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

Abstract

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.