Toxicity Testing for Drug Development Using Human Intestinal Organoids and IntestiCult™
Drug-induced gastrointestinal toxicity remains a major challenge in preclinical drug development and is a leading cause of late-stage drug attrition. Accurately identifying intestinal toxicity early in the drug discovery process can help researchers make more informed decisions and prioritize the most promising therapeutic candidates. However, conventional in vitro models, including immortalized cell lines such as Caco-2, lack the cellular diversity and tissue architecture of the human intestine, while animal models do not always predict human-specific responses.
Human intestinal organoids provide a physiologically relevant in vitro model for drug toxicity testing and safety assessment. When incorporated into new approach methodologies (NAMs), intestinal organoids can improve the evaluation of therapeutic candidates by providing human-relevant data earlier in the drug development process. This supports the broader shift toward optimizing reliance on animal testing in preclinical safety evaluation. These self-organizing three-dimensional (3D) cultures contain multiple intestinal epithelial cell types, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, and recapitulate key structural and functional features of the intestinal epithelium. As a result, they provide a robust platform for investigating epithelial barrier function, nutrient transport, tissue homeostasis, and responses to xenobiotics.
This protocol describes a standardized workflow for performing toxicity assays using human intestinal organoids cultured in IntestiCult™, either first-generation IntestiCult™ Organoid Growth Medium or IntestiCult™ Plus. The medium supports robust organoid expansion and reproducible maturation with consistent lot-to-lot performance, helping researchers generate consistent, high-quality data for drug toxicity testing and safety assessment. While this protocol provides a reliable framework for organoid-based toxicity assays, experimental parameters, including organoid seeding density, compound concentration, and exposure duration, may be optimized to meet the requirements of specific research applications.
Materials and Reagents
- IntestiCult™ Media:
- IntestiCult™ Plus Organoid Growth Medium (Catalog #06010)
- IntestiCult™ Organoid Growth Medium (Human; Catalog #06010)
- 25% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)
- DMEM/F-12 with 15 mM HEPES (Catalog #36254)
- Corning® Matrigel® Matrix, Growth Factor Reduced (GFR), Phenol Red-Free (e.g. Corning 356231)
- 24-Well Organoid Culture Plate (Catalog #200-0561) or Costar® 24-Well Flat-Bottom Plate, Tissue Culture-Treated (Catalog #38017)
- 96-Well Organoid Culture Plate (Catalog #200-0562) or Costar® 96-Well Flat-Bottom Plate, Tissue Culture-Treated (e.g. Sigma CLS 3595)
- 70 µm Reversible Strainer (Catalog #27216)
- Y-27632 (Catalog #72302)
Protocol
A. Preparation of Materials and Reagents
- Prepare complete IntestiCult™ Medium as described in the relevant product information sheet (PIS).
- Prepare DMEM + 1% BSA as described in the relevant product information sheet.
- Pre-warm 24- and 96-well Tissue Culture-Treated plates at 37°C for 24 hours prior to use.
B. Expansion of Organoids
- Expand organoids in a 24-well plate according to standard protocols as described in the relevant PIS. Estimate 1 x 50 μL dome in a 24-well plate to seed 8 x 10 μL domes of a 96-well plate.
NOTE: This is an estimate only and should be adjusted based on organoid size and density. Final seeding density will be 120 fragments per 10 μL dome.
- Grow organoids for 7 to 10 days, until they have reached full size and are ready to passage.
- Follow standard protocols for passaging intestinal organoids present in the relevant PIS until step C.15 for IntestiCult™ Plus or step B.2. for IntestiCult™ Organoid Growth Medium. Adjust the final step to seed approximately 100 - 120 fragments per 10 μL dome.
- Centrifuge the sample at 300 x g for 5 minutes.
- Remove as much supernatant as possible and store on ice.
- Resuspend the organoid pellet in an appropriate amount of Matrigel® (10 μL per well to be seeded) and mix gently.
- Remove a pre-warmed 96-well plate from the 37°C incubator.
NOTE: If using Organoid Culture Plates, they do not need to be pre-warmed.
- Remove 10 μL of the organoid-Matrigel® solution from the tube and slowly, while keeping the pipette completely upright, add the 10 μL droplet to the center of the target well.
- Repeat for the remainder of the samples.
- Place the plate back in the 37°C incubator.
- Incubate for at least 15 minutes to allow for the Matrigel® to polymerize.
- Gently add 100 μL of IntestiCult™ Plus Start Medium or IntestiCult™ Organoid Growth Medium to each well.
C. Drug Treatment Protocol and Toxicity Testing
A 5-day treatment period allows for prolonged exposure, thereby demonstrating smaller growth inhibition effects. A longer growth phase and shorter treatment phase will highlight acute toxicity, but little growth inhibition.
Growth Phase
- Grow organoids in a 96-well plate:
- For 2 days with IntestiCult™ Plus Start Medium, then when replacing medium, switch to IntestiCult™ Plus Balance Medium for an additional 2 days.
- For 2 days with IntestiCult™ Organoid Growth Medium.
Treatment Phase
- Prepare fresh aliquots of each medium/drug concentration to be tested, including a solvent control.
- On Day 4, replace the media in each well with the appropriate test medium (IntestiCult™ Plus Balance medium plus the test article).
- Replace the medium again on Day 5 and 6 with a fresh test medium.
- Prepare fresh aliquots of each medium/drug concentration to be tested, including a solvent control.
- On Day 2, replace the medium in each well with the appropriate test medium and incubate for 2 days.
- Replace the medium again on Day 4 and 6 with a fresh test medium.
Analysis Phase
- On the final day of the selected treatment phase, image each well using either a Zeiss or STEMvision™ imager. Cultures can be maintained for up to two weeks.
- Follow standard protocols for Promega CellTiter-Glo® 3D:
- Thaw reagent overnight.
- Replace media in each well with 100 μL pre-warmed DMEM/F12.
- Add 100 µL CellTiter-Glo® 3D reagent to each well.
- Incubate at room temperature (RT; 15 - 25°C) for 10 minutes.
- Mix each well vigorously with a pipettor (or a multichannel pipette) to resuspend the Matrigel® dome.
NOTE: Ensure that domes are completely suspended by inspecting each well visually. If complete or partial domes remain, repeat suspension.
- Transfer suspensions to an opaque white assay plate.
- Incubate at RT for 30 minutes.
- Measure luminescence as specified in the CellTiter-Glo® 3D technical manual.
Additional Resources
References
- Vlachogiannis G et al. (2018) Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science. 359(6378): 920–26.
- Lynch S et al. (2019) Stem cell models as an in vitro model for predictive toxicology. Biochem J. 476(7): 1149-1158
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