Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses (OVs) hold promise as candidates for cancer therapy due to their ability to selectively infect and kill tumor cells. The effectiveness of oncolytic viral therapy, however, depends critically on evaluating the infectivity of ex vivo living tissue specimens prior to treatment. A detailed instruction on how to infect living tissue ex vivo with an oncolytic virus and measure the resulting viral activity is provided by this methodology. Viral replication and tissue infectivity may be precisely measured by following this methodology, which paves the way for the creation of potent oncolytic viral therapies.

Experimental Materials and Recommended Services

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Oncolytic viruses	These viruses have the potential to be used in cancer therapy since they only infect and destroy cancer cells while preserving healthy ones.	Oncolytic Virus Service Oncolytic Virus RFP Reporter Oncolytic Virus Reporter Genes
Reporter transgenes	Genes that code for proteins or enzymes with readily detectable and quantifiable activities or expression	Clones Gene Synthesis and Cloning Service Reporter Stable Cell Lines
U2OS cell line	A human osteosarcoma cell line is widely utilized in biology and biomedical research because of its well-characterized characteristics and simplicity of cultivation.	U2OS cell line Reporter Stable Cell Lines
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Procedure

1. Tissue Preparation:

a. Tissue collection: Take newly removed tissue samples from operations and place them in DMEM (Dulbecco's Modified Eagle Medium), which has 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) solution added to it.

b. Instrument Sterilization: All metal forceps and disposable blades should be sterilized before tissue processing by immersing them for at least five minutes in a 70% ethanol solution.

c. The culture plate should be prepared as follows: c. Fill each well of a 24-well plate with 2 ml of DMEM medium supplemented with 10% FBS, 1% PS, and 0.1% Amphothericin B.

d. Sampling the Core Biopsy: To preserve sterility, put the tissue sample in a sterile 15 cm petri dish with the cover inverted, using sterile forceps. Gather many tissue cores from various areas of the tissue samples using a 2 mm biopsy punch.

e. Core Division: Using a sterile razor blade, cut each tissue core into four equal parts, making sure that each part is the same size.

f. Core Placement: Fill each well of the prepared 24-well plate with 1.5 ml of DMEM medium by using a pipette to insert each quadrant of the tissue core into a different well. This minimizes bias while enabling representative tissue sample.

Fig. 1 illustrates the tissue sampling and sectioning protocol, where 2 mm cores are divided into quarters and distributed randomly into wells A1-A4. After the initial alamar blue reading, tissue from well A1 is transferred to C1, with a second reading after the 72-hour virus incubation. Wells A4 and B4 receive Luciferin supplementation post-infection. (doi: 10.3791/2854) Figure 1. Overview of the tissue sampling / sectioning protocol. (Diallo, J., et al., 2011)

2. Assessment of Tissue Viability:

a. Incubate the Alamar Blue dye by adding 25 μL to wells A1 and A2 of the culture plate. The plate should be incubated for one hour at 37°C in a humidified incubator with 5% CO₂.

b. Fluorescence Measurement: After incubation, fill six wells of a 96-well plate with 3 × 100 μL of medium from each of the wells C, A1, and A2. With excitation at 530 nm and emission at 590 nm, use a fluorescence plate reader to measure the fluorescence signal.

c. Transfer tissue fragments from well A1 to well C1, which is filled with new DMEM medium, to initiate tissue infection. As a control, leave well A2 uninfected. Infect wells A3 and A4 with 10⁶ plaque-forming units (PFU) of an oncolytic virus expressing the required transgenes.

d. Alamar Blue Reassessment: To evaluate tissue viability following infection, perform the Alamar Blue test on wells C1 and D1 72 hours after the first infection.

3. Visualization of Transgene Expression:

a. GFP Expression Imaging: Using a dissecting microscope equipped with fluorescence capture, remove the culture medium from the tissue sections to observe GFP transgene expression.

b. Luciferase Expression Imaging: Transfer 5 μL of 10 mg/ml luciferin substrate into wells A4 and B4, thoroughly mix, and let sit at room temperature for 5 minutes. Using an In Vivo Imaging System (IVIS), image tissue wells to see luciferase expression.

c. Quantification of Luminescence: Use IVIS imaging software to analyze light signals and measure the amounts of luciferase expression.

4. Viral Titration by Plaque Assay:

a. Tissue homogenization: To release viral particles, homogenize infected tissue samples in phosphate-buffered saline (PBS). Homogenates should be kept at -80°C until further examination.

b. Plating Cells: After plating U2OS cells on 6-well plates, allow them to grow to 95% confluency.

c. Virus Dilution: Apply diluted virus solutions to plated U2OS cells after serially diluting the viral stock in serum-free medium.

d. Plaque Formation: Cover cells with carboxymethyl cellulose (CMC) solution and let them sit for 48 hours after the initial incubation. Use methanol-acetic acid fixative to fix cells and Coomassie blue solution to stain them, respectively.

e. Plaque Counting: Count viral plaques to determine viral titers, considering dilution factors, and calculate plaque-forming units per milliliter (PFU/ml) or gram (PFU/g) of tissue.

In conclusion, ex vivo infection of live tissue with oncolytic viruses provides a unique opportunity to assess tissue infectivity and viral replication prior to initiating therapy. Researchers can learn more about the efficacy of oncolytic viral therapy by measuring viral titers, carefully processing tissue, and assessing tissue viability. The development of customized cancer treatment programs is facilitated by this technique, which offers a standardized way to evaluate tissue infectivity. Oncolytic viral-based cancer therapy techniques may undergo significant improvements as a result of the protocol's ongoing use and refinement.

* For research use only. Not intended for any clinical use.

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