- Aggressive chemotherapy in childhood often results in testicular damage and consequently jeopardizes future fertility. The presence of spermatogonial cells (SPGCs) in the testes of prepubertal cancer patient boys (PCPBs) can be used to develop future strategies for male fertility preservation. In the present study, we examined the presence of SPGCs in testes of chemotherapy-treated PCPBs and their ability to develop spermatogenesis in vitro using a three-dimensional (3D) culture system. Seven testicular biopsies were obtained from chemotherapy-treated PCPBs and one from a patient with β-thalassemia major. Isolated testicular cells were cultured in a methylcellulose culture system (MCS)-containing StemPro enriched with growth factors for 5-15 weeks. The presence of pre-meiotic, meiotic and post-meiotic cells was examined by immunofluorescence staining (IF) and/or RT-PCR analysis. We observed SPGCs in the examined testicular biopsies. Isolated testicular cells cultured in MCS developed into colonies and contained pre-meiotic, meiotic and post-meiotic cells. Furthermore, we identified sperm-like cells that had developed from testicular cells of a PCPB. Our results demonstrate for the first time, the presence of biologically active SPGCs in testicular biopsies of chemotherapy-treated PCPBs, and their capacity to develop in vitro to different stages of spermatogenesis including the generation of sperm-like cells. This study may open the way for new therapeutic strategies for fertility preservation of PCPBs and for azoospermic patients. Key words: Chemotherapy; Radiotherapy; Leukemia, Male infertility; Pre-pubertal cancer patient boys; Spermatogenesis; Testis; In vitro culture; spermatogonial cells.