Cryopreservation of human testicular diploid germ cell suspensions

Abstract

For patients with threatened fertility, preservation of it is a major concern. Although promising results have been obtained in animal models using testicular germ cell suspensions, in humans, it is crucial to first develop an efficient method of cryopreservation to be able to apply to transplantation. Thus, four reliable and available cryopreservation techniques in any fertility centre were tested to cryopreserve an enriched fraction of diploid germ cells isolated from human testicular biopsies. The protocols were evaluated based on cell viability, and the results showed significant differences between the four methods. The semen and tissue cryopreservation methods appeared to be inadequate for diploid germ cell suspensions, and programmed slow freezing gave significantly lower results than open pulled straw vitrification; the latter was found to be the protocol that best preserved cell viability. The vitrification of isolated human diploid germ cells is innovative and constitutes valuable information for cryopreservation in cases of transplants or in vitro maturation.

A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue

“…This finding has been reported in previous studies, indicating that vitrification may eventually become the procedure of choice for the preservation of immature testicular tissue (Curaba et al, 2011;Baert et al, 2012;Poels et al, 2012;Sá et al, 2012). Over the last decade, different vitrification protocols using different cryoprotectants, concentrations and exposure times, support equipment, as well as testicular tissue preparation time have been used (Curaba et al, 2011;Gouk et al, 2011;Baert et al, 2012;Poels et al, 2012;Sá et al, 2012;Liu et al, 2013).…”

“…Several animal (Curaba et al, 2011;Gouk et al, 2011;Baert et al, 2012;Poels et al, 2012;Liu et al, 2013) and human (Curaba et al, 2011;Baert et al, 2013;Poels et al, 2013;Sá et al, 2012) vitrification in the cryopreservation of prepubertal testicular tissue with encouraging results. These studies have shown that the preservation of testicular cells and tissue through vitrification has been at least as effective as traditional slow freezing protocols (Curaba et al, 2011;Baert et al, 2012;Sá et al, 2012). Despite the encouraging results, the search for an optimal cryopreservation protocol capable of successfully and predictably restoring fertility to prepubertal males is still ongoing (Baert et al, 2013).…”

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

“…(2012)Rhesus monkeyMature10% DMSOInsulated container −80°C, LN 2 58 ± 4Recipient testicular colonizationHermann et al . (2007)VitrificationHumanAdult(1)1.1 M DMSO,1.34 M EG, 10%HSAopen pulled straw (OPS) vitrification56 ± 24No functional assay performedSá et al . (2012)(2)0.67 M S,2.3 M DMSO, 3.0 M EG, 10%HSAN/AHumanAdult1.28 M DMSO 25% FCS−1°C/min, LN 2 75 ± 4No functional assay performedYango et al .…”

“…Thus, cancer cell-free TCSs would be the best option to preserve and restore fertility for these patients (Sá et al ., 2012). Currently, OPS vitrification, including high concentrations of DMSO (1.1 to 2.8 M), non-penetrant CPAs and 10% HSA has resulted in the highest cell viability.…”

“…Comparisons of functional assays using fresh to cryopreserved samples indicate loss of SSCs during the cryopreservation procedure. Thus, the effectiveness of the cryopreservation procedures is critical and should be improved (Izadyar et al, 2002a,b; Shinohara et al ., 2002; Frederickx et al ., 2004; Baert et al, 2012; Hermann et al ., 2012; Jahnukainen et al, 2012; Sá et al, 2012; Pacchiarotti et al, 2013; Sato et al ., 2013). …”