Biology of the Testis (BITE) laboratory, Genetics Reproduction and Development (GRAD) research group, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Laboratory for Molecular and Cellular Therapy (LMCT), Translational Oncology Research Center (TORC), VUB, Brussels, Belgium.
Biofabrication. 2024 Aug 14;16(4). doi: 10.1088/1758-5090/ad618f.
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 comparing-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 CD45immune 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 × 10cells under static conditions. Switching to-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) and generation ofH2AXspermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to the-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.
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缺失。利用琼脂糖微井从青春期前的 C57BL/6 J 睾丸细胞中创建 TO。首先强调的是通过比较支持小鼠 TO 生成的-MEM + 10% 无血清替代物 (KSR) 培养基，在最初的 2 周重组阶段提高生殖细胞的存活率，到三种优化的培养基 (1-3)。还测试了细胞密度和培养动力学，以重现与睾丸相似的组织学特征。在优化生殖细胞存活率和细胞组织后，评估生长因子和免疫调节的影响，通过 CD45 免疫细胞耗竭或地塞米松 (DEX) 补充，以增强随后的分化阶段的精子发生。睾丸细胞自行重新组织成类似于睾丸解剖单位的类器官，其特征是一个由间质包围的管状结构。培养基 1-3 在重组阶段证明对类器官生长更有利，培养基 3 中的 TO 显示生殖细胞数量（7.4%±4.8%）与对照（9.3%±5.3%）相当。此外，在静态条件下，37%±30%的细胞显示出有组织的组织学。在分化阶段切换到-MEM + 10% KSR 可将形成效率提高到 85%±7%，同时增加生殖细胞数量、睾酮产生（3.1±0.9ng/ml）和产生 H2AX 精子样细胞（步骤 8-11，总细胞的 1.2%±2.2%）。向-MEM 中添加分化因子可将精子样细胞数量增加到 2.9%±5.9%，通过对 CREM、过渡蛋白 1 和花生凝集素的阳性染色得到证实。尽管这些仍然是二倍体，核成熟不规则。DEX 补充没有额外的效果，免疫细胞耗竭对 TO 的形成有不利影响。TO 的可操作性在研究男性不育和探索疗法方面具有优势，可扩展性可实现高通量化学筛选，并减少生殖毒性和药物发现研究中动物的使用。
