Lentivirus Production

Dissolve Polyethyleneimine [Polysciences PN 23966] by swirling using de-ionized H₂O heated to 80°C and using ~90% of volume required to reach a final concentration of 1 [1 μg/μl].

Cool to Room temperature.

Set to 7.0 by adding HCl.

Add de-ionized H₂O to reach final concentration of 1µg/µL.

Filter using a 0.22 μm membrane and divide into aliquots. Store in -20°C freezer.

Thaw in 37°C water bath until precipitates have fully dissolved.

Store thawed aliquot in fridge [4°C].

Re-dissolve precipitates by incubating at 37°C before use if needed.

Make media, warm in water bath.

Seed cells on Thursday [0.9 x 10⁶] or Friday [1.8 x 10⁶] before [25 ml media]. This will yield approx. 20 million cells per T175. Seed 8-9 flasks for a 6 virus batch.

Wash with 10mL [on the roof, then submerge carefully].

2mL, 0h 5m 0s in incubator, hit on the side.

10mL, spin 400x g .

Resuspend in 5mL, count, [80 ul PBS + 10 ul cell susp, i.e. 1:10 dil].

Add the right number of cells to each flask [see above], plus 10mL - 20mL .

Warm media in water bath.

Wash with 10mL [on the roof, then submerge carefully].

2mL, 0h 5m 0s in incubator, hit on the side.

Add 5mL - 7mL to each flask to neutralize trypsin.

Rinse flasks and transfer cells to a [or two] 50 ml tube.

Spin down at 400x g,20°C .

Resuspend cells in appropriate volumne [about 20mL].

Count.

Add 20mL to each flask [DMEM Glutamax + 10% FBS + Penicillin 100 U/ml + Streptomycin 100 U/ml].

Plate 12.5 per T175 flask. Need 12 for 6 virus batch.

Add the calculated cell suspension to the flask with the media.

Check to see how confluent cells are [should preferably be 75% - 90%].

Make sure you have all calculations done prior to starting, including the volumne of PEI to be used [102 μm - 132 μm].

Mix packaging vectors [pMDL, psRev and pMD2G] and transfer vector in a 50 ml tube with serum free media [DMEM+P/S] or PBS, so that the total volumne [including PEI] becomes 3.6 ml.

Add PEI and mix by vortexing for a few seconds.

Incubate the DNA/PEI mix at Room temperature for 0h 15m 0s .

Change media in flasks and add 16.2mL .

Add 1.8mL to each T175 flask, mix gently over cells by tilting the flask.

Check that they are alive and if they have the right flurophore [if applicable].

Collect media from the two flasks for each virus into one 50 ml tubes.

Spin the supernatant at 800x g,4°C .

While tubes are in the centrifuge, prepare in hood the requirements for the next step. Also, rinse the now empty culture flasks with Virkon and discard.

Remove the 50 ml tubes from centrifuge, and in the hood, pour the supernatant into open syringe with attached filter over the labelled Beckman UC tube. After each virus/50 ml tube, change the syringe and filter. Ensuring that the discarded syringe and filter are placed and rinsed in Virkon.

Put Beckman UC tubes into their chambers using forceps.

To avoid collapsing the tubes, fill up sample tubes with media. Fill up counterweight tubes with DPBS.

Balance the weight of the tubes carefully, down to 5 ug difference.

Clean everything in the hood thoroughly. Any plastics that have been used must be rinsed with Virkon before discarding. Spray pipettes, racks, pipette gun etc. with ethanol. Clean hood surfaces with Virkon first, then with ethanol.

Turn off the hood fully and then put the UV on.

Put down rotor, spin it.

Put down all 6 chambers into centrifuge [you do not have to weigh the empty ones].

Check settings of centrifuge:

• 19500x g,4°C

• Enter to save

• Ensure Acceleration is set to max

• Ensure Deceleration is set to max

Wait until centrifuge reaches full speed before leaving.

Set alarm for when centrifugation is done, don´t let the tubes stand longer.

Deceleration of centrifuge takes approx. 0h 5m 0s. After it reaches 3000G, you can release the vacuum to increase the speed of deceleration.

Take out Beckman UC tubes using forceps, and pour out liquid into Virkon.

Place tuned upside down on paper to dry, be careful to remove all liquid, also from edges and walls.

Take a bottle of cold PBS from freezer and resuspend pellet in 80µL [75 ul - 100 ul is ok]

Seal tubes with Parafilm, vortex and put into fridge, leave them there for at least 2h 0m 0s - but preferably .

Aliquot virus [10 ul/tube] and store -80°C.

Viruses can now be titrated and/or used.