Lentiviral Vector Production

Advanced lipid nanoparticle technology for superior lentiviral production in adherent cultures
Invitrogen Lipofectamine 3000 Transfection Reagent is a highly efficient, cost-effective tool for lentiviral production. This versatile reagent enables high viral titers even with genes that are large or difficult to package.

In a side-by-side comparison between Lipofectamine 3000 Reagent, Lipofectamine 2000 Reagent and PEI, Lipofectamine 3000 reagent–mediated lentiviral production was ~5–10 times greater than that obtained from the other transfection reagents (Figure 5).

Learn more about Lipofectamine 3000 for lentiviral production in adherent cells

Transfection

Is my CTS LV-MAX transfection reagent supposed to be cloudy?
It is normal to see some turbidity and cloudiness in LV-MAX transfection reagent. Lipid transfection reagents are sensitive to low temperature; if you store them at temperatures lower than the recommended 2–8°C storage conditions or if the reagent freezes it will precipitate, leading to low transfection efficiency or inactivity.

I'm interested only in the transfection reagent. Can I purchase it separately from the transfection kit?
Not currently. The LV-MAX Transfection Kit comes with three components that are not sold a la carte. The system components were specifically optimized to work as a system for maximal viral vector titer. We can however customize different ratios of the supplement, enhancer and transfection reagent.

Have you tried other commercially available transfection reagents (i.e., PEI) in your system?
Yes, we have tried a full range of transfection reagent formulations while developing the system. LV-MAX Transfection Reagent is compatible with the suspension Viral Production Cells to support transfection in a high cell density format and offers higher performance than other transfection reagents.

LV-MAX is more expensive than PEI-based transfection reagent. How can you claim it’s a low-cost option?
The cost per viral particle is much lower using LV-MAX system compared to PEI-based method due to the robustness of the LV-MAX system. Because of the high efficiency of the complete system, less material and volumes are needed to make the same amount of virus, which also reduces labor and ancillary consumable reagents (plasmids, purification etc.).

Can I transfect before passage 5 (P5)?
We recommend that cells be used for lentiviral production following passage 5 to allow for full recovery of the cells from the cryopreservation.

Enhancer addition time is suggested to be anytime between 5–14 hours after transfection, how critical is it that we stay within this time frame window?
Enhancer addition was tested over the entire rage of 5–14 hours and was found to perform with equivalent efficiency when added anytime during that range of time. The enhancer formulation coupled with the broad window of addition time was specifically optimized to allow the user flexibility in schedule and start time of the assay without affecting performance.

Production

What comes with the CTS Viral Production Cells?
We have extensive documentation to demonstrate lineage history, viral clearance, and traceability of our GMP manufactured process. Under CDA, we can share our Certificate of Analysis and Table of Contents. Full access to documentation package is available through outlicensing@thermofisher.com.

Can I use the CTS Viral Production Cells or CTS LV-MAX system for clinical trials or commercial use?
Please refer to our standard sales terms and conditions and limited use label license for specific products. For further inquiries, please contact us at outlicensing@thermofisher.com.

My cells are initially low viability post thaw, is this normal?
The protocol recommends culturing the cells to passage 5 (P5) so that the cells recover sufficiently prior to starting the protocol for LV production. It is normal to see variance in viability upon thaw, but this should normalize during recovery passages.

What density should I be seeding the cells?
Depending on whether you are doing a 3 day or 4-day culture, we have outlined the differences in seeding densities in our LV-MAX user guide manual. We also have specific guidance around seeding densities to be used for lentiviral vector production.

Why do we have to dilute the cells once more before transfection? Is that necessary?
This is a necessary step to replenish nutrients to the cells and ensure that the cells are at the correct density to achieve the concentrations needed for optimal transfection efficiency. Proper cell density at time of transfection ensures maximal performance of the system.

Do you have recommendations for how to measure non-fluorescent LV titer?
In our user guide manual, we provide a protocol for titer measurement using antibiotic selection.

Why is there no media exchange requirement in the LV-MAX protocol and the cells remain in the same media throughout the two-day transfection? What data do you have to check on the effect of media exchange?
We have done comprehensive studies on the media changes. The LV-MAX production medium is specially designed to support the health of the Viral Production Cells cultured at high-density. Therefore, there’s no need for media exchanges.

The Viral Production Cells that come with the starter LV-MAX Lentiviral Production System are supposed to be good in passage 5–20. Does this refer to passages 5–20 post-thaw, or should we consider the number of passages the cells went through before cryopreservation?
The passages should be counted upon thaw and discarded when they reach passage 20 post-thaw.

Should I harvest at 24 hours and 48 hours? What about waiting for 72 hours? What’s the best harvest method?
Our system has been optimized to provide maximal lentiviral production at 48 hours post transfection. Waiting until 72 hours is not recommended since existing particles within the media will infect the production cells and can reduce the titer.

Commercialization

What is the intended claim for these products?
LV-MAX products are intended for research use only while our CTS LV-MAX products are intended for research use or manufacture of cell, gene or tissue-based products. Please refer to individual product for further details on the intended use claim.

What are the major differences between LV-MAX and the CTS LV-MAX products?
Performance between the two systems is equivalent. In contrast to the research use only LV-MAX products, CTS LV-MAX products have an additional intended use for cell and gene therapy applications and complimented with a more robust regulatory support package to enable clinical therapeutic applications. This means that to the best of our ability, we keep our CTS product specifications and release requirements up to date in accordance with the latest regulatory guidance specific for this intended use.

Do I need to obtain licensing rights if I just want to try the system?
Please refer to our standard sales terms and conditions and limited use label license for specific products. For additional information, please contact us at outlicensing@thermofisher.com.

Have you tried other media like HEK 293 expression media with your system?
Yes, we have tried other media and have seen reduced performance. For maximal LV vector yield, we recommend using LV-MAX medium as part of the entire LV-MAX system.

What scale have you tested for scale up so far? Do you have protocols you can share?
We continually develop large scale protocols to support clinical and commercial LV vector production. Please contact cellculturesupport@thermofisher.com  or the latest information.

What is the recommended ratio of plasmids to use in the system?
We recommend using the lentiviral packaging plasmids to the transfer plasmids at a ratio of 3:2.

Have you tried other plasmids with your systems?
The LV-MAX production system is designed to be compatible with any format of plasmid DNA. The quality of plasmids (e.g., purity), ratio of the plasmids, and vector design all contribute to viral titer. Please contact us at cellculturesupport@thermofisher.com for suggestions.

I need support in designing my plasmid constructs. Who should I reach out to?
Please contact our GeneArt department for assistance at geneartsupport@thermofisher.com.