Wistar rats immature testicular tissue vitrification and heterotopic grafting

Abstract

ObjectiveTo evaluate the efficiency of two vitrification protocols for rat immature testicular tissue and heterotopic transplantation.MethodsTwenty-four pre-pubertal Wistar rats were divided into three groups (n=8). After orchiectomy, testicular fragments (3mm) from Groups 1 and 2 were vitrified with different cryoprotectant concentration solutions, using sterile inoculation loops as support. After warming up, the fragments were submitted to cell viability assessment by Trypan blue and histological evaluation. Vitrified (Groups 1 and 2) and fresh (Group 3) fragments were grafted to the animals periauricular region. After 8 weeks of grafting, the implant site was histologically analyzed.ResultsThe viability recovery rate from Group 1 (72.09%) was higher (p=0.02) than that from Group 2 (59.19%). Histological analysis showed similar tubular integrity between fresh fragments from Groups 1 and 3. Group 2 samples presented lower tubular integrity. We ran histological analyses in the grafts from the Groups. In all groups, it was possible to see the implant site, however, no fragment of testicular tissue or signs of inflammation were histologically found in most samples from Groups 1 and 3. In one sample from Group 2, we found degenerated seminiferous tubules with necrosis and signs of an inflammatory process. In another sample from Group 2, we found seminiferous tubules in the implant site.ConclusionThe vitrification of pre-pubertal testicular tissue of rats showed little damage to cell viability through histological analysis when we used cryoprotectants in a lower concentration. Heterotopic transplantation could not preserve the structural organization of the testicular tissue.

Comparison of the effects of three cryoprotectants on the cryopreservation of mouse subcutaneous tissue under different conditions

“…The cryopreservation of stem cells and embryos has been successful (10,11). The cryopreservation of rat testicular tissue (12), rat, pig and human islet tissue (13)(14)(15)(16) and liver tissues of mice, rats, dogs, monkeys and humans has also been successful (17). Cryopreservation is not only conducive to the accumulation of donor tissue, but also significantly reduces its immunity (18).…”

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system

“…[7][8][9] Scientists recommend cryopreservation of testicular tissue prior to cytotoxic therapies for preserving fertility in pre-pubertal cancer patients. 10 In vitro maturation (IVM) of spermatogonial stem cells obtained from cryopreserved pre-pubertal testicular tissues via two-dimensional (2D) and three-dimensional (3D) culture systems is the best potential procedure for the generation of functional sperm. 11,12 2D culture systems cannot effectively simulate the organized structure of the testicular microenvironment.…”

“…Organoid culture technology is a simple and effective strategy to recognize the unknown mechanisms responsible for SSCs proliferation and differentiation 7–9 . Scientists recommend cryopreservation of testicular tissue prior to cytotoxic therapies for preserving fertility in pre‐pubertal cancer patients 10 …”