Sertoli Cells pp 287-294 | Cite as

Cryopreservation of Human Testicular Tissue by Isopropyl-Controlled Slow Freezing

  • Yoni BaertEmail author
  • Jaime Onofre
  • Dorien Van Saen
  • Ellen Goossens
Part of the Methods in Molecular Biology book series (MIMB, volume 1748)


Tissue cryopreservation uses very low temperatures to preserve structurally intact living cells in their natural microenvironment. Cell survival is strongly influenced by the biophysical effects of ice during both the freezing and the subsequent thawing. These effects can be controlled by optimizing the fragment size, type of cryoprotectant, and cooling rate. The challenge is to determine cryopreservation parameters that suit all cell types present in the tissue. Here we describe a quick and convenient protocol for the cryopreservation of testicular tissue using an isopropyl-insulated freezing device, which was validated in both a mouse and a human model.


Mouse Human Testicular tissue Cryopreservation Isopropyl Slow freezing 



This work was supported by a PhD grant from the Agency for Innovation by Science and Technology and research grants from the European Commission Research and Innovation Marie Sklodowska Actions (MSCA)s Seventh Framework program (FP7-People-2013-ITN-603568), the Flemish League against Cancer-Public Utility Foundation, the Scientific Research Foundation Flanders (FWO), University Hospital Brussels (fund Willy Gepts), and the Vrije Universiteit Brussel (Methusalem grant). Y.B. and D.V.S. are postdoctoral fellows of the FWO.


  1. 1.
    Yavin S, Arav A (2007) Measurement of essential physical properties of vitrification solutions. Theriogenology 67:81–89CrossRefPubMedGoogle Scholar
  2. 2.
    Mazur P (1970) Cryobiology: the freezing of biological systems. Science 168:939–949CrossRefPubMedGoogle Scholar
  3. 3.
    Karlsson JOM, Toner M (1996) Long-term storage of tissues by cryopreservation: critical issues. Biomaterials 17:243–256CrossRefPubMedGoogle Scholar
  4. 4.
    Avarbock MR, Brinster CJ, Brinster RL (1996) Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 2:693–696CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Nugent D, Meirow D, Brook PF, Aubard Y, Gosden RG (1997) Transplantation in reproductive medicine: previous experience, present knowledge and future prospects. Hum Reprod Update 3:267–280CrossRefPubMedGoogle Scholar
  6. 6.
    Thuwanut P, Srisuwatanasagul S, Wongbandue G, Tanpradit N, Thongpakdee A, Tongthainan D, Manee-in S, Chatdarong K (2013) Sperm quality and the morphology of cryopreserved testicular tissues recovered post-mortem from diverse wild species. Cryobiology 67:244–247CrossRefPubMedGoogle Scholar
  7. 7.
    Onofre J, Baert Y, Faes K, Goossens E (2016) Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation. Hum Reprod Update 22:744–761CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Shinohara T, Inoue K, Ogonuki N, Kanatsu-Shinohara M, Miki H, Nakata K, Kurome M, Nagashima H, Toyokuni S, Kogishi K et al (2002) Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination. Hum Reprod 17:3039–3045CrossRefPubMedGoogle Scholar
  9. 9.
    Curaba M, Poels J, van Langendonckt A, Donnez J, Wyns C (2011) Can prepubertal human testicular tissue be cryopreserved by vitrification? Fertil Steril 95:2123.e9–2123.12Google Scholar
  10. 10.
    Keros V, Hultenby K, Borgstrom B, Fridstrom M, Jahnukainen K, Hovatta O (2007) Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment. Hum Reprod 22:1384–1395CrossRefPubMedGoogle Scholar
  11. 11.
    Wyns C, Van Langendonckt A, Wese F-X, Donnez J, Curaba M (2008) Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue. Hum Reprod 23:2402–2414CrossRefPubMedGoogle Scholar
  12. 12.
    Picton HM, Wyns C, Anderson RA, Goossens E, Jahnukainen K, Kliesch S, Mitchell RT, Pennings G, Rives N, Tournaye H et al (2015) A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Hum Reprod 30:2463–2475CrossRefPubMedGoogle Scholar
  13. 13.
    Valli H, Gassei K, Orwig KE (2015) Stem cell therapies for male infertility: where are we now and where are we going? Bienn Rev Infertil 4:1017–1039Google Scholar
  14. 14.
    Baert Y, Goossens E, Van Saen D, Ning L, in’t Veld P, Tournaye H (2012) Orthotopic grafting of cryopreserved prepubertal testicular tissue: in search of a simple yet effective cryopreservation protocol. Fertil Steril 97:1152-7–1152-2CrossRefGoogle Scholar
  15. 15.
    Baert Y, Van Saen D, Haentjens P, in’t Veld P, Tournaye H, Goossens E (2013) What is the best cryopreservation protocol for human testicular tissue banking? Hum Reprod 28:1816–1826CrossRefPubMedGoogle Scholar
  16. 16.
    Baert Y, Braye A, Struijk RB, van Pelt AMM, Goossens E (2015) Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics. Fertil Steril 104:1244-1252–1244-1254CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Yoni Baert
    • 1
    Email author
  • Jaime Onofre
    • 1
  • Dorien Van Saen
    • 1
  • Ellen Goossens
    • 1
  1. 1.Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative MedicineVrije Universiteit Brussel (VUB)BrusselsBelgium

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