人类原发性睾丸细胞器官的开发一直面临着挑战，主要是因为哺乳动物睾丸细胞组织结构的复杂性和培养方法的限制。本研究中，我们通过人类原发性睾丸细胞制备了睾丸器官样结构。随后，我们评估了干细胞因子（SCF）对睾丸器官模型中细胞分化和凋亡的影响。我们从3名脑死亡捐赠者中获取了睾丸细胞。通过免疫细胞化学（ICC）、RT-PCR和流式细胞术对人类精原干细胞（SSCs）进行了特性鉴定。我们使用悬滴培养法从原发性睾丸细胞中制备了睾丸器官样结构，并将其分为三组进行培养：对照组、实验组1（用FSH和维甲酸（RA）处理）和实验组2（用FSH、RA和SCF处理），培养时间为五周。我们通过RT-qPCR方法评估了SCP3（同源染色体联会复合体蛋白3）作为减数分裂基因和PRM2（精子后减数分裂标记物）以及凋亡基因Bax（Bcl-2相关X蛋白）和Bcl-2（B细胞淋巴瘤2）的表达情况。此外，我们还通过免疫组化学方法鉴定了PRM2的表达情况。SCP3、PRM2和Bcl-2的相对表达在培养五周后实验组2中最高，而BAX的表达水平在实验组2中较低。免疫组化学分析显示，在实验组2中，PRM2作为精子后减数分裂标记物的表达最高，优于二维培养和对照组，但实验组1和实验组2之间不存在显著差异。形态学评估显示，器官样结构是紧凑的球形结构，周边区域由未特异性的细长成纤维细胞状细胞组成。我们的研究结果表明，睾丸器官培养系统促进了精原干细胞（SSC）的分化，尤其是在SCF存在的情况下。发展出的器官样结构能够重现干细胞微环境的许多重要特性。©2023国际人工器官和移植中心（ICAOT）和Wiley Periodicals有限公司。

Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model.The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC).Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells.Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.© 2023 International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.