{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T04:58:40Z","timestamp":1758776320023,"version":"3.44.0"},"reference-count":41,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T00:00:00Z","timestamp":1758758400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Endocrinol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Prepubertal testicular organotypic culture serves as an <jats:italic>in vitro<\/jats:italic> model for research in regenerative medicine, developmental biology, and toxicology. However, the selection of culture systems for diverse applications and purposes lacks explicit rationale or standardized guidelines.<\/jats:p><\/jats:sec><jats:sec><jats:title>Objective<\/jats:title><jats:p>To evaluate the developmental status of germ cells and Sertoli cells in prepubertal testicular organotypic cultures using different media and tools, and to provide evidence for model standardization.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and Methods<\/jats:title><jats:p>Immature testicular tissues (ITTs) were isolated from prepubertal mice and cultured in combinations of three media (Medium 1\/2\/3, M1\/2\/3) and two tools (agarose gel pillars, tissue culture inserts) at 34\u00b0C with 5% CO<jats:sub>\u2082<\/jats:sub> for 8 days. After culture, tissue morphology and cell status were assessed via histological analysis (hematoxylin-eosin [HE] staining) and immunofluorescence. Quantification was performed using ImageJ, and statistical analysis was conducted with GraphPad Prism.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Over the 8-day culture period, ITTs survived and grew in all systems, maintaining the structural integrity of seminiferous tubules, with tubular diameter increasing over time. Spermatogonia remained viable within the tubules, the number of germ cells increased, and both germ cells and spermatogonia exhibited proliferative activity. Sertoli cells were well preserved, and spermatogonia differentiated into meiotic spermatocytes in the tubule lumen. Compared with M1, M2 and M3 significantly enhanced the proliferation rates of germ cells and spermatogonia, with M2 showing the highest efficiency. Notably, M2 yielded the greatest number of differentiated spermatocytes, whereas M1 better preserved spermatogonial populations despite its lower proliferation rate.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study clarifies the differential effects of culture systems on the development of germ cells and Sertoli cells in ITTs, and provides a standardized organotypic platform for investigating spermatogenesis, male infertility, and potential therapeutic interventions.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fendo.2025.1664628","type":"journal-article","created":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T04:18:18Z","timestamp":1758773898000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Organotypic culture system for prepubertal mice testicular tissue: A comparative study"],"prefix":"10.3389","volume":"16","author":[{"given":"Liping","family":"Deng","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinyi","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xunwei","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hui","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yintao","family":"Cheng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Honggang","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,9,25]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1007\/s11626-020-00500-2","article-title":"Special review series on 3D organotypic culture models: Introduction and historical perspective","volume":"57","author":"Hayden","year":"2020","journal-title":"In Vitro Cell Dev Biol - Animal"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1007\/s11626-020-00539-1","article-title":"In vitro three-dimensional organotypic culture models of the oral mucosa","volume":"57","author":"Klausner","year":"2021","journal-title":"In Vitro Cell Dev Biol - Animal"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1007\/s11626-020-00526-6","article-title":"Human small intestinal organotypic culture model for drug permeation, inflammation, and toxicity assays","volume":"57","author":"Markus","year":"2020","journal-title":"In Vitro Cell Dev Biol - Animal"},{"key":"B4","doi-asserted-by":"publisher","first-page":"116355","DOI":"10.1016\/j.bone.2022.116355","article-title":"A new ex vivo model of the bone tissue response to the hyperglycemic environment - The embryonic chicken femur organotypic culture in high glucose conditions","volume":"158","author":"Ara\u00fajo","year":"2022","journal-title":"Bone"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1002\/pbc.22000","article-title":"Normal testicular function and spermatogenesis","volume":"53","author":"Shalet","year":"2009","journal-title":"Pediatr Blood Cancer"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.3389\/fphys.2018.01413","article-title":"Haploid germ cells generated in organotypic culture of testicular tissue from prepubertal boys","volume":"9","author":"de Michele","year":"2018","journal-title":"Front Physiol"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1093\/humupd\/dmx036","article-title":"Testicular organoids: a new model to study the testicular microenvironment in vitro","volume":"24","author":"Alves-Lopes","year":"2018","journal-title":"Hum Reprod Update"},{"key":"B8","doi-asserted-by":"publisher","first-page":"RD23216","DOI":"10.1071\/RD23216","article-title":"The limitations of testicular organoids: are they truly as promising as we believe","volume":"36","author":"Mecca","year":"2024","journal-title":"Reprod Fertil Dev"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-020-0283-z","article-title":"In vitro testicular organogenesis from human fetal gonads produces fertilization-competent spermatids","volume":"30","author":"Yuan","year":"2020","journal-title":"Cell Res"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1093\/molehr\/gaw051","article-title":"Irinotecan metabolite SN38 results in germ cell loss in the testis but not in the ovary of prepubertal mice","volume":"22","author":"Lopes","year":"2016","journal-title":"Mol Hum Reprod"},{"key":"B11","doi-asserted-by":"publisher","first-page":"1045","DOI":"10.1016\/j.fertnstert.2018.07.018","article-title":"Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue","volume":"110","author":"Medrano","year":"2018","journal-title":"Fertil Steril"},{"key":"B12","doi-asserted-by":"publisher","first-page":"RP85562","DOI":"10.7554\/eLife.85562","article-title":"Steroidogenesis and androgen\/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice","volume":"12","author":"Moutard","year":"2023","journal-title":"Elife"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1093\/humupd\/dmaa050","article-title":"Fertility preservation for prepubertal boys: lessons learned from the past and update on remaining challenges towards clinical translation","volume":"27","author":"Wyns","year":"2021","journal-title":"Hum Reprod Update"},{"key":"B14","doi-asserted-by":"publisher","first-page":"1773","DOI":"10.1038\/s41598-018-19761-9","article-title":"Chemotherapy drugs cyclophosphamide, cisplatin and doxorubicin induce germ cell loss in an in vitro model of the prepubertal testis","volume":"8","author":"Smart","year":"2018","journal-title":"Sci Rep"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1016\/j.toxrep.2020.12.023","article-title":"Chemotherapy induced damage to spermatogonial stem cells in prepubertal mouse in vitro impairs long-term spermatogenesis","volume":"8","author":"Lopes","year":"2021","journal-title":"Toxicol Rep"},{"key":"B16","doi-asserted-by":"publisher","first-page":"12105","DOI":"10.1038\/s41598-023-39304-1","article-title":"Generation of rat offspring using spermatids produced through in vitro spermatogenesis","volume":"13","author":"Matsumura","year":"2023","journal-title":"Sci Rep"},{"key":"B17","doi-asserted-by":"publisher","first-page":"3458","DOI":"10.1038\/s41598-021-82792-2","article-title":"Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control","volume":"11","author":"Matsumura","year":"2021","journal-title":"Sci Rep"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.3389\/fphys.2022.884122","article-title":"Microfluidic and static organotypic culture systems to support ex vivo spermatogenesis from prepubertal porcine testicular tissue: A comparative study","volume":"13","author":"Kanbar","year":"2022","journal-title":"Front Physiol"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1111\/andr.13325","article-title":"Complete meiosis in rat prepubertal testicular tissue under in vitro sequential culture conditions","volume":"11","author":"Saulnier","year":"2023","journal-title":"Andrology"},{"key":"B20","doi-asserted-by":"publisher","first-page":"7407","DOI":"10.1038\/s41598-022-11286-6","article-title":"Achievement of complete in vitro spermatogenesis in testicular tissues from prepubertal mice exposed to mono- or polychemotherapy","volume":"12","author":"Delessard","year":"2022","journal-title":"Sci Rep"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1093\/molehr\/gaw047","article-title":"In vitro differentiation of rat spermatogonia into round spermatids in tissue culture","volume":"22","author":"Reda","year":"2016","journal-title":"Mol Hum Reprod"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(93)81072-5","article-title":"The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis","volume":"65","author":"Griess","year":"1993","journal-title":"Biophys J"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1007\/s11626-020-00437-6","article-title":"The air-liquid interface culture of the mechanically isolated seminiferous tubules embedded in agarose or alginate improves in vitro spermatogenesis at the expense of attenuating their integrity","volume":"56","author":"Gholami","year":"2020","journal-title":"In Vitro Cell Dev Biol Anim"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0283773","article-title":"In vitro spermatogenesis in isolated seminiferous tubules of immature mice","volume":"18","author":"Feng","year":"2023","journal-title":"PloS One"},{"key":"B25","doi-asserted-by":"publisher","first-page":"1930","DOI":"10.3390\/cells13231930","article-title":"The role of plzf in spermatogonial stem cell maintenance and differentiation: mapping the transcriptional dynamics and key interactions","volume":"13","author":"Ghasemi","year":"2024","journal-title":"Cells"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0053976","article-title":"SALL4 expression in gonocytes and spermatogonial clones of postnatal mouse testes","volume":"8","author":"Singh","year":"2013","journal-title":"PloS One"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.0030198","article-title":"Meiotic pairing and segregation of achiasmate sex chromosomes in eutherian mammals: the role of SYCP3 protein","volume":"3","author":"de la Fuente","year":"2007","journal-title":"PloS Genet"},{"key":"B28","doi-asserted-by":"publisher","first-page":"12354","DOI":"10.1038\/s41598-023-39323-y","article-title":"Culture-space control is effective in promoting haploid cell formation and spermiogenesis in vitro in neonatal mice","volume":"13","author":"Hashimoto","year":"2023","journal-title":"Sci Rep"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1093\/molehr\/gax017","article-title":"Establishment, maintenance and functional integrity of the blood\u2013testis barrier in organotypic cultures of fresh and frozen\/thawed prepubertal mouse testes","volume":"23","author":"Rondanino","year":"2017","journal-title":"MHR: Basic Sci Reprod Med"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1097\/MED.0000000000000370","article-title":"Fertility restoration with spermatogonial stem cells","volume":"24","author":"de Michele","year":"2017","journal-title":"Curr Opin Endocrinology Diabetes Obes"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1242\/dev.132761","article-title":"The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia","volume":"143","author":"Lovelace","year":"2016","journal-title":"Development"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.4103\/aja.aja_103_18","article-title":"PLZFposc-KITpos-delineated A1\u2013A4-differentiating spermatogonia by subset and stage detection upon Bouin fixation","volume":"21","author":"Fan","year":"2019","journal-title":"Asian J Andrology"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1038\/nature09850","article-title":"In vitro production of functional sperm in cultured neonatal mouse testes","volume":"471","author":"Sato","year":"2011","journal-title":"Nature"},{"key":"B34","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1152\/physrev.00013.2015","article-title":"Spermatogenesis: the commitment to meiosis","volume":"96","author":"Griswold","year":"2016","journal-title":"Physiol Rev"},{"key":"B35","doi-asserted-by":"publisher","first-page":"167","DOI":"10.1038\/s41467-023-43871-2","article-title":"Derivation of human primordial germ cell-like cells in an embryonic-like culture","volume":"15","author":"Esfahani","year":"2024","journal-title":"Nat Commun"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.3389\/fendo.2022.895528","article-title":"Roles of spermatogonial stem cells in spermatogenesis and fertility restoration","volume":"13","author":"Diao","year":"2022","journal-title":"Front Endocrinology"},{"key":"B37","doi-asserted-by":"publisher","first-page":"163","DOI":"10.2741\/4891","article-title":"Spermatogonial stem cells: A story of self-renewal and differentiation","volume":"26","author":"Subash","year":"2021","journal-title":"Front Biosci (Landmark Ed)"},{"key":"B38","doi-asserted-by":"publisher","first-page":"1251","DOI":"10.1038\/s41467-019-09182-1","article-title":"Staged developmental mapping and X chromosome transcriptional dynamics during mouse spermatogenesis","volume":"10","author":"Ernst","year":"2019","journal-title":"Nat Commun"},{"key":"B39","doi-asserted-by":"publisher","first-page":"5683","DOI":"10.1038\/s41467-020-19414-4","article-title":"Single-cell analysis of developing and azoospermia human testicles reveals central role of Sertoli cells","volume":"11","author":"Zhao","year":"2020","journal-title":"Nat Commun"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1080\/14737159.2021.1924060","article-title":"Common markers of testicular Sertoli cells","volume":"21","author":"You","year":"2021","journal-title":"Expert Rev Mol Diagn"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.3389\/fendo.2023.1112834","article-title":"Throughout in vitro first spermatogenic wave: Next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants","volume":"14","author":"Dumont","year":"2023","journal-title":"Front Endocrinology"}],"container-title":["Frontiers in Endocrinology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fendo.2025.1664628\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T04:18:19Z","timestamp":1758773899000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fendo.2025.1664628\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,25]]},"references-count":41,"alternative-id":["10.3389\/fendo.2025.1664628"],"URL":"https:\/\/doi.org\/10.3389\/fendo.2025.1664628","relation":{},"ISSN":["1664-2392"],"issn-type":[{"value":"1664-2392","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,25]]},"article-number":"1664628"}}