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Browsing by Author "David, Susana"

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  • 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
    Publication . David, Susana; Mandal, Manoj; Anes, Elsa; Pires, David
    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.
    2025-01-24Conference object Open access Show more
  • 2023-11-23Conference poster not in proceedings Open access Show more
  • Development and characterisation of a 3D cell culture model of the tuberculosis granuloma
    Publication . Pires, David; David, Susana; Mandal, Manoj; Soderberg, Julia; Anes, Elsa
    Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Mtb) that results in 1.6 million deaths yearly. The TB granuloma is the hallmark cellular structure of latent TB that contains the spread of infection1 . More comprehensive in vitro models of TB that better resemble the cellular and immunoregulatory complexity of the granuloma would facilitate the study of the interplay between the bacteria and the different immune system cells2 . We aim to generate an in vitro, 3D cell culture model of the TB granuloma that can be easily implemented using readily available commercial reagents and materials. A commercial encapsulation system based on sodium cellulose sulphate (NaCS) and Poly (diallyldimethylammonium chloride) (PDADMAC)3 was used to generate small capsules containing human peripheral blood mononuclear cells (PBMC) in the presence of GFPexpressing Mtb H37Rv and maintained in culture for several weeks. The 3D structure formed by the cells inside and outside the capsules was evaluated by fluorescence microscopy and flow cytometry to distinguish the different cell types, and how they are organised inside the sphere and to measure cell survival and bacteria replication. The results show that human PBMCs readily form 3D cellular aggregates around infected cells and that cells cultivated outside the capsules are attracted and surround the capsules in response to infection. The model could be maintained for several weeks before bacteria-induced cell necrosis. PBMC’s viability remained stable, with more than 80 % live cells following two weeks of culture. Moreover, adding an exterior layer of cells helped control bacterial replication, suggesting relevant communication between cells inside and outside the capsules to control the infection.
    2023-12-01Conference object Open access Show more