Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.