具有高度分室的管状管腔结构及其冷冻保存的小鼠睾丸类器官的产生

睾丸类器官具有维持男性生育能力甚至恢复男性不育症的巨大潜力。但是，现有的关于具有特定于睾丸的结构和功能的器官的研究很少，并且有许多局限性。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导致复苏或再生器官的特性丧失，从而使它们不适合临床或研究需求。在本文中，我们研究了小鼠年龄和细胞数对低吸附板中睾丸细胞自我聚集的影响。各种培养基组成，培养系统和细胞数被用于培养细胞球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器官的自组织。我们确定了针对睾丸细胞，细胞球和组织的适当冷冻保存条件。随后，比较了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，并通过组织学和免疫荧光染色进行比较和评估。结果表明，由2周龄小鼠的30×104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腔内结构和细胞分布高度相似的类器官。这种转化发生在使用琼脂糖水凝胶培养系统中的10％基因敲除血清替代剂（KSR）的α-MEM培养基中的14天内进行。此外，将Sertoli细胞紧密连接以形成血液测尾屏障。管状面积，生殖细胞，Sertoli细胞和周围肌动物细胞的相对速率分别为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与由α-MEM组成的冷冻冷冻速度缓慢冷冻，该冷冻保护剂由α-MEM组成10％二甲基亚氧化二甲基（DMSO）。对于所有含有5％DMSO和5％乙二醇（EG）的冷冻冻结剂对于所有不同体积的睾丸细胞球是最佳的。与由冷冻睾丸组织和细胞球产生的睾丸类器官相比，冻结睾丸细胞在维持器官分化特性和细胞细胞相互作用方面被证明是最有效的。这项研究的发现有助于一种在体外培养方案中的“通用”睾丸类器官，并有望在青春期前癌症患者和成年不育患者中保存和恢复生育能力和恢复。

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.