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AAV Production in HEK293T Cells

Introduction You may also like...

This protocol can be used to produce AAV from one Five Chambers Cell-Stack (CS5)  (3,180 cm2 - the same AAV Viral Service
surface area as 21 x T-175 flasks). Cell stacks provide an efficient means to scale-up without having to handle a
large number of T-175 flasks. Viral Vector Guides

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Last Update: October 10, 2023
Molecular Biology Reference
Workflow Timeline

Day 0: Seed cells in CS2

Day 2: Seed cells in CS5
Day 3 (am): Transfect cells
Day 7 (am): Harvest cells

Equipment
Class II, Type A2 Biological Safety Cabinet Pipet controller
0.5–10 µL single channel pipette Hazardous waste container
2–20 µL single channel pipette pH meter
20–200 µL single channel pipette Stir plate
200–1000 µL single channel pipette Magnetic stir bar
Hemocytometer or cell counter Sonicator
Ice bucket Ear protection
CO2 incubator Vortex

Reagents
Adherent HEK293T cells (ideally AAV-293T clones)
DMEM high glucose, Corning 10-013-CV
*Pro-Tip* While adherent, these cells are very loosely
DMEM, low glucose (1 g/L) glucose, sodium pyruvate, Corning 10-
attached to the dish surface and should be handled
014-CV (optional)
carefully. Avoid touching the cells when replacing media.
7.5% sodium bicarbonate, Corning 25-035-CI (optional)
T-175 flask, Corning 430825, 175 cm2 1 M HEPES, HyClone SH30237.01 (optional)
Cellstack 5, Corning 3319, 3180 cm2 L-alanyl-L-glutamine (or alternative stable glutamine such as
Cellstack 2, Corning 3269, 1272 cm2 glutaGRO, Corning 25-015-CI)
Heat-inactivated FBS (HI-FBS) Opti-MEM, Thermo Fisher 31985-070
0.05% Trypsin/EDTA (e.g. TrypLE, Thermo Fisher, 12605010)
*Pro-Tip* Different brands and lots of FBS can promote or 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV
inhibit transfection. Test a variety of brands and lots of FBS (cations can affect the attachment of adherent cells)
to find one suitable for your protocols. FBS can be 1 mg/mL Polyethylenimine (PEI) 25 kDa MW
purchased already heat-inactivated, or it can be inactivated
in the lab by heating to 56 °C for 30 minutes. *Pro-Tip* Other transfection reagents may be used in this
protocol, but their conditions must be optimized.
0.45 μm polyethersulfone (PES) filter system, Nalgene, 565-0010
(or for larger volumes 1000 mL 0.45 µm Rapid-Flow PES Filtration
Plasmids for transfection: pHelper; pRC (Rep-Cap), plasmid
Unit, Nalgene 167-0045)
expressing your gene of interest
*Pro-Tip* Do not use filters made of materials other than Triton X-100
PES. AAV particles stick to many other surfaces, but do not Benzonase/DNAse I (Millipore 71205-3)
stick to PES. Using a PES filter will maximize titer. 40% Polyethylene Glycol 8000 (PEG) + 0.5 M NaCl
Cell lysis buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl2)
Reagent Preparation

DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such as glutaGRO)
To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of glutaGRO 11 mL of 200 mM L-alanyl-L-
glutamine. Store at 4 °C.
D1 + 0.1 M sorbitol (optional): DMEM low glucose + 1% v/v HI-FBS + 0.1 M sorbitol
In our hands, we have found that a lower glucose, lower FBS media causes less filter clogging during harvesting. The addition of sorbitol
has been shown to increase viral titers by up to 1.8-fold (Rego et al., 2018 ).
To prepare D1 + 0.1 M sorbitol, add 57 mL of 1.46 M sorbitol, 7.5 mL HI-FBS, and 7.5 mL 100X glutaGRO, 7.5 mL of 7.5% Sodium
Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose.
1 mg/mL polyethylenimine (PEI) solution:
Dissolve 100 mg of PEI powder into 100 mL of deionized water.
While stirring, slowly add hydrochloric acid until the solution clears.
Check the pH of the solution and use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0.

*Pro-Tip* The pH of this solution will drift pretty rapidly upon addition of acid or base. Add only a few drops at a time. Allow them
to mix and recheck the pH to prevent over or undershooting the desired pH.

Allow the solution to mix for 10 min and then recheck the pH to ensure that it has not drifted.
Filter the solution through a 0.22 μm membrane.
Aliquot 500–1000 μL into sterile tubes.
Store the tubes at -80 °C.
After thawing, the solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working stock.
40% POLYETHYLENE GLYCOL (PEG) 8000 solution:
Dissolve 400 g of PEG 8000 and 24 g of NaCl into deionized water and adjust to a final volume of 1000 mL.
Stir at RT until fully dissolved.

*Pro-Tip* This step is challenging due to the high viscosity of PEG. Stirring under medium heat will promote faster dissolution.

Adjust the pH to ~7.4.

Autoclave or sterile filter.

*Pro-Tip* Stirring during the cooling period is recommended or the solution may separate into phases.

Aliquot and store at 4 °C.

Cell Lysis Buffer: 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl2
Add the following to the 2 L sterile bottle: 1836 mL deionized water + 100 mL of 1 M Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4
mL of 1 M Magnesium Chloride
Close the bottle and mix by inverting multiple times
Adjust pH to ~8.5
Filter sterilize through a 0.22 μm membrane.
Store at 4 °C.

Considerations Before You Start

The health of the HEK293T cells is critical for optimal AAV yield.

Do not overgrow your cells. Pass the cells twice a week during the maintenance phase and do not allow cells to reach 100% confluence (80–90%
is ideal).
Pass and plate the cells the day before the transfection.
Thaw a new vial of cells after 30 passages.

Procedure

1. Trypsinize and resuspend the HEK293T cells from 2 x T-175 flasks. Cells should be at ~80% confluence. For each T-175 flask:
Aspirate culture media and rinse once with 10 mL of PBS.
Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min.
Neutralize trypsin by adding 10 mL of DMEM Complete.
Pipet back and forth vigorously multiple times to obtain a single cell suspension (no clumps of cells).
2. Pool cells from 2 x T-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix.
3. Seed all cells in 1 CS2. Return to incubator for 48 h.
4. Trypsinize and resuspend cells from the CS2. Cells should be at ~80% confluence.
 Aspirate culture media and rinse once with 60 mL PBS.
Aspirate PBS and add 35 mL of 0.05% Trypsin/EDTA.
Wait ~2-3 minutes for cells to detach.
Gently tap the sides of the CS2 to help detach the cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile bottle.
Rinse the CS2 with 100 mL of DMEM complete medium and pool with the cells harvested in the previous step.
Pipette back and forth or shake vigorously to obtain a single cell suspension (no clumps).
Count cells using a hemocytometer or cell counter.
5. Seed 350 million cells from the CS2 into one CS5 with a total volume of 850 mL. Return to the incubator for 24 h–36 h.
6. Proceed with transfection:
Calculate the amount of each plasmid needed to have a 1:1:1 molar ratio with 2 mg total DNA per CS5

Plasmid Plasmid Size (bp) DNA Concentration (μg/μL) Volume of DNA (μL)

RepCap 7,265 1.00 727.6

pHelper 11,854 1.00 1,185

Transfer Plasmid 5,842 1.00 584.2

Total bp 24,961

In total, we would like 2 mg of DNA or 2000 μg.

Using the total number of base pairs from all three plasmids, we can determine the total μg/bp we need to achieve a 1:1:1 molar ratio of
each plasmid:

2000 μg / 24,961 bp = 0.08 μg/bp

Therefore, for each plasmid we need:

Sample Calculations

RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg

Volume Needed: 582.1 μg / 1.0 μg/μL = 582.1 μL

pHelper: 0.08 μg/bp × 11,854 bp = 949.8 μg

Volume Needed: 949.8 μg / 1.0 μg/μL = 949.8 μL

Transfer Plasmid: 0.08 μg/bp × 5,842 bp = 468.1

μg

Volume Needed: 468.1 μg / 1.0 μg/μL = 468.1 μL

Aliquot 100 mL of OptiMEM into a sterile 250 mL bottle.

Aliquot 774 mL of DMEM + 2% HI-FBS into a sterile 1 L bottle.
Add each plasmid DNA into the bottle containing the Opti-MEM. Mix well.
Add 4 mL of PEI (1:2 μg DNA to μg PEI ratio). Shake the bottle up/down vigorously for 30 sec (it’s okay to make bubbles).
Incubate at RT for 15 min. Note that longer incubation times can reduce transfection efficiency.
Add the OptiMEM + DNA + PEI solution to the bottle containing 774 mL of DMEM + 2% HI-FBS. Mix well.
Take the CS5 out of the incubator and pour the media into a waste container.
Carefully add the OptiMEM + DNA + PEI solution to the CS5. Make sure that all five layers are covered with media. 293T cells are delicate
and detach very easily - media should always be added away from the cells (not poured on them) and can be adjusted by carefully tilting
the CS5 back and forth.

*Pro-Tip* To help distribute the media amongst the five layers, tilt the CS5 such that the media goes toward the cap. If the media
touches the cap, replace it with a new one before putting the CS5 in the incubator.

Return the CS5 to the incubator for 96 h. This incubation time can be adjusted depending upon the serotype. Often AAV2 are harvested at
48–72 h, while other serotypes are harvested at 96–120 h.
Optional: perform a media exchange at ~18 h post transfection. Changing the media can reduce toxicity associated with PEI and
allow for the addition of other components, such as sorbitol and sodium bicarbonate, sorbitol has been found to increase viral titers
by up to 1.8-fold. See the recipe for D1 + 0.1 M sorbitol above.
 Carefully pour off the media into a waste container.
Gently pour new media into the CS5.
Distribute the media evenly between the layers.
Return the CS5 to the incubator for ~72 h.
7. Harvest cells and media by tapping the sides of the CS5. Cells should detach easily.
8. Transfer cells and media into 500 mL conical vessels.
9. Rinse CS5 once with 100 mL of PBS and add to the harvested media and cells.
10. Centrifuge at 3900 rpm for 20 min at 4 °C to pellet the cells.
11. Keep the cell pellet on ice and transfer supernatant to a sterile 500 mL bottle.
12. Process the supernatant as follows:
Filter through a 0.45 μm PES membrane.
Add 40% PEG such that the final PEG concentration is 8%.
Add 25 mL of PEG solution to each 100 mL of supernatant. Split into 2 x 500 mL sterile bottles as needed.
Add stir bar and stir slowly at 4 °C for 1 h, then keep at 4 °C for 3 h without stirring to allow full precipitation. Precipitation of the viruses
can proceed overnight at 4 °C if needed.
Transfer the entire sample to 3 x 500 mL conical bottles and centrifuge at 3900 rpm for 15 min at 4 °C.
Discard the supernatant and resuspend the pellets in a total of 5 mL of cell lysis buffer (recipe above). Pipet back and forth to resuspend
each pellet completely.
Combine resuspended pellets and keep on ice.
13. Process the cell pellet from above as follows:
Resuspend and lyse the cells by adding a total of 12.5 mL of cell lysis buffer. Pipet back and forth to resuspend each pellet completely.
Combine all resuspended cell pellets and sonicate 5 x 1 sec pulses with at least 5 minutes on ice between each pulse, 50% amplitude.
Return to ice between each round of sonication to avoid overheating of the sample. Mix well between rounds of sonication.
Sonicate until no live cells can be seen when stained with Trypan Blue.
Pellet cell debris by centrifugation at 3900 rpm for 10 min at 4 °C.
Transfer the cleared lysate to the tube containing the resuspended virus from step 12 above.
14. Add 50 units of benzonase per mL of viral suspension (you should have ~18 mL from steps 12 & 13). Benzonase is an endonuclease that will
degrade any residual DNA carried over from the packaging process.
15. Incubate at 37 °C for 45 min.
16. You are now ready to purify your prep. The clarified supernatant can be kept overnight at 4 °C before proceeding with the purification protocol.

Sample Data

Figure 1: HEK293T cells at various confluencies. This AAV production protocol should be started with cells that are ~80% confluent (center panel).
These cells were imaged with a 10X objective and were split 2 days before these images were taken.