Summary Some bacteria have coevolved to establish symbiotic or pathogenic relationships with plants, animals or humans. With human association, the bacteria can cause a variety of diseases. Thus, understanding bacterial phenotypes at the single‐cell level is essential to develop beneficial applications. Traditional microbiological techniques have provided great knowledge about these organisms; however, they have also shown limitations, such as difficulties in culturing some bacteria, the heterogeneity of bacterial populations or difficulties in recreating some physical or biological conditions. Microfluidics is an emerging technique that complements current biological assays. Since microfluidics works with micrometric volumes, it allows fine‐tuning control of the test conditions. Moreover, it allows the recruitment of three‐dimensional (3D) conditions, in which several processes can be integrated and gradients can be generated, thus imitating physiological 3D environments. Here, we review some key microfluidic‐based studies describing the effects of different microenvironmental conditions on bacterial response, biofilm formation and antimicrobial resistance. For this aim, we present different studies classified into six groups according to the design of the microfluidic device: (i) linear channels, (ii) mixing channels, (iii) multiple floors, (iv) porous devices, (v) topographic devices and (vi) droplet microfluidics. Hence, we highlight the potential and possibilities of using microfluidic‐based technology to study bacterial phenotypes in comparison with traditional methodologies.
Macrophages play an essential role in the process of recognition and containment of microbial infections. These immune cells are recruited to infectious sites to reach and phagocytose pathogens. Specifically, in this article, bacteria from the genus Mycobacterium, Salmonella and Escherichia, were selected to study the directional macrophage movement towards different bacterial fractions. We recreated a three-dimensional environment in a microfluidic device, using a collagen-based hydrogel that simulates the mechanical microarchitecture associated to the Extra Cellular Matrix (ECM). First, we showed that macrophage migration is affected by the collagen concentration of their environment, migrating greater distances at higher velocities with decreasing collagen concentrations. To recreate the infectious microenvironment, macrophages were exposed to lateral gradients of bacterial fractions obtained from the intracellular pathogens M. tuberculosis and S. typhimurium. Our results showed that macrophages migrated directionally, and in a concentration-dependent manner, towards the sites where bacterial fractions are located, suggesting the presence of attractants molecules in all the samples. We confirmed that purified M. tuberculosis antigens, as ESAT-6 and CFP-10, stimulated macrophage recruitment in our device. Finally, we also observed that macrophages migrate towards fractions from non-pathogenic bacteria, such as M. smegmatis and Escherichia coli. In conclusion, our microfluidic device is a useful tool which opens new perspectives to study the recognition of specific antigens by innate immune cells.
Extravasation of circulating cells is an essential process that governs tissue inflammation and the body's response to pathogenic infection. To initiate anti-inflammatory and phagocytic functions within tissues, immune cells must cross the vascular endothelial barrier from the vessel lumen to the subluminal extracellular matrix. In this work, we present a microfluidic approach that enables the recreation of a three-dimensional, perfused endothelial vessel formed by human endothelial cells embedded within a collagen-rich matrix. Monocytes are introduced into the vessel perfusate, and we investigate the role of luminal flow and collagen concentration on extravasation. In vessels conditioned with the flow, increased monocyte adhesion to the vascular wall was observed, though fewer monocytes extravasated to the collagen hydrogel. Our results suggest that the lower rates of extravasation are due to the increased vessel integrity and reduced permeability of the endothelial monolayer. We further demonstrate that vascular permeability is a function of collagen hydrogel mass concentration, with increased collagen concentrations leading to elevated vascular permeability and increased extravasation. Collectively, our results demonstrate that extravasation of monocytes is highly regulated by the structural integrity of the endothelial monolayer. The microfluidic approach developed here allows for the dissection of the relative contributions of these cues to further understand the key governing processes that regulate circulating cell extravasation and inflammation.
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