{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T12:12:17Z","timestamp":1781871137498,"version":"3.54.5"},"reference-count":42,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2025,3,8]],"date-time":"2025-03-08T00:00:00Z","timestamp":1741392000000},"content-version":"vor","delay-in-days":7,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2025,3]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Tuberculosis (TB) remains one of the leading infectious causes of death worldwide. Persistent bacterial populations in specific microenvironments within the host hamper efficient TB chemotherapy. Caseum in the necrotic core of closed granulomas and cavities of pulmonary TB patients can harbor high burdens of drug\u2010tolerant <jats:italic>Mycobacterium tuberculosis<\/jats:italic> (MTB) bacilli, making them particularly difficult to sterilize. Here, we describe protocols for the generation of a surrogate matrix using lipid\u2010rich macrophages to mimic the unique composition of caseum <jats:italic>in vivo<\/jats:italic>. Importantly, this caseum surrogate induces metabolic and physiological changes within MTB that reproduce the nonreplicating drug\u2010tolerant phenotype of the pathogen in the native caseous environment, making it advantageous over alternative <jats:italic>in vitro<\/jats:italic> models of nonreplicating persistent (NRP) MTB. The protocols include culture of THP\u20101 monocytes, stimulation of lipid droplet accumulation, lysis and denaturation of the foamy macrophages, inoculation and preadaptation of MTB bacilli in the caseum surrogate, and evaluation of drug bactericidal activity against the NRP population. This novel <jats:italic>in vitro<\/jats:italic> model is being used to screen for potent bactericidal antimicrobial agents and to identify vulnerable drug targets, among a variety of other applications, thereby reducing our reliance on <jats:italic>in vivo<\/jats:italic> models. \u00a9 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Caseum surrogate preparation from \u03b3\u2010irradiated <jats:italic>M. tuberculosis<\/jats:italic>\u2013induced foamy THP\u20101 monocyte\u2013derived macrophages (THPMs)<\/jats:p><jats:p><jats:bold>Alternate Protocol 1<\/jats:bold>: Caseum surrogate preparation from stearic acid\u2013induced THPMs<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Generation of nonreplicating persistent <jats:italic>M. tuberculosis<\/jats:italic> and drug susceptibility testing<\/jats:p><jats:p><jats:bold>Alternate Protocol 2<\/jats:bold>: Higher\u2010throughput drug susceptibility screening using caseum surrogate<\/jats:p>","DOI":"10.1002\/cpz1.70118","type":"journal-article","created":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T08:52:13Z","timestamp":1741769533000},"source":"Crossref","is-referenced-by-count":2,"title":["A Physiologically Relevant <i>In Vitro<\/i> Model of Nonreplicating Persistent <i>Mycobacterium tuberculosis<\/i> in Caseum"],"prefix":"10.1002","volume":"5","author":[{"given":"Min","family":"Xie","sequence":"first","affiliation":[{"name":"Center for Discovery and Innovation Hackensack Meridian Health  Nutley New Jersey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paulina","family":"Osiecki","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation Hackensack Meridian Health  Nutley New Jersey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suyapa","family":"Rodriguez","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation Hackensack Meridian Health  Nutley New Jersey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"V\u00e9ronique","family":"Dartois","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation Hackensack Meridian Health  Nutley New Jersey"},{"name":"Department of Medical Sciences 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