti transfer plasmid back bone selection marker)[4] Select culture medium final concentration 10 μg/mL
Day 1 (morning) – infect HT1080 cells
1. Four hours before infecting cells, seed a 6-well plate according to the followingtable:
Cells Density Volume of media
HT1080 210,000 cells/well 2 mL/well
Note: Cells are ~30% confluent at time of infection.
2. Two hours before infection, thaw the virus on ice.
Note: Do not accelerate the lentiviral vector thawing process because it candecrease the titer of the virus.
3. Prepare dilution medium by combining 20 mL of fresh culture medium with16 µL of 10 mg/mL Polybrene (final concentration of 8 µg/mL), then vortex tomix.
Proceduralguidelines
Requiredmaterials notsupplied
Perform lentiviralvector titration
Chapter 6 Titer lentiviral vectorTiter using antibiotic selection 6
LV-MAX™ Lentiviral Production System User Guide 31
4. For a virus sample, prepare seven microcentrifuge tubes and label them as T1, T2to T7.
5. Add 180 µL of prepared dilution medium to T1, then add 900 µL to T2 throughT7.
6. Add 20 µL of virus sample, crude lentivirus, or concentrated virus aliquot to T1(101 dilution), then invert the tube to mix.
T1 T2 T3 T4 T5 T6 T7
100 µL
180 µL
10 10 10 10 10 10
900 µL 900 µL 900 µL 900 µL
20 µL
Medium
Dilution
LVVs
100 µL 100 µL 100 µL 100 µL 100 µL
2 3 4 5 6 107
900 µL900 µL
7. Use a new tip to transfer 100 µL of T1 dilution to T2, then invert the tube to mix(102 dilution).
8. Use a new tip to transfer 100 µL of T2 dilution to T3, then invert the tube to mix(103 dilution).
9. Repeat Step 8 to complete dilutions T4 to T7.
Note: If your virus was concentrated, you may need more dilutions.
10. Replace the culture medium in A1, A2, A3, B1, and B2 of the prepared 6-wellplate of HT1080 cells with 1,500 µL of prepared dilution medium, then 2,000 µLto B3 as a negative control (‘Neg Ctrl’)
11. Pipet 500 µL of diluted virus into each respective well, starting with T3,according to the following figure.
LVVs infection grid
T3A
1 2 3
3
B
500 µLT4
500 µLT5
4 x10
64 x10 74 x10
44 x10
CtrlNeg
54 x10500 µL
T6500 µL
T7500 µL
Final viral dilution
T3A
1 2 3
B
T4 T5
T6 T7CtrlNeg
12. Ensure even distribution of the diluted virus across each well by moving theinfected 6-well plate left, right, backward, then forward several times.
Chapter 6 Titer lentiviral vectorTiter using antibiotic selection6
32 LV-MAX™ Lentiviral Production System User Guide
13. Centrifuge the infected cell plate at 900 × g (using a swinging bucket centrifuge)at room temperature for 30 minutes.
14. Incubate the infected cell plate overnight.
Day 2 (morning) – change medium, then incubate cells
1. Remove the medium from each well, then add 2 mL/well of fresh HT1080 culturemedium (without Polybrene).
Note: When replacing medium, always start from the Negative Control andwork backwards to minimize the amount of virus carried over.
2. Incubate cells for 24 hours.
Day 4 – start antibiotic selection
1. Prepare HT1080 culture medium with antibiotic, such as Blasticidin with a finalconcentration of 10 µg/mL or Puromycin with a final concentration of 1 µg/mL.
Note: Select antibiotic based on your pLenti-transfer back bone selection marker.
2. Replace the culture medium in each well with 2 mL of prepared selectionmedium, then incubate for 48 hours.
3. Repeat Step 2 until the ‘NegCtrl’ well has no cells left.This is an approximately 10-day process.
Day 13 – stain with Crystal Violet
1. Prepare the Crystal Violet staining solution: 1% Crystal Violet dissolved in a 10%ethanol/H20 solution.
2. Remove the antibiotic selection medium from the 6-well plate, then wash eachwell with 2 mL of PBS.
3. Add 1 mL of prepared Crystal Violet staining solution to each well, then incubatethe plate for 20 minutes at room temperature.
4. After 20-minutes incubation, remove the staining solution from the 6-well plate.Store the staining solution because it can be reused.
5. Wash the stained 6-well plate with water several times until a clear backgroundappears.
1. Visually count the number of stained colonies per well.
2. Use the following formula to calculate the titer (Transforming Units/mL) of thevirus stock:Titer = (Number of discrete colonies × dilution factor) / Volume of inoculumExample:Count = 30 colonies stained in the 4 × 106 dilution well (B-1) and 5 coloniesstained in the 4 × 107 dilution well (B-2)Volume of diluted virus = 2 mL
Calculate thelentiviral titer
Chapter 6 Titer lentiviral vectorTiter using antibiotic selection 6
LV-MAX™ Lentiviral Production System User Guide 33
Chapter 6 Titer lentiviral vectorTiter using antibiotic selection6
34 LV-MAX™ Lentiviral Production System User Guide
Scaling up tables
Tables for scaling up preset and custom sized production volumes
This section contains two tables for use in scaling up the production volume.
The first table (Table 6 on page 36) displays all the required volumes for each reagent based on the presetproduction vessel sizes that can be used.
The second table (Table 7 on page 37) displays the formula for each of the required volumes for each reagentbased on the custom production volume to be used. Use the formula in the table for each reagent to calculateyour specific volume, then input that volume in the space provided under the correct heading.
An explanation of the scaling up table headings are in the following list:• Production vessel: The vessel used for culturing the lentiviral vector (ensure the vessel size is large enough
for the production volume plus all other reagents).• Production volume: This is the initial volume of media and cells combined.• Number of cells: The number of cells required.• Volume of media: The amount of media to dilute the cells in.• Supplement: The volume of LV-MAX™ Supplement required.• Opti-MEM I: The volume of Opti-MEM™ I Reduced Serum Medium required for tube 1 (DNA).• Packaging: The amount of packaging plasmid required for tube 1 (DNA).• Transfer: The amount of transfer plasmid required for tube 1 (DNA).• Opti-MEM I: The volume of Opti-MEM™ I Reduced Serum Medium required for tube 2 (TfxR).• LV-MAX™ Transfection Reagent: The amount of LV-MAX™ Transfection Reagent required for tube 2 (TfxR).• Enhancer: The amount of LV-MAX™ Enhancer required.
A
LV-MAX™ Lentiviral Production System User Guide 35
WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.
· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, and so on). To obtain SDSs, see the “Documentationand Support” section in this document.
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LV-MAX™ Lentiviral Production System User Guide 39
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below. Consult the relevant SDSfor specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by thechemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.
· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste
container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)
· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of
according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.
Appendix C SafetyChemical safetyC
40 LV-MAX™ Lentiviral Production System User Guide
Biological hazard safety
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. Conduct all work in properly equipped facilitieswith the appropriate safety equipment (for example, physical containmentdevices). Safety equipment can also include items for personal protection, suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields, safetyglasses, or goggles. Individuals should be trained according to applicableregulatory and company/ institution requirements before working withpotentially biohazardous materials. Follow all applicable local, state/provincial,and/or national regulations. The following references provide generalguidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf
· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
Appendix C SafetyBiological hazard safety C
LV-MAX™ Lentiviral Production System User Guide 41
Visit thermofisher.com/support for the latest service and support information.• Worldwide contact telephone numbers• Product support information
– Product FAQs– Software, patches, and updates– Training for many applications and instruments
• Order and web support• Product documentation
– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you haveany questions, please contact Life Technologies at www.thermofisher.com/support.
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42 LV-MAX™ Lentiviral Production System User Guide