TITLE: Efficient third generation lentiviral particle production
AUTHORS: Michelle Newbery, Simon          Maksour, Amy Hulme, Neville Ng, Mirella          Dottori, Lezanne           Ooi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The overexpression of a gene of interest by third generation lentiviral particle generation systems is a critical process in molecular biology, cell biology and gene therapy research. While many lentiviral protocol production methods have been discussed in literature, this protocol takes into account previously established optimisations with the aim to minimise user handling time, cost, and maximise practical yield. This protocol allows for at least 6 days of consecutive viral particle collection without compromising HEK293T cell culture or viral production efficiency, and can be easily and cost effectively reproduced in basic cell culture laboratories. </div></div>

[STEPS]
?. [Lentiviral transfection]
Maintain HEK293T cells in 0.15 mL/cm2 (e.g. 12 mL in 75 cm2 flask) animal product free FreeStyle 293 Expression Medium, foetal serum free DMEM/F12 + 2 mM GlutaMAX + 5% Knock Out Serum Replacement or DMEM/F12 + 2 mM GlutaMAX + 5% FBS. Include 50 U/mL Penicillin and Streptomycin to reduce risk of bacterial contamination if necessary.Dissociate and subculture HEK293T to ~70% confluency per virus of interest. Viral particle production can be performed within at least 15 passages without loss of yield.
?. [Lentiviral transfection]
For each lentiviral plasmid containing gene of interest and packaging plasmid vectors, calculate reagent volumes required for 12 µg gene of interest vector, 6 µg pMDLg/pRRE (Addgene #12251), 3 µg pRSV-Rev (Addgene #12253) and 3 µg pCMV-VSV-G (Addgene #8454) and 60 µg PEI per 75 cm2 of HEK293T culture.Maximal transfection efficiency by PEI complex is typically obtained with higher proportions of PEI:DNA (Figure 1). We observed no differences in transfection with KSR or FBS -based medium.
?. [Lentiviral transfection]
Prepare PEI-DNA solution in 1.5 mL of DMEM/F12 and incubate for  at ambient temperature.
?. [Lentiviral transfection]
Add PEI-DNA complex solution to cell culture flask(s) and return to incubator . Note PEI-DNA complex transfection can occur without usage of low serum transfection medium products. PEI-DNA complex precipitate should be microscopically visible as ~0.3 µm objects (Figure 2).
?. [Lentiviral transfection]
Each day collect medium in an appropriate sized sterile container (e.g. 50 mL centrifuge tubes or 120 mL specimen collection tubes), and store at . Proceed to viral particle concentration at selected endpoint (Step 6). We utilise viral particle concentration to avoid prolonged incubation of pluripotent or multipotent stem cells in HEK293T culture medium during viral transduction experiments. If viral particle concentration is not necessary, freeze immediately at . Note a single collection step is performed on day 6 due to negligible difference in loss of yield between day 3 and 6, which avoids multiple rounds of centrifugation. Viral particle collection can be performed for at least up to 7 days.
4 °C
-80 °C
?. [Lentiviral particle concentration]
Sterilise ultracentrifuge tubes and appropriate sized viral particle aliquot tubes (0.2 or 1.5 mL microcentrifuge tubes). For each 75 cm2 flask if collecting 12 mL over 6 days, at least 2 x 36 mL ultracentrifuge tubes are required.
?. [Lentiviral particle concentration]
Centrifuge pooled viral particle supernatant at  to remove cell debris.
Centrifuge: 300 34, 3 min
?. [Lentiviral particle concentration]
Filter supernatant from each tube through a 0.45 µm pore PES filter into corresponding ultracentrifuge tubes.
?. [Lentiviral particle concentration]
Balance ultracentrifuge tubes within 0.1 g by weighing and adding appropriate amount of medium.
?. [Lentiviral particle concentration]
Collect viral particles by centrifugation of filtered viral particle supernatant at .
Centrifuge: 50000 34, 90 min, 4 10
?. [Lentiviral particle concentration]
Carefully transfer tubes on ice, mark pellet position/expected position of pellet and decant slowly to discard supernatant.
?. [Lentiviral particle concentration]
Resuspend pellet as a ~200X concentrate of total supernatant (e.g. 180 µL from 36 mL of supernatant).As lentivirus particles are enveloped and more susceptible to degradation in comparison to unenveloped DNA viruses, the addition of 10% DMSO as a cryoprotectant allows at least 4X freeze-thaw cycles without compromising viral particles and reduces necessity for low volume aliquots (Figure 3).
?. [Lentiviral particle concentration]
Prepare viral particle aliquots of appropriate volumes corresponding to application and freeze at . Viral titre (TU/mL) can be calculated (# transfected cells / volume of virus) based on fluorescent reporter or immunofluorescent staining.
-80 °C