Retour sur le colloque OuestOÏD 2026
Une 2ème édition riche en interactions
Le 1er octobre 2026, le groupe de travail OuestOÏD a organisé une journée dédiée aux organoïdes et autres modèles cellulaires 3D à Nantes, avec le soutien de Biogenouest.
L’occasion pour les membres du réseau OuestOÏD et tous les acteurs de ce domaine de se retrouver autour de modèles cellulaires variés.
Bénédicte Martin, animatrice du réseau, a ouvert le colloque OuestOÏD 2026 au nom du Groupe de travail, en rappelant les objectifs et missions du réseau et son positionnement au sein du GIS Biogenouest.
Les conférences
Enjeux éthiques des modèles cellulaires avancés
par Maxence Gaillard
Section de bioéthique et d’éthique des sciences, UNESCO, Nantes
Nous présentons quelques questions de bioéthique soulevées par les nouveaux modèles cellulaires (notamment les organoïdes). Si la collecte de cellules et de tissu humain suit des procédures généralement bien établies, le développement de biotechnologies de plus en plus complexes pousse à ses limites les modèles de consentement et les mécanismes d’autorisation en vigueur. Dans certains cas, c’est la nature même des nouveaux objets que l’on peut produire en laboratoire qui bouleverse les catégories sur lesquelles reposent nos procédures institutionnelles, mais aussi nos jugements moraux. Les normes réglementaires qui régissent la recherche doivent être mises à jour à l’aune des données de la science, mais aussi en prenant en considération les différents niveaux d’incertitude – sur ce que sont ces objets et ce que l’on peut en savoir, ou quant à leur potentiel statut moral.
Reconstruction du destin cellulaire de la notochorde humaine : intégration multi-omique pour modéliser développement & pathologies
par Anne Camus
Center for Research in Transplantation and Translational Immunology (CR2TI), Nantes
Intervertebral disc degeneration is a leading cause of low back pain, a widespread musculoskeletal condition, and is strongly associated with the disruption of the nucleus pulposus (NP) tissue homeostasis within the heart of the disc. There is currently no effective treatment for disc degeneration. The NP originates from the notochord, an embryonic mesodermal structure that progressively differentiates and adapts to the hypoxic, matrix-rich environment of the developing intervertebral disc [1, 2, 3]. In addition, notochordal cells critically regulate postnatal disc integrity [4]. Bridging basic knowledge gaps in human development is particularly essential concerning the molecular and cellular controls of notochordal cell specification, maturation and viability during embryonic disc formation and beyond. Recapitulating, the developmental path from notochordal progenitors to notochord-derived NP cells in vitro remains challenging [5, 6]. Most differentiation protocols bypass early specification steps and thus fail to fully capture this understudied lineage.
Using a three-dimensional (3D) organoid culture system, we have reconstructed the developmental process from human pluripotent stem cells (hiPSCs) through mesendoderm progenitor cells toward notochord-like cells and NP-like cells, using single-cell transcriptomics and spatial profiling.
Single-cell transcriptomic analysis identifies hiPSCs-derived mesendoderm progenitor cells as a distinct population with a unique gene signature and conserved regulatory networks shared with human embryonic organizer cells. Self-organization in 3D aggregates led to spontaneous endogenous NOTO expression, a key transcription factor that orchestrates the developmental regulatory networks underlying notochordal identity, and promoted their commitment to notochordal fate. However, physical constraints alone were insufficient for full maturation. In contrast, long-term culture sustained by TGF-β1 and GDF-5 signaling [7] triggered robust matrix deposition, along with the acquisition of transcriptomic and proteomic hallmarks of native NP cells and the presence of prominent intracytoplasmic vacuoles by day 28, as characterized by lysosome-related LAMP1 immunostaining. The coexistence of two major cell types found in the native juvenile human disc, i.e. notochord-like cells and NP-like cells, was observed within matured organoids indicating that maturation is progressing toward terminal differentiation.
Collectively, these findings reconstruct a sequential and physiologically relevant differentiation path in a 3D system, tracing the progression from progenitors to notochord-derived NP-like cells. This scalable platform enables the investigation of fundamental mechanisms underlying human disc development and degeneration, while providing a framework to explore the rejuvenating potential of notochord-like cells for disc regeneration.
REFERENCES:
[1] Warin et al., Dev. Dyn., 2026; [2] Paillat et al., Eur Cell Mater., 2023; [3] Reviewed in Warin et al., Development, 2026; [4] Reviewed in Bach et al., Front. Cell Dev. Biol., 2022 ; [5] Colombier et al., Cells, 2020 ; [6] Warin*, Vedrenne* et al., iScience, 2024 ; [6] Colombier et al., Stem Cells, 2016.
Les communications orales
9 communications orales ont été proposées pendant le colloque, portant sur des modèles variés, aussi bien humains qu’animaux.
Olivier Goureau
Institut de la Vision, Paris
Retinal diseases are leading causes of vision loss in developed countries. Degeneration of photoreceptors or the supporting retinal pigment epithelium (RPE) are the major causes of inherited retinal dystrophies, age-related macular degeneration or diabetic retinopathy. The clinical and genetic diversity of these pathologies hampers the development of effective therapies. Despite their value for studying disease mechanisms, animal models often fail to reflect key aspects of human retinal pathophysiology, underlying the need for human models that accurately replicate disease processes.
In this context, retinal organoids derived from pluripotent stem cells represent a powerful culture system developed in the last decade that holds great promise for developmental studies, disease modelling and regenerative medicine. These approaches recapitulate the main steps of retinal development to form laminated 3D retinal organoids containing rod and cone photoreceptors with outer segment-like structures, offering a methodology for studying 3D retina morphogenesis and allowing for reliable in vitro modeling of some retinal degenerative diseases such as photoreceptor-related dystrophies.
Despite significant progress, retinal organoid organization and functionality differ from the adult retina, which is expected given the disparity between in-vitro and in-vivo environments. Retinal organoids lack integration of RPE and a vascular network, limiting nutrient supply and long-term cell viability mainly in the core region of organoids, which transiently contains retinal ganglion cells.. New strategies are currently being developed to generate more complex retinal organoids by incorporating retinal pigment epithelium (RPE) capable of interacting with photoreceptors, as well as iPSC-derived vascular-like structures. Furthermore, assembloïd technology is being developed to generate retino-thalamic circuits that promote retinal ganglion cell survival, thereby providing more physiologically relevant models for studying optic neuropathies such as glaucoma.
David Reboutier
IGDR, Rennes
In association with the cornea, the lens contributes to the formation of sharp images on the retina. Lens opacification, or cataract, is the leading cause of blindness worldwide. Currently, the most effective treatment for cataract is the surgical removal of the opacified lens, followed by its replacement with an artificial intraocular lens. Although highly effective, this approach is costly and can be associated with post-operative complications.
To identify alternative treatments that could prevent or even reverse cataract formation, it is essential to develop relevant organoid models. Our work shows that culturing mouse lens epithelial cells under defined three-dimensional culture conditions enables the rapid generation of large numbers of organoids. Interestingly, these organoids display remarkable optical properties and recapitulate many aspects of healthy and pathological lens organization at the tissue, cellular, and transcriptomic levels.
The aim of this presentation is to describe how we characterized this novel model, which appears to be a particularly promising tool for studying lens physiology and pathology and for identifying new therapeutic approaches to cataract.
Fabienne Archer
OrganoZOO, Lyon
Dans le cadre du PEPR Elevage Durable (ED) piloté par INRAE, une nouvelle plateforme nationale d’organoïdes issus d’animaux d’intérêt en agriculture et faune sauvage vient d’être créée. Baptisée OrganoZOO, cette plateforme a pour premier objectif de mettre à disposition des modèles aboutis, issus d’un réseau de laboratoires maîtrisant, développant et distribuant des modèles complexes de culture in vitro (2D, 3D, Organoïdes). Le second objectif sera de contribuer à la complexification des modèles existants (intégration cellules immunes, innervation, modèle fluidique) et au développement de nouveaux modèles (autres organes, autres espèces). Ces développements seront réalisés soit en lien avec les projets spécifiques du PEPR ED, soit en réponse à des demandes extérieures (collaborations ou prestations de services). La plateforme contribuera par ailleurs à la diffusion de connaissances (tutorats, formations). Cette nouvelle plateforme s’appuie sur le CRBanim, pilier animal de l’Infrastrusture RARe et s’inscrit dans le réseau national des plateformes Organoides RIBBON.
Pierre Layrolle
TONIC, Toulouse
Despite the occurrence of many skeletal pathologies, in vitro 3D bone models are still not available for testing drug treatments. Bone has a complex and dynamic structure composed of a mineralized extracellular matrix, where osteoblasts, osteocytes and multinucleated osteoclasts interact in a coordinated manner to regulate bone formation, maintenance, and resorption. Bone tissue is also vascularized by peculiar H-type vessels that couple angiogenesis and osteogenesis. Reproducing the dynamic interplay between bone-forming osteoblasts, bone-resorbing osteoclasts, and vascular networks remains one of the biggest challenges in bone tissue engineering. Current in vitro bone constructs fail to capture this complexity, resulting in limited translational potential. Here, we report the development of a human bone tissue that exhibits bone formation, resorption, and vascular network formation by self-assembling primary cells and biphasic calcium phosphate (BCP) particles and its integration into a microfluidic chip. We have previously demonstrated that human bone marrow derived mesenchymal stromal cells (hBMSC) seeded and cultured on BCP spontaneously differentiated into mature osteoblasts and produce an abundant extracellular matrix forming 3D osteoid constructs [1,2]. This model was further enriched with endothelial cells that formed an early network of H-type vessels. Peripheral blood-derived monocytes (PBMC) were also introduced to form multinucleated osteoclasts coupling bone formation and resorption. Cell densities, ratio, sequences and miniaturization were continuously optimized allowing their integration into microfluidic chips for 3D physiological perfusion cultures. This strategy of assembling human primary cells with BCP particles successfully generated an in vitro bone organ that recapitulates early vascular organization and remodeling opening the possibility of studying physiopathology and regenerative therapies.
Eléa Boucard
MOBIDIC, Rennes
Follicular lymphoma is an indolent B-cell neoplasm that is highly dependent on its microenvironment. To better understand the bidirectional interactions between tumor microenvironment and malignant B-cells, models are required. Yet, animal studies have proven difficult to implement to fully replicate and understand these interactions. In contrast, two-dimensional in vitro models are easier to develop but do not recapitulate physical cues and 3D architecture found in tissues.
To overcome the limitations of two-dimensional cell culture, we have developed an innovative high throughput 3D culture approach based on cell encapsulation to study stroma-B-cell interactions and replicate the tumoral niche. Alginate was used to encapsulate lymphoma B-cells in a core-shell system using a microfluidic device. Our system enabled the high-speed generation of monodisperse microcapsules (diameter of 213.3±8.8µm, CV 4.2%) and displaying high viability of a lymphoma cell line over 7 days (72.5%). Additionnally, the culture platform has demonstrated compatibility with several cell types. Finally, permeability studies showed that the shell is permeable to small molecules and does not impede cellular needs.
In conclusion, this culture platform combines rapid and robust microcapsules generation with high versatility. It is suitable with a wide range of cell culture media and cell lines with a tunable alginate shell.
Grégory Menchon
NorDiC, Rouen
Le corticosurrénalome (CCS) est une tumeur maligne rare et agressive dont le pronostic reste souvent sombre. Pendant longtemps, la recherche préclinique s’est appuyée sur des cultures cellulaires en 2D (monocouches). Celles-ci manquent cruellement de pertinence biologique, car elles ne reproduisent ni la structure tridimensionnelle ni la complexité du microenvironnement tumoral. Ces dernières années, des progrès notables ont permis de développer des modèles 3D (tels que des sphéroïdes ou des matrices extracellulaires enrichies en collagène de type I). Ces structures in vitro restaurent des profils de sécrétion hormonale plus fidèles à la réalité physiologique et montrent une résistance accrue aux traitements médicamenteux (comme le mitotane), se rapprochant ainsi de la réalité clinique.
Malgré ces avancées, les modèles 3D actuels restent limités car ils se concentrent principalement sur les cellules tumorales seules. Or, la réponse aux traitements et la progression tumorale dépendent fortement du microenvironnement immunitaire. Dans ce but, nous tentons de concevoir des modèles 3D hétérotypiques, indispensables pour décrypter les mécanismes d’évasion immunitaire du CCS et identifier de nouvelles cibles thérapeutiques efficaces.
Par ailleurs, notre laboratoire s’appuie sur des technologies de pointe telles que la microscopie à feuille de lumière (light-sheet microscopy). Cet outil de choix permet d’explorer en profondeur la structure de nos modèles et de caractériser avec précision l’infiltration lymphocytaire. En combinant imagerie haute résolution et préservation tissulaire, cette approche met en lumière le potentiel de cette technologie pour décrypter les dynamiques cellulaires et les interactions au sein de microenvironnements complexes.
Christophe Blanquart
CRCI2NA, Nantes
BACKGROUND
Pleural mesothelioma (PM) is a rare and aggressive cancer mainly caused by asbestos exposure. Although dual immune checkpoint blockade (anti-PD-1/anti-CTLA-4) is approved as first-line therapy, its clinical benefit remains limited. We previously demonstrated that the attenuated Schwarz strain of measles virus (MV) exhibits oncolytic activity against PM cells. However, preclinical evaluation is hindered by the non-permissiveness of rodent models to MV. To address this limitation, we developed a humanized 3D “vascularized mesothelioma-on-chip” (VMOC) model using microfluidic technology.
METHODS
Commercial IdenTX microfluidic chips (AIM Biotech) were used. The model consists of two perfusable endothelial-lined vessels flanking a central microvascular network (MVN), generated using human umbilical vein endothelial cells (HUVECs) embedded in fibrin and co-cultured with PM cells and primary human lung fibroblasts (hLFs). The model and MV oncolytic activity were characterized by confocal microscopy, multiplex ELISA, single-cell RNA sequencing, 3’ RNA sequencing, and luminescent assays.
RESULTS
We confirmed the presence of adherens and tight junctions within the endothelial compartment. Injection of 70 kDa FITC-dextran demonstrated functional connectivity between parental vessels and the MVN. Distinct cell subpopulations, resembling those observed in vivo, were identified across all cell types in the VMOC. Following MV administration through the endothelial network, PM cell infection and death were observed, along with strong activation of the type I interferon pathway and increased production of inflammatory mediators.
CONCLUSION
The VMOC model enables in vitro investigation of MV infection and tumor microenvironment reprogramming, providing a valuable platform to better understand treatment responses.
Dima Ghannoum
ICO, Angers
La prise en compte des facteurs physiques des tumeurs cancéreuses constitue une étape importante lors de la reproduction de tumeurs in vitro pour la médecine personnalisée. L’architecture tissulaire désigne la disposition tridimensionnelle des cellules et de leur matrice extracellulaire (MEC). Cette architecture est altérée lors de l’émergence et de la progression d’un cancer, entraînant de nombreux changements aberrants des phénotypes cellulaires et de la distribution des éléments du microenvironnement. Les principales contraintes mécaniques en lien avec l’architecture d’un tissu cancéreux comprennent une pression solide tumorale élevée, un stress de cisaillement des fluides et une augmentation de la rigidité de la MEC.
Le cancer du sein, qui touche principalement les femmes (avec 1 % des cas chez les hommes) et a causé 694 000 décès dans le monde en 2024, est connu pour sa grande hétérogénéité et une augmentation de la rigidité de sa MEC qui est multipliée par un facteur de 2 à 10 par rapport au tissu normal. La rigidité de la matrice extracellulaire induit un processus de mécanotransduction qui régule la réponse du noyau cellulaire, incluant sa morphologie, l’expression génique et les modifications épigénétiques.
Une rigidité accrue peut constituer une barrière limitant la délivrance efficace des médicaments, et l’hétérogénéité de la MEC complique le traitement en induisant une hétérogénéité phénotypique intra- et inter-tumorale. Par conséquent, notre postulat est que connaître et reproduire l’architecture de chaque type de cancer du sein est indispensable pour créer des tumeurs mammaires personnalisées à partir de cellules de patients.
Je présenterai tout d’abord la phase d’étude de l’organisation des tumeurs de patientes scindée en fonction des compartiments de cellules cancéreuses et du microenvironnement tumoral, sur des coupes histologiques. En particulier, j’expliquerai l’importance de prendre en compte le lien entre l’hétérogénéité phénotypique, mesurée à l’aide de la morphométrie nucléaire, et l’hétérogénéité microenvironnementale. Dans un deuxième temps, je présenterai des méthodes de culture en 3D de cellules cancéreuses dérivées de patientes qui font varier la composition et la rigidité des MEC et les effets sur l’organisation des cellules et leur prolifération. Les résultats de ces études vont permettre de développer à court terme des tumeurs directement issues de cellules de patientes pour une évaluation rapide de la réponse à des thérapies existantes, et à moyen terme, des tumoroïdes mammaires pour l’évaluation préclinique de nouvelles molécules thérapeutiques.
Chloé Terciolo
INCIT, Nantes
Our team has identified a novel microbiota-induced Treg subset (DP8α) associated with good prognosis in Inflammatory bowel disease (IBD). The aim of this project is to the develop an original ex vivo human preclinical model to develop a novel cell-based immunotherapy approach to treat IBD using the regulatory properties of DP8α regulatory T cells (Treg). Our project proposes (1) to improve IBD modeling by establishing an autologous co-culture of Tregs cell and patient-derived intestinal organoids (PDIOs) (2) To characterize the morphological and functional phenotype of healthy and IBD organoids and to investigate the mechanisms of crosstalk between epithelial and immune cells.
For each sample, organoids and Tregs cells were established from surgical samples corresponding to non-inflamed colonic zone from IBD or non-IBD donor. We established several PDIOs and analyzed expression levels and/or localization (RT-qPCR and immunofluorescence) of various markers, such as epithelial cell types (MUC2, LYZ, CHGA), proliferation/apoptosis (KI-67, cleaved-caspase-3) and intercellular junctions (E-cadherin, ZO-1). The presence of various epithelial subtypes and intercellular junction markers in both IBD and non-IBD organoids were validated and attested the differentiation status of PDIOs. Treatment of the PDIOs with an inflammatory cytokines cocktail (TNF-/IL1-/IFN-) correlated with a dose dependent up-regulation of a key mediator of inflammation (IL-8), a down-regulation of epithelial markers and the loss of membranous ZO-1. Once the phenotype established, PDIOs was culture with Treg cells labeled by a cell tracer to analyze their interaction over time by time-lapse microscopy and by immunofluorescence.
This model faithfully recapitulates the physio pathological features of the mature intestinal barrier and the physiological complexity of the human gut, including dynamic interactions between the gut epithelial cells, the immune cells.
Les posters et présentations flash
Une session posters était proposée pendant le colloque, avec une présentation flash en 3 minutes des posters le matin. Les participant.es ont également eu l’occasion d’échanger avec les présentatrices des posters pendant les différentes pauses de la journée.
Neuf chercheures ont ainsi pu présenter leurs travaux : Emilie Navarro, Marion Desdouits, Julie Bourseguin, Pauline Maisonnasse, Mélanie Marquis, Morgane Biette, Caroline Vignes, Lila Moreau, et Lise Joliot.
Le prix poster, récompensant à la fois la qualité du poster, sa présentation, et la présentation flash a été attribué à :
Lila Moreau
de l’Institut de Génétique et Développement de Rennes.
Lila est étudiante en première année de doctorat dans l’équipe GeDiNe et a époustouflé le jury !
Bravo à elle pour ce beau travail de communication scientifique, récompensé par un chèque de 200 euros remis par la société Elveflow.
Retrouvez ici les résumés des posters :
Encapsulation process of muscle stem cells in alginate matrices to enhance their in vivo post-transplant properties
Identification of a stem cell population derived from human muscle (hMuStem) and the demonstration of its regenerative potential have led to new cell therapy proposals for muscle diseases [1]. However, the ability of these adult stem cells to survive and integrate into the host tissue is a limiting factor with regard to their overall therapeutic impact. Emergence of tissue biomimetic approaches based on encapsulating cells in biocompatible and biodegradable biomaterials offers new opportunities to overcome the limitations of cell therapy in terms of viability and potentiation of effects. Initial in vitro results have enabled us to validate the possibility of encapsulating hMuStem cells in alginate matrices without altering the biological properties associated with their commitment in the myogenic program [2]. It was shown that macroscopic hydrogels obtained by molding methods possess mechanical, structural and diffusion properties compatible with hMuStem cells. However, their large size limits their therapeutic applications to subcutaneous implants. For this reason, transposition of the encapsulation method to microfluidic approaches seems essential to achieve micrometric matrices more suitable with transplantation protocols [3]. To improve the efficiency of hMuStem cell delivery, we have developed a microfluidic chip enabling the efficient encapsulation of cells in micrometer-scale alginate matrices. In parallel, we assessed the impact of this encapsulation process on the morphology and viability of hMuStem cells.
Innovations en matière de modèles in vitro pour mieux étudier les virus respiratoires porcins : Vers une meilleure compréhension des interactions cellulaires
L’étude des interactions fondamentales entre cellules immunitaires et cellules de tissus lors d’infection virales demandent un perfectionnement des systèmes de culture in vitro. En effet, les modèles les plus souvent utilisés consistent en des lignées cellulaires immortalisées mises en contact avec un virus auquel elles sont susceptibles.
De même, face à la demande croissante des acteurs socio-économiques pour des produits immuno-actifs commercialisables, il est impératif de développer des méthodes de criblage in vitro robustes pour évaluer efficacement ces extraits avant d’entreprendre des essais in vivo coûteux.
Pour répondre à ces demandes, dans le cadre des infections respiratoires chez le porc, nous avons développé la culture de cellules primaires, et raffiné nos systèmes de culture en intégrant le dialogue
entre cellules épithéliales et cellules immunitaires. Nous avons mis en place 2 systèmes de culture que nous avons comparés : i) des cellules épithéliales primaires différenciées en interface air-liquide (ALI) qui conserve une barrière épithéliale permettant une compartimentalisation alvéole/interstitium et ii) des coupes vitales de tissus pulmonaires (PCLS) conservant l’architecture native du poumon. À ces 2 systèmes nous avons ajouté des Macrophages Alvéolaires autologues. Ces cocultures ont été challengées par des infections avec le virus du syndrome dysgénésique et respiratoire porcin (SDRPv),
infectant exclusivement les AM, et le coronavirus respiratoire porcin (PRCv) infectant exclusivement les cellules épithéliales. Les productions virales ainsi que les réponses cytokiniques précoces/innées ont été mesurées en RT-qPCR. Les cytokines produites seront aussi dosées en ELISA multiplex et comparées à la réponse induite in vivo dans les lavages bronchoalvéolaires d’animaux challengés par ces deux mêmes virus. Ces modèles pourront être appliqués aux infections grippales, ainsi qu’aux coïnfections grippe/coronavirus afin d’utiliser le porc comme modèle des réponses immunitaires respiratoires chez l’être humain (Bertho Mol Immunol 2021).
Spheroid and microfluidic models to explore the tumor microenvironment complexity in lung and malignant pleural mesothelioma
Introduction: Responsible for an estimated 1.8 million deaths per year, lung cancer is one of the leading causes of cancer incidence and mortality worldwide, with non-small-cell lung cancers (NSCLC) accounting for 80-85% of cases. Another aggressive thoracic cancer, namely pleural mesothelioma (PM), originates from the pleura, the double layered protective tissue that surrounds the lungs and is characterized by a low median survival rate. Over the past decade, immunotherapies have revolutionized patient outcome but only a small fraction of NSCLC and PM patients responds to treatments. The tumor microenvironment (TME) is an important parameter mediating such resistance, and new alternative in vitro models are required to test anti-tumor treatments.
Methods: We established two models to mimic the TME and their cellular interactions. A 3D multicellular complex spheroid (MCTS) model was achieved and encompass NSCLC cells, primary human lung fibroblasts (hLFs), primary endothelial cells (ECs), and monocyte-derived macrophages. A second model of vascularized mesothelioma-on-chip model (VMOC) was developed using microfluidic chip embedding PM cells, hLFs, primary ECs in a sophisticated TME. Those 3D models were characterized by combining multi-omics approaches including single-cell RNA sequencing (scRNA-seq), meta-analysis, imaging and various functional assays to dissect cell-cell interactions within the TME.
Results: We first showed that various NSCLC cell lines induced profound EC alterations at the transcriptomic, proteomic and kinomic levels with the induction of pro-inflammatory pathways that reflect their activation state.
We then documented that cell heterogeneity in 3D MCTS and VMOC was much more complex than 2D cultures, with several cell states identified. Particularly, several subclusters of MCTS-ECs were distinguished, among which an inflammatory subcluster that was absent from standard 2D culture but present in freshly-isolated ECs from NSCLC biopsies. The stromal compartment was also particularly heterogeneous, with a specific VMOC-fibroblast subcluster that may result from the EC differentiation via the EndMT. Finally, within MCTS macrophages show privileged cell-cell interaction with ECs and express markers of pro-tumor M2-like perivascular macrophages. All those cell states have been described to bear particular therapeutic importance in cancer.
Conclusion: We established various in vitro 3D models that allow to better delineate the precise function of cell state within the TME, and how they could modulate tumor immunity. In the future, those models will be relevant to mimic patient response to anti-cancer therapies.
Integrated Live Imaging and Automated Analysis Pipeline for Studying Dynamic Interactions Between Enteroid Organoids (eIO) and T Cells in 3D Matrigel – Horizon Europe MITI2 Project
Organoids derived from patient samples are an innovative model for the development of new therapeutics. These models offer an alternative to animal testing and better replicate human physiology than animal models or 2D cells culture. Human organoid models are increasingly being used for preclinical research in the field of immunotherapy.
The objective of the European MITI2 Project (Microbiota Induced T regs for Inflammatory Bowel Disease Immunotherapy) is to propose a new therapy against Crohn’s disease based on the adoptive transfer of regulatory T cells. To test the protective role of regulatory T cells, we used an innovative model of co-culture of enteroid organoids and regulatory T cells in 3D Matrigel. The goal of this work is to develop an integrated pipeline for acquiring and analyzing live images of these co-cultures, enabling the quantification of cellular interactions and apoptosis and suitable for high-throughput experimentation. Image acquisition using the Zeiss Cell Discoverer 7 automated microscope in wide-field microscopy includes co-cultures of eIO (brightfield) and T cells (Cell Tracker CM-DiL) with NucView (caspase-3 indicator) in Matrigel droplets. Parameters are optimized (X2.5 objective, short exposure/low intensity to minimize phototoxicity). Z-stack (500 μm range, 20 μm intervals) and time-lapse imaging are used for dynamic observation.
The automated analysis (Arivis) includes a deep learning-based instance segmentation model (trained on 200 annotated organoids in Arivis Cloud) for precise 3D eIO detection. A Blob Finder algorithm is used for T cell segmentation which offers a quick method for segmenting small round objects such as cells, using watershed-based object separation. High-throughput quantification of cellular interactions, distances, speeds, contact times, and apoptosis (NucView+ events) is performed.
Lanthanide-based near-infrared optical imaging agents associated with microglia for the study of neuroinflammation in 2D/3D models
Recent advances in the development of human cellular models of the central nervous system offer unique opportunities for the study of neuropathologies. These new tools enable the specific analysis of neurons and glial cells interaction in three-dimensional (3D) organoids [1]. However, the potential of this technology is limited by several major imaging constraints associated with conventional fluorophores: low resolution, limited photostability, emission in the visible range which is disturbed by autofluorescence and that induces strong light diffraction strongly reducing the resolution of 3D model images. The spectral overlap of the broad emission bands of organic fluorophores prevents multiplex measurements. We aim to overcome these constraints with new near-infrared (NIR) luminescent imaging agents to access the complex processes of cell dynamics in 3D cultures. Our innovative approach is based on lanthanide-based metallacrowns (Ln(III)-MC) that are resistant to photobleaching and emit as narrow NIR bands, the wavelengths of which do not vary with experimental conditions and which minimize light scattering [2]. For the needs of the present project, luminescent Ln(III)-based MCs possess several advantages, in particular: (i) highest values of quantum yield values for NIR-emitting Ln(III) molecular complexes leading to high detection sensitivity; (ii) rapid, large-scale imaging agent production via a one-step self-assembly and (iii) easy functionalization with chromophoric groups allowing the tuning of the Ln(III) excitation wavelength to achieve satisfactory tissue penetration [3].
With the financial support of the MITI CNRS program (Fongi_NeuroNIR, 2025–2026), we have demonstrated the efficient labelling of delicate and functionally complex cells, such as murine primary microglia, using the Er(III) MCs derivatives of dimeric Nd(III) or Er(III)/Ga(III) MCs (up to λem = 1524 nm). By adapting the protocols previously validated, our first collaborative work revealed that the labelling was not only effective and non-cytotoxic, but also persistent in primary culture of murine microglia for at least two weeks under both 2D and 3D conditions. Furthermore, our initial functional analysis demonstrated that the staining did not affect either microglia phagocytic activity or inflammatory responses under basal conditions or after immune activation. Primary murine microglia have also been dissociated, labelled in suspension and used to colonize murine-derived neurospheroids, establishing an innovative functional model for tracking cell mobility and future 3D organoid applications.
Bio-banque d’organoïdes issus de la faune sauvage : un outil prometteur dans la compréhension et la lutte contre les agents pathogènes zoonotiques
Les espèces sauvages constituent des réservoirs naturels pour de nombreux agents pathogènes et jouent un rôle majeur dans l’émergence des maladies zoonotiques, à l’origine d’environ 70 % des infections humaines émergentes. Afin de mieux comprendre les mécanismes impliqués dans les interactions hôte-pathogène, les organoïdes constituent un modèle expérimental particulièrement pertinent. Contrairement aux cultures cellulaires bidimensionnelles conventionnelles, les organoïdes offrent un système tridimensionnel reproduisant l’architecture, la diversité cellulaire et plusieurs fonctions physiologiques de leurs tissus d’origine. Leur utilisation s’inscrit pleinement dans le cadre du principe des 3R (Remplacer, Réduire, Raffiner), en proposant une alternative innovante permettant de diminuer le recours à l’expérimentation animale.
Ce projet vise à mettre au point et à caractériser des modèles d’organoïdes intestinaux et pulmonaires dérivés de plusieurs espèces animales, incluant à la fois des petits mammifères utilisés en recherche et des mammifères appartenant à la faune sauvage. En parallèle, des bio-banques ont été établies avec succès pour ces différentes espèces, fournissant ainsi des ressources biologiques précieuses pour de futures études sur les agents pathogènes associés à la faune sauvage et les maladies zoonotiques.
À plus long terme, ces modèles pourraient servir de base à la modélisation de maladies digestives ou respiratoires, à l’étude des relations hôte / pathogène incluant la compréhension des différences de sensibilité et de réponse immunitaire entre les espèces. Ainsi, la mise en place de ces bio-banques contribue à élargir les outils disponibles pour l’étude des maladies zoonotiques et à améliorer notre compréhension des phénomènes d’émergence infectieuse.
Conditioning strategies to enhance functional maturation of human EMT (Engineered Muscle Tissues)
The study of muscle diseases such as Duchenne muscular dystrophy (DMD) currently relies on animal models (mdx mouse, GRMD dog, DMDmdx rat). Although these models enable an integrated view of disease mechanisms and preclinical testing of therapies, their translation to patients is limited by interspecies differences and ethical concerns that call for reduced animal use. In this context, more predictive human in vitro models are needed.
Engineered Muscle Tissues (EMTs), 3D bioengineered muscle constructs, represent a promising alternative as they partially recapitulate the native muscle microenvironment and allow several functional assessments. However, current EMTs remain immature and do not fully reproduce adult skeletal muscle characteristics or disease phenotypes. To enhance EMT maturation and improve their relevance for DMD modelling, two key maturation cues: mechanical stretch and electrical stimulation, were investigated using a specific experimental device developed by our team. EMTs were generated from either immortalized human myoblasts or myogenic progenitors derived from human induced pluripotent stem cells (hiPSCs). Following mechanical stretching, EMTs were subjected to chronic electrical conditioning mimicking in vivo muscle innervation. After ten days of differentiation, contractile force was measured. Electrical conditioning increased force production in EMTs derived from immortalized myoblasts, whereas a decrease was observed in hiPSC-derived EMTs. Despite these differences, both models exhibited reduced dependence on extracellular calcium, indicating improved functional maturation. These findings highlight the importance of optimizing the combination of cell source and conditioning protocols to generate EMTs that resemble adult skeletal muscle and robustly model DMD, enabling future evaluation of innovative therapeutic approaches.
Human intestinal organoids to measure infectious norovirus in the environment and foods
Norovirus, a viral genus in the Caliciviridae family, is the main cause of viral gastroenteritis in humans. These viruses can infect all age groups and are transmitted through the fecal-oral route, directly from person to person or through contaminated foods or water. Shed in high concentrations in the stool of infected people, noroviruses are present in human sewage and can contaminate surface or coastal waters upon sewage spill-over or ineffective treatment. When this water is used for food production such as vegetable irrigation or shellfish aquaculture, foods become contaminated in turn. Thus, norovirus is often implicated in foodborne outbreaks. For almost 50 years, human norovirus could not be cultivated in vitro and data on its persistence as infectious particles are lacking. In 2016, norovirus replication was achieved in human intestinal stem cell-derived cultures in 3D (organoids).
Here, we show how we first acquired this technique to study the persistence of infectious norovirus in seawater. Then, we optimized methods to allow the elution of infectious virus from contaminated oysters and lettuces, and adapted the organoid culture and infection system to quantify viral infectious titers from these food matrices. These results demonstrate the added value of the human intestinal organoid platform for food and environmental virology, while highlighting the next challenges.
A human hiPSC model of holoprosencephaly to identify SHH-dependent transcriptomic signatures
Holoprosencephaly (HPE) is a rare developmental disorder characterized by incomplete separation of the cerebral hemispheres, resulting from impaired patterning of the embryonic forebrain. A broad phenotypic spectrum is observed, ranging from severe forms characterized by cyclopia to milder midline defects. Multiple genes have been implicated in HPE, with Sonic Hedgehog (SHH), a key morphogen required for forebrain and midline patterning, being the most frequently affected. Despite major advances in genetic testing, the significant genetic heterogeneity of HPE limits molecular diagnosis to approximately 30% of patients.
Human-induced pluripotent stem cells (hiPSCs) provide a unique opportunity to generated disease-relevant neural cell types and recapitulate early developmental stages that are otherwise inaccessible in humans. Our long-term objective is to identify transcriptomic and multi-omic signatures associated with HPE to better understand disease mechanisms and ultimately improve molecular diagnosis. We previously established a 12-day monolayer differentiation protocol to generate anterior ventral neuroectodermal cells from hiPSCs, with a controlled activation of SHH signalling during the first days of differentiation. These cells acquire an early ventral forebrain midline identity, corresponding to a developmental territory critically affected in HPE, and thus recapitulate the developmental window during which HPE arises. This model is also highly sensitive to graded pharmacological reduction of the SHH signaling.
Here, we present a robust and shortened differentiation protocol of differentiation, providing an optimized in vitro model of early HPE pathogenesis. By using CRISPR-engineered cell lines with either heterozygous or homozygous SHH mutations, we show that our model captures molecular alterations associated with reduced SHH expression levels. Moreover, we are able to identify different levels of SHH pathway disruption. Together, these results confirm the establishment of a human developmental model for investigating HPE, and provide a solution to the identification of disease-specific transcriptomic signatures associated with altered SHH signaling. Integration of this approach with patient-derived hiPSCs models, including organoid systems, should ultimately enable the development of diagnostic signatures to complement genomic data and improve diagnostic rates for patients with HPE.
Le groupe de travail OuestOÏD
La journée s’est terminée par une conclusion de Yannick Arlot, membre du Groupe de travail, qui a remercié l’ensemble des intervenant.es et des participant.es et qui a insisté sur la bonne dynamique du Groupe de travail OuestOÏD.
Le groupe de travail OuestOÏD est composé de 9 personnes originaires des différents sites de recherche du réseau :
Laurent Griscom (BIM3D, Rennes), Bénédicte Martin (Biogenouest), Yannick Arlot (IGDR, Rennes), Grégory Menchon (NorDiC, Rouen), Gaëlle Friocourt (LATIM, Brest), Anne Gaignerie (ION/MicroPICell, Nantes), Maud Contrant (ANSES, Ploufragan), Dévina Ung (iBrain, Tours), et Sophie Lelièvre (ICO, Angers).
Nos partenaires et soutiens
