Virus production in HEK293T

This protocol describes how to produce lentivirus and retrovirus in HEK293T cells, including a final concentration step.

Estimated time

4-6 days. Note that on the transfection day, you must change the media 6-8 hours after the transfection.

Scale and virus type selection

  • Decide on a scale to perform the virus production. This method gives virus titers roughly high enough to infect an equal-area plate initially; each subsequent freeze-thaw cycle will reduce virus titer by roughly 50%. A 10cm dish is sufficient to infect about one and a half 96-well plates. Look up relevant values from the scale table.

  • Decide on a virus type; this determines your packaging and envelope plasmids. Look up the packaging and envelope plasmids in the virus type table.

  • For lentiviruses, use this excel template.

Scale:

Scale

Area (cm^2)

Seeding amount (cells)

Total DNA mass (ug)

Collection media volume (mL)

Concentrated volume (μL)

10cm

56.7

6.0 x 10^6

24

6.5

200

6-well

9.6

1.0 x 10^6

4.1

1.25

40

If using a different virus production scale, the total DNA mass should be scaled by surface area. The collection media volume should be roughly half of the standard amount of media used for that scale.

Virus type:

Type

Packaging plasmid

Envelope plasmid

Lentivirus

psPAX2 (pKG362)

pMD2.G (pKG096)

Retrovirus

pIK-MLVgp (pKG015)

pHDMG (pKG022)

Virus Production

The following is given for a 10cm-dish production scale. For other scales, substitute the correct seeding amounts/total DNA mass/other values from the scale table.

Day 0

  1. Seed 293T cells onto a gelatin-coated virus production plate/dish. For a 10cm dish, this should be 6 million cells (use the calculated seeding amount for other scales).

Day 1

  1. Co-transfect your cells with transfer, packing, and envelope plasmids.

  2. Prepare a mastermix of PEI and knockout DMEM. It is helpful to prepare a 110% master mix (include 10% extra mix) to account for pipetting losses. Ensure that you add the PEI to the KO-DMEM and not in the other order.

    1. Use the total number of micrograms of DNA (24 for a 10cm dish) times the experimentally-determined PEI ratio to get the number of microliters of 1mg/mL PEI you need to add. In other words, the PEI ratio is a PEI:DNA mass ratio.

    2. We use 1.33 mL of KO-DMEM per 10cm dish, but the transfection seems relatively insensitive to this ratio.

  3. After waiting for 10-15 minutes, mix the following amounts of DNA with the PEI-KO DMEM master mix. You can add the master mix to the DNA or add the DNA to aliquoted master mix.

    Scale

    Mass Transfer (ug)

    Mass Packaging (ug)

    Mass Envelope (ug)

    Ratio

    1

    1

    2

    10cm

    6

    6

    12

    6-well

    1.02

    1.02

    2.05

  4. After waiting an additional 10-15 minutes, add the mixture dropwise and evenly around the plate. After adding, be sure to gently rock the plate side-by-side to mix.

    Important

    The six-to-eight hour timing is important for this protocol. We use a relatively large amount of transfected DNA, which means we are also adding a high concentration of PEI. Media changing after six-to-eight hours can greatly reduce transfection-related cell death.

  5. Six-to-eight hours after transfection, replace the media on the plates with HEPES-buffered DMEM. Use the collection volume of media, which is 6.5mL for a 10cm dish.

    The recipes to prepare both the 1M stock solution of HEPES and the HEPES-buffered DMEM can be found here.

Warning

Viral particles may be present after the first media change. Be sure to use proper PPE (i.e., lab coats, disposable sleeves) and wipe down the hood with Pre-Empt after use, from here on!

Day 2, 3, … (n - 1)

  1. 18-24 hours after the last media change, collect the media, replacing it with the same volume of fresh HEPES-buffered DMEM. The collected media from separate days can be stored in the same 50mL conical tube. Keep the collected media at 4°C.

Note

Two collections give high enough virus titer to infect the vast majority (70%+) of cells. However, a third collection can also be taken to further increase titer.

Day (n)

  1. On the last day of collection, either:

    1. Proceed to the Virus Concentration protocol if you want to store your virus long-term.

    2. Filter the collected virus using a 0.45um filter, and keep the unconcentrated virus at 4°C for up to a few days. The virus media can be used instead of normal DMEM in order to directly infect cells.

      For example, 100 µL of unconcentrated virus media (plus 0.1µL polybrene) can be added to each well of a 96-well plate to perform an infection.

Note

Unconcentrated virus appears to lead to more cell death than concentrated virus does. Therefore, concentrating the virus is advised.

Virus Concentration

Estimated time

1 hour in-TC time, 1 day overnight time

  1. Filter collected virus through a 0.45µm syringe filter into a 50ml conical. This eliminates any 293T cells that may have been carried over or collected in the media.

  2. Add 1/3 volume of Lenti-X concentrator (e.g., for 30ml virus, add 10ml Lenti-X). Mix by inverting several times.

  3. Store at 4°C overnight.

Note

If you plan to infect plated cells the next day after concentrating the virus, seed cells on gelatin-coated plates the same day as virus filtration and concentrator addition. Typically, this is 10K cells/well of a 96-well plate.

  1. The next day, centrifuge at 1500 x g at 4°C for 45 minutes (use the lower centrifuge). Be sure to use the caps/lids on the centrifuge buckets.

  2. Aspirate the supernatant. There will be a little liquid left; this is okay.

  3. Resuspend the pellet gently in the remaining liquid and transfer the entire volume to an Eppendorf tube.

  4. Add media to the Eppendorf tube to reach the desired final volume suggesed in the Concentrated volume column of the above table.

  5. Use or store the concentrated virus. a. To use: Infect according to the Transduction of concentrated virus protocol. b. To store: Store all or any extra virus remaining after infection in a cryovial at -80°C.