Organoid screening in drug discovery

Why organoids are used in screening

Conventional two-dimensional cell culture is inexpensive and simple to run, but a monolayer of cells does not reproduce the architecture of a tissue, the arrangement of cell types within it, or the polarity and lumen formation that shape how a compound reaches its target.

Three-dimensional organoids grown from stem cells self-organize into structures that carry several of the cell types and some of the architecture of the tissue they derive from. Because they can be established from human cells, they allow a compound to be tested against human tissue in culture rather than against a rodent surrogate. What they produce is a measurement in that culture system. It is evidence that informs a decision; it is not a prediction of what a compound will do in patients, and no in vitro system on its own establishes clinical efficacy or safety.

Automated and high-throughput workflows

What an automated workflow involves

Organoid culture is labor-intensive and sensitive to handling, which is why screening at any scale is normally automated. An integrated system typically combines an incubator that can be accessed by a robot, liquid handling for media exchange and compound addition, a high-content imaging instrument, and scheduling software that moves plates between them. The point of the automation is consistency: an organoid culture that is fed and imaged on an irregular schedule produces variability that is difficult to separate from a compound effect.

Image analysis

Automated image analysis, including machine learning approaches, is used to track organoid shape, size and viability over time from brightfield and fluorescence images. The advantage over a single endpoint measurement is that the same wells can be followed across a time course, so a compound that slows growth can be distinguished from one that kills. What is being quantified is the response of the culture, not a treatment effect in a patient.

Working at scale

Moving from 96-well to 384-well plates is what makes a screen of any size feasible, and high-throughput screening platforms are used to run apoptosis, cytotoxicity and proliferation readouts across large compound sets. Organoids complicate this: they are embedded in a gel matrix, they vary in size within a well, and they take days to weeks to establish. A realistic organoid screen is usually smaller than a conventional cell-line screen, and the design question is which compounds are worth putting into the more informative but slower system.

A documented example: a compound identified by organoid screening

Intestinal inflammation

A phenotypic screen in human intestinal organoids identified glycyrrhizin as a compound that protects against cell death induced by tumor necrosis factor. The reported mechanism is inhibition of downstream caspase-8 signaling, which is required for that form of apoptosis, and the same compound reduced intestinal inflammation in a mouse model of colitis induced by dextran sulfate sodium.^[S4] The published account describes recovery of body weight in the mouse model as a tendency rather than a demonstrated difference, and this article follows that wording.^[S4] The example is useful because it shows the sequence that makes organoid screening credible: a hit found in human cells, a mechanism identified, and a confirmation in an animal model before any claim about disease is made.

What to establish before placing an organoid screen

• {'items': ['The source of the organoids. Whether they are derived from induced pluripotent stem cells, from adult tissue, or from patient tumors changes what the result means and what consent and provenance documentation exists for the material.', 'The matrix and the medium. Organoid growth depends on the extracellular matrix preparation and the growth factor composition, and both vary between lots. Ask how lot variation is controlled and whether the same lot will cover the whole screen.', 'The readout and how a hit is defined. Viability by adenosine triphosphate measurement, image-based growth rate, and marker expression can disagree with each other. Establish the primary endpoint before the screen, not after.', 'The controls. A cytotoxic compound and a vehicle control on every plate are the minimum; without them plate-to-plate drift cannot be separated from compound effect.', 'What happens to the data. Image-based screens generate large files. Agree who holds them, in what format, and for how long, since a regulatory question years later may require going back to the raw images.']}

Imaging organoids in a gel matrix

Airway organoids grown from human lung epithelial cells in a growth-factor-containing matrix illustrate the imaging problem common to all organoid work. The cultures are followed in brightfield while they grow, then stained and imaged by automated confocal microscopy through the gel, since the organoids cannot be removed from it without disrupting them. Analysis reconstructs the three-dimensional object and measures cell morphology, viability and differentiation markers. The depth of the gel, and the fact that organoids settle at different heights within it, are the practical limits on how much of a plate can be imaged in a given time.

Establishing and reading out an organoid culture

Establishing the culture

Human induced pluripotent stem cells are directed down a lineage and then allowed to self-organize. Material taken from patient tissue or epithelium is dissociated, treated enzymatically, and cultured in medium containing the relevant growth factors. In both cases the cells are embedded cold in an extracellular matrix preparation and the gel is set at 37 degrees Celsius, which supports the three-dimensional structure and allows the polarity and lumen formation seen in the tissue of origin to develop.

Endpoints

Viability is commonly measured by adenosine triphosphate quantification, and image analysis is used alongside it to follow growth rate and change in size over the course of an experiment. Combining the two matters because they answer different questions: a compound that stops organoids growing and a compound that kills them can produce a similar single-timepoint viability reading, and only the time course separates them. Growth-rate metrics normalized to a control are used for this purpose, and any such metric should be defined in the protocol, since the normalization determines what the number means.

Where organoids are heading

Patient-derived organoids and personalized approaches

Organoids derived from an individual patient tumor carry that tumor genetic alterations, and several groups are investigating whether the response of such a culture to a panel of agents corresponds to the response of the patient. This is an active research question rather than an established capability, and it sits outside the nonclinical work this site covers. For a drug developer, the near-term value of patient-derived organoids is different: a panel of them spanning several genetic backgrounds gives an early read on whether a compound effect is confined to particular tumor genotypes.

Organoids and the reduction of animal use

Organoids are one of the approaches used in efforts to replace, reduce and refine animal use, the framework known as the three Rs. In the United States that work is coordinated federally through the interagency center for alternative toxicological methods at the National Institute of Environmental Health Sciences, which develops and evaluates alternatives to animal use for safety testing.^[S3] The Food and Drug Administration describes new approach methodologies as innovative testing methods used to assess the safety, efficacy and quality of regulated products, and consistently frames its programme as reducing, replacing or refining animal testing rather than ending it.^[S2] Separately from these method questions, animal work in the United States is governed by registration with the Department of Agriculture under the Animal Welfare Act and, for federally funded research, an assurance filed with the Office of Laboratory Animal Welfare; accreditation by AAALAC International is a voluntary programme reassessed every three years and is not a regulatory requirement.^[S6][S7][S8]

A common misreading of the 2022 law

The provision often called the FDA Modernization Act 2.0 is section 3209 of Public Law 117-328, signed on December 29, 2022.^[S1] It replaced the phrase "preclinical tests (including tests on animals)" in the Federal Food, Drug, and Cosmetic Act with "nonclinical tests," and it defines a nonclinical test as one that may include cell-based assays, organ chips and microphysiological systems, computer modeling, other nonhuman or human biology-based methods, and animal tests. Animal testing is listed in that definition, not removed from it. The law did not make animal studies unnecessary, and it did not oblige the agency to accept any particular alternative method for any particular purpose.

Outsourcing organoid work

Organoid culture is a specialized skill, and establishing it internally takes months of work that is separate from the programme it is meant to support. Placing the screen with a contract laboratory that already runs these cultures is a reasonable alternative, and it lets a sponsor treat organoid data as one input into the decision about which compounds go into animal studies. It does not remove the need for those studies, and the points listed earlier in this article are the ones to settle in the statement of work before the screen begins.

About this article

This is an independent editorial article for people who commission nonclinical work in the United States. It describes methods, not products, and it does not recommend any laboratory or supplier. Statements attributed to a published study or a government source carry that source with the date it was accessed. Results in organoid cultures inform decisions in drug development; they do not establish that a compound is effective or safe in people. Last reviewed: September 3, 2026.

References

1. Public Law 117-328 (Consolidated Appropriations Act, 2023), Division FF, Title III, Sec. 3209 — Animal Testing Alternatives, amending section 505(i) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 355(i)); approved December 29, 2022. https://www.govinfo.gov/content/pkg/PLAW-117publ328/html/PLAW-117publ328.htm (accessed 2026-09-03)
2. FDA — New Approach Methodologies (NAMs), Science and Research Special Topics. https://www.fda.gov/science-research/science-and-research-special-topics/new-approach-methodologies-nams (accessed 2026-09-03)
3. NICEATM — NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NIEHS/NIH). https://ntp.niehs.nih.gov/whatwestudy/niceatm (accessed 2026-09-03)
4. Takahashi et al. Organoid phenotypic screening identified glycyrrhizin that confers protection against tumor necrosis factor-induced cell death. Stem Cell Reports. 2026;21(5):102891 (PMID 41997150). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13163214/ (accessed 2026-09-03)
5. AAALAC International — FAQs. https://www.aaalac.org/accreditation-program/faqs/ (accessed 2026-09-03)
6. USDA APHIS — Animal Welfare Act: research facilities must obtain a registration; Animal Care inspections. https://www.aphis.usda.gov/awa/public-search (accessed 2026-09-03)
7. Public Health Service Policy on Humane Care and Use of Laboratory Animals (Animal Welfare Assurance submitted to NIH OLAW). https://grants.nih.gov/policy-and-compliance/policy-topics/animal-welfare/laws-regulations/phs-policy (accessed 2026-09-03)