{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T14:58:00Z","timestamp":1774018680678,"version":"3.50.1"},"reference-count":50,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T00:00:00Z","timestamp":1773964800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000030","name":"Centers for Disease Control and Prevention","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000030","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>\n                    Tuberculomas are the conglomeration of tuberculous granulomas into structurally organized three-dimensional (3D) masses that result from\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    infection and represent one of the more severe morphological forms of tuberculosis (TB). Several\n                    <jats:italic>in vitro<\/jats:italic>\n                    models that mimic human TB granulomas have been reported to decipher complex host-pathogen interactions and to discover new prophylactic and therapeutic interventions. They serve as ethical bridge approaches to human studies. However, these models need improvements in generating well-organized granuloma lesions, classic tuberculoma structures, and relevant microenvironments. They are impractical for screening extensive chemical and genetic libraries owing to their low throughput, limited scalability, batch-to-batch variability, and high costs. Here, we describe a \u2018mycobacteria-in-spheroid\u2019 co-culture workflow in a standard 96-well plate format that generates a robust 3D cell culture model. This model reproduces key attributes and microenvironments in human tuberculomas and can be scaled up as a high-throughput screening (HTS)-compatible bioplatform. The tuberculoma-like structures generated encompass organized, florid granulomatous foci and exhibit solid, necrotic, and cavitary morphologies. This model can be developed using freshly isolated human primary cells or a monocytic cell line with virulent mycobacteria. The platform combines the entire workflow, from generation to imaging of tuberculoma-like structures,\n                    <jats:italic>in situ<\/jats:italic>\n                    . It permits the serial quantitation of drug efficacy and monitoring of lesion resolution over several days to weeks following a single treatment. Additionally, we outline a methodology for adopting this workflow for cryo-preservation, enhancing its potential for commercial application. The ease of generation, pliability, cryo-shelf stability, and reproducibility of the bioplatform make it ideal for HTS applications and for implementation in discovery programs for TB and other granulomatous diseases.\n                  <\/jats:p>","DOI":"10.3389\/fimmu.2026.1695558","type":"journal-article","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T13:18:10Z","timestamp":1774012690000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Methodology for a freshly engineered or cryo-preserved 3D tuberculoma bioplatform for studying tuberculosis biology and high-content screening of therapeutics"],"prefix":"10.3389","volume":"17","author":[{"given":"Suraj B.","family":"Sable","sequence":"first","affiliation":[{"name":"Division of Tuberculosis Elimination, National Center for HIV, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention","place":["Atlanta, GA, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Allison","family":"Kline","sequence":"additional","affiliation":[{"name":"Division of Tuberculosis Elimination, National Center for HIV, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention","place":["Atlanta, GA, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wen","family":"Li","sequence":"additional","affiliation":[{"name":"Division of Tuberculosis Elimination, National Center for HIV, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention","place":["Atlanta, GA, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James E.","family":"Posey","sequence":"additional","affiliation":[{"name":"Division of Tuberculosis Elimination, National Center for HIV, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention","place":["Atlanta, GA, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,20]]},"reference":[{"key":"B1","volume-title":"Global Tuberculosis Report 2025","year":"2025"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1038\/nri3211","article-title":"Revisiting the role of the granuloma in tuberculosis","volume":"12","author":"Ramakrishnan","year":"2012","journal-title":"Nat Rev Immunol"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-immunol-032712-100022","article-title":"The formation and function of granulomas","volume":"36","author":"Pagan","year":"2018","journal-title":"Annu Rev Immunol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1016\/j.molmed.2021.11.004","article-title":"Understanding the tuberculosis granuloma: the matrix revolutions","volume":"28","author":"Elkington","year":"2022","journal-title":"Trends Mol Med"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-024-01124-3","article-title":"How macrophage heterogeneity affects tuberculosis disease and therapy","volume":"25","author":"Russell","year":"2025","journal-title":"Nat Rev Immunol"},{"key":"B6","doi-asserted-by":"publisher","first-page":"798","DOI":"10.1016\/S0096-5588(20)31545-2","article-title":"Pulmonary tuberculomas; pathogenesis, diagnosis, and management","volume":"20","author":"Culver","year":"1950","journal-title":"J Thorac Surg"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1136\/thx.6.3.238","article-title":"Tuberculoma of the lung","volume":"6","author":"Moyes","year":"1951","journal-title":"Thorax"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1164\/artpd.1958.78.3.403","article-title":"Tuberculoma of the lung","volume":"78","author":"Sochocky","year":"1958","journal-title":"Am Rev Tuberc"},{"key":"B9","doi-asserted-by":"publisher","first-page":"795","DOI":"10.1016\/S1473-3099(05)70299-1","article-title":"Intracranial tuberculomas developing while on therapy for pulmonary tuberculosis","volume":"5","author":"Nicolls","year":"2005","journal-title":"Lancet Infect Dis"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.rmcr.2013.04.003","article-title":"Cerebral tuberculomas - A clinical challenge","volume":"9","author":"Monteiro","year":"2013","journal-title":"Respir Med Case Rep"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1183\/09031936.04.00087304","article-title":"Response of pulmonary tuberculomas to anti-tuberculous treatment","volume":"23","author":"Lee","year":"2004","journal-title":"Eur Respir J"},{"key":"B12","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1186\/s13244-021-01138-8","article-title":"Active pulmonary tuberculosis: something old, something new, something borrowed, something blue","volume":"13","author":"Wetscherek","year":"2022","journal-title":"Insights Imaging"},{"key":"B13","doi-asserted-by":"publisher","first-page":"691","DOI":"10.1038\/nri.2017.69","article-title":"Heterogeneity in tuberculosis","volume":"17","author":"Cadena","year":"2017","journal-title":"Nat Rev Immunol"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1164\/rccm.202101-0032OC","article-title":"Micro-computed tomography analysis of the human tuberculous lung reveals remarkable heterogeneity in three-dimensional granuloma morphology","volume":"204","author":"Wells","year":"2021","journal-title":"Am J Respir Crit Care Med"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20221392","article-title":"Spatial mapping reveals granuloma diversity and histopathological superstructure in human tuberculosis","volume":"220","author":"Sawyer","year":"2023","journal-title":"J Exp Med"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(20)30148-1","article-title":"Cavitary tuberculosis: the gateway of disease transmission","volume":"20","author":"Urbanowski","year":"2020","journal-title":"Lancet Infect Dis"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1038\/nm.3937","article-title":"The association between sterilizing activity and drug distribution into tuberculosis lesions","volume":"21","author":"Prideaux","year":"2015","journal-title":"Nat Med"},{"key":"B18","doi-asserted-by":"publisher","first-page":"e1002773","DOI":"10.1371\/journal.pmed.1002773","article-title":"Tuberculosis drugs\u2019 distribution and emergence of resistance in patient\u2019s lung lesions: A mechanistic model and tool for regimen and dose optimization","volume":"16","author":"Strydom","year":"2019","journal-title":"PloS Med"},{"key":"B19","doi-asserted-by":"publisher","first-page":"1046","DOI":"10.1016\/j.cels.2021.08.004","article-title":"Systematic measurement of combination-drug landscapes to predict in vivo treatment outcomes for tuberculosis","volume":"12","author":"Larkins-Ford","year":"2021","journal-title":"Cell Syst"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-022-00731-y","article-title":"Anti-tuberculosis treatment strategies and drug development: challenges and priorities","volume":"20","author":"Dartois","year":"2022","journal-title":"Nat Rev Microbiol"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-022-00734-z","article-title":"Host-directed immunotherapy of viral and bacterial infections: past, present and future","volume":"23","author":"Wallis","year":"2022","journal-title":"Nat Rev Immunol"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2004.00371.x","article-title":"An in vitro dual model of mycobacterial granulomas to investigate the molecular interactions between mycobacteria and human host cells","volume":"6","author":"Puissegur","year":"2004","journal-title":"Cell Microbiol"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1038\/sj.icb.7100019","article-title":"An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection","volume":"85","author":"Birkness","year":"2007","journal-title":"Immunol Cell Biol"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02537-14","article-title":"Characterization of host and microbial determinants in individuals with latent tuberculosis infection using a human granuloma model","volume":"6","author":"Guirado","year":"2015","journal-title":"mBio"},{"key":"B25","doi-asserted-by":"publisher","first-page":"e53657","DOI":"10.1371\/journal.pone.0053657","article-title":"Human granuloma in vitro model, for TB dormancy and resuscitation","volume":"8","author":"Kapoor","year":"2013","journal-title":"PloS One"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.21283","article-title":"Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model","volume":"6","author":"Tezera","year":"2017","journal-title":"Elife"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1165\/rcmb.2016-0321OC","article-title":"A novel in vitro human granuloma model of sarcoidosis and latent tuberculosis infection","volume":"57","author":"Crouser","year":"2017","journal-title":"Am J Respir Cell Mol Biol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.3389\/fbioe.2020.00931","article-title":"A modular microscale granuloma model for immune-microenvironment signaling studies in vitro","volume":"8","author":"Berry","year":"2020","journal-title":"Front Bioeng Biotechnol"},{"key":"B29","doi-asserted-by":"publisher","first-page":"e0055221","DOI":"10.1128\/mSphere.00552-21","article-title":"Establishment of a patient-derived, magnetic levitation-based, three-dimensional spheroid granuloma model for human tuberculosis","volume":"6","author":"Kotze","year":"2021","journal-title":"mSphere"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.03705-14","article-title":"High-content screening technology combined with a human granuloma model as a new approach to evaluate the activities of drugs against Mycobacterium tuberculosis","volume":"59","author":"Silva-Miranda","year":"2015","journal-title":"Antimicrob Agents Chemother"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02073-16","article-title":"A bioengineered three-dimensional cell culture platform integrated with microfluidics to address antimicrobial resistance in tuberculosis","volume":"8","author":"Bielecka","year":"2017","journal-title":"mBio"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.21769\/BioProtoc.2418","article-title":"Bioelectrospray methodology for dissection of the host-pathogen interaction in human tuberculosis","volume":"7","author":"Tezera","year":"2017","journal-title":"Bio Protoc"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jiz020","article-title":"In vitro granuloma models of tuberculosis: potential and challenges","volume":"219","author":"Elkington","year":"2019","journal-title":"J Infect Dis"},{"key":"B34","doi-asserted-by":"publisher","first-page":"e3820","DOI":"10.21769\/BioProtoc.3820","article-title":"Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-Host Interaction","volume":"10","author":"Arbues","year":"2020","journal-title":"Bio Protoc"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1002\/jbm.a.34946","article-title":"Oxygen diffusion and consumption in extracellular matrix gels: implications for designing three-dimensional cultures","volume":"102","author":"Colom","year":"2014","journal-title":"J BioMed Mater Res A"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1002\/term.2656","article-title":"Assessing glucose and oxygen diffusion in hydrogels for the rational design of 3D stem cell scaffolds in regenerative medicine","volume":"12","author":"Figueiredo","year":"2018","journal-title":"J Tissue Eng Regener Med"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.727508","article-title":"Extracellular matrix-induced GM-CSF and hypoxia promote immune control of mycobacterium tuberculosis in human in vitro granulomas","volume":"12","author":"Arbues","year":"2021","journal-title":"Front Immunol"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.820134","article-title":"In the thick of it: formation of the tuberculous granuloma and its effects on host and therapeutic responses","volume":"13","author":"Cronan","year":"2022","journal-title":"Front Immunol"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.57.030502.091033","article-title":"The secret lives of the pathogenic\u00a0mycobacteria","volume":"57","author":"Cosma","year":"2003","journal-title":"Annu Rev Microbiol"},{"key":"B40","doi-asserted-by":"publisher","first-page":"1757","DOI":"10.1016\/j.cell.2021.02.046","article-title":"A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization","volume":"184","author":"Cronan","year":"2021","journal-title":"Cell"},{"key":"B41","doi-asserted-by":"publisher","first-page":"693","DOI":"10.1016\/S1074-7613(02)00475-2","article-title":"Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos","volume":"17","author":"Davis","year":"2002","journal-title":"Immunity"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1172\/JCI57235","article-title":"In vitro differentiation of human macrophages with enhanced antimycobacterial activity","volume":"121","author":"Vogt","year":"2011","journal-title":"J Clin Invest"},{"key":"B43","doi-asserted-by":"publisher","first-page":"71ps6","DOI":"10.1126\/scitranslmed.3001847","article-title":"Tuberculosis immunopathology: the neglected role of extracellular matrix destruction","volume":"3","author":"Elkington","year":"2011","journal-title":"Sci Transl Med"},{"key":"B44","doi-asserted-by":"publisher","first-page":"827","DOI":"10.1016\/j.immuni.2022.04.004","article-title":"Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control","volume":"55","author":"Gideon","year":"2022","journal-title":"Immunity"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1101\/2025.08.27.672670","article-title":"Identification and characterization of host-directed therapeutics for tuberculosis using a versatile human 3D tuberculoma bioplatform","author":"Sable","year":"2025","journal-title":"bioRxiv"},{"key":"B46","doi-asserted-by":"publisher","first-page":"110826","DOI":"10.1016\/j.celrep.2022.110826","article-title":"T cell transcription factor expression evolves over time in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques","volume":"39","author":"Grant","year":"2022","journal-title":"Cell Rep"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.3390\/biomedicines9091209","article-title":"In vitro miniaturized tuberculosis spheroid model","volume":"9","author":"Mukundan","year":"2021","journal-title":"Biomedicines"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.1515\/bmc-2021-0013","article-title":"3D host cell and pathogen-based bioassay development for testing anti-tuberculosis (TB) drug response and modeling immunodeficiency","volume":"12","author":"Mukundan","year":"2021","journal-title":"Biomol Concepts"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.59961","article-title":"A lung-on-chip model of early Mycobacterium tuberculosis infection reveals an essential role for alveolar epithelial cells in controlling bacterial growth","volume":"9","author":"Thacker","year":"2020","journal-title":"Elife"},{"key":"B50","doi-asserted-by":"publisher","first-page":"e1012295","DOI":"10.1371\/journal.ppat.1012295","article-title":"Advances in an in vitro tuberculosis infection model using human lung organoids for host-directed therapies","volume":"20","author":"Kim","year":"2024","journal-title":"PloS Pathog"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1695558\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T13:18:12Z","timestamp":1774012692000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1695558\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,20]]},"references-count":50,"alternative-id":["10.3389\/fimmu.2026.1695558"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2026.1695558","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,20]]},"article-number":"1695558"}}