Hello Jyoti Sawant

1. Preparation of HEK293T cells: What is the optimal cell density at the time of transduction?

Target cells should be approximately 70% confluent for lentiviral transduction.

2. MOI (Multiplicity of Infection): How do I determine the appropriate MOI for HEK293T cells to achieve high transduction efficiency without excessive viral load?

Perform a pilot experiment using a reporter virus on your target cell line to determine the optimal MOI for your experiment. You should select a few MOI’s to choose the optimum value. For instance, using a GFP Lentivirus, design a range of MOIs to use, let’s say, 5 conditions ranging from MOIs 1, 2, 5, 10 and 15. A good starting point would be to reference commonly used MOI for 293 cells and devise the range around the suggested MOI. It is typically better to test a lower MOI range to avoid cytotoxicity at the higher MOIs. Infect your target cells and evaluate the fluorescence after 48 -72 hours post infection (generally used for lentivirus). Record the fluorescence at the various MOIs to determine your transduced cell percentage. You will select the minimum MOI at which all cells express the transgene.

3. Enhancing transduction efficiency: Are there any additives like polybrene, protamine sulfate, or others that can improve transduction rates? What are the recommended concentrations?

Polybrene is used to enhance transduction efficiency in the range of 3–10 µg/ml. Polybrene neutralizes the charge interactions between the cell surface and viral particles, which improves infection efficiency. If you feel that polybrene is toxic to your cell line, you can substitute protamine sulfate for polybrene.

4. Infection conditions: What are the optimal incubation time, temperature, and media conditions during and after transduction?

After 70% confluency, change to fresh culture media containing anywhere between 3- 10 μg/mL polybrene. Add lentiviral particle solution after you have selected the optimal MOI for infection. Incubate cells for 18-20 hours at 37°C, 5% CO2 in a humidified incubator. Fresh media should be added 24 hours after infection. If viral toxicity is observed, reduce the infection time to 4–20 hours.

5. Post-transduction care: How soon should I replace the media after transduction, and when should I expect to observe transgene expression (e.g., fluorescence or protein expression)?

Remove the virus-containing media and replace with fresh media 24 hours after infection. If viral toxicity is observed in your cell line, you may decrease the infection time to between 4 to 20 hours. Do not add antibiotics until at least 24 hours after infection to allow for sufficient expression of the antibiotic resistance gene.

The transgene expression can be observed 48-72 hr after transduction by fluorescence or Western Blot.

6. Troubleshooting: If transduction efficiency is low, what steps can I take to optimize the process?

There are several factors you need to consider if the transduction efficiency is low.

a. Make sure cells are in low passage (for instance, passage number for HEK293 should be between 3 and 16), and have not been overgrown before subculturing.

b. Make sure your cells are no more than 70% confluent before transduction.

c. Concentrate your viral stocks using ultracentrifugation into smaller volume. Store at -80°C and do not thaw unnecessarily. Aliquot your virus into separate stocks and only thaw an aliquot when you are about to use it. Please note studies have shown that there is a 25% loss of viral titer with each freeze-thaw cycle.

d. It is important that you optimize the MOI for your target cell line.

e. Monitor the health status of your cells prior to transduction to ensure that the cells are contaminant-free (no mycoplasma) and at least 90% viable.

f. Optimize the concentration of polybrene because it depends on cell type (usually 3–10μg/ml) and may need to be empirically determined.

g. You should allow 72-96 hours for recombinant protein expression before performing your assay as this will ensure that there is sufficient accumulation of your desired protein or development of antibiotic resistance.

h. If you feel that the reagents are toxic to your cells, you may change the media much earlier before 24 hours (at least 4 hours) after transduction. Alternatively, use a different target cell line which is less sensitive to the toxicity of your reagents.

Good Luck!

Dear Jyoti,

Malcom's advices are of real interest! these are good advices, and I would consider especially the third point concerning Transduction efficiency.

To this end, I would like to propose you to try our LentiBlast Premium Transduction enhancer.

LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.

It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience.

LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for your experiment.

Some examples of publications with LentiBlast Premium:

• HEK293: Brown AL. Sci Rep . 2021 Oct 25;11(1):21008.
• Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
• Hematopoietic Stem Cells: Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
• HMEC: Nassour J. Nature . 2023 Feb;614(7949):767-773.
• Myeloid progenitor: Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
• T lymphocytes CD4+: Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
• THP-1: Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.

you can have more infos at: https://ozbiosciences.com/lentivirus-retrovirus/113-418-lentiblast-premium-transduction-enhancer.html#/12-capacity-500_l

Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGateor directly at: [email protected]; I will be glad to send you a sample !

best regards,

Cedric