Setup and Analysis of Human Tonsil and Adenoid-Based Immune Organoids for Studying T and B Cell-Mediated Adaptive...

In recent years, demand for human-based in vitro models has grown substantially. In this regard, human secondary lymphoid organ (SLO)-derived ex vivo immune organoids are gaining recognition, as they provide a platform for studying highly differentiated, tissue-specific immune cell subsets found in SLOs, such as lymph nodes, tonsils, or spleen. These systems support the study of adaptive immune responses mediated by T and B cells, e.g., due to the presence of T follicular helper cells and germinal center B cells within these immune organoids. Here, we describe procedures for the isolation of human tonsil- or adenoid-derived immune cells, their cryopreservation and thawing, the establishment of immune organoids in vitro, and a detailed flow cytometry protocol for longitudinal phenotypic profiling of the immune organoids. In summary, these protocols serve as a foundation for downstream functional assays and complementary analyses in a human-first approach. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cell isolation from human tonsils and adenoids Basic Protocol 2: Thawing of cryopreserved tonsil and adenoid cells and establishment of immune organoids Basic Protocol 3: Flow cytometry for longitudinal phenotypical profiling of human tonsil/adenoid-derived immune organoids.