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View additional product information for CTS™ Opti-MEM™ I Medium - FAQs (A4124802, A4124801)
35 product FAQs found
We recommend using V-bottom 96-well plates to harvest the supernatant containing lentiviral particles as they allow for easier detection of the cell pellets after centrifugation of the cultures.
We recommend maintaining shaking cultures in a separate incubator from all other incubators to avoid vibration effects. Alternatively, we recommend using a small, quiet shaker such as the Thermo Scientific CO2 Resistant Shaker (Cat. No. 88881102) for convenience and best performance.
We generally recommend culturing the CTS Viral Production Cells under 8% CO2 conditions. We found that cells cultured in 5% CO2 grew slower and were more prone to more cell clumping than when grown in 8% CO2.
The CTS LV-MAX lentivirus production system is a fully-optimized complete system in which the cells, media, and transfection reagent work together to generate high-titer lentivirus. The CTS LV-MAX Production Medium is specifically designed to work with the system, to support high density suspension cell growth of CTS Viral Production Cells.
Although 3-4 passages before transfection are sufficient for most cell lines recovering directly from liquid N2 storage, we found that CTS Virus Production Cells passaged to P5 showed the highest performance in lentivirus particle production and reproducibility between batches. Therefore, we recommend passaging the cells to P4 at the end of a given week for lentivirus production the following week.
For optimal cell recovery from liquid N2 storage, CTS Viral Production Cells should be passaged to passage 2 (P2) (from the original vial) and up to P3 before banking, to ensure good cell health and viability. Cells that demonstrate good cell health (i.e., ˜26 hr doubling time, round cell morphology, and minimal clumping) may be banked at P2 or P3.
Passaging the CTS Viral Production Cells every 3-4 days allows for cell growth rates to progress from lag to log phase, thus ensuring highest cell viability after splitting cultures. We recommend passaging the cells at a density of no lower than ˜0.35x10E6 viable cells/mL as low cell densities maintained in the lag phase for long periods, will eventually lead to poor overall cell growth. We do not recommend culturing the cells past 4 days.
The cell culture densities for CTS Viral Production Cells are as follows:
- 0.35x10E6 and 0.55x10E6 viable cells/mL (splitting cell density for 4 day and 3 day culture, respectively)
- 3.5x10E6 to 5x10E6 viable cells/mL (subculture cell density)
- Log-phase is from 0.35x10E6 to 6.5x10E6 viable cells/mL.
When propagating lentiviral expression plasmids, it is important to propagate them in cells that will minimize any sequence rearrangements or unwanted recombination due to the long terminal repeat (LTRs) sequences and large size of the vector backbone ( ˜10 Kb). To minimize these events, we recommend using Stbl3 cells for stable propagation of lentiviral expression plasmids.
We recommend using either the GFP method or antibiotic-based method described in the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018450_CTS_LV-MAX_LentiviralProdSys_UG.pdf). You may try other viral titering methods, however, we do not have protocols for methods other than by GFP or antibiotics.
Low lentiviral titer could be due to many different factors such as non-optimized lentiviral expression vector design, low transfection efficiency, incorrect cell density, suboptimal culture conditions (e.g., incubator, shaker speed, vessel type, media contamination), purification strategy, and titer method. To best track your workflow from culturing your cells to titering the lentivirus, we recommend using a positive control such as the Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector (Cat. No. V37006).
This could mean that the seeding or starting cell density was too low. The CTS LV-MAX Supplement relies on high cell density. Refer to the CTS LV-MAX Lentiviral Production System manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018450_CTS_LV-MAX_LentiviralProdSys_UG.pdf) for cell density recommendations.
Anti-clumping reagents could potentially inhibit transfection efficiency and are generally not recommended. If needed, add no more than 0.1% Pluronic F-68
The CTS LV-MAX lentivirus 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 lentiviral titer. Please contact us at techsupport@thermofisher.com for suggestions.
The formulation of the CTS LV-MAX Enhancer coupled with the broad window of enhancer addition time was specifically optimized to allow user flexibility in schedule and start time of the protocol without affecting performance. For optimal results, we recommend adhering to the suggested time window for enhancer addition.
The CTS LV-MAX Production Medium is specially designed to support the health of Viral Production Cells cultured at high-density. We have performed comprehensive studies in-house and have determined that there is no need for media change during the protocol.
Please refer to page 31 of the user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018450_CTS_LV-MAX_LentiviralProdSys_UG.pdf) for a protocol for lentiviral titer measurement using antibiotic selection.
The sequences of the LV-MAX Lentiviral Packaging Mix plasmids are published on our website; however, the ratio of the plasmids in the mix is confidential.
We recommend using the lentiviral packaging plasmids and transfer plasmids at a ratio of 3:2.
Our system has been optimized to provide maximal lentiviral production at 48 hr post transfection. Waiting until 72 hr is not recommended since existing lentiviral particles within the media will infect the production cells and can reduce the titer.
The passages should be counted upon thawing, and the cells should be discarded when they reach passage 20 post-thaw.
We recommend that CTS Viral Production Cells be used for lentiviral production only following passage 5 to allow for full recovery of the cells from the cryopreservation; CTS Viral Production Cells are sensitive upon thawing and need the extra passages for recovery.
This is a necessary step to replenish nutrients and ensure that the cells are at the correct density to achieve the concentration needed for optimal transfection efficiency. It is crucial that the cells are at the proper cell density during the time of transfection to ensure maximal performance.
Depending on whether your culture will be 3 or 4 days, we have outlined the differences in seeding densities in the product manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018450_CTS_LV-MAX_LentiviralProdSys_UG.pdf). We also have specific guidance for seeding densities to be used for lentiviral vector production.
We continually develop large-scale protocols to support clinical and commercial lentiviral vector production. Currently, we have a 2 L bio-reactor protocol that is available from techsupport@thermofisher.com. We are also working on a 5 L and 50 L scale.
It is okay to see variation in viability upon thawing, but this should normalize during subsequent passages. We recommend culturing the cells as described in chapter 2 of the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018450_CTS_LV-MAX_LentiviralProdSys_UG.pdf) so that the cells recover sufficiently and achieve >90% viability before starting the protocol for CTS lentviral production.
We have tried other media and have seen reduced performance. For maximal lentiviral vector yield, we recommend using the CTS LV-MAX Production Medium.
The CTS LV-MAX Transfection Kit is specifically designed to work synergistically with the CTS LV-MAX Production Medium and CTS Viral Production Cells to provide maximal lentiviral titer. Mixing and matching reagents is not recommended, as this will decrease the overall performance.
The CTS LV-MAX Transfection Kit contains three components, the transfection reagent, supplement, and enhancer, that are not sold as stand-alone items. The system components were specifically optimized to work as a complete system to obtain maximal lentiviral titer. We can however customize different ratios of the transfection reagent, supplement, and enhancer - for further information, please send an email to customorders@thermofisher.com.
We can provide protocol guidance for how to detect transfection reagent components under CDA. Please contact our outlicensing@thermofisher.com for information.
For downstream purification of lentivirus under serum-free conditions, we recommend using tangential flow filtration. Please contact techsupport@thermofisher.com for the suggested protocol. In order to maintain the integrity of lentiviral vectors, we do not recommend using ultracentrifugation under serum-free conditions.
The intended use, validation, and protocol optimization varies among the different cell types. Expi293F Cells and FreeStyle 293-F Cells were specifically developed and validated for transient protein production while Viral Production Cells and CTS Viral Production Cells were designed and validated for lentiviral vector manufacturing. The commonality between the Expi293F Cells, Viral Production Cells, and CTS Viral Production Cells is that they were originally derived from FreeStyle 293-F lineage. Both Expi293F and Freestyle 293F cells come with regulatory support intended for bio-production applications whereas CTS Viral Production Cells (cGMP banked) are intended for cell and gene therapy applications, and complemented with a more robust regulatory support package to enable clinical gene 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.
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.
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 the documentation package is available through outlicensing@thermofisher.com.
Please refer to our standard sales terms and conditions and limited use label license. For further inquiries, please send an email to outlicensing@thermofisher.com.