Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of an in vitro culture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for the in vitro maintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 10⁶ cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 μm; the mean number of organoids was 5.57 ± 1.60/10⁵ μm² on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression of C-KIT but reduced the expression of GFRα1 at day 28 when compared to those without hormone supplements (p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishing in vitro spermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.

中文翻译：

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体外三维（3D）睾丸类器官培养系统，使用冷冻/解冻的新生牛睾丸组织进行有效的生殖细胞维持和繁殖

患有癌症的青春期前男孩的生育能力需要在性腺毒性治疗之前冷冻保存未成熟的睾丸组织（ITT）。然而，人类 ITT 活检中的生殖细胞数量有限，因此需要开发一种体外使用冻融 ITT 进行生殖细胞扩增的培养系统。在这里，我们生成了睾丸类器官体外冻融两周龄新生牛 ITT 的生殖细胞的维持和扩增。我们研究了不同的细胞接种密度、培养血清、接种方法和促性腺激素补充剂对富集性母细胞的维持和增殖的影响。我们的结果表明，冻融的新生儿 ITT 中富集的生殖母细胞和睾丸细胞可以在适当的基于基质胶的培养环境中在三天内自组装成球状类器官。为了实现青春期前睾丸类器官的最佳形成，建议接种密度为 1 × 10 ^{6 个细胞/孔，而不是其他密度。}该策略产生平均直径为 60.53 ± 12.12 μm 的类器官；第11天类器官的平均数量为5.57±1.60/10 ⁵ μm ^{2 。}冻融后类器官的活力保持在79.75±2.99%。在培养基中添加胶质细胞源性神经营养因子、成纤维细胞生长因子2和白血病抑制因子，增加了类器官中KI67阳性增殖细胞的比例，提高了C-KIT但减少了表达肾小球滤过率α1第 28 天与未补充激素的人相比（p< 0.05）。此外，在培养基中补充促卵泡激素和睾酮有助于维持显着较高的活力（p< 0.05) 在第 28 天的 ITT 类器官中。这些类器官可以冷冻保存并根据需要解冻。ITT 类器官的成功生成为建立体外精子发生、繁殖人类生殖细胞、研究睾丸生理学和生殖细胞肿瘤的起源，以及测试新药在未来临床应用中的毒性。