P-798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis

This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated byin vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements.Participants/materials, setting, methodsPost-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP-9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα-1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR).Main results and the role of chanceThe viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (<10% SSCs) and enriched SSCs(>50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit ...
Source: Human Reproduction - Category: Reproduction Medicine Source Type: research