A 3d cell culture model of the tuberculosis granuloma that can be applied for host genetic studies in the context of a multicellular immunologic response to infection

Introduction: The granuloma is an inflammatory infiltrate of mononuclear cells. Some bacterial infections are characterized by the formation of granulomas as part of the immune response to contain the infection. Granuloma models have contributed valuable insights into the genetic basis of granuloma formation during infection. For example, IFNGR1 and IFNGR2 variants have been found to disrupt the immune response, resulting in impaired granuloma formation and increased susceptibility to diseases by Mycobacterium sp. More easily implemented comprehensive models would facilitate the study of the different immune mechanisms and help identify new diseaseassociated genes. Our objective is to generate an in vitro 3D cell culture model using human primary cells and microspheres to generate a stratified granuloma model for future use in genetic, immunological and drug discovery studies. Methods: A commercial system was used to encapsulate human peripheral blood mononuclear cells (PBMC) infected with GFP-expressing M. tuberculosis and maintained in culture for several weeks. The cellular constituents of these granulomas and their organization were characterized by fluorescence microscopy and flow cytometry as well as the viability of the cells and the extent of bacterial replication in factor of time. Results: The results demonstrate a ready recruitment of cells towards infected macrophages, leading to the formation of densely populated aggregates. These aggregates maintained cell viability for several weeks and displayed an enhanced control of bacterial replication compared to the more common monolayer infection models. Moreover, the capsules can be easily disrupted when required to isolate genetic material for further analysis. Conclusion: The proposed 3D model resembles some structural and cellular characteristics of the tuberculosis granuloma and maintains its stability beyond more common 2D models of infection. These preliminary results demonstrate that this model can be used to further explore the determinants of granuloma formation and host response to infection.