Testicular Organoid Formation in Microwell Culture

Testicular organoids present an exciting 3D in vitro platform to bridge the gap between 2D culture and animal models in male reproduction research, allowing studies on testicular cell-cell interactions, morphogenesis, development, and the spermatogonial stem cell microenvironment in conditions that are more physiologically relevant. Therefore, research with testicular organoids offers opportunities for fertility preservation, disease modeling, and high throughput reproductive toxicity screening. Our laboratory has developed a simple and reproducible protocol using microwell plates, which facilitate the aggregation of single cells and promote the generation of thousands of homogenous organoids that recapitulate testicular cytoarchitecture and functions. In this protocol, a testicular cell suspension is obtained by enzymatic digestion of immature testes and centrifuged into pyramid-shaped microwells, where cells will aggregate and form organoids after a few days in culture. Here we detail our standard protocol for the generation of porcine testicular organoids, which can also be applied to other mammalian species.