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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/226129
Mapping the structure and function of whole-mount retinal organoids
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Cell therapy is a therapeutic avenue for retinal degenerative diseases. Photoreceptortransplantation faces challenges in obtaining high-quality and mature photoreceptors that caneffectively integrate and function, facilitating regeneration. For designing therapies we will produceretinal organoids (RO) from ihPSC. To enhance our understanding of RO maturation in a 3D anddynamic context, we aim to characterize the structure and function of retinal neurons. This will helpidentifying the optimal timing for harvesting photoreceptors for subsequent implantation. In thiswork, we focus on the characterization of RO derived from ihPSC from healthy and vision-impairedindividuals, specifically Retinitis Pigmentosa type 25. We tracked calcium activity in 3D with light-sheet fluorescence microscopy (LSFM). Thisoptical configuration allows fast tracking of dynamics (10 Z-planes at 5 Hz) occurring on a volumetricscale (600x830x100μm) with minor photo-toxic effects. For visualizing changes in the calciuminfluxes, we used viral vectors encoding for GCaMP6s. Changes in fluorescence intensity werequantified and analysed in a 3D fashion to determine the pattern and frequency of calcium waves inthe neuronal network. Subsequently to the life dynamic recordings, and to characterize thestructure of whole-mount mature ROs (up to day 250), we optimized an optical clearing method(FluoClear BABB) in combination with improved antibody permeabilization. We imaged in high-resolution the calcium activity of RO derived from healthy and vision-impaired donors and quantified differences in the functional communities of neurons by usingtransfer entropy. Using our clearing protocol, we identified the morphology and population of thethree neuron-path that provide the direct route for visual information transmission: cone and rodphotoreceptors, bipolar and ganglion cells. RO are promising in vitro models for studying retinal diseases. Our findings shed morelight on the temporal, spatial and functional organization of retinal cells within the organoid. Our LSFM can be applied to study fast events occurring in mm range samples with cellular resolution
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CUNQUERO, Marina, ISLA-MAGRANE, Helena, CASTRO-OLVERA, Gustavo, SORIANO I FRADERA, Jordi, MARSAL, Maria, MATEOS, Nicolas, ZUFIAURRE, Maddalen, GARCIA-ARUMI, Josep, DUARRI, Anna, LOZA-ALVAREZ, Pablo. Mapping the structure and function of whole-mount retinal organoids. _Investigative Ophthalmology & Visual Science_. 2024. Vol. 65, núm. 7. [consulta: 20 de febrer de 2026]. ISSN: 0146-0404. [Disponible a: https://hdl.handle.net/2445/226129]