Abstract
The introduction of stem cell-based technologies for the derivation of three-dimensional retinal tissues, the so-called retinal organoids, offers many new possibilities for vision research: Organoids facilitate studies on retinal development and in vitro retinal disease modeling, as well as being valuable for drug testing. Further, retinal organoids also provide an unlimited cell source for cell replacement therapies. Here, we describe our protocol for efficiently differentiating large, stratified retinal organoids from mouse embryonic stem cells: unbiased manual dissection of the developing retinal organoid at an early stage into three evenly sized neuroepithelial portions (trisection step) doubles the yield of high-quality organoids. We also describe some useful applications of the protocol, e.g., generation of rod- or cone-enriched retinal organoids, AAV transfection, and cell birth dating. In addition, we provide details of how to process retinal organoids for single organoid gene expression analysis, immunohistochemistry, and electron microscopy.
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Acknowledgments
This work was supported by funding programs for DZNE Helmholtz and TU Dresden CRTD (M.K.), DFG (KA2794/3-1; SPP1738) (M.K.), and MedDrive TU Dresden UKD-Medical Faculty (M.K.). We thank Sara Oakeley (Basel, Switzerland) and Felix Wagner (DZNE, Dresden) for helpful comments on the manuscript.
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Organoid trisection using single-step cutting technique (MP4 58363 kb)
Organoid trisection using two-step cutting technique (MP4 91134 kb)
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Völkner, M., Kurth, T., Karl, M.O. (2019). The Mouse Retinal Organoid Trisection Recipe: Efficient Generation of 3D Retinal Tissue from Mouse Embryonic Stem Cells. In: Weber, B.H.F., Langmann, T. (eds) Retinal Degeneration. Methods in Molecular Biology, vol 1834. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8669-9_9
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DOI: https://doi.org/10.1007/978-1-4939-8669-9_9
Publisher Name: Humana, New York, NY
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