Protocol for the generation and differentiation of thymic epithelial organoids from adult mouse thymus tissue

Here, we present a protocol for generating long-term expandable thymic epithelial cell (TEC) organoids from adult mouse thymus. We describe the steps for tissue isolation, dissociation, 3D culture matrix embedding, organoid maintenance, and passaging. We then detail procedures for TEC differentiation into cortical and medullary TEC lineages using defined growth factors, and techniques for analyzing organoids, including flow cytometry and qPCR. This protocol provides a valuable tool for studying TEC development and function. For complete details on the use and execution of this protocol, please refer to Lim et al.¹.