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. 2020 Nov 20;10(22):e3820.
doi: 10.21769/BioProtoc.3820.

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

Ainhoa Arbués  1   2 , Michael Kammüller  3 , Damien Portevin  1   2
Affiliations

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

Ainhoa Arbués et al. Bio Protoc. .

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 dormant-like 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.

Keywords: Dormancy; Granuloma; Host; In vitro model; Mycobacterium; Resuscitation; Tuberculosis.

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Conflict of interest statement

Competing interestsDP received the funding to implement this model from Novartis AG. The funders initiated the study design, but had no role in data collection and analysis. MK is a full-time employee of Novartis.

Figures

Figure 1.
Figure 1.. Schematic representation of the 3D in vitro granuloma model.
A. Mtb-infected PBMCs are embedded in an extracellular matrix (ECM). Formation of granuloma-like structures can be observed after 7-8 days. B. PBMCs can be released from the ECM by collagenase digestion. C. Subsequent treatment with Triton X-100 allows the retrieval of Mtb.
See this image and copyright information in PMC

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