OREO

Induced Pluripotent Stem Cells (iPS) can generate 3D retinal organoids that contain all major types of retinal cells and a distinct stratification close to the in vivo morphology. These organoids are therefore very promising for disease modeling purposes, as well as possible cell therapy strategies in patients with retinal degeneration. In particular, the use of organoids is envisaged to facilitate understanding of the cellular mechanisms leading to retinitis pigmentosa (RP), a group of inherited neurodegenerative diseases that cause selective cell death of photoreceptors. The mechanisms leading to cell death remain unknown and no adequate treatment for RP is currently available. Retinal degeneration, and in particular RP, can be targeted via “disease in a dish” modeling methods. The idea is to create a degenerative process that resembles RP, with progressive apoptosis of photoreceptors. Although 2D RP models have been proposed, an organoid-based 3D RP model would be closer to reality. Drugs can then be tested on this RP model to monitor their effectiveness and identify potential drug candidates for clinical trials. High-resolution imaging tools are essential for performing non-destructive quality control on these 3D cell cultures. At present, the development of organoids is ensured by cultivating a series of samples, a number of which are sacrificed at each control stage. The imaging process requires fixing, slicing and performing multiple fluorescent labeling procedures on organoids to determine the many cell types present, and the correct development of the 3D culture. If non-destructive live imaging were available for these samples, a single organoid could be monitored over time, allowing direct observation of the development and behavior of each sample. However, real-time imaging techniques for use in regenerative medicine are currently limited to 2D cultures. Full Field Optical Coherence Tomography (FFOCT) is a non-invasive, high-resolution approach to Optical Coherence Tomography (OCT). FFOCT, developed by members of the project group, allows non-invasive imaging of ocular tissues with three-dimensional micrometric resolution. We recently added a complementary contrast mechanism to FFOCT to access functional information. Dynamic FFOCT detects intracellular movement to display a contrast based on cellular metabolism, an indicator of cell viability. In addition, our technique can combine fluorescence and FFOCT channels in superposition to allow the localization of fluorescent cells in their 3D microenvironment. We hypothesize that we will be able to i) create a 3D organoid-based model of RP, and ii) detect photoreceptor degeneration and recovery in this model, non-invasively and label-free, with FFOCT, thus reproducing the quantitative information generally available only by immunofluorescence. The multidisciplinary OREO project will push the limits of current practice on three fronts - technological, computational and biological - by developing a multimodal FFOCT imaging tool for use in a clean room environment; developing computational tools for the quantification and automatic classification of cells; and developing a new 3D model of RP and its isogenic control that will be tracked over time with FFOCT.