Abstract
Cryopreservation of immature testicular tissue before chemo/radiotherapy is the only option to preserve fertility of cancer-affected prepubertal boys. To avoid reintroduction of malignant cells, development of a transplantable scaffold by decellularization of pig immature testicular tissue (ITT) able to support decontaminated testicular cells could be an option for fertility restoration in these patients. We, therefore, compared decellularization protocols to produce a cytocompatible scaffold. Fragments of ITT from 15 piglets were decellularized using three protocols: sodium dodecyl sulfate (SDS)-Triton (ST), Triton-SDS-Triton (TST) and trypsin 0.05%/ethylenediaminetetraacetic acid (EDTA) 0.02%-Triton (TET) with varying detergent concentrations. All protocols were able to lower DNA levels. Collagen retention was demonstrated in all groups except ST 1%, and a significant decrease in glycosaminoglycans was observed in the TST 1% and TET 1% groups. When Sertoli cells (SCs) were cultured with decellularized tissue, no signs of cytotoxicity were detected. A higher SC proliferation rate and greater stem cell factor secretion were observed than with SCs cultured without scaffold. ST 0.01% and TET 3% conditions offered the best compromise in terms of DNA elimination and extracellular matrix (ECM) preservation, while ensuring good attachment, proliferation and functionality of human SCs. This study demonstrates the potential of using decellularized pig ITT for human testicular tissue engineering purposes.
Highlights
Thanks to progress in the field of cancer therapy, more than 80% of children survive their disease in Europe [1]
extracellular matrix (ECM) scaffolds obtained after tissue decellularization are being increasingly considered for development of artiffiicial organ structures able to mimic organ functions once they have been recolonized bbyyoorgrgaann-s-sppeecicfiificcceclelsll.sD. ecDeellcuellalurilzaartiizoantioisndiesfindedfinaesda pasroacepssroecneasbslienngacbelilnl gancdeldleabnrids dreembroivsarle, mwohvilael,mwaihnitlaeinminagintthaeinsitnrugctuhreesatnrudctcuormepaonsditioconmofpothseitiEoCnMof[2t6h]e
By means of H and E staining, we showed that human Sertoli cells (SCs) were able to attach to pig immature testicular tissue (ITT) decellularized with ST 0.01%, TST 0.1% or trypsin 0.05%/ethylenediaminetetraacetic acid (EDTA) 0.02%-Triton (TET) 3% (Figure 6A) while maintaining expression of GATA4 and vimentin until the end of the culture (Figure 6B,C)
Summary
Thanks to progress in the field of cancer therapy, more than 80% of children survive their disease in Europe [1]. Cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells (SSCs) is an ethically accepted approach proposed to these boys with the aim of restoring their fertility once they reach adulthood [2,3]. Three methods are conceivable to restore fertility from stored ITT: autotransplantation, in vitro maturation, or SSC transplantation following selective cell isolation. These different approaches are all supported by results obtained in animals, yielding offspring from in vitro-produced mouse spermatozoa [4] or following testicular tissue grafts even in larger animals including non-human primates [5]. Since the start of clinical application of ITT cryopreservation [2,3,13,14,15], many boys have reached the age at which fertility becomes an increasing concern, so development of techniques allowing them to father children is an ever more pressing matter
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