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View additional product information for LV-MAX™ Lentiviral Production System - FAQs (A35684)
24 product FAQs found
The LV-MAX and CTS LV- MAX products have equivalent performance. 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 are complemented 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.
Our 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 the individual products for further details on the intended use claim.
Yes. Please provide your details here (https://www.thermofisher.com/us/en/home/global/forms/life-science/lentiviral-vector-production-get-info.html) and mention your interest in our lentivirus production services. A member of our services team will contact you shortly.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Lentiviral particles packaged in our LV-MAX Production Medium will be stable at 4 degrees C for 24 hours and will then begin to lose activity. For long-term storage, we recommend storing at –80 degrees C.
We strongly recommend the human fibrosarcoma line HT1080 (ATCC, Cat. No. CCL-121) as the “gold standard” for reproducibly titering lentivirus. However, you may wish to use the same mammalian cell line to titer your lentiviral stocks as you will use to perform your expression studies (e.g., if you are performing expression studies in a dividing cell line or a non-primary cell line). If you have more than one lentiviral construct, we recommend that you titer all of the lentiviral constructs using the same mammalian cell line.
Spinoculation increases lentiviral transfection efficiency and provides higher viral titer than without the spin step. If you don't spin, you can expect to get 5- to 10-fold less lentiviral titer.
We recommend a weight-to-weight ratio of 2:3 transfer plasmid:packaging vectors; if needed use a 3:3 weight-to-weight ratio.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
In the user guide (http://tools.thermofisher.com/content/sfs/manuals/MAN0017000_LV_MAX_ViralProductionSystem_UG.pdf ), we have provided recommendations based on our development of the kit. If you need to optimize further for your specific need, we recommend a weight-to-weight ratio of transfer plasmid:packaging vectors at 1:3, 2:3, and 3:3.
The critical incubation time is the one for the diluted LV-MAX Transfection Reagent. Prior to combining with diluted DNA, the diluted LV-MAX Transfection Reagent should be incubated for only 1 min, and not more than 4 mins.
Yes, that should be fine.
No, these cells are derived from Freestyle 293F, and have been adapted to our LV-MAX Production Medium.
Yes, all components of the LV-MAX Production System are animal origin-free. However, if Opti-MEM I Reduced Serum Medium is used as the DNA-LV-MAX Transfection Reagent complexation medium, it will no longer be animal origin-free.
Yes, all components of the LV-MAX Production System are serum-free.
In the LV-MAX Production System, only the LV-MAX Production Medium is manufactured according to cGMP guidelines. However, we do offer our CTS LV- MAX products (CTS LV-MAX Production Medium, CTS Viral Production Cells, and CTS LV-MAX Transfection Kit) that are all manufactured according to cGMP guidelines.
The Viral Production Cells are not cGMP-banked. However, we offer the CTS Viral Production Cells that are cGMP banked and optimized to work with the CTS LV-MAX Production Medium and CTS LV-MAX Transfection Kit.
We have not optimized the LV-MAX Production System for AAV production. However, the Viral Production Cells contain the right genetic elements for producing AAV.
We have identified 3 key protocols that will address most customers' needs. In the user guide (http://tools.thermofisher.com/content/sfs/manuals/MAN0017000_LV_MAX_ViralProductionSystem_UG.pdf ), you will find protocols for 1 mL in a 2 mL 96-well deep block, 5-30 mL in 50 mL conical tubes, and 30 mL-1 L in various shaker flask sizes. For larger-scale needs, we have developed a bioreactor protocol for up to 2 L. if you are interested, please email us at cts@thermofisher.com for details.
No, we've done the work for you. You will need to determine the best production cell density under your cell counting system. We recommend trying 3 different cell densities, i.e., 3 x 10E6, 4 x 10E6, and 4.5 x 10E6 cells/mL to determine the optimal cell density.
No. In addition to the LV-MAX System, you will need Opti-MEM I Reduced Serum Medium (Cat. No.31985088) as the DNA-LV-MAX Transfection Reagent complexation medium. You will also need a pLenti transfer plasmid and pLenti packaging mix. Please see the user guide (http://tools.thermofisher.com/content/sfs/manuals/MAN0017000_LV_MAX_ViralProductionSystem_UG.pdf ) for a complete list of recommended materials and equipment.
You may use an alternative shaker, but it must be CO2 resistant. If you use a non-digital shaker, you will need to optimize the shaker speed based on general observation. You must ensure that the cells do not settle but maintain suspended. You will need to generate your own cell growth curve to ensure you exceed 90% cell viability.
While we currently don't have data for prior generation lentiviral packaging systems, the LV-MAX Lentiviral Production System should be compatible with them but may require further optimization.
Several factors are known to affect viral yield:
- Production cell density: We suggest a cell density of 4 x 10E6 viable cells/mL in our protocol, but since different cell counting methods may give different counting results, we recommend that you optimize your production cell density based on your cell counting system. We recommend trying 3 different cell densities, i.e., 3 x 10E6, 4 x 10E6, and 4.5 x 10E6 cells/mL to determine the optimal cell density.
- Quality of the plasmid: Make sure that your plasmids are endotoxin-free and of high purity.
- Protocol: Following our cell culture protocol to establish your culture is the key to successfully use our LV-MAX System.
- Size of the gene: The size of the gene you are interested in will impact viral particle yield; the larger the size of the gene, the lower will be the viral particle yield.
Note: While viral titer yields are highly dependent on production volume, the specific gene being expressed, and its length, our R&D team as well as external collaborators have consistently seen an increase in viral titer that is equivalent to or 10-fold higher when using LV-MAX Lentiviral Production System as compared to adherent PEI, or suspension PEI methods.
Viral Production Cells can be frozen directly in cryogenic medium consisting of 90% LV-MAX Production Medium plus 10% DMSO. Here is the protocol:
- Bank cells at passage 3 when cell density reaches ˜3.0-4.5 x 10E6 viable cells/mL at >95% viability.
- Centrifuge the cells at 100 x g (low speed) for 5 mins to pellet. Discard the spent medium.
- Prepare freezing medium: e.g., 10 mL of freezing medium is 9 mL of LV-MAX Production Medium + 1 mL DMSO; mix well and sterilize by 0.2 µm filtration.
- Resuspend the cell pellet in the prepared freezing medium.
- Dilute the cells to a final density of 10 x10E6 viable cells/mL and aliquot 1 mL per cryovial.
- Freeze the cells at -80 degrees C for 1 day.
- Transfer frozen vials to liquid nitrogen for long-term storage.
LV-MAX Transfection Reagent, enhancer, and supplement are specifically designed to work synergistically with the LV-MAX Production Medium and Viral Production Cells to provide maximal lentiviral titer. Mixing and matching reagents is not recommended, as this will decrease the overall performance.