Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Summary

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Abstract

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Introduction

Patient-derived tumor organoids (PDOs) are rapidly emerging as a robust model system to study cancer development and therapeutic responses. PDOs are three-dimensional (3D) cell culture systems that recapitulate the complex genomic profile and architecture of the primary tumor^1,2. Unlike traditional two-dimensional (2D) cultures of immortalized cancer cell lines, PDOs capture and maintain intratumoral heterogeneity^3,4, making them a valuable tool for both mechanistic and translational research. Although PDOs are becoming an increasingly popular model system, commercially available reagents and analysis methods for cellular effects that are compatible with PDO cultures are limited.

The lack of robust methods to analyze subtle changes in treatment response hinders clinical translation. The gold standard cell health reagent in 3D cultures, CellTiter-Glo 3D, utilizes ATP levels as a determinant of cell viability^5,6. While this reagent is useful for endpoint assays, there are several caveats, most notably the inability to use samples for other purposes after completion of the assay.

Live-cell imaging is a sophisticated form of kinetic microscopy that, when combined with fluorescent reagents, has the capacity to quantify a variety of cell health readouts within PDO models, including apoptosis^7,8,9 and cytotoxicity¹⁰. Indeed, live-cell imaging has been integral to high throughput screening of compounds in 2D platforms^11,12. Systems such as the Incucyte have made the technology affordable and thus accessible to research groups in a variety of settings. However, the application of these systems to analyze 3D cultures is still in its infancy.

Herein, we describe a method to assess drug response in PDO models of cancer using multiplexed live-cell imaging (Figure 1). Through analysis of bright field images, changes in PDO size and morphology can be kinetically monitored. Furthermore, cellular processes can be simultaneously quantified over time using fluorescent reagents, such as Annexin V Red Dye for apoptosis and Cytotox Green Dye for cytotoxicity. The methods presented are optimized for the Cytation 5 live-cell imaging system, but this protocol may be adapted across different live-cell imaging platforms.

Protocol

Studies using human tumor specimens were reviewed and approved by the University of Iowa Institutional Review Board (IRB), protocol #201809807, and performed in accordance with the ethical standards as laid down in the 1964 Helsinki Declaration and its later amendments. Informed consent was obtained from all subjects participating in the study. Inclusion criteria include a diagnosis of cancer and the availability of tumor specimens.

1. Plating intact PDOs in a 96-well plate

1. Prepare reagents.
   1. Preheat 96-well plates at 37 °C overnight and thaw BME overnight at 4 °C.
   2. Prepare full organoid culture media optimized for culturing the cancer type of interest. Specific culture media used for experiments shown herein are provided in Supplementary Table 1.
      NOTE: Media components may need to be modified for different tumor types. For example, the organoid culture media is supplemented with 100 nM estradiol for gynecologic tumors¹³. Prepared media is stable at 4 °C for 1 month. For long-term storage, aliquot the media into 50 mL tubes and store at -20 °C.
2. Prepare two separate aliquots of organoid culture media at 4 °C and 37 °C. For example, if 60 wells are being plated in a 96-well plate, use 6 mL of warm organoid culture media and 150 µL of ice-cold organoid culture media.
3. Prepare organoid wash buffer: Supplement 1x PBS with 10 mM HEPES, 1x Glutamax, 5 mM EDTA, and 10 µM Y-27632. Store at 4 °C
4. Harvest PDOs cultured in a 24-well plate. Perform all steps on ice or at 4 °C unless otherwise noted.
   1. Aspirate media from each well using a vacuum line system.
   2. Add 500 µL of ice-cold organoid wash buffer and gently pipette 2-3x using a 1000 µL pipettor. Incubate the plate on ice for 10 min.
   3. Transfer the contents of each well to a 50 mL conical tube. To ensure that all the PDOs are in suspension, rinse each well with an additional 300 µL of organoid wash buffer and transfer to the 50 mL conical tube. Centrifuge for 5 min at 350 x g at 4 °C
   4. Aspirate the supernatant from the BME/organoid pellet using a vacuum line system, leaving ~ 5 mL remaining in the tube. Add 20 mL of organoid wash buffer and gently resuspend the pellet using a 10 mL serological pipette. Incubate on ice for 10 min.
   5. Centrifuge for 5 min at 350 x g at 4 °C. Aspirate the supernatant with a vacuum line system, taking care not to disrupt the PDO pellet.
5. Plating PDOs in a 96-well plate: Perform all steps on ice unless otherwise noted.
   1. Resuspend the PDO pellet in an appropriate amount of ice-cold organoid culture media to create a PDO suspension. Transfer PDO suspension to an ice-cold 1.5 mL microcentrifuge tube.
      NOTE: To calculate the amount of organoid culture media, determine the number of wells to be plated in a 96-well plate, taking into consideration that PDOs are plated in a 5 µL dome in a 1:1 ratio of organoid culture media and BME. For example, when plating one 96-well plate and using only the inner 60 wells, the total amount of PDO suspension needed will be 300 µL: 150 µL organoid culture media and 150 µL BME. For models that exhibit optimal growth at different percentages of BME, the ratio of BME: media may be modified in this step, though it is important to standardize the ratio across all assays for each specific model. To account for pipetting error, add 10% volume to each component.
   2. Count the number of PDOs.
      1. Transfer 2.5 µL of the PDO suspension to an ice-cold 1.5 mL microcentrifuge tube and mix with 2.5 µL of BME. Transfer the 5 µL mixture onto a clean glass microscope slide. Do not coverslip the slide. The mixture will solidify into a dome.
      2. Visualize using a bright field microscope at 4x. Count the number of PDOs in the 5 µL mixture; the goal is to have roughly 25-50 PDOs per 5 µL dome.
         NOTE: If the desired density is not achieved in the test mixture, adjust the final volume of the PDO suspension either by adding more organoid culture media or centrifuging the PDO suspension and resuspending the PDO pellet in a lower volume of ice-cold organoid culture media. Regardless of how the PDO suspension is modified in this step, the final ratio of BME: PDO suspension in step 1.5.3. should be 1:1.
   3. Using a 200 µL pipettor with wide bore tips, carefully mix PDO suspension with an equal amount of BME to achieve a 1:1 ratio of organoid culture media to BME. Avoid bubbles, which will disrupt the integrity of the domes. 
   4. Using a 20 µL pipettor, seed 5 µL domes into the center of each well of a prewarmed 96-well plate, seeding only the inner 60 wells. To ensure equal distribution of the PDOs, periodically pipet the contents of the 1.5 mL tube with a 200 µL pipettor with wide-bore tips.
   5. After all wells have been seeded, place the lid on the plate and gently invert. Incubate the inverted plate at 37 °C for 20 min in the tissue culture incubator to allow domes to solidify.
      NOTE: Inverting the plate ensures that the BME/organoid culture media dome retains the 3D structure to provide adequate room for PDO formation.
   6. Flip the plate so that it is sitting with the lid up and incubate for 5 min at 37 °C.

2. Treatments and the addition of fluorescent dyes for multiplexing

1. While BME domes are solidifying in the 96-well plates, prepare dilutions of fluorescent live-cell imaging reagents. Specific parameters for multiplexing Annexin V Red Dye and Cytotox Green Dye are given herein.
2. Fluorescent reagent preparation (Day -1): Calculate the appropriate volume of organoid culture media based on the number of wells to be treated, assuming each well will be treated with 100 µL of dye-dosed media. Dilute dye in pre-warmed organoid culture media to the desired concentration.
   NOTE: The total amount of media needed will vary depending on the experiment. Add 10% to the final volume to account for pipetting error. For example, to treat the inner 60 wells of a 96-well plate, prepare 6.6 mL of dye-dosed media (Table 1).
3. Treat each well with 100 µL of 2x dye-dosed organoid culture media. Add 200 µL of sterile 1x PBS to the outer empty wells of the plate. Incubate at 37 °C overnight.
   NOTE: PBS in the peripheral wells decreases the evaporation of media from the inner wells.
4. Addition of drugs/treatment agents (Day 0): Prepare drugs in pre-warmed organoid culture media at a 2x concentration in a volume of 100 µL per well.
   NOTE: DMSO can be toxic to cells at high concentrations. A concentration of 0.1% DMSO is not exceeded in the experiments performed in this study. In addition to drugs, some fluorescent reagents are distributed as a DMSO solution. It is important to account for total DMSO concentration when working with such reagents.
5. Add 100 µL of 2x dye-dosed media to each well; avoid creating bubbles.

3. Setting up imaging parameters

1. Place plate in Cytation 5. Open Gen5 software. Click New Task > Imager Manual Mode. Select Capture Now and input the following settings: Objective (select desired magnification); Filter (select microplate); Microplate format (select number of wells); and Vessel type (select plate type). Click Use Lid and Use slower carrier speed. Click OK.
   NOTE: Vessel Type: Be as specific as possible when selecting information about the plate because the software is calibrated to the specific distance from the objective to the bottom of the plate for each plate type and thickness of the plastic.
   ​Slower Carrier Speed: Select this box to avoid disrupting domes when loading/unloading plates.
2. Create a Z-Stack that will image the entire BME dome.
   1. Select a well of interest to view (left panel, below histogram).
   2. Select the Bright Field channel (left panel, top). Click Auto-expose and adjust Settings as needed.
   3. Set the bottom and top of the Z-Stack: Expand Imaging Mode tab (left panel, middle). Check the Z-Stack box. Using the course adjustment arrows (left panel, middle), click the down adjustment until all PDOs have come into and then out of focus and are fuzzy. Set this as the bottom of the Z-Stack. Repeat in the opposite direction using the course adjustment arrows to set the top of the Z-Stack.
   4. To ensure that the Z-Stack settings are appropriate for other wells of interest, select another well (left panel, below histogram) and visualize the top and bottom of the Z-Stack.
   5. To manually enter the focal positions, click the three dots next to the fine adjustment (left panel, top). A window will open; type in the top Z-Stack value (found in the left panel, center, under Imaging Mode). Repeat for the bottom Z-Stack value. Adjust as necessary to capture the desired Z range by repeating step 3.2.3. If adjustments were necessary, select another well to verify settings.
3. Set the exposure settings for the fluorescent channel(s). Settings are described for two fluorescent channels (GFP & TRITC). The specific number of fluorescent channels will depend on the experiment and which fluorescent cubes are installed in the live-cell imaging system.
   NOTE: If the signal intensity is anticipated to be significantly higher at the end of the experiment, users should consider performing test experiments to determine the optimal exposure settings at the end of the experiment that can then be applied when setting up the initial parameters.
   1. Expand the Imaging Mode tab (left panel, middle) and open Edit Imaging Step. A pop-up window will appear.
   2. Under Channels, click on the bubble for the desired number of channels. Designate one channel for bright field and additional channels for each fluorescent channel. In this example, Channel 1 = Bright Field; Channel 2 = GFP; Channel 3 = TRITC. Using the drop-down Color menus, select the appropriate setting for each channel. Close the editing window by clicking OK.
   3. Set up each fluorescent channel.
      1. Switch the channel to GFP (left panel, top).
      2. Click Auto-expose (left panel, top). Expand the Exposure tab (left panel, middle) and adjust the exposure settings to minimize the background signal.
      3. Copy exposure settings to the Image Mode tab following steps 3.3.3.3-3.3.3.6.
      4. Click on the Copy icon next to the Edit Imaging Step box. Click Edit Imaging Step, which will open another window.
      5. Under the GFP channel, click the Clipboard icon in the Exposure line to add the Illumination, Integration Time, and Camera Gain settings to the channel.
      6. Repeat Steps 3.3.3.4-3.3.3.5 for the TRITC channel. Click OK to close the window.
4. Set up the image preprocessing and Z-projection steps to automate image preprocessing.
   1. Click on the Camera icon (left panel, bottom corner). A new window will open.
   2. Under Add Processing Step (left panel, bottom), click on Image Preprocessing. A new window will open.
   3. On the Bright Field tab, deselect Apply Image Preprocessing.
   4. For each Fluorescent Channel tab, make sure Apply Image Preprocessing is selected. Deselect Use same options as channel 1 and click OK. The window will close.
   5. Under Add Processing Step, click on Z Projection. A new window will open. If desired, adjust the slice range (e.g., to narrow the Z range). Close the window by selecting OK.
5. Create protocol.
   1. Click Image Set in the toolbar. In the drop-down menu, click Create experiment from this image set. The Imaging Window will close, and the Procedure Window will open.
      NOTE: The parameters selected in Imager Manual Mode will automatically be loaded into the new window, whereby an experimental protocol can be created.
   2. Set the temperature and gradient: Click Set Temperature under the Actions heading (left). A new window will open. Select Incubator On and manually enter the desired temperature under Temperature. Next, under Gradient, manually enter 1. Close the window by selecting OK.
      NOTE: Creating a 1 °C gradient will prevent condensation from forming on the lid of the plate.
   3. Designate wells to the image.
      1. Double-click on the Image Step under Description. Click Full Plate (right corner, top). This will open the Plate Layout window.
      2. Highlight wells of interest using the cursor. Click OK. If desired, check Autofocus Binning and Capture Binning boxes. Click OK to close the window.
         NOTE: Binning will require exposure adjustment, as described in step 3.3.3.2 above. Please refer to the Discussion section for specific scenarios in which this feature may be used.
   4. Set intervals for kinetic imaging.
      1. Click on Options under the Other heading (left). Check the Discontinuous Kinetic Procedure box.
      2. Under Estimated Total Time, enter the run time for the experiment (e.g., 5 days). Under Estimated Interval, enter the interval to image the plate (e.g., every 6 h).
      3. Click Pause after each run to allow time for the plate to be transferred to the incubator. Close the window by selecting OK.
   5. Update data reduction steps.
      1. Click OK to close the Procedure Window. A tab will open to update data reduction steps. Select Yes. Double-click on Image Preprocessing. Click through the different channels to verify settings and click OK.
      2. Double-click on Z Projection. Click through the different channels to verify settings. Click OK. Then, click OK again to close the Data Reduction Window.
   6. Format the plate layout.
      1. Open the Plate Layout Wizard and designate well types following steps 3.6.2-3.6.3.
      2. Click on the Plate Layout icon in the toolbar (left corner, top) to open the Plate Layout Wizard.
      3. Check the boxes next to the well types used in the experiment. Under Assay Controls, enter the number of different control types using the arrows. Click Next to open the Assay Control #1 window.
      4. Set assay control well conditions following steps 3.6.5-3.6.8.
      5. On the Assay Control #1 window, enter the control label in the Plate Layout ID box. If desired, enter the full name in the adjacent box. Select the number of replicates for the respective control condition using the arrows.
      6. If using multiple concentrations or a dilution series within the control, click Define dilutions/concentrations and use the drop-down menu to select the Type. Enter values for each concentration/dilution in the table.
         NOTE: The auto function can be used if concentrations change by a consistent increment.
      7. Select the Color tab in the toolbar. Choose desired text color and background color for control in the drop-down menu. Click Next.
      8. Repeat as necessary with additional controls.
      9. Set sample well conditions following 3.6.10-3.6.12.
      10. On the Sample Setup page, enter the sample ID Prefix (e.g., SPL). Select the number of replicates using the arrows. If using samples with varying treatment concentrations, select Concentrations or Dilutions in the Type drop-down menu. Enter dilutions/concentrations in the table and enter units in the Unit box.
      11. Select Identification Fields in the toolbar. Enter the desired Category Name(s) (e.g., sample ID, drug) in the table.
      12. Select the Color tab in toolbar. Select a different color for each treatment group/sample. Click Finish. This will open the Plate Layout page.
          NOTE: The numbers on the left side correlate with the different sample numbers.
      13. Assign Sample IDs following steps 3.6.14-3.6.16.
      14. Select SPL1 from the left panel. Use the cursor to select wells.
          NOTE: Autoselect tools can be adjusted in the serial assignment box. The number of replicates and orientation of the layout can be pre-designated.
      15. Repeat with other samples to complete the plate layout. Once satisfied, click OK.
      16. In the File toolbar, select Sample IDs. Fill in Sample ID columns with the appropriate information for each sample (e.g., drug type). Press OK.
   7. Save the protocol.
      1. In the toolbar, click File > Save Protocol as. Select the location to save the file. Enter a file name. Click Save to close the window.
      2. Click File > Exit in the toolbar. A tab will open to save changes to Imager Manual Mode. Select No.
      3. A tab will open to save changes to Experiment 1. Select No. A tab will open to update the protocol definition. Select Update. Close the software.
6. Import the protocol into BioSpa OnDemand and finish setting up the Experiment.
   1. Open the BioSpa OnDemand (scheduling software) software.
   2. Select an available slot in the software.
   3. Remove the plate from the live-cell imaging system. Click Open Drawer to access the appropriate slot in the scheduling software and insert the plate. Click Close Drawer.
      NOTE: This step can be performed at any point once the protocol has been created in step 3.5.7 above. However, the plate must be in the Cytation 5 to perform a timing run in the below Step 3.6.4.3.
   4. Import the Protocol.
      1. Under the Procedure Info tab, select User in the drop-down menu. Next to the Protocol slot, click Select > Add a new entry.
      2. Next to the Protocol slot, click Select. This will open a new window to navigate to the desired Protocol in the file architecture. Click Open to import the Protocol into the scheduling software.
      3. Enter the amount of time needed to image the plate. Click OK to close the Gen5 Protocol List window.
         NOTE: This step is especially important when running several experiments at a time. To determine the time needed to image the, click Perform a timing run now. Click OK.
   5. Set imaging intervals and schedule the experiment.
      1. Under Interval, enter the imaging interval designated in step 3.5.4.
      2. Under Start Time Options, select When Available. Under Duration, select Fixed or Continuous.
         NOTE: A specific start time can be designated instead of running the protocol at the next available time. Selecting Fixed Duration will set a specific endpoint for the experiment and requires the user to designate an experimental timeframe. Continuous Duration will allow the experiment to run with no endpoint and can only be ended by a user stopping the experiment.
      3. Click Schedule Plate/Vessel. This will open the Plate Validation Sequence. A tab will open with the proposed first read time. Click Yes to accept this schedule.

4. Image analysis in Gen5 software (Figure 2)

1. Open Image Analysis module.
   1. Open Gen5. In the Task Manager, select Experiments > Open. Select the experiment to open the file. Click Plate > View in the toolbar.
   2. Change Data drop-down menu to Z Projection. Double-click on a well of interest. Select Analyze > I want to setup a new Image Analysis data reduction step. Click OK.
2. Cellular analysis
   1. Primary mask
      1. Under Analysis Settings, select Type: Cellular Analysis and Detection Channel: ZProj[Tsf[Bright Field]] (left panel, center).
      2. Click Options. This will open the Primary Mask and Count page. In the Threshold box, check Auto and click Apply. Click the Highlight Objects box (right panel, bottom) to show objects within the designated threshold. Adjust as necessary to include objects of interest.
         NOTE: Threshold settings are based on pixel intensity. For example, if the threshold is set to 5000, pixels with an intensity greater than 5000 will be included in the analysis.
      3. Under Object Selection, designate the minimum and maximum object size (µm). Adjust as necessary to exclude cellular debris/single cells.
         NOTE: PDO size may vary significantly between different models and types. Use the measuring tool in the Gen5 software to determine the minimum and maximum PDO size thresholds for each model. Users may choose a smaller minimum PDO size threshold relative to the value provided by the measuring tool in order to prevent the exclusion of PDO fragments at later time points after drug treatment.
      4. To limit the analysis to a certain region of the well, deselect Analyze entire image and click Plug. In the Image Plug window, use the drop-down menu to select Plug shape. Adjust the size and position parameters as necessary to fit the region of interest.
         NOTE: It is important to maximize the number of PDOs within the plug while also excluding PDO-free areas to minimize background. Designate a plug size that will consistently capture the majority of the objects of interest across replicates. Generating a plug that also excludes the edges of the dome is important as it excludes any objects that may appear distorted due to the refraction of light from the extreme curvature of the dome around the edges. Include primary edge objects may also be deselected to capture only entire PDOs within the plug.
   2. Subpopulation analysis. An example of subpopulation designation is provided in Figure 3.
      1. Click on Calculated Metrics in the Cellular Analysis toolbar. Click Select or create object level metrics of interest (right corner, bottom). Under Available Object metrics, select metrics of interest (e.g., circularity) and click the Insert button. Click OK.
         NOTE: The morphology and density of each PDO model will determine the best metrics of interest to distinguish the subpopulation.
      2. Click on Subpopulation Analysis in the Cellular Analysis toolbar. Click Add to create a new subpopulation. A pop-up window will open.
      3. If desired, enter a name for the subpopulation. Under Object Metrics, select a metric of interest and press Add Condition. In the Edit Condition window, enter parameters for the chosen Object Metric. Repeat with additional metrics as necessary.
         NOTE: Parameters may be adjusted manually or set using the finder tool. For example, to exclude debris, users could add Area as an Object Metric and select objects smaller than 800. Circularity as an Object Metric is routinely used, and any objects with a circularity greater than 0.2-0.5 are included, depending on the model.
      4. In the table at the bottom of the window, check the desired results to display. Click OK > Apply.
      5. To view the objects within the subpopulation, use the Object Details drop-down menu (right panel, center) to select the subpopulation. Objects that fall within the parameters will be highlighted in the image.
         NOTE: To change the highlight colors of the primary mask and subpopulation, click Settings (right panel, bottom).
      6. To adjust subpopulation parameters, reopen the Subpopulation Analysis window from the Cellular Analysis toolbar. Select the subpopulation and click Edit. Click Add Step.
         ​NOTE: This will apply the same analysis to all wells within the experiment at all time points. In the drop-down menu on the Matrix page, different metrics can be selected for individual viewing.

5. Exporting data from Gen5 to Excel

1. To customize a data file for export, select the Report/Export Builders icon in the toolbar. In the pop-up window, click New export to Excel.
2. On the Properties page of the pop-up window, select Scope > Plate and Content > Custom. Click on the Content option in the toolbar. Click Edit Template, which will open the Excel program.
3. Within the spreadsheet, select Add-ins > Table > Well Data. Hover over the various selections to see options for export. Select metric of interest (e.g., Object Mean[ZProj[Tsf[TRITC]]]).
   NOTE: Plate layout can be added to the spreadsheet analysis template by selecting Add-ins > Protocol Summary > Layout.
4. An Edit window will open. In the Wells box, designate the wells for export either by Well-ID or Well #. Select OK. A template will be loaded into the spreadsheet file. Close spreadsheet. The template is automatically saved.
5. Click OK on the New export to Excel window and close the Report/Export Builders window.
6. Click the Export icon in the Gen5 toolbar. Check the box next to the desired export file. Click OK. Gen5 will automatically populate the spreadsheet template and open the file in Excel.

6. External data analysis

1. Open the Export file (.xlsx) in Excel.
2. For each well, divide each individual value by the 0:00 time point value for that well. This will set time point 0 equal to 1, and each value beyond that will be relative to the initial reading.
3. Open a new file in the data analysis software. Select the XY layout option.
   NOTE: In this protocol, GraphPad Prism (version 9.5.1) was used.
4. Input labels for each data group. Copy and paste the time points and corresponding normalized values for each treatment group into the Prism table. A graph for the data will be automatically generated and can be found under Graphs.

Results

Our objective was to demonstrate the feasibility of using multiplexed live-cell imaging to assess PDO therapeutic response. Proof of concept experiments were performed in two separate PDO models of endometrial cancer: ONC-10817 and ONC-10811 (see Supplementary Figure 1 and Supplementary Figure 2 for ONC-10811 data). Apoptosis (annexin V staining) and cytotoxicity (Cytotox Green uptake) were kinetically monitored in response to the apoptosis-inducing agent, staurosporine. Specifically, PD...

Discussion

PDO cultures are becoming an increasingly popular in vitro model system due to their ability to reflect cellular responses and behaviors². Significant advances have been made in PDO generation, culture, and expansion techniques, yet methods to analyze therapeutic responses have lagged. Commercially available 3D viability kits are lytic endpoint assays, missing out on potentially valuable kinetic response data and limiting subsequent analyses by other methods⁸. Emerging stud...

Materials

Name	Company	Catalog Number	Comments
1.5 mL microcentrfuge tube	Dot Scientific Inc	1008113
15 mL conical centrifuge tube	Sarstedt	62.554.100
554 NM LED Cube	Agilent	1225012
96-well plate	Corning Costar	3596	Prewarmed to 37 °C
96-well plate	Agilent	204626-100	Prewarmed to 37 °C
A83-01	Tocris	2939	Final concentration is 500 nM (component of organoid culture media)
Advanced DMEM/F-12	Gibco	12634-010	component of organoid culture media
B27 Supplement	Gibco	17504044	Final concentration is 1x (component of organoid culture media)
BioTek BioSpa 8 Automated Incubator	Agilent	BIOSPAG-SN	Tabletop incubator; BioSpa OnDemand scheduling software comunicates with Gen5 to transfer plates between the BioSpa and the Cytation 5 for imaging (this protocol uses version 1.01.10)
BioTek Cytation 5 Cell Imaging Multimode Reader	Agilent	CYT5PW-SN	Plate reader; Gen5 software is used for this device (this protocol uses version 3.12.08)
Cultrex UltiMatrix Reduced Growth Factor Basement Membrane Extract	R&D Systems	BME001-10
Daunorubicin HCl	Sigma-Aldrich	S3035	Reconstituted in DMSO
Dimethyl sulfoxide	Sigma-Aldrich	D2438
EDTA (0.5 M)	Thermo Fisher	AM9260G
Forskolin	Tocris	1099	Final concentration is 10 µM (component of organoid culture media)
Glutamax	Gibco	35050-061	Final concentration is 1x (component of organoid culture media)
HEPES	Gibco	15630-080	Final concentration is 10 mM (component of organoid culture media)
Human EGF, Animal-Free Recombinant Protein	Gibco	AF-100-15-1MG	Final concentration is 0.5 ng/mL (component of organoid culture media)
Human FGF-10 Recombinant Protein	Gibco	100-26-1MG	Final concentration is 10 ng/mL (component of organoid culture media)
Human R-Spondin 1 Recombinant Protein	Gibco	120-38-5UG	Final concentration is 250 ng/mL (component of organoid culture media)
Hydrocortisone Stock Solution	StemCell Technologies	7926	Final concentration is 500 ng/mL (component of organoid culture media)
Imaging Filter Cube- GFP	Agilent	1225101
Imaging Filter Cube- TRITC	Agilent	1225125
Imaging LED GFP/CFP	Agilent	1225001
Incucyte Annexin V Red Dye	Sartorius	4641	Reconstituted in organoid culture media
Incucyte Cytotox Green Dye	Sartorius	4633	DMSO solution
N-Acetyl-L-cysteine	Sigma-Aldrich	A7250	Final concentration is 1.25 mM (component of organoid culture media)
Nexcelom Bioscience ViaStain AOPI Staining Solution	Fisher-Scientific	13366169	Add 1:50 volume
Nicotinamide	Sigma-Aldrich	N0636	Final concentration is 10 mM (component of organoid culture media)
Noggin	R&D Systems	6057-NG	Final concentration is 100 ng/mL (component of organoid culture media)
Penicillin-Streptomycin	Gibco	15140122	Final concentration is 10 units/mL (component of organoid culture media)
Phosphate Buffered Saline (1x)	Gibco	14190-144
Primocin	InvivoGen	ant-pm-05	Final concentration is 100 µg/mL (component of organoid culture media)
Recombinant Human Heregulinβ-1	Pepro Tech	100-03	Final concentration is 37.5 ng/mL (component of organoid culture media)
Staurosporine solution from Streptomyces sp.	Sigma-Aldrich	S6942
TrypLE Express	Life Technologies	12604013
Y-27632, CAS 331752-47-7	Sigma-Aldrich	688000	Final concentration is 5 µM (component of organoid culture media)
β-Estradiol	Sigma-Aldrich	E2758	Final concentration is 100 nM (component of organoid culture media)