Organoid validation

Organoids are three-dimensional (3D) cell clusters derived entirely from primary tissues, embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs). They can faithfully recapitulate the architecture and function of native organs in vitro. Organoids possess the capacity for self-renewal and self-organization, and exhibit organ-specific functions that closely resemble those of their tissues of origin.

The concept of organoids can be traced back to 1907, when Wilson and colleagues reported that dissociated sponge cells were capable of reaggregating and regenerating an entire organism. In 1987, researchers demonstrated that cultured mammary epithelial cells could differentiate into the characteristic ductal structures in vitro^[1]. Thirty years later, organoid technology was recognized as the "Method of the Year 2017" by Nature Methods. Since then, organoid models representing a wide range of organs and diseases have been successfully established and widely adopted, significantly accelerating advances in basic research, drug discovery, precision medicine, and numerous other biomedical applications.

The establishment of organoid models primarily relies on two categories of tools: organoid culture tools and organoid validation tools.

Organoid culture tools include recombinant proteins, small-molecule inhibitors, extracellular matrix hydrogels (such as Matrigel), and specialized culture media. Directed differentiation is achieved by supplementing cultures with specific recombinant proteins or small-molecule inhibitors, while extracellular matrices and culture media provide the essential biochemical and structural support required for organoid growth, as well as microenvironments that mimic those of different native tissues.

Organoid validation tools primarily include antibodies, genomic analysis, transcriptomic/epigenomic sequencing, and other molecular characterization techniques. To verify that cultured organoids faithfully retain the genetic characteristics of their parental tissues, genomic and transcriptomic analyses are employed to assess their molecular profiles. In addition, antibody-based assays are used to detect the expression of key biomarkers within organoids, thereby confirming that they preserve the histopathological features and cellular identity of the original tissue.

Organoid validation can be broadly categorized into three complementary dimensions:

Histopathological validation – including hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and related morphological analyses.

Transcriptional and epigenetic validation – including RNA sequencing, ATAC-seq, DNA methylation profiling, and other multi-omics approaches.

Functional biological validation – including both in vitro and in vivo functional assays to evaluate physiological and pathological responses.

Collectively, these validation strategies provide a comprehensive assessment of organoid fidelity. They confirm the morphological and phenotypic similarity between organoids and their corresponding native tissues, verify the expression of tissue-specific biomarkers, characterize cellular identity and composition, reveal the spatial organization of distinct cell populations, and evaluate molecular and functional homology with the original organ. Together, these approaches ensure that organoids faithfully recapitulate the structural, molecular, and functional characteristics of native tissues, supporting their reliability for disease modeling, drug discovery, and translational research.

Figure 1. Schematic overview of the integrated workflow for tumor organoid establishment, genetic engineering, and validation.^【2】