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Establishing a 3D cell culture model of the tuberculosis granuloma as a drug-discovery platform

I Araújo(2) R Ferreira(1) R Pinheiro(1) E Anes(2) P JG Bettencourt(1) S David(2,3) D Pires(1,2,3)

1:Center for Interdisciplinary Research in Health, Católica Medical School, Universidade Católica Portuguesa, Rio de Mouro, 2635-631, Portugal.; 2:Host-Pathogen Interactions Unit, Research Institute for Medicines, iMed.ULisboa, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal.; 3:Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), Lisbon, Portugal.

The tuberculosis (TB) granuloma is the hallmark cellular structure of latent TB. More comprehensive in vitro models that better resemble the complexity of the granuloma would facilitate the discovery of more active drugs in the granuloma environment and provide a better understanding of bacteria and immune cell interplay.

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 generated small spheres containing human peripheral blood mononuclear cells (PBMC) infected with GFP-expressing Mtb H37Rv. The results show that human PBMCs readily form 3D cellular aggregates around infected cells. The model could be maintained for several weeks before bacteria-induced cell necrosis. Using different cell types inside or outside the spheres resulted in distinct bacterial replication, demonstrating the contribution of each cell type and suggesting relevant communication between cells inside and outside the spheres to control the infection. The expression of inflammatory genes and the generation of reactive oxygen and nitrogen intermediates increased with the multiplicity of infection. Moreover, bacteria in the 3D model depict increased resistance to isoniazid and rifampicin and increased susceptibility to pyrazinamide when compared to the usual 2D cell culture model.

In conclusion, the 3D infection model resembles some of the structural features and drug-susceptibility profile expected in the TB granuloma and significantly improves the duration of infection experiments. This model shows promise for future use in drug discovery studies.

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