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Use of a three-layer gradient system of cells for rat testicular organoid generation


We have recently developed a 3D culture system that allows the reorganization of rat primary testicular cells into organoids with a functional blood–testis barrier, as well as the establishment and maintenance of germ cells. The innovative aspect of our model, the three-layer gradient system (3-LGS), comprises cells combined with Matrigel placed between two layers of Matrigel without cells, which creates a gradient of cells and allows the reorganization of testicular cells into organized structures after 5–7 d in culture. This reorganization is not observed when testicular cells are suspended in only one layer of Matrigel, the methodology used in the majority of the protocols for generating organoids. The model can be applied to follow and quantify cell migration during testicular organoid formation, and to explore the role of growth factors and the toxic effects of drugs and environmental contaminants on germ cell maintenance and blood–testis barrier integrity. The 3-LGS is a robust and reproducible method that requires a small volume of Matrigel and a low number of cells (16 μl and 132,000 cells, respectively), enabling and facilitating high-throughput analysis of germ-to-somatic cell associations in vitro.

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Figure 1: Schematic representation of the 3-LGS setup.
Figure 2: Seminiferous tubule collection and digestion.
Figure 3: 3-LGS setup.
Figure 4: Testicular organoid collection.
Figure 5: Whole-mount stained organoid mounting.
Figure 6: Testicular organoid formation, organization and function.


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We are grateful to A. Osman for support with confocal microscopy and to L. Lopes for assistance with the graphic design of the schematic illustration in this protocol. We thank L. Zhang, J. Fontana and I. Eggertsen for technical support in the animal facilities, as well as V. Pampanini, H. Albalushi and M. Kurek for acquisition and editing of photographic work. We are also grateful for all the funding given to this project by the HKH Kronprinsessan Lovisas förening för barnasjukvård, Frimurare Barnhuset in Stockholm; the Paediatric Research Foundation, Jeanssons Foundation (JS2014-0091); Sällskapet Barnavård in Stockholm; The Swedish Research Council (2012-6352); the Emil and Wera Cornells Foundation; the Samariten Foundation; the Swedish Childhood Cancer Foundation (PR2016-0124, TJ2016-0093) and the EU-FP7-PEOPLE-2013-ITN (603568) 'Growsperm'.

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Authors and Affiliations



J.P.A.-L. developed the method. J.P.A.-L. and J.-B.S. designed the protocol. J.P.A.-L. performed the experiments and the acquisition of the results. J.P.A.-L., J.-B.S. and O.S. interpreted the results. O.S. and J.-B.S. obtained funding. J.P.A.-L. wrote the protocol, and all authors critically reviewed and approved the final version of the protocol.

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Correspondence to João Pedro Alves-Lopes.

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Alves-Lopes, J., Söder, O. & Stukenborg, JB. Use of a three-layer gradient system of cells for rat testicular organoid generation. Nat Protoc 13, 248–259 (2018).

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