TITLE: AAV Production in HEK293T Cells
AUTHORS: Addgene the nonprofit plasmid repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used for AAV production in HEK293T cells. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/aav-production-hek293-cells/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqii7esn.png" /></div></div>

[STEPS]
?. [Procedure]
Trypsinize and resuspend the HEK293T packaging cells from 2 x T-150 flasks. Cells should be at ~80% confluence. For each T-150 flask:Aspirate culture media and rinse once with  of PBS.Aspirate PBS and add  of 0.05% Trypsin/EDTA. Wait ~.Neutralize trypsin by adding  of HI-FBS.
10 ml
4 ml
4 ml
?. [Procedure]
Pool cells from 2 x T-150 flasks. Adjust volume to  with DMEM complete media and mix.
200 ml
?. [Procedure]
Seed all cells in 1 CF2. Return to incubator for .
?. [Procedure]
Trypsinize and resuspend cells from the CF2. Cells should be at ~80% confluence.Aspirate culture media and rinse once with 60 mL PBS.Aspirate PBS and add   of 0.05% Trypsin/EDTA.While waiting for the cells to lift-up, aliquot   of HI-FBS to a sterile   plastic bottle.Gently tap the sides of the CF2 to help detach the cells, then transfer the cells into the bottle containing   of HI-FBS to neutralize the trypsin.Rinse the CF2 with   of DMEM complete medium and pool with the cells harvested in the previous step.Pipette back and forth vigorously to obtain a single cell suspension (no clumps).Aspirate PBS and add  of 0.05% Trypsin/EDTA.While waiting for the cells to lift-up, aliquot  of HI-FBS to a sterile  plastic bottle.Gently tap the sides of the CF2 to help detach the cells, then transfer the cells into the bottle containing  of HI-FBS to neutralize the trypsin.Rinse the CF2 with  of DMEM complete medium and pool with the cells harvested in the previous step.Add  of complete medium (final volume ).
35 ml
5 ml
100 ml
5 ml
30 ml
435 ml
500 ml
?. [Procedure]
Seed 250 million cells from the CF2 into 1 CF5. Return to incubator for  - .
?. [Procedure]
Proceed with transfection:Calculate the amount of each plasmid needed to have a 1:1:1 molar ratio with  total DNA per CF5Aliquot  of OptiMEM into a sterile  bottle.Aliquot  of DMEM + 2% HI-FBS into a sterile  bottle.Add the plasmid DNA into the bottle containing the Opti-MEM. Mix well.Add   of PEI (1:3 μg DNA to μg PEI ratio). Shake the bottle up/down vigorously for   (it’s okay to make bubbles).Incubate at room temperature for  . Note that longer incubation times can reduce transfection efficiency.Add the OptiMEM + DNA + PEI solution to the bottle containing   of DMEM + 2% HI-FBS. Mix well.Take the CF5 out of the incubator and pour the media into a waste container.Carefully add the OptiMEM + DNA + PEI solution to the CF5. 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 CF5 back and forth.Add  of PEI (1:3 μg DNA to μg PEI ratio). Shake the bottle up/down vigorously for  (it’s okay to make bubbles).Incubate at room temperature for . Note that longer incubation times can reduce transfection efficiency.Add the OptiMEM + DNA + PEI solution to the bottle containing  of DMEM + 2% HI-FBS. Mix well.Take the CF5 out of the incubator and pour the media into a waste container.Carefully add the OptiMEM + DNA + PEI solution to the CF5. 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 CF5 back and forth.Return the CF5 to the incubator for . This incubation time can be adjusted depending upon the serotype. Often AAV2 are harvested at -, while other serotypes are harvested at  -.
2 mg
In total we would like 2.5 mg of DNA or 2,500 μgUsing 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:        2,500 μg/ 24,961 bp = 0.10 μg/bpTherefore, for each plasmid we need:        2,500 μg/ 24,961 bp = 0.10 μg/bp
Sample Calculations
RepCap: 0.10 μg/bp × 7,265 bp = 727.6 μg
Volume Needed: 727.6 μg/ 1.0 μg/μl = 727.6 μl
pHelper: 0.10 μg/bp × 11,854 bp = 1185.4 μg
Volume Needed: 1185.4 μg/ 1.0 μg/μl = 1185.4 μl
Transfer Plasmid: 0.10 μg/bp × 5,842 bp = 584.2 μg
Volume Needed: 584.2 μg/ 1.0 μg/μl = 584.2 μl
176 ml
250 ml
350 ml
500 ml
7.5 ml
350 ml
*Pro-Tip*To help distribute the media amongst the five layers, tilt the CF5 such that the media goes toward the cap. If the media touches the cap, replace it with a new one before putting the CF5 in the incubator.
?. [Procedure]
Harvest cells and media by tapping the sides of the CF5 on a soft surface. Cells should detach easily.
?. [Procedure]
Transfer cells and media into  conical tubes (or  conical vessels).
50 ml
500 ml
?. [Procedure]
Rinse CF5 once with of PBS and add to a new  conical tube.
20 ml
50 ml
?. [Procedure]
Centrifuge at 3,000 g or max speed on a tabletop centrifuge for  at  to pellet the cells.
4 °C
?. [Procedure]
Keep the cell pellet on ice and transfer supernatant to a sterile 500 mL bottle.
?. [Procedure]
Process the supernatant as follows:Filter through a 0.45 μm PES membrane.Add   of PEG solution to each   of supernatant. Split into 2 x   sterile bottles as needed.Add stir bar and stir slowly at   for  , then keep at   for   without stirring to allow full precipitation. Precipitation of the viruses can proceed overnight at   if needed.Transfer the entire sample to   conical tubes and centrifuge at 2,818 g for   at  .Discard the supernatant and resuspend the virus (small pellet) in   PBS + 0.001% pluronic F68 + 200mM NaCl. Pipet back and forth to resuspend the virus completely.Keep resuspended pellet on ice.Add  of PEG solution to each  of supernatant. Split into 2 x  sterile bottles as needed.Add stir bar and stir slowly at  for , then keep at  for  without stirring to allow full precipitation. Precipitation of the viruses can proceed overnight at  if needed.Transfer the entire sample to  conical tubes and centrifuge at 2,818 g for  at .Discard the supernatant and resuspend the virus (small pellet) in  PBS + 0.001% pluronic F68 + 200mM NaCl. Pipet back and forth to resuspend the virus completely.
25 ml
100 ml
500 ml
4 °C
4 °C
4 °C
50 ml
4 °C
10 ml
?. [Procedure]
Process the cell pellet as follows:Resuspend and lyse the cells by adding  of PBS + 0.001% pluronic F68 + 200mM NaCl and sonicating 4 x 1 sec pulses with at least  on ice between each pulse, 50% amplitude. Return to ice between each sonication to avoid overheating of the sample.Pellet cell debris by centrifugation at 3,220 g for  at .
10 ml
4 °C
?. [Procedure]
Add 50 units of benzonase per mL of viral suspension in PBS (you should have ~ from steps 12 & 13). Benzonase is an endonuclease that will degrade any residual DNA carried over from the packaging process.
20 ml
?. [Procedure]
Incubate at  for .
37 °C
?. [Procedure]
Transfer the viral suspension to centrifuge tubes and centrifuge at  at  for.
4 °C
Centrifuge: 2415 34
?. [Procedure]
Transfer the clarified supernatant to a new tube and proceed with purification.
Note: an aliquot of the solution can be kept for qPCR to determine the vector loss due to the purification. Please note that reagents presents in the media may impair PCR efficiency.
?. [Procedure]
You are now ready to purify your prep. The clarified supernatant can be kept overnight at  before proceeding with the purification protocol.
4 °C