Abstract：
This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.
摘要：
这项研究提出了一种双相方法来克服当前睾丸类器官（TO）培养的局限性，包括组织学异质性，生殖细胞丢失和精子发生的缺乏。使用琼脂糖微孔从青春期前C57BL/6J睾丸细胞产生TOs。第一个重点是通过比较α-MEM+10%KSR培养基，在最初的2周重组阶段提高生殖细胞存活率，已知在小鼠中支持TO生成，到三个优化的介质(1-3)。还测试了细胞密度和培养动力学以重建与睾丸的组织学相似性。＆＃xD;优化生殖细胞存活和细胞组织后，通过CD45+免疫细胞清除或补充地塞米松(DEX),评估了生长因子和免疫调节在随后分化阶段促进精子发生的作用.睾丸细胞自我重组为类似睾丸解剖单元的类器官，以一个被间质包围的管状结构为特征。培养基13在重组阶段被证明具有优良的类器官生长，培养基3中的TOs表现出与对照（9.3±5.3％）相当的生殖细胞数量（7.4±4.8％）。此外,37±30%显示在静态条件下来自32×103个细胞的组织组织学。在分化阶段切换到α-MEM+10%KSR将形成效率提高到85±7%，随着生殖细胞数量的增加，睾酮产生（3.1±0.9ng/mL）和γH2AX+精子细胞样细胞的产生（步骤8-11，占总数的1.2±2.2％）。在α-MEM中添加分化因子可使精子细胞样细胞数量增加到2.9±5.9％，通过CREM阳性染色证实，TP1和PNA。虽然,这些仍然是二倍体，核成熟不规则。DEX补充没有额外的效果，和免疫细胞消耗不利地影响TO形成。＆＃xD;TOs的可操作性在研究男性不育和探索疗法方面提供了优势，具有可扩展性，可实现高通量化学筛选，并减少动物在生殖毒性和药物发现研究中的使用。
