Generation of Organotypic Testicular Organoids from Rat and Pig Primary Cells in Microwell Culture.

Organoids are three-dimensional structures formed by multiple cell types that mimic the function of the respective organ in vivo. Testicular organoids are formed by testicular somatic cells and germ cells that retain tissue-specific architecture and function. Especially in male reproductive biology, testicular organoids present a platform to study cell-cell interactions, tissue development, and the germ-cell niche microenvironment. This not only holds opportunities for male fertility preservation, but it can also facilitate high-throughput drug and toxicity screening. The microwell aggregation technique presents a simple and reproducible approach for generating a large number of homogenous organoids in pyramid-shaped wells. Testes from prepubertal animals are digested, and the obtained testicular starting cell population is centrifuged into the wells and cultured to form organoids. Here, we describe our standard protocol for the generation of testicular organoids from primary rat or pig testicular cells in microwell culture, highlighting the difference between the two species and providing a strategy to enrich for germ cells.