Thermo Fisher Scientific LV-MAX User Manual
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 E.0
For Research Use Only. Not for use in diagnostic procedures.
Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008
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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 Description
E.0 5 January 2021 Update of reagent volumes in protocol.
D.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 accept the 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.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 5
Product description ............................................................. 5
Contents and storage ............................................................ 5
System components ............................................................. 6
Viral production cells ........................................................ 6
Growth medium ............................................................ 6
Transfection supplement .................................................... 6
Transfection reagent ......................................................... 6
Production enhancer ....................................................... 6
Required materials not supplied ................................................... 7
■
CHAPTER 2 Thaw and establish Gibco™ Viral Production Cells ................ 8
Guidelines for handling cells ...................................................... 8
Required materials not supplied ................................................... 8
Thaw Gibco™ Viral Production Cells ............................................... 9
Subculture Gibco™ Viral Production Cells .......................................... 9
Required materials .......................................................... 9
Passage Gibco™ Viral Production Cells ....................................... 10
Cryopreserve Gibco™ Viral Production Cells ....................................... 11
Cryopreserve cells ......................................................... 11
■
CHAPTER 3 Produce lentiviral vector ............................................ 12
Procedural guidelines ........................................................... 12
Equipment guidelines .......................................................... 12
Required materials ............................................................. 12
Optimized transfection conditions ................................................ 13
Transfect Gibco™ Viral Production Cells ........................................... 13
Transfect cells ............................................................. 15
Harvest the lentiviral vector ...................................................... 16
Titer the lentiviral vector ......................................................... 16
LV-MAX
™
Lentiviral Production System User Guide
3
Contents
■
■
CHAPTER 4 Produce recombinant lentiviral vector in a 3-L stirred
tank bioreactor ..................................................................... 17
Procedural guidelines ........................................................... 17
Guidelines for scaling up reactions ............................................... 17
Required materials ............................................................. 17
Transfect Gibco™ Viral Production Cells ........................................... 18
Transfect cells ............................................................. 19
CHAPTER 5 Titer lentiviral vector ................................................ 21
Titer using GFP expression ...................................................... 21
Procedural guidelines ...................................................... 21
Required materials not supplied ............................................. 21
Perform lentiviral vector titration ............................................. 22
Calculate the lentiviral titer .................................................. 23
Titer using antibiotic selection ................................................... 24
Procedural guidelines ...................................................... 24
Required materials not supplied ............................................. 24
Perform lentiviral vector titration ............................................. 24
Calculate the lentiviral titer .................................................. 26
■
APPENDIX A Related products .................................................. 28
Related products .............................................................. 28
■
APPENDIX B Safety ............................................................... 29
Chemical safety ................................................................ 30
Biological hazard safety ......................................................... 31
■
APPENDIX C Documentation and support ...................................... 32
Customer and technical support ................................................. 32
Limited product warranty ........................................................ 32
4
LV-MAX™ Lentiviral Production System User Guide
1
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 lentiviral vector production system based on
transient transfection of high-density, suspension HEK293F cells adapted to a chemically defined,
serum-free and protein free medium. The system is available in both Research Use and Cell Therapy
Systems (CTS) options to enable a seamless transition from discovery to production.
The Gibco™ LV-MAX™ Lentiviral Production System provides cells, production medium, supplement,
transfection reagent, and enhancer to produce high titer lentiviral vectors.
Product information
Contents and storage
Reagents provided in the kit are sucient for 300 mL of lentiviral production volume.
Table 1 LV-MAX™Lentiviral Production System Starter Kit (Cat No. A35684)
Component
Viral Production Cells
LV-MAX™ Production Medium A3583401 1 L
LV-MAX™ Transfection Kit
•
LV-MAX™ Supplement
•
LV-MAX™ Transfection Reagent
•
LV-MAX™ Enhancer
[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]
(1 X 107 cells/mL) A35347 2 × 1 mL Liquid nitrogen
Cat. No. Amount Storage
A35346 1 Kit
•
15 mL
•
2 × 0.9 mL
•
12 mL
•
2°C to 8°C
•
Protected from
light
[2]
LV-MAX™ Lentiviral Production System User Guide
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Chapter 1 Product information
1
System components
System components
The following section provides descriptions of the components in the Gibco™ LV-MAX™ Lentiviral
Production System.
Viral production cells
Gibco™ Viral Production Cells are derived from the HEK293F cell line, and are 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
Growth medium
LV-MAX™ Production Medium is a complete, chemically defined, animal origin-free, serum-free, proteinfree formulation, developed for growth and transfection of Gibco™ Viral Production Cells. This medium
is ready-to-use and does not require the addition of supplements.
Transfection supplement
LV-MAX™ Supplement is a chemically defined, animal origin-free, serum-free, protein-free formulation
designed to control cell growth during transfection and increase lentiviral vector production without
compromising cell viability.
Transfection reagent
LV-MAX™ Transfection Reagent is uniquely designed for high eciency co-transfection of multiple
plasmids into Gibco™ Viral Production Cells, with low toxicity.
Production enhancer
LV-MAX™ Enhancer is a chemically defined, animal origin-free, serum-free, protein-free formulation that
is designed to boost lentiviral vector production in Gibco™ Viral Production Cells.
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LV-MAX™ Lentiviral Production System User Guide
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Table 2 Materials required for lentiviral vector production
Item Source
CO2 resistant orbital shaker 88881101
Adjustable micropipettors MLS
Laboratory mixer (Vortex mixer or equivalent) MLS
3 mm orbital shaker MLS
Equipment and reagents to determine cell density and viability MLS
LV-MAX™ Lentiviral Packaging Mix A43237
Opti-MEM™ I Reduced Serum Medium 31985088
Chapter 1 Product information
Required materials not supplied
1
Material for ryopreservation
DMSO MLS
Cryovials MLS
Plastics
2-mL sterile 96-deep well block MLS
2-mL sterile 96-deep well block (v-bottom) MLS
96-well round bottom plate MLS
Nunc™ 50-mL conical tube
Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterile
125 mL 4115-0125
250 mL 4115-0250
1 L 4115-1000
2 L 4115-2000
3 L 4115-2800
339653
LV-MAX™ Lentiviral Production System User Guide
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Thaw and establish Gibco™ Viral
2
Guidelines for handling cells
IMPORTANT! Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at
–80°C.
•
Avoid subjecting cells to short-term, extreme temperature changes.
•
After storing cells in liquid nitrogen following receipt on dry ice, allow the cells to remain in liquid
nitrogen for 3–4 days before thawing.
•
For all cell manipulations, mix cells by gentle swirling and avoid vigorous shaking/pipetting.
•
For routine cell culture maintenance, subculture cells every 3–4 days when they reach 3.5–5.5 × 10
cells/mL (see “Subculture Gibco™ Viral Production Cells” on page 9). Do not subculture cells that
have not reached early log phase growth of ≥3.5 × 106 cells/mL.
Production Cells
6
Required materials not supplied
•
Gibco™ Viral Production Cells
•
125-mL Erlenmeyer Flask (e.g., Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom:
Sterile for culturing Viral Production Cells, Cat. No. 4115-0125)
•
Orbital shaker (e.g., MaxQ™ HP Tabletop Orbital Shaker, Cat. No. SHKE416HP)
•
Temperature and CO2 controlled incubator (e.g., Large-Capacity Reach-In CO2 Incubator, Cat. No.
3950)
•
Reagents and equipment to determine cell viability (e.g., hemocytometer with trypan blue or cell
counter)
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LV-MAX™ Lentiviral Production System User Guide
Chapter 2
Thaw and establish Gibco™ Viral Production Cells
Thaw Gibco™ Viral Production Cells
1.
Remove the vial of cells from liquid nitrogen and swirl in a 37°C water bath for 1 to 2 minutes to
thaw the cells rapidly until only a small amount of ice remains.
Note: Do not submerge the vial in the water.
2.
Just before the cells are completely thawed, decontaminate the vial by wiping it with 70% ethanol
before opening it in a laminar flow hood.
3.
Use a 2-mL or 5-mL pipette, to transfer the entire contents of the cryovial into a 125-mL
polycarbonate, disposable, sterile, vent-cap Erlenmeyer shaker flask containing 30 mL of LV-MAX
Production Medium pre-warmed to 37°C.
4.
Incubate the cells in a 37°C incubator with ≥80% relative humidity, and 8% CO2 on an orbital
shaker platform.
Note: Set the shake speed to 125±5 rpm for shakers with a 19 mm shaking diameter, 120±5 rpm
for shakers with a 25 mm shaking diameter and 95±5 rpm for shakers with a 50 mm shaking
diameter.
Thaw Gibco™ Viral Production Cells
2
™
5.
Culture cells for 3–4 days post-thaw and then determine viable cell density and percent viability.
Cell viability should be ≥90% 3–4 days post-thaw, with viable cell density typically >1 × 106 viable
cells/mL; if viability is not >90%, incubate cells for up to an additional 3 days to reach optimal
density. Subculture cells when the viable cell density reaches 1–3 × 106 viable cells/mL.
Subculture Gibco™ Viral Production Cells
Gibco™ Viral Production Cells are capable of achieving high cell densities; therefore, it is important that
cells attain a minimum density of 3.5–5.5 × 106 viable cells/mL at the time of subculturing.
Required materials
•
Gibco™ Viral Production Cells cultured in LV-MAX™ Production Medium
•
LV-MAX™ Production Medium, pre-warmed to 37°C
•
Opti-MEM™ I Reduced Serum Medium
•
Disposable, sterile Erlenmeyer flasks
•
Orbital shaker (e.g., MaxQ™ HP Tabletop Orbital Shaker, Cat. No. SHKE416HP)
•
Temperature and CO2 controlled incubator (e.g., Large-Capacity Reach-In CO2 Incubator, Cat. No.
3950)
•
Reagents and equipment to determine cell viability (e.g., hemocytometer with trypan blue or cell
counter)
LV-MAX™ Lentiviral Production System User Guide
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Chapter 2
2
Subculture Gibco™ Viral Production Cells
Thaw and establish Gibco™ Viral Production Cells
Passage Gibco™ Viral Production Cells
1.
Use the viable cell density to calculate the volume of cell suspension required to seed a new shake
flask according to the recommended seeding densities in Table 3 and the recommended culture
volumes in Table 5.
Table 3 Recommended seeding densities for routine cell culture maintenance
Sub-culture timing Recommended seeding density
For cells ready 3 days post-subculture 0.5 × 106 viable cells/mL
For cells ready 4 days post-subculture 0.3 × 106 viable cells/mL
™
Table 4 Recommended Vi-CELL
XR Cell Counting Settings
Parameter
Minimum diameter 5 Cell brightness (%) 85
Maximum diameter 50 Cell sharpness 100
Number of images 50 Viable cell spot brightness (%) 65
Aspirate cycles 3 Viable cell spot area (%) 5
Trypan blue mixing cycles 3 Minimum circularity 0
Decluster degree Medium
Value Parameter Value
Table 5 Recommended volumes for routine cell culture maintenance in vented, non-baed
flasks
Flask size
125 mL 30–35 mL
250 mL 60–70 mL
500 mL 120–140 mL
1 L 240–280 mL
2 L 480–560 mL
2.8–3 L 720–840 mL
[1]
If using volumes outside of the recommended range, it is critical to ensure that all cell growth (i.e., doubling times), health (i.e.,
cell diameter, viability), and expression levels remain consistent with control conditions. Cell performance is decreased if cell
health is compromised.
Culture volume
[1]
Parameter
125±5 rpm (19 mm shaking diameter)
120±5 rpm (25 mm shaking diameter)
95±5 rpm (50 mm shaking diameter)
90±5 rpm
85±5 rpm
80±5 rpm
10
2.
Transfer the calculated volume of cells to fresh, pre-warmed LV-MAX™ Production Medium in a
shake flask.
LV-MAX™ Lentiviral Production System User Guide
Chapter 2 Thaw and establish Gibco™ Viral Production Cells
Cryopreserve Gibco™ Viral Production Cells
3.
Incubate flasks in a 37°C incubator with ≥80% relative humidity, and 8% CO2 on an orbital shaker
platform until cultures reach a density of 3.5–5.5 × 106 viable cells/mL.
Note: Cells subcultured at densities outside of the early log-phase growth window may show
longer doubling times and lower titers over time. Modify the initial seeding density to attain the
target cell density of 3.5–5.5 × 106 viable cells/mL at the time of subculturing.
4.
Repeat Steps 1–3 to maintain or expand cells for transfection.
Cryopreserve Gibco™ Viral Production Cells
Gibco™ Viral Production Cells can be frozen directly in LV-MAX™ Production Medium with 10% DMSO.
Alternatively, conditioned cryopreservation medium consisting of 45% fresh LV-MAX™ Production
Medium, 45% conditioned LV-MAX™ Production Medium, and 10% DMSO can be used.
Cryopreserve cells
1.
Centrifuge cells that have attained a viable cell density of 3.5−5.5 × 106 viable cells/mL and cell
viability at 300 × g for 5 minutes. Discard the supernatant without disturbing the cell pellet.
2
2.
Add ice cold LV-MAX™ Production Medium with 10% DMSO to the cell pellet, then resuspend the
cell pellet by gentle pipetting.
3.
Dilute the cells to a final density of 1 × 107 viable cells/mL in 1 mL total volume of 90% fresh
LV-MAX™ Production Medium with 10% DMSO.
4.
Freeze the cells in an automated or manual controlled-rate freezing apparatus following standard
procedures.
For ideal cryopreservation, the freeze rate should decrease by 1°C per minute.
5.
Transfer the frozen vials to liquid nitrogen for long-term storage.
LV-MAX™ Lentiviral Production System User Guide
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