Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-Host Interaction

Abstract

Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma's environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the singular advantage of recapitulating dormantlike Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments, which have not been reported in other published protocols to generate in vitro granulomas.

Soluble immune mediators orchestrate protectivein vitrogranulomatous responses acrossMycobacterium tuberculosiscomplex lineages

“…PBMCs from four healthy blood donors were infected independently with the selected MTBC isolates, and subsequently embedded within an extracellular matrix (ECM) composed of collagen and fibronectin, the main components of the lung extracellular matrix, as previously reported (Arbues et al, 2020b). Briefly, an ECM solution composed of 950 μl/ml of collagen (PureCol, 3 mg/ml; Advanced BioMatrix), 50 μl/ml of PBS 10X, 4 μl/ml of human fibronectin (1 mg/ml; Sigma-Aldrich), and 10 μl of 1 N NaOH (Sigma-Aldrich) was prepared and incubated at 4°C for 90 min.…”