Human pancreatic duct organoids are three-dimensional culture systems that can be derived from normal or diseased tissue. They provide a long-term, genetically stable, and well-characterized culture system for efficacy and toxicity testing of potential therapeutic candidates for diseases such as cancer and cystic fibrosis. In particular, patient-derived, pancreatic ductal adenocarcinoma (PDAC) organoids have been shown to predict chemotherapy sensitivity in pancreatic cancer patients and therefore serve as a promising model system to advance precision medicine. Culturing organoids in a high throughput format enables us to capture donor-specific drug responses or to test multiple treatments simultaneously.

Below, we describe a protocol for setting up pancreatic drug testing in high throughput formats using a metabolic activity assay for cell toxicity readout. The protocol was optimized for PDAC organoids maintained in PancreaCult™ Organoid Growth Medium (Human). For organoid establishment, cryopreservation, and expansion protocols, please see the Product Information Sheet and Technical Manual.

Materials

• Wash Medium: DMEM/F-12 + 15mM Hepes (Catalog #36254) + 1% BSA
• PancreaCult™ Organoid Growth Medium (Human) (Catalog #100-0781)
• Optional for PDAC cultures: PGE2 (Catalog #72192)
• Optional for PDAC cultures: Human Recombinant EGF (Catalog #78006.1)
• ROCK Inhibitor Y-27632 (Catalog #72302)
• Matrigel® (Corning® Catalog #356231, ≥ 8 mg/mL protein)
• CellTiter-Glo® 3D Cell Viability Assay (Promega Catalog #G9681)
• White-Walled 96-Well Plate (e.g. VWR Catalog #82050-726)
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• Gentle Cell Dissociation Reagent (GCDR; Catalog #100-0485)
• 15 mL Conical Tubes (Catalog #38009)
• Tissue Culture-Treated Flat-Bottom (e.g. Catalog #38017)
• 384- or 96-Well Flat-Bottom Culture Plates (e.g. Catalog #200-0480)
• Optional: Antibiotics (e.g. Gentamicin)
• Pipette-Aid and Sterile Serological Pipettes (5, 10, and 25 mL)
• 200 µL and 1000 µL Pipettors
• 8- or 12-Channel Multi-Pipette or Robot
• Sterile Low-Retention Tips 200 µL, 1000 µL
• 70% Ethanol Solution
• Incubator with Atmospheric O₂ and 5% CO₂ at 37°C
• Biosafety Cabinet
• Vortex Mixer
• 37°C Water Bath
• Microscope
• Plate Reader
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Preparation

1. Expand pancreatic organoids in 40 μL Matrigel® domes as described in the Technical Manual.
   Note: Seeding one 96-well plate requires ~ 8 - 9 densely grown 40 μL domes
2. Warm a 24-well plate in the 37°C incubator overnight.
3. Prepare complete PancreaCult™ Organoid Growth Medium (Human) as described in the Technical Manual.
4. Prepare wash medium.
5. Thaw Matrigel® on ice.
6. On the day before the drug screen readout (Day 3), thaw the CellTiter-Glo® 3D reagent overnight at 2 - 8°C.

Pre-Passaging (Day -1)

After 5 - 7 days of culture, pancreatic cancer organoids often show an accumulation of shedded cells and are variable in sizes. To optimize organoid health, growth rate, and size for drug assays, we, therefore, recommend a 1:1 pre-passaging step using GCDR digestion.

1. Break up Matrigel® domes in spent medium by scraping and pipetting up and down gently in each well with a P1000. Collect up to 6 domes per 15 mL conical tube and place the tube on ice. Rinse each well with 500 μL wash medium and collect in the corresponding tube.
2. Top up 15 mL tube to 12 mL with cold wash medium and pipette up and down 10 times with a Pipette-Aid.
3. Centrifuge at 300 g for 5 minutes at 2 - 8°C.
   Note: If a large Matrigel® layer is visible above the organoid pellet, repeat steps 2 and 3 three times to further dilute and remove the Matrigel®.
4. Carefully aspirate the supernatant with a pipettor, then resuspend the pellet in 1 mL GCDR and pipette five times with a P1000.
5. Add 5 mL GCDR for a total volume of 6 mL GCDR per 15 mL tube, then pipette up and down five times or vortex tube briefly.
6. Place the tube on ice in a vertical position for 5 minutes, ensuring that the ice covers the total liquid volume.
7. Centrifuge at 300 x g for 5 minutes at 2 - 8°C.
8. Aspirate the supernatant, resuspend the pellet in the wash medium and pool the pellets into one 15 mL tube to a total volume of 1 mL.
   Example: For three pellets of the same cell line, add 700 μL of wash medium to the first pellet and resuspend. Adjust the P1000 volume to 800 μL to take up all tube content, then transfer into the next tube and resuspend the second pellet. Adjust the P1000 volume to 900 μL to collect all content and transfer it into the third tube. Resuspend the third pellet and check that the total volume is 1000 μL. If so, proceed to step 8, if not, top up to 1000 μL.
9. Break larger organoids into fragments by triturating four times with a P1000 while resting the tip on the bottom of the 15 mL tube, to create increased shear force.
   Note: There should be some resistance when pipetting up and down. The recommended organoid/fragment size is 50 - 200 μm.
10. Add an additional 3 mL wash medium per 15 mL tube for a total volume of 4 mL, and pipette up and down gently to mix.
11. Centrifuge at 300 x g for 5 minutes at 2 - 8°C.
12. Aspirate the supernatant and resuspend the pellet in 40 μL Matrigel® per dome harvested.
    Example: From 18 wells harvested, seed 18 new domes by resuspending pellet in 800 μL Matrigel® to account for some material loss in tips.
13. Remove the pre-warmed 24-well plate from the incubator and immediately seed 40 µL Matrigel® per well.
    Note: Alternatively, multiple domes can also be seeded into each well of a pre-warmed 6-well plate.
14. Transfer the plate to the incubator to solidify the domes for 10 minutes.
15. Optional for PDAC: supplement PancreaCult™-OGM with PGE2.
16. Add 500 μL of PancreaCult™-OGM with PGE2 per well and add PBS to any unused wells.
17. After 24 hours, organoids are ready to be seeded for the drug screening assay.

Figure 1. Pre-Passaging of Pancreatic Cancer Organoid Culture

(A) Representative images of pancreatic cancer organoids grown for 7 days in 24-well dome cultures before passaging and (B) 0 and 18 hours after (1:1) passaging into fresh domes to remove debris (right panels). Scale bar = 800 μm.

Drug Screening (Day 1-4)

Before you start: Drug efficacies and toxicities can vary widely depending on the type of drug, its delivery, and the organoid line used (e.g. normal or diseased). Our protocol uses 3 days of drug exposure with daily medium changes to minimize drug degradation effects and avoid unrelated organoid decline due to extended culture times without medium changes. It is recommended to test and optimize drug dilution ranges and exposure times to achieve robust dose-response curves.

1. Prepare concentrated stock solutions of the compounds to be tested in the appropriate solvent (e.g. DMSO). Ensure that drugs are fully dissolved.
2. Dilute stock solutions using a titration series in their appropriate solvent to generate a 10-point dilution series. Use these dilutions as 100X stocks.
   Note: DMSO concentrations of up to 1% did not affect PDAC organoid viability during the assay. It is recommended to determine the optimal titration range of a drug empirically.
3. Prepare experimental plate layout. Use 4 replicate wells per compound concentration, solvent control (solvent only), and untreated control (medium only). This setup allows for two compounds to be screened per 96-well plate. Refer to Figure 2 for a sample plate layout.
   Note: Screens can also be performed in 384-well plates. We recommend maintaining a seeding density of 300 organoids per 384-well for the CellTiter-Glo® 3D viability assay to ensure good assay sensitivity.
   

   Figure 2. Sample Cytotoxicity Screening Plate Layout

4. Perform a 1 in 100 dilution of the 100X stock compounds or solvent in PancreaCult™ OGM to prepare fresh media for screens. Prepare sufficient volumes for all wells that will be treated.
5. Transfer the 96-well plate(s) of organoids to be treated to the biosafety cabinet.
6. Aspirate and discard the medium in each well using a multichannel pipette.
7. Add 100 µL of the appropriate medium supplemented with compounds or solvents (prepared in step 4) to their corresponding wells, or 100 µL complete PancreaCult™ OGM to the untreated control wells.
8. Incubate at 37°C and 5% CO₂ for 24 hours.
9. Repeat steps 4 - 8 two more times, for a total of three treatments using freshly prepared medium.
10. Analyze cell viability 24 hours after the last compound treatment, for a total treatment duration of 72 hours.

Figure 3. PDAC Organoids Cultured in 96-Well Plate for Drug Toxicity Assay

(A) Representative images of pancreatic cancer organoids in dome cultures, immediately (Day 0) and 24 hours post-seeding into a 96-well plate (Day 1). Note that deflated organoids at Day 0 reform into cystic organoids within 24 hours. (B) Organoid morphology after 24 hours and 72 hours of treatment with DMSO control (top row) or 5-Fluorouracil (5-FU, bottom row) using daily medium changes. Scale bar = 200 μm.

Cell Viability Assay (Day 4)

1. On the day before the drug screen readout, thaw CellTiter-Glo® 3D reagent overnight at 2 - 8°C.
2. On Day 4 (72 hours after the initial addition of the drugs), warm DMEM/F-12 to room temperature before use.
3. Equilibrate the 96-well plate of treated organoids to room temperature for approximately 20 minutes.
4. Mix CellTiter-Glo® 3D bottle gently by inverting to obtain a homogeneous solution.
5. In a conical tube, combine mixed CellTiter-Glo® 3D reagent and DMEM/F-12 at a 5:3 ratio (i.e. 50 µL CellTiter-Glo® 3D + 30 µL DMEM/F-12 per well). Mix gently by inverting or pipetting up and down slowly. Prepare sufficient mixture for all wells and use at room temperature.
   Note: The manufacturer recommends a 1:1 dilution which is reduced in this protocol to account for the volume of the Matrigel® and any medium left in the well.
6. Without touching the domes, aspirate and discard the medium in each well using a multichannel pipette.
7. Swiftly add 80 µL of CellTiter-Glo® 3D + DMEM/F-12 mix to each well using a multichannel pipette.
8. Pipette up and down several times to dissociate and lyse the organoids and Matrigel®. Avoid introducing bubbles.
9. Incubate the plate on a heated benchtop orbital shaker at 25°C and 120 rpm for 30 minutes.
10. Transfer 55 µL of the contents of each well to a white 96-well plate.
11. Record luminescence.
12. Plot non-linear regression curves using the log of the compound concentration and measured relative luminescence unit (RLU) values (as a percentage of the solvent control) to generate dose-response curves and determine the IC50.

Figure 4. Using PDAC Organoids as a Tool for Drug Toxicity Evaluation In Vitro

Dose-response curves and IC50 values for two PDAC organoid lines treated 72 h hours with Irinotecan (A) or 5-Fluorouracil (B) in Matrigel® layer or dome cultures. n = 2, Mean ±SD.