STUDY QUESTION Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)?SUMMARY ANSWER Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy.WHAT IS KNOWN ALREADY Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples. Organoids hold promise in providing fundamental early insights in creating such platforms. However, the generation of TOs that closely resemble the innate testis, to enable a thorough monitoring of the necessary steps for germ cell differentiation and somatic functionalities, remains a challenge.STUDY DESIGN, SIZE, DURATION We used a Matrigel-based three-layer gradient culture system to generate human TOs and to reveal whether chemotherapy exposure affects TO formation capacity and the functionality of pre- and peri-pubertal testicular somatic cells. Testicular cells of 11 boys (aged 7.7 +/- 4.1 (mean +/- SD) years) were assessed for TO formation in relation to previous chemotherapy exposure and SOX9 expression in histological sections of paraffin-embedded testicular tissue samples collected on the day of biopsy and compared with testicular tissue samples obtained from 28 consecutive patients (aged 6.9 +/- 3.8 (mean +/- SD) years). All 39 patients were part of the fertility preservation project NORDFERTIL; an additional 10 samples (from boys aged 5.5 +/- 3.5 (mean +/- SD) years, without an underlying pathology) in an internal biobank collection were used as controls.PARTICIPANTS/MATERIALS, SETTING, METHODS We obtained 49 testicular tissue samples from boys aged 0.8-13.4 years. Fresh samples (n = 11) were dissociated into single-cell suspensions and applied to a three-layer gradient culture system for organoid formation. Histological sections of another 28 samples obtained as part of the fertility preservation project NORDFERTIL, and 10 samples from a sample collection of a pathology biobank were used to evaluate the effects of prior exposure to alkylating agents on testicular samples. Testicular organoid formation was defined based on morphological features, such as compartmentalized structures showing cord formation, and protein expression of testicular cell-specific markers for germ and somatic cells was evaluated via immunohistochemical staining. Hormone secretion was analysed by specific enzyme-linked immunosorbent assays for testosterone and anti-M & uuml;llerian hormone (AMH) production.MAIN RESULTS AND THE ROLE OF CHANCE Our results revealed that 4 out of 11 prepubertal testicular samples formed TOs that showed compartmentalized cord-like structures surrounded by interstitial-like areas and increasing levels of both testosterone as well as AMH over a 7-day culture period. We observed that SOX9 expression was correlated positively with TO formation. Moreover, exposure to alkylating agents before biopsy was inversely correlated with SOX9 expression (P = 0.006).LARGE SCALE DATA N/A.LIMITATIONS, REASONS FOR CAUTION Due to the limited amount of material available, only 11 out of the 39 pre- and peri-pubertal testicular tissue samples could be used for the organoid formation experiments. The testicular tissue samples obtained from a sample collection of the internal biobank of Department of Pathology, Karolinska University Hospital were considered normal and included in the study if no testicular pathology was reported. However, detailed information regarding previous medical treatments and/or testicular volumes of the patients included in this biobank was not available.WIDER IMPLICATIONS OF THE FINDINGS Our observations suggest that SOX9 expression may serve as a putative indicator of TO formation, indicating a critical role of Sertoli cells in promoting organoid formation, seminiferous tubule integrity, and testicular function in pre- and peri-pubertal testicular tissue.STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.), Finnish Cancer Society (K.J.), Finnish Foundation for Paediatric Research (K.J.), Swedish Research Council (2018-03094; 2021-02107) (J.-B.S.), and Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2020-00335; 2021-00073; 2022-00317) (J.-B.S. and K.J.). Y.C. and Y.Y. received a scholarship from the Chinese Scholarship Council. J.P.A-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. R.T.M. was supported by a UKRI Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health was supported by an MRC Centre Grant (MR/N022556/1). The authors declare no competing interests.