Dec 31, 2019

Public workspaceAAV Production in HEK293T Cells V.2

DOI
dx.doi.org/10.17504/protocols.io.bawpifdn
  • 1Addgene
Addgene The Nonprofit Plasmid Repository
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DOI: dx.doi.org/10.17504/protocols.io.bawpifdn
External link: https://www.addgene.org/protocols/aav-production-hek293-cells/
Protocol Citation: Addgene The Nonprofit Plasmid Repository 2019. AAV Production in HEK293T Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bawpifdn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 31, 2019
Last Modified: December 31, 2019
Protocol Integer ID: 31407
Abstract
This protocol is used for AAV production in HEK293T cells. To see the full abstract and additional resources, visit the Addgene protocol page.

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.




Guidelines

Workflow Timeline


Day 0: Seed cells in CF2
Day 2: Seed cells in CF5
Day 3 (am): Transfect cells
Day 7 (am): Harvest cells
Materials


Equipment

  • Biosafety cabinet
  • Pipetman
  • Pipettors
  • Pipettes
  • Incubator
  • Sterile stir bars


Reagents

  • Adherent HEK293T cells (ideally AAV-293T clones)
Note
*Pro-Tip*
While adherent, these cells are very loosely attached to the dish surface and should be handled carefully. Avoid touching the cells when replacing media.

  • T-150 flask (Corning, 150 cm2)
  • Five chamber cell stack (CF5, Corning, 3180 cm2)
  • Heat-inactivated FBS (HI-FBS)
Note
*Pro-Tip*
Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one suitable for your protocols. FBS can be purchased already heat inactivated or it can be inactivated in the lab by heating to Temperature56 °C ℃ for Duration00:30:00 .

  • 0.45 μm polyethersulfone (PES) filter system
Note
*Pro-Tip*
Do not use filters made of materials other than PES. AAV particles stick to many other surfaces, but do not stick to PES. Using a PES filter will maximize titer.

  • DMEM high glucose with L-glutamine and 1 mM pyruvate
  • Opti-MEM
  • 0.05% Trypsin/EDTA
  • 1X PBS pH 7.4
  • 1 mg/mL Polyethyenimine (PEI) 25 KDa MW
Note
*Pro-Tip*
Other transfection reagents may be used in this protocol, but their conditions must be optimized.


Reagent Preparation

DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine. To a Amount500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and Amount11 mL of 200 mM L-alanyl-L-glutamine. Store at Temperature4 °C .


1 mg/mL polyethylenimine (PEI) solution:
  • Dissolve Amount100 mg of PEI powder into Amount100 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.
Note
*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 Duration00:10:00 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 Temperature-80 °C .
  • After thawing, the solution can be stored at Temperature4 °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 ddWater and adjust to a final volume of 1,000 mL.
  • Stir at room temperature until fully dissolved.
Note
*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.
Note
*Pro-Tip*
Stirring during the cooling period is recommended or the solution may separate into phases.

  • Aliquot and store at Temperature4 °C .









Safety warnings
See SDS (Safety Data Sheet) for safety warnings and hazards.
Before start
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
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 Amount10 mL of PBS.
  • Aspirate PBS and add Amount4 mL of 0.05% Trypsin/EDTA. Wait ~Duration00:02:00 .
  • Neutralize trypsin by adding Amount4 mL of HI-FBS.
  • Pipet back and forth vigorously multiple times to obtain a single cell suspension (no clumps of cells).
Pool cells from 2 x T-150 flasks. Adjust volume to Amount200 mL with DMEM complete media and mix.

Seed all cells in 1 CF2. Return to incubator for Duration48:00:00 .

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 Amount35 mL of 0.05% Trypsin/EDTA.
  • While waiting for the cells to lift-up, aliquot Amount5 mL of HI-FBS to a sterile Amount100 mL plastic bottle.
  • Gently tap the sides of the CF2 to help detach the cells, then transfer the cells into the bottle containing Amount5 mL of HI-FBS to neutralize the trypsin.
  • Rinse the CF2 with Amount30 mL 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).
  • Add Amount435 mL of complete medium (final volume Amount500 mL ).
Seed 250 million cells from the CF2 into 1 CF5. Return to incubator for Duration24:00:00 - Duration36:00:00 .


Proceed with transfection:
  • Calculate the amount of each plasmid needed to have a 1:1:1 molar ratio with Amount2 mg total DNA per CF5
Note
  • In total we would like 2.5 mg of DNA or 2,500 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:
2,500 μg/ 24,961 bp = 0.10 μg/bp
  • Therefore, for each plasmid we need:
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

  • Aliquot Amount176 mL of OptiMEM into a sterile Amount250 mL bottle.
  • Aliquot Amount350 mL of DMEM + 2% HI-FBS into a sterile Amount500 mL bottle.
  • Add the plasmid DNA into the bottle containing the Opti-MEM. Mix well.
  • Add Amount7.5 mL of PEI (1:3 μg DNA to μg PEI ratio). Shake the bottle up/down vigorously for Duration00:00:30 (it’s okay to make bubbles).
  • Incubate at room temperature for Duration00:15:00 . Note that longer incubation times can reduce transfection efficiency.
  • Add the OptiMEM + DNA + PEI solution to the bottle containing Amount350 mL 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.
Note
*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.

  • Return the CF5 to the incubator for Duration96:00:00 . This incubation time can be adjusted depending upon the serotype. Often AAV2 are harvested at Duration48:00:00 -Duration72:00:00 , while other serotypes are harvested at Duration96:00:00 -Duration120:00:00 .







Harvest cells and media by tapping the sides of the CF5 on a soft surface. Cells should detach easily.
Transfer cells and media into Amount50 mL conical tubes (or Amount500 mL conical vessels).


Rinse CF5 once with Amount20 mL of PBS and add to a new Amount50 mL conical tube.



Centrifuge at 3,000 g or max speed on a tabletop centrifuge for Duration00:15:00 at Temperature4 °C to pellet the cells.


Keep the cell pellet on ice and transfer supernatant to a sterile 500 mL bottle.
Process the supernatant as follows:
  • Filter through a 0.45 μm PES membrane.
  • Add Amount25 mL of PEG solution to each Amount100 mL of supernatant. Split into 2 x Amount500 mL sterile bottles as needed.
  • Add stir bar and stir slowly at Temperature4 °C for Duration01:00:00 , then keep at Temperature4 °C for Duration03:00:00 without stirring to allow full precipitation. Precipitation of the viruses can proceed overnight at Temperature4 °C if needed.
  • Transfer the entire sample to Amount50 mL conical tubes and centrifuge at 2,818 g for Duration00:15:00 at Temperature4 °C .
  • Discard the supernatant and resuspend the virus (small pellet) in Amount10 mL PBS + 0.001% pluronic F68 + 200mM NaCl. Pipet back and forth to resuspend the virus completely.
  • Keep resuspended pellet on ice.
Process the cell pellet as follows:
  • Resuspend and lyse the cells by adding Amount10 mL of PBS + 0.001% pluronic F68 + 200mM NaCl and sonicating 4 x 1 sec pulses with at least Duration00:15:00 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 Duration00:15:00 at Temperature4 °C .
  • Transfer the cleared lysate to the tube containing the resuspended virus from step 12 above.




Add 50 units of benzonase per mL of viral suspension in PBS (you should have ~Amount20 mL from steps 12 & 13). Benzonase is an endonuclease that will degrade any residual DNA carried over from the packaging process.

Incubate at Temperature37 °C for Duration00:45:00 .


Transfer the viral suspension to centrifuge tubes and centrifuge at Temperature4 °C at Centrifigation2415 x g forDuration00:10:00 .

Transfer the clarified supernatant to a new tube and proceed with purification.
Note
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.

You are now ready to purify your prep. The clarified supernatant can be kept overnight at Temperature4 °C before proceeding with the purification protocol.