The overall goal of the following experiment is to observe the replication of oncolytic vaccinia virus in fresh tissues obtained following surgery. This is achieved by sampling the tissue for cell culture to minimize bias due to tissue heterogeneity. As a second step tissue viability is assessed, which ensures the quality of the starting material prior to infection with virus.
Next viral trend gene expression is monitored and viral. Titus are determined. In order to assess whether the live surgical specimen can support productive vaccinia infection, results are obtained that show vaccinia replication in tissues based on the observation of fluorescent protein or bioluminescence reporter expression and increased viral titer over time.
Hi, I'm Jean Simone Diallo. The implications of this technique extend toward therapy of cancer cause they can lead to the identification of patients that may be well-suited for treatment using oncolytic viruses. Hi, I'm Naomi, though.
This method can provide insight into the infect ability of tumor tissues using oncolytic vaccinia virus. It can also be applied to other oncolytic viruses such as vesicular, stomatitis virus, and likely other viruses such as herpes simplex virus, adenovirus, and Rio virus with minor modifications. Hi, I'm Dominic.
Generally, individuals new to this method may struggle because the tissues can be difficult to core reproducibly and cutting the cores into small and even pieces may be a challenge. Hi, I'm John Bell demonstrating today's procedure will be two bright young graduate students in my lab, Naomi DeSilva and Dominic Wa.And assisting them will be Derek Butterwick, a surgical resident from the Ottawa Hospital Working in the lamina flow cell.Culturehood. Use a two millimeter biopsy punch to obtain cause of tissue from various regions of the tissue sample.
Deposit each core in the lid of an upturned Petri dish containing PBS with the help of a pipette using forceps to adjust the sample if required. Leave enough space between each core so that they can easily be cut along the horizontal axis. Use a sterilized razor blade to split each tissue core into four even quarters.
Then use a pipette tip to place each quarter into four separate wells of a 24 well plate containing two milliliters of media. Repeat using each quarter of a second core and placing these quarters into the same four wells of the 24 well plate. Continue in this manner until all of the tissue cores have been plated.
Once all of the tissue sample cores have been deposited in the wells, add 25 microliters of alamar blue to one of the wells containing tissue and a control well and incubate for at least one hour at 37 degrees in a humidified incubator with 5%carbon dioxide. After the incubation time has elapsed, remove three 100 microliter aliquots from each alamar blue treated well and deposit them in separate wells of a 96 well plate. Repeat with two further 100 microliter aliquots from the same wells.
Read the alamar blue signal using a fluorescent plate reader and record the data. Use a pipette tip to transfer each piece of tissue from the alamar blue treated wells to a clean media containing well of the 24 well plate. Avoid transferring large amounts of media with the sample.
Next infect one of the tissue containing wells would tend to the six plaque forming units of GFP expressing vaccinia virus diluted in 25 microliters of media. Then infect another well would tend to the six plaque forming units of luciferase expressing vaccinia virus. Incubate the plate at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide 72 hours after infection.
Add 25 microliters of alamar blue to the previously assay sample. Incubate for one hour at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide. Then remove three 100 microliters samples and analyze in a fluorescent plate reader as before.
First aspirate the media from the GFP expressing vaccinia virus treated well and washed twice with PBS directly in the well. Then using a fluorescence capable dissection microscope. Take a phase contrast image of the tissue piece at an appropriate resolution of times 40.
Next, switch to fluorescent mode and using A GFP capable filter. Visualize GFP expression in the GFP transduced tissue. Finally, use a fluorescent filter for another wavelength as far as possible from that of the transgene of interest.
And take a picture of background fluorescence. Next, collect the tissue pieces using pipette tips. Weigh the tissue.
This measurement will be used to calculate the titer of the virus in forming units per gram at the end of the procedure. Store the tissue at minus 80 degrees Celsius for future titering Pipette five microliters of a 10 milligram per milliliter Lucifer substrate into the well that was transduced with luciferase expressing vaccinia virus and into a well containing only tissue culture. Media incubate for five minutes at room temperature.
Load the plate into the in vivo imaging system and set the exposure time to five seconds. Take a picture of the luciferase expressing vaccinia virus transduced wells. Repeat the procedure with a shorter exposure time if the image appears saturated or use a longer exposure time if no signal is observed.
Set up a six well plate containing 1 million live U2 OS cells per well in full medium and incubate overnight in a 37 degrees Celsius 5%carbon dioxide humidified incubator so that the wells reach 95%co fluency the next day. Retrieve the previously weighed infected tissue samples. Transfer the samples to a five milliliter polystyrene round bottom centrifuge tube and add one milliliter of PBS.
Use a tissue homogenizer to homogenize the tissue and release the viral particles. Then make one in 10 serial dilutions of the tissue homogenate viral suspension in serum free media by transferring 100 microliters of the tissue homogenate viral suspension to 900 microliters of serum free media and repeating as necessary. Then aspirate the media from the six well plate containing the U2 OS cells.
Pipette 500 microliters of each serial dilution into a well of the plate. Return the plate to the tissue culture incubator for one hour, gently rocking the plates every 15 minutes to prevent drying of the cells while the cells are incubating warmer bottle of two times DMEM containing 20%FBS and an aliquot of 3%CMC solution in a 37 degree water bath. Once warmed, make a one to one mixture of 3%CMC to two times DMEM plus 20%FBS to achieve a final concentration of 1.5%CMC and 10%FBS.
Following the incubation. Aspirate the media from the U2 OS cells and replace it with two milliliters of the 1.5%CMC 10%F-B-S-D-M-E-M mixture per well return the plate to the tissue culture incubator for further 48 hours. After 48 hours, add two milliliters of methanol acetic acid fixative to the CMC media mixture in each, well incubate the plate at room temperature for 10 minutes in the tissue culture hood.
Once the incubation time has elapsed, aspirate the solution from each. Well rinse each well with tap water to remove any remaining fixative. Next, pipette two milliliters of kumasi blue solution into each.
Well then place the plate on a shaking platform and incubate room temperature for 30 minutes while shaking at low speed bead, aspirate the kumasi blue solution from the well and rinse with tap water. Leave the uncovered plate in the hood to dry for about an hour. Count the viral plaques at the dilution step where between 10 and 100 plaques are visible.
Multiply the number of plaques counted by the dilution used, and then multiply the resulting number by two to give a titer in plaque forming units per milliliter. Further, divide this number by the weight initially measured for the sample to report tis in plaque forming units per gram. This graph illustrates tissue viability as measured by alma blue's signal intensity at two hours and 72 hours post collection and shows that both normal and tumor tissue can remain metabolically active over a period of at least 72 hours.
These representative data show the tighter of vaccinia virus collected pergram of tissue sample at two and 72 hours post infection. This indicates that the virus is undergoing a productive viral life cycle that leads to self amplification and spread. This image illustrates a typical luminescence signal obtained from patient vaccinia virus.
Luciferase infected tissue samples imaged using the in vivo imaging system. Regions of intense luminescence appear red according to the luminescence scale bar on the right comparison of the phase contrast and GFP channel fluorescence microscopy images demonstrate the proportion of the tissue transduced by vaccinia virus GFP. The absence of signal in the red channel indicates the specificity of the fluorescence signal.
While attempting this procedure, it is important to remember to use fresh tissue and sample representative areas of it as much as possible Following this procedure. Other methods like tissue fixation immunohistochemistry, DNA or RNA extractions can be performed in lieu of tittering in order to answer additional questions like, what specific tissue structures is the virus infecting and what is the impact of the virus on gene expression? After watching this video, you should have a good understanding of how we process samples for ex vivo infections with vaccinia virus, how we follow the spread of the virus through the expression of GFP or Luciferase Transgenes.
And finally, how we tighter vaccine a virus from process samples. Don't forget that working with live viruses can be hazardous depending on the strain and precautions, such as wearing gloves and working in appropriate laminar flow should always be taken while performing this procedure.
