PDOs are current state-of-the-art models with significant advances, but important limitations remain. First, the layer of complex sugars (glycans) decorating the cells and mucus is not well understood, despite its essential role demonstrated on the interaction with microbiota and pathogens and its importance as a cancer biomarker and therapeutic target. Second, the cellular microenvironment is essential for maintaining a healthy epithelium, interacting with immune cells, and shaping how cancer develops and responds to therapy. However, incorporating key microenvironmental components, such as immune cells, fibroblasts and vasculature, into PDO cultures is still in its early stages.
Glycomics is an emerging field whose specialized analytical techniques are still being developed. Within TOP-GUT we used established growth factor cocktails to drive differentiation towards mucus-producing secretory cell lineages and analyzed the secreted mucus by cutting-edge mass spectrometry. We also adapted spatial proteomics to visualize the local immune cell composition along the GI tract. Together, these approaches begin to build a region-resolved glycan and immune cell atlas of the human GI tract in vitro, providing a basis for further mechanistic analysis in glycosylation, host defence and disease.
The cellular microenvironment is largely defined by the physical scaffold surrounding the cells (extracellular matrix), the neighbouring support cells (such as fibroblasts) and the presence of small blood vessels. In TOP-GUT, we have fine-tuned biological hydrogels to support healthy and cancer-associated fibroblast, the formation of (perfusable) vasculature, and the development of crypt-villi-like structures. PDOs and co-cultures have been incorporated into two commercially available OoC platforms and a third, new chip is being developed, that better captures the three-dimensional architecture of the intestinal wall. Overall, this work advances PDOs from simple epithelial spheres towards complex, vascularized and stromal-rich GI tissue models.
Besides the understanding and development of more advanced in vitro models, ethical and regulatory routes must be considered to enable their clinical use in personalized medicine. One TOP-GUT project specifically addresses European and national legislations, using the Norwegian Directorate of Health’s regulations on stem cell-based embryo models as a case study to show how conceptual definitions can shape legal boundaries. Similar conceptual discussions are emerging for organoids. However, unlike embryo models, GI PDOs are structurally and functionally more limited and not viewed as embryo-like or organism-level hybrid entities. At the same time, PDOs are a novel technology and terminology; definitions and specific guidelines for clinical purpose and research are still being developed. Through its legal and ethical work, TOP-GUT contributes to shaping this emerging regulatory framework.
