Edmonds Maxwell E, Forshee Micah D, Woodruff Teresa K
Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University.
Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University;
J Vis Exp. 2020 Oct 7(164). doi: 10.3791/61403.
Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.
睾丸类器官为体外研究睾丸发育、精子发生和内分泌学提供了一种工具。为了创建睾丸类器官，已经开发了几种方法。这些方法中的许多都依赖细胞外基质（ECM）来促进从头组织组装，然而，在组织的仿生形态和功能方面，不同方法之间存在差异。此外，已发表方法的直接比较很少。在这里，通过研究类器官生成方案的差异并给出相应结果进行直接比较。描述了四种典型的生成方法：（1）无ECM的二维培养，（2）有ECM的二维培养，（3）无ECM的三维培养，以及（4）有ECM的三维培养。使用三个主要基准来评估睾丸类器官的生成。这些基准是细胞自组装、主要细胞类型（支持细胞、间质细胞、生殖细胞和睾丸周细胞）的包含情况，以及适当分区的组织结构。在所测试的四种环境中，二维ECM培养和无ECM的三维培养产生的类器官内部形态与天然睾丸最相似，包括管状细胞与间质细胞类型的从头分区、管状结构的发育以及已建立的长期内分泌功能。所有研究方法都使用了未分选的原代小鼠睾丸细胞悬液，并使用了常见的可获取培养资源。这些睾丸类器官生成技术为体外睾丸器官发生和生理学的研究计划提供了一个高度可获取且可重复的工具包。
