• Mouse intestinal organoid

    Low magnification TEM image of a KO mouse intestinal organoid for AP-1m2 (Ophélie Nicolle - Aurélien Bidaud-Meynard)

  • Mouse intestinal organoid

    TEM image of polarized enterocytes of a WT mouse intestinal organoid (Ophélie Nicolle - Aurélien Bidaud-Meynard)

  • Mouse intestinal organoid

    TEM image of a cytoplasmic microvillous inclusion of an intestinal organoid from KO mice for Munc18-2 (Ophélie Nicolle - Aurélien Bidaud-Meynard)

  • TESTING
    Blastocyst-like 3D structure

    Yellow: tight junctions/ZO-1 marking; Magenta: adherens junctions/CDH1 labeling; Cyan: aPKC/apical domain molecule labeling); from Kagawa et al., 2021.

  • Blastoid

    Obtained from human iPSCs (Red: EPI-like epiblast cells/NANOG marking; Green: PrE-like primitive endoderm cells/GATA4 marking; Blue: TE-like trophectoderm cells/GATA3 marking)

  • B4_blastocyst

    Blue: NANOG marking; Green: NR2F2 marking; Magenta: RXRA marking).

Interest in 3D models

The progress of recent decades in terms of stem cell culture and differentiation, as well as 3D culture, has enabled the emergence of organoids, intermediate models between monolayer cell cultures and in vivo models. These three-dimensional models make it possible to get closer to physiological conditions, and have the advantage of being produced in the same species, unlike animal models, which will increase the knowledge of molecular events in tissue pathology and the predictability of molecules in development.

These booming technologies meet the requirement for reduction of animal models according to the 3R principle (FC3R).

Organoids

The definition of organoids is still debated.

Today, an organoid is defined as a three-dimensional cellular structure, whose cells are capable of self-organization, self-renewal, and differentiation into different cell types of the original tissue, capable of ensuring at least one of the functions of this tissue, and having preserved the identity of the organ or tissue from which they come. The organoid partially synthesizes the complexity of the original tissue, mimicking its architecture and some of its specific functions.

However, the identity is not complete and we cannot strictly speak of a mini-organ, given the absence of other physiological actors such as the vascular and immune systems.

Mouse intestinal organoid

Mouse intestinal organoid presenting several lumens (Blue: nuclei; green: F-actin; red: ZO-1) by Maxime Mahé

Types of 3D Cell Models

Depending on the scientific question raised, researchers have different 3D culture models of varying complexity, ranging from:

  • Spheroids produced mainly from cell lines and consisting of three-dimensional objects obtained by preventing the attachment of cells to the culture support and promoting their co-aggregation. We can also find in this category heterotypic spheroids with co-culture with stromal cells (CAF, immune cells, vascular or lymphatic endothelial cells, adipocytes, pericytes) to integrate elements of the microenvironment;
  • Organoids derived from multipotent adult stem cells from the tissue studied, consisting of a single lineage type;
  • Organoids derived from pluripotent stem cells (induced or embryonic) that can reconstitute the different cell lineages of the tissue studied.
Multi-cellular tumor spheroid (MCTS) from Malignant Pleural Mesothelioma

Multi-cellular tumor spheroid (MCTS) of Malignant Pleural Mesothelioma including macrophages (magenta) in confocal microscopy by Christophe BLANQUART

Cancer research may involve:

  • Multicellular tumor spheroids (MTS), derived from established cell lines, under conventional culture conditions;
  • Tumorospheres produced from dissociated tumor tissue;
  • Tumoroids developed from primary tumors or patient-derived xenografts (PDX), after dissociation and purification of tumor cells, in culture conditions close to those used for the culture of organoids (matrix and specific growth factors) and optimized to preserve self-renewal and differentiation of cancer stem cells.
  • “Normal” organoids subject to tumorigenic alterations.

Applications

Organoids make it possible to follow the dynamics of organ development, and their transformation, and are therefore of major interest in research in developmental biology, in physio-pathology, for the development and evaluation of therapies, for medicine precision and regenerative medicine.

Kidney Cell Spheroid

Blue: nuclei; Red: Junctions/ZO-1 marking). By Yannick Arlot

Issues

The obstacles and obstacles to the production of organoids concern:

  • Large scale production
  • Reproducibility/Standardization of protocols
  • Control of genomic instability of stem cells
  • Presence of residual stem cells after differentiation
  • Stem cell immunogenicity

The analysis and characterization of organoids and other 3D models raise technical problems for:

  • preparation of samples before analysis by:
    • Cytometry or transcriptomics (dissociation)
    • Genomics (multiplexing)
    • 3D imaging (transparency)
  • the processing and management of the voluminous data generated by these analyses.