睾丸类器官在维持男性生育能力甚至恢复男性不育方面具有巨大潜力。然而，现有的关于生成具有睾丸特异性结构和功能的类器官的研究很少，并且存在许多局限性。睾丸类器官冷冻保存的研究更加有限，不适当的冷冻保存方法可能会导致复苏或再生的类器官特性丧失，使其不适合临床或研究需要。在本文中，我们研究了小鼠年龄和细胞数量对睾丸细胞在低吸附板中自聚集成球体的影响。使用不同的培养基成分、培养系统和细胞数量将细胞球培养14天以形成睾丸类器官，并通过组织学和免疫荧光染色评估类器官的自组织。我们确定了睾丸细胞、细胞球和组织的适当冷冻保存条件。随后，通过组织学和免疫荧光染色对源自冷冻保存的睾丸组织、睾丸细胞和睾丸细胞球的类器官进行比较和评估。结果表明，由2周龄小鼠的30×104个睾丸细胞组成的睾丸细胞球能够形成与天然小鼠睾丸组织的管腔结构和细胞分布高度相似的类器官。使用琼脂糖水凝胶培养系统，在含有 10% 敲除血清替代物 (KSR) 的 α-MEM 培养基中孵育 14 天，发生这种转化。此外，支持细胞紧密相连，形成血睾屏障。肾小管面积、生殖细胞、支持细胞和管周肌样细胞的相对比率分别为36.985%±0.695、13.347%±3.102、47.570%±0.379和27.406%±1.832。原代睾丸细胞的最佳冷冻保存方案涉及使用由 α-MEM 和 10% 二甲基亚砜 (DMSO) 组成的冷冻保护剂缓慢冷冻。对于所有不同体积的睾丸细胞球，使用含有 5% DMSO 和 5% 乙二醇 (EG) 的冷冻保护剂缓慢冷冻是最佳选择。与由冷冻睾丸组织和细胞球产生的睾丸类器官相比，冷冻睾丸细胞被证明在维持类器官分化特征和细胞间相互作用方面最有效。这项研究的结果有助于建立“通用”睾丸类器官体外培养方案，在青春期前癌症患者和成年不孕患者的生育力保存和恢复方面具有广阔的应用前景。版权所有 © 2024。由爱思唯尔公司出版。

Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ± 0.695, 13.347 % ± 3.102, 47.570 % ± 0.379, and 27.406 % ± 1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.Copyright © 2024. Published by Elsevier Inc.