A Streamlined, Label-Free Real-Time 50% Tissue Culture Infectious Dose (TCID50) Assay using Impedance for Automated Viral Titer Quantification

The scope of our research was to develop a method for accelerating viral quantification process, reducing hands-on time, and enabling a high throughput real-time cytopathic effect tracking. Our protocol addresses lack of simple, fast, safe, and highly consistent method compared with conventional wire titer assays. To begin measuring the impedance background of a biosensor plate, use a multi-channel pipette to carefully transfer 50 microliters of prewarmed media into each well of the plate.

Use the reverse pipetting technique to minimize bubble formation and ensure consistent volume dispensation. Place the plate into the plate station, then launch the system software and select the virology module. Enter relevant notes in the experiment note tab and input cell information, including cell name and cell number in the cell sub-tab of the layout tab after selecting the corresponding wells.

Next on the schedule tab, click add a step to create step one designated for plate background measurement. Then click start to initiate the recording. Remove the plate from the plate station and place it in the laminar hood for cell seeding.

Add two milliliters of trypsin to detach the permissive cells from the T-75 flask. After counting the cells, adjust the cell suspension to the desired concentration. Pipette 100 microliters of the cell suspension to each well of the biosensor plate.

After incubating it for 30 minutes at room temperature, transfer the plate into the plate station inside the incubator at 37 degrees Celsius with 5%carbon dioxide. Navigate to the schedule tab and add step two using the default recording settings of 100 sweeps and a 15-minute interval. To prepare virus serial dilution, calculate the total volume of medium needed for dilution.

Warm the medium in a 37-degree Celsius water bath for 30 minutes. Rapidly thaw an aliquot virus stock vial in the water bath for one to two minutes until all ice crystals have melted. Prepare tenfold serial dilution of the virus by transferring 20 microliters of virus stock into a 1.5 milliliter microcentrifuge tube containing 180 microliters of pre-warmed media at each step.

Pipette up and down to thoroughly mix the virus with the medium. To equilibrate the virus dilutions, incubate the diluted virus samples at 37 degrees celsius with 5%carbon dioxide for 20 minutes prior to inoculation. Next on the layout tab of the software, select the wells designated for viral titer treatment.

Enter the initial dilution value of 0.001. Enter 10 is the dilution factor, and select the direction of dilution from left to right. Then enter the virus inoculation volume and click apply to save the treatment information.

Assign negative control wells by selecting the appropriate wells and checking negative control under well type in the treatment tab. To inoculate the virus, click pause in the software to stop data acquisition. Remove the plate from the plate station and place it in the laminar hood.

Using a multi-channel pipette, carefully remove the culture media from each well. Gently add 50 microliters of the virus dilution onto the top of the cell monolayer, avoiding disruption of the cells. Place the plate back into the plate station.

On the schedule tab, add step three using the default recording settings to resume impedance recording. After two hours of virus inoculation, pause the recording and remove the plate from the plate station to the laminar hood. With a multi-channel pipette, slowly add 150 microliters of prewarmed medium to each well avoiding disruption of the cell monolayer.

Return the plate to the plate station and resume recording by clicking start. To calculate the tissue culture infectious dose 50, navigate to the data analysis tab and add all assay wells to the plot chart. In the parameter section, select TCID50 from the dropdown list to initiate calculation.

Choose bar chart to calculate TCID50 at a specific time point, or select time-dependent TCID50 to generate a TCID50 time course over the desired period. In uninfected HEK 293A cells, the normalized cell index increased over the first 48 hours post-inoculation and then plateaued, indicating continued cell proliferation followed by confluence. In adenoviral GFP infected HEK 293A cells, the normalized cell index dropped below the uninfected control approximately 50 hours post inoculation, and declined to zero by 100 hours indicating virus-induced cytopathic effects.

Viral green fluorescent protein expression increased inversely as the normalized cell index decreased during the first 100 hours post-inoculation in adenoviral GFP infected HEK 293A cells. Live cell imaging showed continued cell growth during the first 48 hours post-infection, followed by reduced confluency and increased green fluorescent protein signal as viral infection progressed with loss of viable cells observed by approximately 100 hours. Adenoviral GFP infection induced a dose-dependent decrease in the normalized cell index with higher virus concentrations causing earlier and steeper declines across replicate wells.

At a 10 raised to the power of 8 dilution of adenoviral GFP, the normalized cell index exhibited high variability across replicate wells indicating partial cytopathic effects. The adenoviral GFP TCID50 increased over time and reached a plateau by approximately 150 hours post-inoculation. In MDCK cells infected with influenza A virus, impedance measurements showed dose-dependent changes in the normalized cell index compared to uninfected controls.

Our protocol offers a fully automated label-free, real-time objective, viral quantification solutions based on impedance technology and supported by this integrated virology software. Current challenges are prolonged exposure to viruses and reliance on the staining with those toxic reagents required in those conventional wire titer assays and subjectivity and analyst of variability associated with label-free imaging based methods.