Recombinant Adenoviruses are wonderful tools used by the scientists for high efficiency,
large gene sequence transfers into almost any cell type.
Whether it is for in vitro or in vivo experiments,
transduction of dividing or non-dividing cells,
recombinant adenoviruses can be exactly what you need to achieve large - scale, high transduction efficiency.
In our previous video,
we covered the basics of adenovirus packaging,
starting from cloning your gene of interest into abm’s Adenovirus expression system,
followed by transfection of DNA into HEK293 cells,
and leading to the collection of a low titer adenoviral “seed stock”.
You can find the link to that video in the description below.
In this video, we will cover the basics of:
High-titer viral amplification with adherent or suspension HEK293 cells,
Virus purification using iodixanol gradient ultracentrifugation, or Cesium chloride density gradient ultracentrifugation
Virus titration by end-point dilution assay.
Let’s get started!
abm’s adenovirus expression vector is designed without the early viral transcription units,
E1 and E3 proteins, and is therefore replication-defective.
As a result, recombinant adenovirus packaging requires cell lines expressing the E1 gene
such as HEK-293 adherent cells or suspension cells.
To generate high-titer adenovirus,
we will infect HEK293 cells with pre-made seed stocks as discussed in our previous video.
We will use 1011 PFU/ml as an example in the following procedure,
however more or less cells can be adopted for a lower or higher titer desired.
Prepare 20 plates of HEK293 cells in 15 cm cell culture dishes
at 75% confluent at the time of seed stock infection.
Replace the old media from each plate with 20mL of fresh complete media (DMEM 5% FBS, 1% P/S)
before inoculating with 1ml of seed stock per plate.
Incubate until the cells exhibit complete cytopathic effect,
where >90% of cells have detached from the plate.
This process should take approximately 3 to 5 days.
Collect cells and supernatant using a cell scraper
and centrifuge at 3500 rpm for 20 minutes to pellet the cells.
Resuspend the cells in 500ul of 1x gradient buffer per plate.
Resuspended cells can be stored at -80’C if purification will not proceed immediately after cell collection.
Freeze-thaw the cell pellet three times by alternating between dry ice and a 37’C water bath.
Vortex briefly after each free-thaw cycle.
The freeze-thaw step will release the adenovirus from the cells into the supernatant.
Centrifuge the lysate at 10,000 rpm for 10 minutes,
and transfer the supernatant (which now has all the viruses) into a new 50ml conical tube.
Your supernatant is now ready for virus purification.
If you are using suspension cells,
prepare 100ml of cells at a density of 1.5 million/ml one day before seed stock inoculation.
Inoculate suspension cells at a ratio of 1:200.
For example, 0.5ml of virus per 100ml of cells,
and wait 3 to 5 days for CPE to complete.
After complete CPE is achieved,
harvest the cells and media in centrifuge bottles.
Centrifuge the cells and media at 7500 rpm for 10 minutes to pellet the cells.
Resuspend cell pellets in 10 ml of gradient buffer and perform 3 cycles of freeze/thaw.
Spin at 10,000 rpm for 10 minutes to clarify the supernatant.
Your supernatant is now ready for virus purification.
Prepare the iodixanol density gradient in ultracentrifuge tubes by adding the reagents in the following order:
8.0ml of 15% iodixanol, 5.0ml of 25% iodixanol, 5 ml of 40% iodixanol, and 5ml of 58% iodixanol.
Overlay your supernatants onto the iodixanol gradient; take care to not disturb the gradient layers.
Centrifuge at 60,000 rpm for 1 hour at 10’C.
Use an 18-gauge needle and syringe to release pressure at the top of the tube.
Carefully extract the virus solution using the same needle from between the 25%/40% iodixanol layers.
Next, transfer virus preparations into a pre-wet dialysis bag
and dialyze in a 4-6L beaker or flask against 1x PBS or the final desired buffer.
Place the set up on a magnetic stirrer overnight at 4’C
Next day, discard all the PBS, add fresh 4L of PBS and continue buffer exchange for another 1.5 hours.
Transfer dialyzed virus into a 30kDa MWCO spin column and concentrate the virus to a desired volume.
Finally, pass the virus through a 0.22um syringe filter.
Add glycerol to a final concentration of 5% and mix thoroughly
Aliquot and store at -80'C.
Another method to purify high titer adenovirus particles is to use the cesium chloride density gradient ultracentrifugation method.
Prepare the CsCl gradient tubes in ultracentrifuge tubes.
Add 10mL of 1.4g/mL CsCl solution to the ultracentrifuge tube.
Slowly overlay 10 ml of 1.25g/ml CsCl solution, followed by 10 ml of virus supernatant.
Centrifuge at 60,000 rpm for 1 hour at 10’C.
The lowest band between the 1.25 and 1.4g/ml layers in the tube contains intact virus particles.
A second upper band may be observed which contains defective virus particles, do not extract this upper band.
To extract the virus band, use a 22-gauge needle and syringe,
pierce a hole at the top of the tube to release air pressure,
and then insert the needle 1 cm below the lower virus band.
Carefully extract the intact virus with the bevel up.
Transfer the virus particles to a 30kDa MWCO spin column and concentrate the virus to the desired volume.
Finally, filter the viral solution with a 0.22 um syringe filter.
Add glycerol to a final concentration of 5% and mix thoroughly.
Aliquot and store at -80C.
Now that you have your purified sample, the last step is to calculate the virus titer
In each well of a 96 well plate,
seed 293 cells in 100ul of complete media so that they will be 10-20% confluent the next day.
The following day,
prepare a 1/1000 dilution by diluting 10ul purified adenovirus stock in 10ml of complete media.
Vortex for 5 seconds.
Make a series of 10 additional dilutions, each by a factor of 10
Label columns 1 through 10 of the 96-well plate with the corresponding dilutions, increase from left to right.
The last two columns of the plate will serve as uninfected negative controls.
For uninfected negative controls, add 100uL of fresh complete media to each well of the last two columns of the plate
in order to measure uninfected cell viability.
Next,
carefully pipette 100ul of the diluted virus to each well of the corresponding column on the 96 well plate
8 wells per column which equals 8 replicates
Each column of the plate should be infected with the corresponding dilution.
Incubate the plate for 10 -14 days and monitor cytopathic effect (CPE) every 3 to 4 days.
The column infected with higher concentration of virus should exhibit CPE by day 4.
On day 14,
record whether a cell is CPE positive or CPE negative.
Wells showing any sign of CPE are given a score of 1,
and wells showing no CPE are scored as 0.
Calculate titer using the Spearman-Karber equation:
Multiply the value obtained by 10 to obtain virus titer in TCID50/ml
Then, multiply by 0.69 to convert to plaque-forming units (PFU)/ml.
You have now successfully isolated high titer adenovirus particles
and calculated the virus titer for downstream applications!
To learn more about adenovirus,
check out our Learning Resources at abmgood.com!