SISTEMA DE INFORMACIÓN (SIM)

2.10.1 Co-transfection of Ad293 cells for viral production

Ad293 cells were plated out onto a 25 cm3 flask and incubated overnight. Cells were 60% confluent on the day of transfection. DNA plasmids were transfected into Ad293 cells using the FuGene protocol (2.7.11). The two DNA plasmids required for the co-transfection (pBHGlox and pDC315_MEGF10 constructs) were mixed at a ratio of 1:2. The transfection mix was added in a drop wise manner directly to the media of a T25 of Ad293 cells and incubated at 37°C, 5% CO2overnight. Cells were inspected visually for GFP expression using a fluorescent microscope (Nikon) using the 10x objective.

2.10.2 Adenoviral amplification

Transfected Ad293 cells were grown to confluence with most of the cells showing GFP expression. After 48 hrs cells were harvested using a cell scraper and used to seed two 75 cm3 tissue culture flasks. The cells were incubated at 37°C, 5% CO2 and inspected daily for viral production, indicated by the infected cells becoming round and detaching from the surface. After 10 days the cells showed at least 50% cytopathic effect and were harvested using a cell scraper. Cells were pelleted by centrifugation at 2000 x g for 10 min before the supernatant was aspirated and the pellet

Virus was released from the cells using a freeze-thaw method. Ethanol was added on top of dry ice and the cells frozen by floating the Eppendorf tube on top of the ethanol for 5 min. Cells were thawed in a beaker of room

temperature water and once defrosted the cells were mixed by vortex. The process was repeated three times. The cells were centrifuged at 5000 xg for 10 min to pellet the cell debris, leaving the adenovirus in the supernatant. A 25 cm2

for 2 hr. 5ml of media was then added and the cells incubated for 3 days until the viral infection caused the cells to round up and become detached from the dish. The virus was harvested as described previously.

The virus was amplified three times in Ad293 Cells to increase the adenoviral titre:

Amplification Flasks of Ad293 Cells Amount of Virus

1 1xT25

2 2xT75

3 5xT75

After 50% of the cells had begun to show signs of infection, with the cells becoming round and detatching from the dish, in the third amplification, the cells were centrifuged and the pellet frozen at -80°C with the media from the flask before purification of the adenovirus.

2.10.3 Adenopack adenovirus purification

Adenovirus purification was performed using the Vivapure Adenopack 100 kit (Sartorius Stedim Biotech) following the manufacturer’s instructions. Briefly, the infected cells and media were defrosted and the cells freeze- thawed, as described previously. The cells were centrifuged at 3500 xg for 15 min to pellet the cell debris and the viral supernatant was transferred to a

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1 ml of culture) was added to the viral supernatant, mixed and incubated at 37°C for 30 min. For purification of the virus, a retort stand and clamp was prepared in a tissue culture hood and all subsequent steps were performed under sterile conditions. The provided tube set and 50 ml syringe were set

up in the clamp with the feed tube in the viral supernatant. The supernatant was drawn up into the syringe and the one-way valve used to ensure all air was expelled from the syringe and tubing. A Minisart plus was attached to the syringe assembly and the supernatant filtered into a fresh container, leaving a small volume of supernatant to prevent air entering the Minisart. Loading buffer at 1/9 volume of the total filtered supernatant was added and the Minisart discarded. The Adenopack was prepared taking care to remove all air to ensure efficient viral binding. A 10 ml syringe was filled with PBS, connected to the Adenopack and 5 – 6 ml of PBS gently passed through. The syringe plunger was pumped up and down a few times to remove air from the Adenopack before the remaining PBS was passed, through leaving 1ml in the syringe to prevent air entering.

The feed tube from the 50 ml syringe and tube set was placed into the prepared sample solution. The air was removed from the syringe and valve and a wet-to-wet connection was made between the wetted Adenopack unit and the tube outlet. The prepared sample was slowly passed through the Adenopack unit at a rate of approximately 10 ml/min, determined by being able to count single drops. To prevent air entering the system 1 - 2 ml of sample was left in the syringe. The Adenopack was washed by slowly passing through 100 ml of washing buffer using a higher flow-rate, leaving 1- 2 ml in the syringe to prevent air entering the system. To elute the virus, the Adenopack was removed from the tube set and attached to a 10 ml syringe filled with 5 ml elution buffer. The syringe was held over a sterile 15 ml tube and 1 ml of elution buffer was very slowly passed through the Adenopack over 1 - 2 min. The Adenopack was then incubated for 10 min at room temperature before slowly passing the remaining elution buffer through at approximately 1 ml/min.

2.10.4 Storage of adenovirus

The virus was concentrated and the buffer exchanged to storage buffer (20 mM Tris/HCl, 25 mM NaCl, 2.5% glycerol (w/v), pH8). 1 ml of the purified

2.10.5 Viral Titre Assay

The viral titre was calculated using the tissue culture infectious dose 50 (TCID50) method (AdEasyVector System, Qbiogen, Inc.). A 96 well plate was prepared with 1x104 Ad293 cells per well incubated at 37°C, 5% CO2 overnight. The purified virus was serially diluted in media to give dilutions of 10-1to 10-10and one concentration was added to 10 wells per row of the 96 well plate, with the remaining two wells being left as a control without virus. Cells were incubated for 10 days and the numbers of wells showing signs of viral infection (cytopathic effect, CPE.) were counted. The ratio of the number of wells showing cytopathic effect out of the 10 wells set up was calculated for each viral dilution. The sum of the ratios was calculated and denoted as ‘s’. The log10of each viral dilution is denoted as ‘d’.

To calculate the viral titre: T = 101+d(s-0.5)

T = 101+1(7.7-0.5)= 108.2 _{50 is 10}9.2/ml

To calculate the PFU/ml: T = 1x109.2-0.7 = 1x108.5PFU/ml = 3x108PFU/ml