A Shared Framework for Gastrointestinal Organoid Research: It’s Time

Over 15 years ago, landmark studies identified key markers of intestinal stem cells and demonstrated their ability to generate self-organizing epithelial structures using defined niche factors.¹ This discovery opened new avenues in gastrointestinal (GI) research, enabling the growth of intestinal epithelial cultures from mouse and human tissues in 3D formats that retain key features of native epithelia. Nearly every species tested has since shown the capacity to form organoids, highlighting the adaptability of this system.^2,3 The field has rapidly expanded to include diverse stem cell sources—adult, fetal, embryonic, and induced pluripotent—and experimental platforms ranging from spheroids and monolayers to humanized in vivo models.4, 5, 6, 7 Yet with this expansion has come increasing complexity and variability.

As organoid models become central to GI research, inconsistent culture conditions, vague reporting standards, and a lack of benchmarking to in vivo metrics are increasingly impeding progress. This concern was echoed at the 2022 James W. Freston Single Topic Conference: Gastrointestinal Organoids and Engineered Organ Systems in Washington, D.C., and both the 2024 and 2025 FASEB GI Conferences in Banff Canada and Southbridge, Massachusetts, respectively. At each conference, panel-led discussions collectively involving hundreds of scientists reached consensus on the urgent need for some level of standardization. As the field evolves toward more complex co-culture systems that integrate immune, stromal, endothelial, and microbial components, the absence of reproducible protocols and reporting threatens to compound variability. Establishing shared standards and benchmarks now is essential to support reproducibility between studies and support reliable innovation as the technology advances.

Organoid culture systems rely on media formulations designed to balance stem cell maintenance with differentiation, a process highly sensitive to growth factor composition and source.⁸ Media ingredients vary widely across labs from ‘homemade’ conditioned media to commercial recombinant proteins, and extracellular matrices like Matrigel adding further lot-to-lot variability. These factors together lead to significant inter-lab variability and limit reproducibility, as emphasized in the 2018 editorial Taming the Wild West of Organoids.⁹ In addition to these technical variations, organoid cultures often lack systematic benchmarking to in vivo cell states. Although tools such as single-cell transcriptomics and reference datasets like the Human Cell Atlas10, 11, 12 now enable detailed comparisons, most studies do not fully leverage these resources. Variables such as media, matrices, cell sources, and bioengineered platforms are rarely tested against native tissues.^13,14 As a result, interpretability remains limited, particularly in disease modeling and drug screening, as highlighted in the 2022 point-counterpoint Organoid vs Mouse Model: Which is a Better Research Tool to Understand the Biologic Mechanisms of Intestinal Epithelium? and associated rebuttal.^15,16 Further complicating the landscape is the wide range of stem cell sources used to generate organoids, including fetal,¹⁷ neonatal,¹⁸ adult, and induced pluripotent stem cell (iPSC)-derived cells.¹⁹ There is also substantial variation in experimental platforms, from 3D spheroids¹ and 2D monolayers,⁷ to co-cultures,²⁰ microfluidics,²¹ and biomimetic systems,^7,22,23 each of which can shape experimental outcomes in distinct ways. Yet publications often omit key contextual information like nomenclature, passage number, media components, or platform configuration. As highlighted by opinion leaders, inconsistent terminology further complicates comparisons, resulting in a field rich in data but lacking coherence.^9,24

We must ultimately approach these challenges through a coordinated effort involving academic laboratories, journals and publishers, biotechnology and pharmaceutical partners, and funding agencies. Broad implementation will depend on community-wide collaboration and collective accountability across sectors. As a start, we embarked upon a community survey implemented through the American Gastroenterological Association’s social media channels and email listservs from June through September 2025. The survey included 99 respondents with fully completed surveys, with considerably more partial responses, across various career stages, with broad geographic distribution across North America, Europe, Asia, and Australia, though with strongest representation from major United States academic institutions (Figure 1). Roughly 90% of respondents reported that they routinely use (69.6%) or sometimes use (20.5%) organoids in their work. Survey results identified broad themes of high importance to the GI organoid research community, including reporting standards, benchmarking, and shared reference lines/cross-lab replication. We therefore structured our forthcoming Guiding Principles: Standardization of Gastrointestinal Organoid Cultures into a 3-part series as outlined below:

• (1)Guiding Principles: Reporting elements for gastrointestinal organoid research. Grants and publications should include detailed descriptions of media composition (including concentration and source of growth factors), matrix type, stem cell source, passage number, and platform configuration in addition to other study-specific details. These metadata should be treated with the same rigor as sequencing protocols or imaging methods. Journals, journal editors, and reviewers have a key role to play in developing and enforcing reporting standards that promote consistency across the field. Creating open-access templates for reporting will allow easier comparisons across studies and reduce misinterpretations that stem from insufficient methodological detail. Of note, some survey respondents cautioned against prescriptive or rigid reporting standards that could delay or prevent the publication of novel methodologies or findings. We will therefore include consideration of unique circumstances in addition to general guidelines across the field in the “Reporting Elements” article.
• (2)Guiding Principles: Current practices and considerations for benchmarking gastrointestinal organoids. Single-cell RNA sequencing now offers the opportunity to systematically benchmark cultured cells against in vivo references, including atlases of healthy and diseased human tissue. Standardized benchmarking panels—akin to positive and negative controls—should be developed and made publicly available. Where feasible, multi-omic and functional profiling (including proteomics, barrier assays, and morphogenesis metrics) should be used to validate fidelity to native tissue. These metrics will help clarify how media, substrates, and stem cell sources influence cellular identity and function. Some survey respondents expressed concerns about mandating cost- and time-prohibitive benchmarking metrics that will increase barriers for new organoid researchers. As such, the “Benchmarking Metrics” article will distinguish between guidance for resource-intensive research and individual lab research benchmarking practices.
• (3)Guiding Principles: Promoting shared reference lines and cross-lab replication in gastrointestinal organoid research. To minimize variability, the field should adopt a small number of well-characterized reference lines derived from adult, fetal, and iPSC sources. These lines should be made broadly available and used alongside experimental models for benchmarking. Journals and funding bodies should support mechanisms for cross-lab validation of key findings, particularly in preclinical studies that aim to inform therapeutic development. Although some survey respondents already share lines openly, others note regulatory and ethical issues that may prevent widespread sharing of human-derived lines. These issues will be discussed in the “Shared Reference Lines and Cross-lab Replication” article.

Figure 1.

GI organoid survey results. (A) Geographic distribution of survey respondents (n = 99). Circle size and color intensity represent the number of respondents per institution using a colorblind-friendly palette. The majority of participants were based in the United States (n = 87) and Canada (n = 2). The main map shows North America; insets show Europe (n = 8; Germany, United Kingdom, Ireland, Spain, Portugal, Denmark, Belgium, and France), Japan (n = 1), and Australia (n = 1). Institutions with ≥4 respondents are shown in dark blue; 3 respondents in blue; 2 respondents in teal; and 1 respondent in yellow-green. (B) Career stage distribution of survey respondents. The survey respondents were predominantly faculty members, with assistant professors (27.45%) and professors (26.47%) comprising over one-half of participants, followed by associate professors (15.69%), whereas trainees, including students (10.78%) and postdoctoral fellows (9.80%), represented about one-fifth of respondents. “Other” (4; 4.0%) included a research assistant professor, a principal investigator, and associate researcher, and a retired adjunct associate professor.

As GI organoid use expands into new applications and therapeutic development, the need for clear standards is increasingly urgent. Although other groups have published comprehensive guidelines spanning organoid studies across tissues including the GI tract,25, 26, 27, 28 our community survey highlights important GI-specific gaps that will be addressed in the forthcoming Guiding Principles series. Addressing these gaps through coordinated community efforts, expert-driven guidelines, and targeted support from funding agencies are essential for organoid-based discoveries. Establishing consensus now will build a stronger foundation for future innovation and translational impact—it’s time.