We have recently developed a 3D culture system that allows the reorganization of rat primary testicular cells into organoids with a functional blood-testis barrier, as well as the establishment and maintenance of germ cells. The innovative aspect of our model, the three-layer gradient system (3-LGS), comprises cells combined with Matrigel placed between two layers of Matrigel without cells, which creates a gradient of cells and allows the reorganization of testicular cells into organized structures after 5-7 d in culture. This reorganization is not observed when testicular cells are suspended in only one layer of Matrigel, the methodology used in the majority of the protocols for generating organoids. The model can be applied to follow and quantify cell migration during testicular organoid formation, and to explore the role of growth factors and the toxic effects of drugs and environmental contaminants on germ cell maintenance and blood-testis barrier integrity. The 3-LGS is a robust and reproducible method that requires a small volume of Matrigel and a low number of cells (16 μl and 132,000 cells, respectively), enabling and facilitating high-throughput analysis of germ-to-somatic cell associations in vitro.

中文翻译：

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三层细胞梯度系统在大鼠睾丸类器官生成中的应用。

我们最近开发了一种3D培养系统，该系统可以将大鼠原发性睾丸细胞重组为具有功能性血液-睾丸屏障的类器官，并可以建立和维持生殖细胞。我们模型的创新之处在于三层梯度系统（3-LGS），该结构包含与基质胶结合的细胞，该基质置于两层基质胶之间，而没有细胞，从而形成了细胞梯度，并允许睾丸细胞在重组后重组为有组织的结构培养5-7天。当睾丸细胞仅悬浮在Matrigel的一层中时，未观察到这种重组，Matrigel是用于产生类器官的大多数方案中使用的方法。该模型可用于追踪和量化睾丸类器官形成过程中的细胞迁移，并探讨生长因子的作用以及药物和环境污染物对生殖细胞维持和血液-睾丸屏障完整性的毒性作用。3-LGS是一种可靠且可重现的方法，需要少量的Matrigel和少量的细胞（分别为16μl和132,000个细胞），从而能够并促进体外对生殖细胞与体细胞的关联的高通量分析。