Welcome to the Institute of Cellular Biology and Pathology, Nicolae Simionescu from Bucharest, Romania. Here in the Viral Transduction Laboratory, We show a part of the adenoviral particle production using an optimized methodology based on the AdEasy system developed by Professor Vogelstein. The main steps of the technology for producing this adenoviruses are, one, a recombination of pAdTrack containing GFP with the pAdEasy-1 plasmid in BJ5183 bacteria.
Two, packaging the adenoviral particles. Three, amplification of the adenovirus in AD293 cells. Four, purification of the adenoviral particles from cell lysate and culture medium.
Five, viral titration and functional testing of the adenovirus. Here, we will present an important step of the methodology. The purification of the adenoviral particles from cell lysate and culture medium.
The purification of the adenovirus starts with harvesting the virus-producing cells and the medium. AD293 cells were transfected with the recombinant plasmid and as the adenovirus was packaged. Then the adenovirus was amplified by successive larger cultures.
Here, we obtained 25 T175 plates, and we started the purification of the adenovirus from the cell lysate and the culture medium. First, we checked the green fluorescence of the GFP expressed by the transduced cells. If the cells are still attached, we will detach them by tapping the culture dish or by using a long scraper.
The cells and the medium are collected. The flasks are washed with PBS, which is also collected in Falcon tubes. The cells are pelleted by centrifugation.
Keep the cell pellet as well as the medium for further purification of the adenovirus. At this step, the help of a colleague is well appreciated to accelerate the harvesting process. The cell pellet is green due to the GFP expression.
The pellet is resuspended in hypertonic Tris buffer, which would facilitate the disruption of the cells. For cell lysis, we use liquid nitrogen and the dry gas warmed up to 37 degrees. Do not perform more than three cycles since the virus would be damaged.
To increase the efficiency of cell disruption carefully pass the cell homogenize through a 23 gauge syringe needle. Centrifuge the cell lysate at 9, 600 x g for 12 minutes. Pass the supernatant in new tubes and discard the pellets.
Keep the supernatant on ice for further processing by ultracentrifuge. Now, we process the culture medium to isolate the adenovirus released from the cells. For this, first, we weight the necessary amount of ammonium sulfate and add it to the bottle in which the culture medium was collected.
The bottle is shake vigorously until the crystals are completely dissolved. The mixture is incubated at room temperature for two hours and the half. Then the suspension of the precipitate is divided into Falcon tubes.
Centrifuge for 15 minutes at 1, 600 x g. After the centrifugation, the supernatant is discarded and the pellet is resuspended in 10 millimolar Tris buffer. If one cannot continue the purification of the adenovirus with the ultracentrifugation step, the precipitate may be kept for a few days at four degrees, but only after dialysis to remove the ammonium sulfate.
Here, we presented the dialysis step. We use a syringe to introduce the suspension into a previously wetted cassette. The samples are dialyzed against 10 millimolar Tris buffer overnight at four degrees.
The final step of the purification is represented by the ultracentrifugation step on a cesium chloride discontinuous gradient. To form the gradient, first, we pipette three milliliters of the high density solution of cesium chloride. On top of this layer, gently pour three milliliters of low density cesium chloride.
The adenoviral particle suspension from the cell lysate or the culture medium is then overlaid on top of the gradient. The tubes are filled with mineral oil and introduced in the SW41 buckets. After equilibration, the tubes are loaded symmetrically into the rotor, which is introduced in the ultracentrifuge.
The centrifuge runs a 35, 000 RPM and four degrees for 18 hours without break. After ultracentrifugation, the tubes are placed on a stand with a black paper behind in order to harvest the bands. The clear upper phase and the cell debris are discarded in a waste container with a bleaching solution.
The lowest band containing the complete adenovirus is harvested with a pipette tip and collected in a sterile Eppendorf tube. After dialysis, sucrose is added to the viral suspension to a 4%final concentration and viral aliquot are kept frozen at minus 80 degrees. In the results section, we showed the GFP expression in cells transduced with the obtain adenovirus.
48 hours after transduction, GFP is expressed by the transduced cells as shown by fluorescence microscopy. The percentage of the GFP expressing cells is determined by flow cytometry. About 50%of the human and bovine endothelial cells transduced with 25 transduction units per cell are GFP positive.
The same transduction yield was obtained for murine hepatocytes when only five transduction units per cell were used, but this is associated with higher mortality due to the cell sensitivity. Reduced to GFP expression was obtained by transduction of mesenchymal stromal cells due to the low expression of the specific receptors. Concluding remarks.
We optimize these laborious technology to reduce the time, the costs, and the effort needed to obtain the adenoviral particles. The adenovirus prepared is able to infect various cell types and to induced the expression of the gene of interest.
