Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Human pancreatic ductal adenocarcinoma (PDAC) patient-derived organoids were used. Tissue resection fragments were obtained from patients undergoing curative surgery at the Antwerp University Hospital. Written informed consent was obtained from all patients, and the study was approved by the UZA Ethical Committee (ref. 14/47/480). Details related to all materials, reagents, equipment, and software used in this protocol are provided in the Table of Materials. An overview of the workflow is presented in Figure 1. Example data is provided in the supplementary material to reproduce the protocol.

1. Day 0: Preparation of 2- or 3-day old organoids

1. Preheat the microplates at 37 °C overnight and thaw the extracellular matrix (ECM) at 4 °C.
2. Prepare full PDAC organoid culture medium: supplement ADF+++ (Advanced DMEM/F12, 1% glutamine supplement, 1% HEPES, and 1% penicillin/streptomycin) with 0.5 nM WNT surrogate-Fc-Fusion protein, 4% Noggin-Fc Fusion Protein conditioned medium, 4% Rpso3-Fc Fusion Protein conditioned medium, 1x B27, 1 mM N-acetyl cysteine (NAC), 5 mM nicotinamide, 500 nM A83-01, 100 ng/mL FGF10, and 10 nM Gastrin).
3. Establish the PDTOs according to the method of choice.
   NOTE: A detailed protocol is provided by Driehuis et al., which describes the conventional method to establish, culture, and passage PDTOs in ECM domes⁴.
4. Enzymatically dissociate the organoids in ECM domes.
   1. Aspirate the medium and wash 1x with phosphate-buffered saline (PBS). Add dissociation enzyme (e.g., 2 mL in a 6-well microplate) and pipette up and down 10x with a 1 mL pipette to mechanically dissociate the organoids and ECM domes.
   2. Incubate for 10 min at 37 °C, pipette up and down, and check whether the organoids are dissociated to single cells. Repeat this step if necessary.
   3. Collect the cell suspension in a 15 mL tube, add ADF+++ to a volume of 10 mL, centrifuge for 5 min at 450 × g at room temperature, and aspirate the supernatant with a Pasteur pipette and suction pump.
   4. Resuspend the pellet in 100-200 µL of full medium depending on the size of the pellet and count the number of cells using the method of choice. For example: mix 10 µL of the cell suspension + 10 µL of Trypan Blue and count with an automated cell counter.
5. Plate single cells in ECM domes.
   1. Dilute the cell suspension and add 2/3 ECM according to Table 1. Pipette up to ten 20 µL droplets per well in a preheated 6-well plate. Invert the plate and incubate for 30 min at 37 °C.
   2. Overlay with full medium supplemented with 10 µM Y-27632 and incubate for 2-3 days in an incubator.
      NOTE: Ten domes containing 75,000 cells each are usually enough to fill one 384-well microplate at a concentration of 200 organoids/well, excluding the wells at the edge.

2. Days 2 – 3: Harvest and seed 2- or 3-day old organoids

1. Collect intact organoids from the ECM domes.
   NOTE: Organoids tend to stick to plastic surfaces (e.g., tubes, pipette tips). To avoid this, plasticware can be prerinsed with a 0.1% bovine serum albumin (BSA)/PBS solution.
   1. Aspirate the medium and wash 1x with PBS. Add 1-2 mL of cold (4 °C) organoid harvesting solution to a 6-well plate depending on the number of ECM domes and incubate on ice on a shaking platform for 10 min.
   2. Pipette up and down with a 1 mL pipette to dissociate the ECM domes, incubate for an additional 10 min on ice, and visually check under a microscope whether the ECM is dissociated.
   3. Optional: if a more uniform size distribution is preferred, filter the suspension through a 70 µm cell strainer before centrifugation.
   4. Collect the organoids in a 15 mL tube precoated with 0.1% BSA/PBS, add ADF+++ up to 10 mL, and centrifuge for 5 min at 200 × g at 4 °C. Aspirate the supernatant and resuspend the pellet in up to 1,000 µL of full PDAC organoid medium depending on the size of the pellet to obtain a concentration of >6,000 organoids/mL.
   5. Count the organoids using any counting method of choice, preferably an image-based one.
2. Seed the organoids.
   NOTE: See the Table of Materials for the minimum volume/well for two different types of 384-well microplates used in this protocol.
   1. Precool all plasticware at -20 °C or on ice for at least 20 min before use to avoid solidification of the ECM.
   2. Prepare the seeding solution from the 1 mL of organoid stock solution (step 2.1.6) using full medium to seed ~200 organoids per well in 50 µL, the minimum volume used to fill a well. Use Supplementary File 1 to calculate the amount of organoid seeding solution. Add a residual volume of 1,500 µL when using a 25 mL reservoir and multichannel pipette or pipetting robot.
   3. Seed the organoids using a pipetting robot.
      NOTE: Both the seeding solution and microplate need to be cooled at 4 °C during pipetting to avoid solidification of the ECM. Therefore, a 25 mL reservoir and microplate holder were 3D printed to be used in combination with the pipetting robot that can hold cooling elements listed in the Table of Materials. STL files for 3D printing the custom labware (Supplementary File 2 and Supplementary File 3) and custom labware JSON files for the pipetting robot (Supplementary File 4 and Supplementary File 5) are provided.
      1. Design the dispensing protocol using the online Protocol Designer Tool. An example JSON file (Supplementary File 6) is provided, in which the custom labware is already loaded and an eight-channel p300 (Gen2) pipette with corresponding pipette tips is used.
      2. Open the pipetting robot control app, select protocols, click on Import, and drag and drop Supplementary File 6 into the designated field.
      3. Select the imported protocol and place all the labware, including cooling elements and plasticware, in the decks according to the layout shown in the Deck Setup field. Use the left slot for the 25 mL reservoir and cooling element as shown in Supplementary File 7.
      4. Click on Run Protocol and Proceed to Setup. Open the Labware Setup tab, click on Run Labware Position Check, and follow the instructions to calibrate the pipetting robot to the new hardware.
         NOTE: Labware offset data can be stored for later, but it is recommended to run the labware position check before each run.
      5. Fill the 25 mL reservoir (placed on top of the cooling element) with the cooled organoid seeding solution and click on Start Run.
         ​NOTE: The top and bottom wells also become filled with organoid suspension solution due to the use of the eight-channel pipette.
      6. Centrifuge the microplate for 1 min at 100 × g at 4 °C.
      7. Incubate at 37 °C for at least 30 min.
      8. Fill the outer blank wells with at least 50 µL of H₂O to avoid evaporation.
      9. Incubate at 37 °C overnight to remove any bubbles in the well that can interfere with the image analysis.

3. Day 4: Drug treatment and reagent dispensing with digital drug dispenser

1. Create the drug dispensing protocol using the digital drug dispenser control software.
   1. Hover over Plate 1 above the plate layout, select edit plate attributes, and fill in plate type: 384 well, additional volume (µL): 50, and DMSO limit (%): 1.
   2. Add fluids by clicking on the + button next to Fluids. Double-click on the newly created fluid and name it; select class (DMSO-based or aqueous+Tween 20) and concentration.
      NOTE: All drugs and reagents must be dissolved in 100% DMSO or 0.3% Tween-20. A 1-10 mM stock solution can be used, taking into account a maximum DMSO concentration of <1%. Table 2 provides examples of the required dilutions for common fluorescent reagents and therapies.
   3. Plate layout
      1. For drug titration, select wells and click on Titration. For fluid, select the drug of interest, choose the highest concentration (e.g., 2,000 nM), and the lowest concentration (e.g., 10 nM); for replicates, choose a minimum of 2, and choose the desired titration pattern.
         NOTE: The titration pattern will depend on many factors, including how much compound is to be fit in a single plate, whether the wells are to be randomized, and the number of replicates and controls.
      2. For the positive control, select three wells, click on Set Value, and fill in 2 µM staurosporine from 10 mM stock in DMSO, which will induce maximum cell death.
      3. For Cytotox Green, select all the used wells, click on Set Value, and enter 60 nM/well.
         NOTE: The Cytotox Green fluorescent staining indicates cells that have died, and therefore will not interfere with drug response monitoring. Here, no fluorescent marker for live cells is required.
      4. For the negative control and DMSO normalization, select all the wells with an additional four wells for the vehicle control, right-click, select normalization, select normalize fluid class: DMSO-based, and normalize to the highest class volume to obtain an equal DMSO concentration in each well.
         NOTE: DMSO concentrations should be <1%. An example TDD drug titration file (Supplementary File 8) is provided.
      5. Click on the arrow under Run in the top left corner, select Always Simulate, and click on Simulate to identify any errors and obtain the volumes of each drug to be prepared.
         NOTE: To overcome a warning when the initial dispensing volume is too low, "Dispense Warning well of 30 nL or greater is recommended for each fluid on each plate," select two wells on the edge that are filled with water, select Set Value, and enter 10 µM of the drug for which the warning occurs. This primes the drug cartridge with a volume higher than 30 nL. These same wells can be used to prime the DMSO cartridge by setting a normalize to % of total volume value (e.g., 0.5%).
2. Uncheck Always Simulate under the Run button; click on Run to start the drug dispensing protocol and follow the instructions.
3. Apply the sealing membrane to the microplate to prevent evaporation.
4. Incubate the Cytotox Green dye 1-2 h at 37 °C in the incubator and proceed to step 4.

4. Acquire images with the live-cell imager

NOTE: For the growth rate and NDR, a scan at timepoint 0 (T0 = start treatment) must be acquired 1-2 h after adding Cytotox Green.

1. Open the live-cell imager Control software, select Method Editor New, go to file > import, and select the example method XML file (Supplementary File 9). Alternatively, create a new file and select Plate: (CORE384fb_OpticalImaging) – Corning 384 Flat Black (Corning #4588), No lid and No humidity cassette; Application: Images-only; Objective: 4x; Pattern: Central; Check channels Brightfield and Green (Led intensity (%) = 40; Exposure Time (ms) = 200).
   NOTE: The green channel settings work well for a concentration of 60 nM Cytotox Green. The Live viewer option can be used to adjust the focus offset and/or the led settings in real time.
2. Click on Start to initiate scanning at T0.
3. Repeat the scan every 24 h for up to 5 days using the same method. Alternatively, to run the timelapse measurement automatically, adjust the method in the live-cell imager Control software to a kinetic experiment by clicking and dragging the Kinetic Loop tab into the method field. Similarly, the Temperature and Gas tabs have to be dragged into the method field to set the system to 37 °C and 5% CO₂ to ensure the correct conditions within the live-cell imager during the experiment.

5. Image and data analysis

1. Merging and compressing data
   1. The live-cell imager Control software generates a folder for each scan at each timepoint. Create a new folder, copy the individual experiment folders into this new parent folder, and add _0h, _24h, _48h, _72h, _96h, and _120h to the corresponding experiment folder names.
   2. Prepare an XLSX plate map from the digital drug dispenser Control software by right-clicking on the plate map layout from the drug dispensing protocol and copy all the wells; paste the data in an XLSX file. Remove Cytotox Green and staurosporine data and add a matrix for Cell Line and Replicate. Enter ctrl- and ctrl+. See Supplementary File 10 for an example plate map.
   3. Open the Data Compression Tool, click on Göz At, select the parent folder, and click on Run to initiate image data compression. All TIFF image files for the different timepoints are compressed into a single HDF5 for each well in a new datasets folder within the parental folder.
2. Image analysis
   1. Go to the image analysis webapp platform, log in, and click on Add New Project in the Home tab. Enter the project name, continue, select Add New Experiment, and upload the datasets folder containing the HDF5 files.
   2. After uploading, go to the project and experiment folder and click on Upload Platemap for additional functionalities. Click on Run Analysis, select Multi-Organoid analysis, Default Parameters, and click on Analyze to initiate image analysis.
   3. Click on Download Results to download the raw data tables which contain the measurements for each well (e.g., total brightfield area, total fluorescence green area, etc.) and the segmented images/videos to confirm the accuracy of the analysis and further data processing.
3. Growth rate-based drug response metrics and normalized drug response
   1. Select the Raw_NDR.xlsx file from the results folder (plate map required) (Supplementary File 11) and load this into the Official_NDR_7point R-script (Supplementary File 12) to automatically generate GR (normalized to ctrl-) and NDR (normalized to ctrl- and ctrl+) value tables (Supplementary File 13, Supplementary File 14, Supplementary File 15, and Supplementary File 16). GR and NDR values are calculated from the parameter as shown in equation (1) using the R-script (Supplementary File 12).
      Total Survival Area = Total Brightfield Area – Total Green Area    (1)
      Where 0 < NDR <1 = cytostatic effect (growth arrest), and NDR < 0 = cytotoxic response (cell death).
      NOTE: The R-script was adapted from Gupta et al.⁶.
   2. From the clonal_data.xlsx table, retrieve single-organoid response data and plot them as a bubble plot.
   3. Use the Z-factor¹⁰ to assess the drug screen quality of a run (see equation (2)). Discard an experiment with a Z-factor < 0.5.
          (2)