小鼠睾丸类器官的构建及其冷冻保存

睾丸类器官在维持男性生育能力乃至恢复男性不育方面具有巨大潜力。然而，现有关于构建具有特定睾丸结构和功能的类器官的研究仍然稀少且存在许多限制。关于睾丸类器官冷冻保存的研究更为有限，不当的冷冻方法可能导致复苏或再生的类器官失去其特性，从而无法满足临床或科研的需求。本研究探讨了小鼠年龄和细胞数量对低吸附培养板中睾丸细胞自聚集形成球体的影响。采用多种培养基组成、培养系统和细胞数量，培养细胞球体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能力。我们确定了适合睾丸细胞、细胞球体和组织的冷冻保存条件。随后，将冷冻保存的睾丸组织、睾丸细胞及睾丸细胞球体衍生的类器官进行比较和评估。结果显示，来自2周龄小鼠的30×10^4个睾丸细胞组成的细胞球体，在α-MEM培养基（含10%敲除血清替代品KSR）和琼脂糖水凝胶培养系统中，经过14天培养，能形成与天然小鼠睾丸组织高度相似的腔道结构和细胞分布的类器官。此外，Sertoli细胞紧密连接，形成血睾屏障。腔道面积、配子细胞、Sertoli细胞和周小管平滑肌细胞的相对比例分别为36.985% ± 0.695、13.347% ± 3.102、47.570% ± 0.379和27.406% ± 1.832。主要睾丸细胞的最佳冷冻方案为缓慢冷冻，使用含有10%二甲基亚砜(DMSO)的α-MEM作为细胞保护剂。含有5% DMSO和5%乙二醇(EG)的冷冻保护剂对不同体积的睾丸细胞球体均表现出最佳效果。与冷冻睾丸组织和细胞球体相比，冷冻睾丸细胞在维持类器官的分化特征和细胞间相互作用方面表现更优。本研究成果为“通用”睾丸类器官的体外培养提供了基础，有望在青春期前癌症患者及成人不育患者的生育能力保护和恢复中发挥重要作用。

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.