The historical roots of the field stretch back further than many researchers realize. As the tutorial recounts, the earliest experiments in tissue self-organization date to 1907, when Wilson demonstrated that dissociated sponge cells could reassemble into functional organisms. Decades later, work by Holtfreter, Weiss and Taylor established the concept of tissue affinity and the remarkable capacity of embryonic cells to reconstitute organized structures from single-cell suspensions. Steinberg’s differential adhesion hypothesis in 1970 provided a theoretical framework for why cells sort themselves into coherent tissues. These classical observations laid the conceptual groundwork for the modern organoid revolution, which arrived in earnest in 2008 and 2009 with two landmark achievements: Eiraku and colleagues’ self-organized formation of polarized cortical tissue from embryonic stem cells, and Sato and colleagues’ demonstration that single Lgr5-positive intestinal stem cells could build complete crypt-villus structures in vitro without any mesenchymal niche.

Those two founding systems, intestinal and brain organoids, form the backbone of the new tutorial, and the authors deliberately chose them because they represent opposite ends of the organoid spectrum. Adult stem cell-derived intestinal organoids, sometimes called miniguts, are genetically stable, expandable over years and remarkably faithful to the epithelium they came from. Human pluripotent stem cell-derived brain organoids, by contrast, are developmental models that recapitulate embryonic neurogenesis, offering access to human brain biology that no other experimental system can provide. By walking through both, the tutorial illustrates how organoid choice depends entirely on the biological question at hand, whether that question concerns tissue homeostasis in the adult or the choreography of human development.

One of the most technically rich sections of the tutorial addresses cell fate decisions, the process by which stem cells commit to specific differentiated identities. In the intestine, regional and local signaling gradients, particularly the Wnt, BMP and Notch pathways, control where and when cells become absorptive enterocytes, mucus-secreting goblet cells, hormone-producing enteroendocrine cells, or tuft cells. The tutorial highlights how organoids allow researchers to manipulate these gradients with unprecedented precision. Experiments have shown that enteroendocrine cells switch their hormone expression profiles along the crypt-to-villus BMP signaling gradient, and that induced quiescence of Lgr5-positive stem cells enables the differentiation of hormone-producing cells. In brain organoids, timed and combinatorial treatments with extrinsic signals can specify ventral telencephalic identities, while minimizing exogenous signals drives rostral hypothalamic differentiation, demonstrating that the same self-organizing logic governs tissues as different as gut and brain.

The tutorial also devotes substantial attention to decrypting cell-cell communication, one of the most challenging problems in tissue biology. Organoids provide a contained system in which signaling networks can be measured at single-cell resolution using multiplexed single-cell analysis, and in which genetic reporters can mark specific populations in living tissue. Fluorescent gene tagging without double-strand DNA cleavage, ASCL2-responsive minigenes that label stem cell activity, and biosensors that quantify single-cell ERK dynamics have all been deployed in organoid systems. Machine learning tools such as OrganoidTracker now allow researchers to follow cell fate dynamics in space and time across entire organoids, converting what was once a static snapshot technology into a live-imaging platform capable of resolving the oscillatory signaling events that control cell fate decisions during intestinal homeostasis.

Genetic disease modeling represents perhaps the most clinically resonant application, and the tutorial lays out the strategies in detail. CRISPR-Cas9 engineering of organoids has enabled researchers to introduce precise mutations into otherwise healthy tissue, generating isogenic disease models. Sequential introduction of cancer mutations in cultured human intestinal stem cells has recapitulated colorectal cancer progression, while CRISPR-mediated engineering of patient-derived organoids has allowed systematic dissection of tumor evolution. On the repair side, functional correction of the CFTR gene by CRISPR in intestinal stem cell organoids from cystic fibrosis patients demonstrated that gene editing could restore disease-relevant function. The forskolin-induced swelling assay, which measures CFTR channel activity in patient-derived rectal organoids, has matured into a validated diagnostic tool, and high-throughput versions of the assay now support drug repurposing for cystic fibrosis.

Infectious disease research has been transformed by organoids as well. The tutorial documents how human intestinal organoids supported the first productive infection of human gut enterocytes by SARS-CoV-2, and how brain organoids became the central platform for understanding Zika virus-induced microcephaly. Multiple groups showed that Zika depletes neural progenitors through activation of the innate immune receptor TLR3, and organoid-based drug screens identified compounds that combat infection. Microinjection platforms and microfluidic gut-on-chip systems now allow controlled exposure of organoids to defined microbes, including complex anaerobic communities, opening the door to systematic study of host-microbiome interactions that were previously inaccessible in vitro.

The tutorial’s treatment of brain organoids extends into territory that borders on the philosophical. Comparative studies using organoids from human, chimpanzee and macaque cells have identified species-specific differences in progenitor behavior that contribute to brain size, and human-specific genes such as ARHGAP11B, NOTCH2NL and SRGAP2 have been shown to expand cortical neurogenesis when tested in organoid systems. Assembloids, fused organoids representing different brain regions, now model interneuron migration, thalamocortical connectivity and even the ascending sensory pathway. Single-cell brain organoid screening has revealed developmental defects in autism, and patient-derived organoids have exposed aberrant neuronal development in schizophrenia and copy number variant disorders. Chimeric models, in which human organoids are transplanted into mouse brains, have produced vascularized, innervated human tissue that establishes subcortical projections in the host animal.

The authors are candid about the field’s remaining challenges. Organoids lack vasculature, immune cells and mechanical cues from their native environment, limiting their maturation and long-term viability. Batch-to-batch variability, particularly in brain organoids, complicates reproducibility, and stress responses within cortical organoids can impair molecular subtype specification. Matrigel, the animal-derived matrix on which most organoids are grown, remains poorly defined, spurring development of synthetic alternatives and engineered materials whose stiffness can itself direct stem cell behavior. The tutorial highlights emerging innovations, including optogenetic control of gene expression, scaffold-guided morphogenesis that produces homeostatic mini-intestines, microfluidic vascularization, and bioprinting platforms that enable drug screening at single-organoid resolution, as the technologies most likely to close these gaps.

The translational trajectory of the field is already visible. Patient-derived organoid biobanks have predicted treatment responses in metastatic gastrointestinal cancers, tumor organoid and T-cell co-culture systems are being used to evaluate engineered immunotherapies, and glioblastoma organoids now serve as real-time avatars for assessing CAR-T cell responses. Organoid transplantation has repaired colitis in mouse models, alleviated traumatic brain injury and addressed short bowel syndrome in preclinical studies. What the Munich tutorial ultimately conveys is that organoids have crossed a threshold: they are no longer a promising curiosity but a mature experimental ecosystem, one whose protocols, pitfalls and possibilities are now documented well enough that any competent laboratory can begin interrogating biology with them.

Subject of Research: Organoid technologies for interrogating fundamental biology and disease

Article Title: Tutorial: interrogating biology using organoid technologies

Article References: Bugter, J. M., Schafer, S. T., & Rad, R. (2026). Tutorial: interrogating biology using organoid technologies. Nature Protocols. https://doi.org/10.1038/s41596-026-01447-6

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01447-6

Keywords: organoids, intestinal organoids, brain organoids, stem cells, CRISPR, disease modeling, self-organization, cell fate, host-microbe interactions, drug screening, assembloids, Nature Protocols