Cryopreservation of testicular tissue before long term testicular cell culture does not alter in vitro cell dynamics

Cryopreservation of testicular tissue before culture does not influence the cell composition in longterm testicular cell cultures.

Yoni Baert, M.Sc., Aude Braye, M.Sc., Robin B. Struijk, M.Sc., Ans M.M. van Pelt, Ph.D., Ellen Goossens, Ph.D.

Volume 104, Issue 5, Pages 1244-1252


To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation.

Experimental basic science study.

Reproductive biology laboratory.

Testicular tissue with normal spermatogenesis was obtained from six donors.


Main Outcome Measure(s):
Detection and comparison of spermatogonia and/or spermatogonial stem cells (SSC) of fresh and frozen tissues.

Human testicular cells derived from fresh (n = 3) and cryopreserved (n = 3) tissues were cultured for 2 months and analyzed with quantitative reverse-transcription polymerase chain reaction and immunofluorescence. Spermatogonia including SSCs were reliably detected by combining VASA, a germ cell marker, with UCHL1, a marker expressed by spermatogonia. The established markers STAR, ACTA2, and SOX9 were used to analyze the presence of Leydig cells, peritubular myoid cells, and Sertoli cells, respectively. No obvious differences were found between the cultures initiated from fresh or cryopreserved tissues. Single or small groups of SSCs (VASA+/UCHL1+) were detected in considerable amounts up to 1 month of culture, but infrequently after 2 months. SSCs were found attached to the feeder monolayer, which expressed markers for Sertoli cells, Leydig cells, and peritubular myoid cells. In addition, VASA−/UCHL1+ cells, most likely originating from the interstitium, also contributed to this monolayer. Apart from Sertoli cells, all somatic cell types could be detected throughout the culture period.

Testicular tissue can be cryopreserved before long-term culture without modifying its outcome, which encourages implementation of testicular tissue banking for fertility preservation. However, because of the limited numbers of SSCs available after 2 months, further exploration and optimization of the culture system is needed.

  • Yoni Baert

    Thank you for your questions. All 6 cell cultures survided until the 2 months end point. However, we observed some interreplicate variability in the general feeder cell composition, which can be explained by sample effect as all specimens came from unique donors. Characteristics they did share were thriving behaviour of certain somatic cells and a low expansion rate of SSCs. We therefore hypothesize that a high somatic cell/SSC proliferation ratio resulted in a decrease of germ cell markers over time.

  • The authors present preliminary data on successful culture of SSC’s. It is discouraging that the cells did not survive past one month. Is there a reason that the authors can hypothesize? Did all 6 cell cultures survive the same duration?

  • Yoni Baert

    Thanks for your interest in our paper. In this tudy, we didn’t look at the genetic material. However, in a recent study, our collaborators of the University of Amsterdam found that the genomic stability is preserved in the culture system. For further information I refer to Nickkholgh et al. Fertility and Sterility 2014. doi: 10.1016/j.fertnstert.2014.08.022.

  • msamplaski

    This is very encouraging data for fertility preservation. Were the authors able to look at the genetic material after culture? DNA fragmentation, or aneuploidy rates?

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