Thermo Fisher Scientific CTS, LV-MAX User Manual
CTS™ LV-MAX™ Lentiviral Production System
USER GUIDE
For xeno-free lentiviral vector production in suspension cells
for use with:
CTS™ Viral Production Cells
CTS™ LV-MAX™ Production Medium
CTS™ LV-MAX™ Transfection Kit
CTS™ Opti-MEM™ I
Publication Number MAN0018450
Revision B.0
For Research Use or Manufacturing of Cell, Gene, or Tissue-Based
Products. CAUTION: Not intended for direct administration into
humans or animals.
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The information in this guide is subject to change without notice.
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ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0018450
Revision Date Description
B.0 5 January 2021 Update of reagent volumes in protocol.
A.0 28 March 2019 New document.
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©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 5
Product description ............................................................. 5
Required components ........................................................... 5
Required materials not supplied ................................................... 6
System components ............................................................. 8
Viral production cells ........................................................ 8
Growth medium ............................................................ 8
Transfection supplement .................................................... 8
Transfection reagent ......................................................... 8
Production enhancer ....................................................... 8
Opti-MEM ................................................................. 8
■
CHAPTER 2 Thaw and establish CTS™ Viral Production Cells .................. 9
Guidelines for handling cells ...................................................... 9
Required materials not supplied ................................................... 9
Thaw CTS™ Viral Production Cells ................................................ 10
Subculture CTS™ Viral Production Cells ........................................... 10
Required materials ......................................................... 10
Passage CTS™ Viral Production Cells ......................................... 11
Cryopreserve CTS™ Viral Production Cells ......................................... 12
Cryopreserve cells ......................................................... 12
■
CHAPTER 3 Produce lentiviral vector ............................................ 13
Procedural guidelines ........................................................... 13
Equipment guidelines .......................................................... 13
Required materials ............................................................. 14
Optimized transfection conditions ................................................ 14
Transfect CTS™ Viral Production Cells ............................................ 14
Transfect cells ............................................................. 16
Harvest the lentiviral vector ...................................................... 17
Titer the lentiviral vector ......................................................... 17
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LV-MAX™ Lentiviral Production System User Guide
CTS
3
Contents
■
■
CHAPTER 4 Produce recombinant lentiviral vector in a 3-L stirred
tank bioreactor ..................................................................... 18
Procedural guidelines ........................................................... 18
Guidelines for scaling up reactions ............................................... 18
Required materials ............................................................. 18
Transfect CTS™ Viral Production Cells ............................................ 19
Transfect cells ............................................................. 20
CHAPTER 5 Titer lentiviral vector ................................................ 23
Titer using GFP expression ...................................................... 23
Procedural guidelines ...................................................... 23
Required materials not supplied ............................................. 23
Perform lentiviral vector titration ............................................. 24
Calculate the lentiviral titer .................................................. 25
Titer using antibiotic selection ................................................... 26
Procedural guidelines ...................................................... 26
Required materials not supplied ............................................. 26
Perform lentiviral vector titration ............................................. 26
Calculate the lentiviral titer .................................................. 28
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APPENDIX A Scaling up tables .................................................. 30
Tables for scaling up preset and custom sized production volumes ................... 30
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APPENDIX B Related products .................................................. 33
Related products .............................................................. 33
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APPENDIX C Safety ............................................................... 34
Chemical safety ................................................................ 35
Biological hazard safety ......................................................... 36
■
APPENDIX D Documentation and support ...................................... 37
Customer and technical support ................................................. 37
Limited product warranty ........................................................ 37
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CTS™ LV-MAX™ Lentiviral Production System User Guide
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IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
The CTS™ 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 CTS™ LV-MAX™ Lentiviral Production System provides cells, production medium, supplement,
transfection reagent, and enhancer to produce high titer lentiviral vectors.
Product information
Required components
The workflows described in this document are for use with the components in the following table.
Component
CTS™ Viral Production Cells (1 X 107 cells/mL) A3152801 1 × 1 mL Liquid nitrogen
CTS™ LV-MAX™ Production Medium
One of the following CTS™ LV-MAX™ Transfection Kit:
CTS™ LV-MAX™ Transfection Kit:
•
CTS™ LV-MAX™ Supplement
•
CTS™ LV-MAX™ Transfection Reagent
•
CTS™ LV-MAX™ Enhancer
CTS™ LV-MAX™ Transfection Kit:
•
CTS™ LV-MAX™ Supplement
•
CTS™ LV-MAX™ Transfection Reagent
•
CTS™ LV-MAX™ Enhancer
Cat. No. Amount Storage
•
A4124001
•
A4124004
•
A4124002
•
A4124003
A4132601 1 L Production Kit
A4132602 10 L Production Kit
•
•
•
•
•
•
•
•
•
•
1 L
6 × 1 L
10 L
20 L
50 mL
6 mL
40 mL
500 mL
60 mL
400 mL
•
2°C to 8°C
•
Protected from
light
[1]
CTS™ LV-MAX™ Lentiviral Production System User Guide
5
Chapter 1 Product information
1
Required materials not supplied
(continued)
Component Cat. No. Amount Storage
•
A4124801
CTS™ Opti-MEM™ I
[1]
Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at −80°C.
•
A4124802
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 1 Materials required for lentiviral vector production (all methods)
Item Source
CO2 resistant orbital shaker 88881101
•
•
100 mL
500 mL
•
2°C to 8°C
•
Protected from
light
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™ Lentiviral Packaging Mix A43237
For cryopreservation:
DMSO MLS
Cryovials MLS
Table 2 Additional materials required for lentiviral vector production in a 96-deep well block
Item
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
Source
Multi-channel micropipettors MLS
PureLink™ Air Porous Tape 12262010
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CTS™ LV-MAX™ Lentiviral Production System User Guide
Chapter 1 Product information
Required materials not supplied
Table 3 Additional materials required for lentiviral vector production in a 50-mL conical 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
125 mL 4115-0125
250 mL 4115-0250
1 L 4115-1000
Table 4 Additional materials required for lentiviral vector production in a shaker flask
Item Source
Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterile
1
125 mL 4115-0125
250 mL 4115-0250
1 L 4115-1000
2 L 4115-2000
CTS™ LV-MAX™ Lentiviral Production System User Guide
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Chapter 1
1
System components
Product information
System components
The following section provides descriptions of the components in the CTS™ LV-MAX™ Lentiviral
Production System.
Viral production cells
CTS™ Viral Production Cells are derived from the HEK293F cell line, and are adapted to suspension
culture in CTS™ LV-MAX™ Production Medium. These cells can be thawed directly into CTS™ 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
CTS™ LV-MAX™ Production Medium is a complete, chemically defined, animal origin-free, serum-free,
protein-free formulation, developed for growth and transfection of CTS™ Viral Production Cells. This
medium is ready-to-use and does not require the addition of supplements.
Transfection supplement
CTS™ 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
CTS™ LV-MAX™ Transfection Reagent is uniquely designed for high eciency co-transfection of multiple
plasmids into CTS™ Viral Production Cells, with low toxicity.
Production enhancer
CTS™ LV-MAX™ Enhancer is a chemically defined, animal origin-free, serum-free, protein-free
formulation that is designed to boost lentiviral vector production in CTS™ Viral Production Cells.
Opti-MEM
CTS™ Opti-MEM™ I is a xeno-free formulation for use with transfection reagent to facilitate complexation
in viral vector production. It is an improved Minimal Essential Medium (MEM) containing insulin,
transferrin, hypoxanthine, thymidine, and trace elements.
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CTS™ LV-MAX™ Lentiviral Production System User Guide
Thaw and establish CTS™ 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 CTS™ Viral Production Cells” on page 10). 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
•
CTS™ 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)
CTS™ LV-MAX™ Lentiviral Production System User Guide
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Chapter 2
2
Thaw CTS™ Viral Production Cells
Thaw and establish CTS™ Viral Production Cells
Thaw CTS™ 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 CTS
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.
™
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 CTS™ Viral Production Cells
CTS™ 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
•
CTS™ Viral Production Cells cultured in CTS™ LV-MAX™ Production Medium
•
CTS™ LV-MAX™ Production Medium, pre-warmed to 37°C
•
CTS™ Opti-MEM™ I
•
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)
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CTS™ LV-MAX™ Lentiviral Production System User Guide
Passage CTS™ 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 5 and the recommended culture
volumes in Table 7.
Table 5 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 6 Recommended Vi-CELL
XR Cell Counting Settings
Chapter 2
Thaw and establish CTS™ Viral Production Cells
Subculture CTS™ Viral Production Cells
2
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 7 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
2.
Transfer the calculated volume of cells to fresh, pre-warmed CTS™ LV-MAX™ Production Medium in
a shake flask.
CTS™ LV-MAX™ Lentiviral Production System User Guide
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Chapter 2 Thaw and establish CTS™ Viral Production Cells
2
Cryopreserve CTS™ 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 CTS™ Viral Production Cells
CTS™ Viral Production Cells can be frozen directly in CTS™ LV-MAX™ Production Medium with 10%
DMSO. Alternatively, conditioned cryopreservation medium consisting of 45% fresh CTS™ LV-MAX
Production Medium, 45% conditioned CTS™ 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.
Add ice cold CTS™ 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 CTS
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.
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CTS™ LV-MAX™ Lentiviral Production System User Guide
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