An improved organotypic cell culture system to study tissue-resident macrophages ex vivo

Tissue-resident macrophages (TRMs) perform organ-specific functions that are dependent on factors such as hematopoietic origin, local environment, and biological influences. A diverse range of in vitro culture systems have been developed to decipher TRM functions, including bone marrow-derived macrophages (BMDMs), induced pluripotent stem cell (iPSC)-derived TRMs, or immortalized cell lines. However, despite the usefulness of such systems, there are notable limitations. Attempts to culture primary macrophages often require purification of cells and lack a high cell yield and consistent phenotype. Here, we aimed to address these limitations by establishing an organotypic primary cell culture protocol. We obtained long-term monocultures of macrophages derived from distinct organs without prior purification using specific growth factors and tissue normoxic conditions that largely conserved a TRM-like identity in vitro. Thus, this organotypic system offers an ideal screening platform for primary macrophages from different organs that can be used for a wide range of assays and readouts. [Display omitted] •A modified cell culture protocol allows long-term culture of TRM-like cells•Protocols have been developed for brain, liver, peritoneum, and lung•TRM-like cells maintain core features of their TRM counterparts in vivo•TRM-like cells can be used for various functional assays in vitro Efficient cell culture systems, especially for primary TRMs, are rare, and most of them have limitations such as low cell yield or artificial cell activation. In this study, we aimed to address several of these limitations and establish an organotypic cell culture protocol that can be easily established in many labs and applied to many different TRMs. With this cell culture approach, we would like to provide a reliable source for immunologists to study primary TRMs from different organs in vitro. In vitro cultures are essential to study tissue-resident macrophages (TRMs) but are hampered by low cell yields or other limitations. To provide reliable TRM sources in vitro, Aktories et al. established cell culture protocols for TRM-like cells from brain, liver, peritoneum, and lung and subsequently employed TRM-like cells for functional assays.