Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Goldschmidt, R. Some experiments on spermatogenesis in vitro. Proc. Natl. Acad. Sci. USA 1, 220–222 (1915).

    CAS  Article  PubMed  Google Scholar 

  2. 2

    Stukenborg, J.-B. et al. New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells. Mol. Hum. Reprod. 15, 521–529 (2009).

    Article  PubMed  Google Scholar 

  3. 3

    Schlatt, S., de Kretser, D.M. & Loveland, K.L. Discriminative analysis of rat Sertoli and peritubular cells and their proliferation in vitro: evidence for follicle-stimulating hormone-mediated contact inhibition of Sertoli cell mitosis. Biol. Reprod. 55, 227–235 (1996).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Tesarik, J. et al. Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum. Reprod. 13, 2772–2781 (1998).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Cremades, N., Bernabeu, R., Barros, A. & Sousa, M. In-vitro maturation of round spermatids using co-culture on Vero cells. Hum. Reprod. 14, 1287–1293 (1999).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Lee, J.H., Kim, H.J., Kim, H., Lee, S.J. & Gye, M.C. In vitro spermatogenesis by three-dimensional culture of rat testicular cells in collagen gel matrix. Biomaterials 27, 2845–2853 (2006).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Hadley, M.A., Byers, S.W., Suárez-Quian, C.A., Kleinman, H.K. & Dym, M. Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro. J. Cell Biol. 101, 1511–1522 (1985).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Legendre, A. et al. An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential. Biomaterials 31, 4492–4505 (2010).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Baert, Y. et al. Derivation and characterization of a cytocompatible scaffold from human testis. Hum. Reprod. 30, 256–267 (2015).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Baert, Y. et al. Primary human testicular cells self-organize into organoids with testicular properties. Stem Cell Rep. 1, 30–38 (2017).

    Article  Google Scholar 

  11. 11

    Sato, T. et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 471, 504–507 (2011).

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Sato, T., Katagiri, K., Kubota, Y. & Ogawa, T. In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method. Nat. Protoc. 8, 2098–2104 (2013).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Yokonishi, T. et al. In vitro reconstruction of mouse seminiferous tubules supporting germ cell differentiation. Biol. Reprod. 89, 15, 11–16 (2013).

    Article  Google Scholar 

  14. 14

    Reda, A. et al. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture. Mol. Hum. Reprod. 22, 601–612 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Alves-Lopes, J.P., Soder, O. & Stukenborg, J.B. Testicular organoid generation by a novel in vitro three-layer gradient system. Biomaterials 130, 76–89 (2017).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Hagiwara, M., Peng, F. & Ho, C.-M. In vitro reconstruction of branched tubular structures from lung epithelial cells in high cell concentration gradient environment. Sci. Rep. 5, 8054 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Pendergraft, S.S., Sadri-Ardekani, H., Atala, A. & Bishop, C.E. Three-dimensional testicular organoid: a novel tool for the study of human spermatogenesis and gonadotoxicity in vitro. Biol. Reprod. 96, 720–732 (2017).

    Article  PubMed  Google Scholar 

  18. 18

    Abu Elhija, M., Lunenfeld, E., Schlatt, S. & Huleihel, M. Differentiation of murine male germ cells to spermatozoa in a soft agar culture system. Asian J. Androl. 14, 285–293 (2012).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Reda, A. et al. In vitro spermatogenesis – optimal culture conditions for testicular cell survival, germ cell differentiation, and steroidogenesis in rats. Front. Endocrinol. 5, 21 (2014).

    Article  Google Scholar 

  20. 20

    Chapin, R.E. et al. Lost in translation: the search for an in vitro screen for spermatogenic toxicity. Birth Defects Res. B Dev. Reprod. Toxicol. 107, 225–242 (2016).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    de Michele, F. et al. Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue. Hum. Reprod. 32, 32–45 (2016).

    PubMed  Google Scholar 

Download references

Acknowledgements

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'.

Author information

Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to João Pedro Alves-Lopes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/nprot.2017.140

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing