We apply patient-derived organoid models, which are created from actual patient tumor specimens to examine how the tumors respond to drugs. We can also define the molecular characteristics that correlate with response. These results are then used to prioritize the best new therapies to advance in the clinical trials in the future.
One of the main challenges in using patient-derived organoid models is that they are a finite resource. In addition, there are very few off-the-shelf kits to assess drug response in organoid models that preserve the sample in a format that allows for other downstream analyses, such as transcriptomic profiling. We developed a multiplexed approach to genetically monitor therapeutic response in organoid models.
We use fluorescent dyes that are specific to different cellular processes, which provides insight into the mechanism of drug activity in each organoid model. In a single experiment, we collect both functional and timed course data in the samples are available for other assays. This method can be applied to assess response to a broad range of therapeutic modalities beyond small molecule inhibitors, including radiation sensitivity.
We are also continuing to innovate by developing tools for other cellular phenotypes that can be kinetically measured such as Senescence. To begin, take a tube with patient-derived organoids or PDO suspension, in an equal amount of basement membrane extracts, or BME. Using a 20 microliter pipette, seed five microliter domes into the center of each well of a pre-warmed 96-well plate.
After seeding all the wells, place the lid on the plate and gently invert it. Incubate the inverted plate at 37 degrees Celsius for 20 minutes in the tissue culture incubator to allow the domes to solidify. Flip the plate over so that the lid is facing upwards then incubate again.
After adding fluorescent dyes and drugs to the plates containing the PDOs, place the plate in citation five. Start the Gen5 software and in the task manager window, select Imager manual mode and click Capture now. In the settings, specify the objective:Filter, microplate format and vessel type.
Select use lid and Use slower carrier speed and click OK.Then choose a well of interest. Select the Bright Field channel, click on auto expose and adjust the settings as required. Next, to set up the Z-Stack, expand the imaging mode tab, select Z-Stack, and adjust the focus using the course adjustment arrows until all PDOs are out of focus.
Establish this point as the bottom of the Z-Stack and repeat in reverse to set the top. Afterward, set up the fluorescent channels by opening the Edit imaging step. Under channels, select the desired number of channels, designate one channel for the Bright Field and additional channels for each fluorescent channel.
Close the window by clicking OK.Change the dropdown menu to the GFP. To set the exposure for the first fluorescent channel, click Auto expose and adjust the exposure settings under the exposure tab to reduce the background signal. To copy exposure settings to the image mode tab, click the Copy icon and then click Edit Imaging Step to open a new window.
In the GFP channel, click the clipboard icon in the exposure line to add settings for illumination, integration time, and camera gain, to the channel. Now, click on the Camera icon and choose image pre-processing under, Add processing step. On the Bright Field tab, uncheck the Apply image pre-processing option.
For each fluorescent channel tab, ensure Apply image pre-processing, is checked. Deselect Use same options as channel one, and then click OK to close the window. Now click on Z projection under the Add processing step.
Adjust the slice range if necessary, and close the window by clicking OK.To set up the protocol for kinetic imaging, in the toolbar click Image set and from the dropdown menu, choose Create experiment, from this image set. Click on Options under the other heading and check the discontinuous kinetic procedure box. Under estimated total time, enter the runtime for the experiment.
Close the window by clicking OK.Additionally, set plate layout and temperature gradient at this time. Validate the data reduction steps by clicking through the windows. Then navigate to file and click save protocol as, in the toolbar.
Select a save location. Enter a file name and click Save. To begin kinetic imaging, load the protocol into the bio spa, on-demand scheduling software and choose an available slot.
In the procedure info tab, select User from the menu. Next to the protocol slot, click Select and choose, Add a new entry. Now click Select next to the protocol slot.
Click Open to navigate and import the protocol into the scheduling software. Enter the required imaging time for the plate and click OK to close the window. Under Interval, input the desired frequency of imaging.
Select schedule plate, or vessel. To initiate the plate validation sequence, click yes to accept this schedule. To begin, image the fluorescently labeled PDOs using the citation five live cell imaging system.
Then launch the Gen5 software, navigate to experiments and click Open in the Task Manager. Open the desired experiment and select Plate, followed by View, in the toolbar. Then switch the data dropdown menu to Z projection.
Double click on a chosen well, select Analyze, click, I want to set up a new image analysis data reduction step, and click OK.In analysis settings, set type to Cellular analysis and detection channel to Z projection transformed TSF Bright Field. Click Options to open the primary mask and count page. Check Auto in the threshold box, and click OK to close the pop-up window.
Under object selection, define minimum and maximum object sizes to filter out cellular debris or single cells and select other features as desired. Next, click Apply and the masked objects will be highlighted. To set up a subpopulation analysis in the cellular analysis toolbar, click on Calculated metrics.
Then click Select or Create object level metrics of interest at the bottom right corner. From available object metrics, choose Desired metrics such as Circularity and click Insert. Click OK to confirm.
Next, select Subpopulation analysis in the cellular analysis toolbar and click Add to create a new subpopulation. Enter a name for the subpopulation. In the object metrics, click Add condition to add a chosen metric.
Input desired parameters in the edit condition window, and click OK.Select the desired results in the table at the bottom of the window and click OK, followed by Apply. Under object details, select the designated subpopulation to view masking. If satisfied, click OK in the cellular analysis window and click Add step to apply the analysis to all wells.
