A Bioengineered Three-Dimensional Cell Culture Platform Integrated with Microfluidics To Address Antimicrobial Resistance in Tuberculosis

Abstract

Antimicrobial resistance presents one of the most significant threats to human health, with the emergence of totally drug-resistant organisms. We have combined bioengineering, genetically modified bacteria, longitudinal readouts, and fluidics to develop a transformative platform to address the drug development bottleneck, utilizing Mycobacterium tuberculosis as the model organism. We generated microspheres incorporating virulent reporter bacilli, primary human cells, and an extracellular matrix by using bioelectrospray methodology. Granulomas form within the three-dimensional matrix, and mycobacterial stress genes are upregulated. Pyrazinamide, a vital first-line antibiotic for treating human tuberculosis, kills M. tuberculosis in a three-dimensional culture but not in a standard two-dimensional culture or Middlebrook 7H9 broth, demonstrating that antibiotic sensitivity within microspheres reflects conditions in patients. We then performed pharmacokinetic modeling by combining the microsphere system with a microfluidic plate and demonstrated that we can model the effect of dynamic antibiotic concentrations on mycobacterial killing. The microsphere system is highly tractable, permitting variation of cell content, the extracellular matrix, sphere size, the infectious dose, and the surrounding medium with the potential to address a wide array of human infections and the threat of antimicrobial resistance.

Learning with uncertainty for biological discovery and design

“…We further validated these results in a more complex, host-pathogen model. We utilized a previously described assay for monitoring intracellular growth where macrophages are infected with luxMtb and luminescence is measured as a proxy of bacterial growth [69], [70] (Methods).…”

“…We utilized wild-type H37Rv and H37Rv expressing an integrated copy of the luxABCDE cassette which enables mycobacteria to endogenously produce light [70]; monitoring luminescence of the latter strain has been demonstrated to correlate well with the standard colony forming unit assay [69].…”

A modular microscale granuloma model for immune-microenvironment signaling studiesin vitro

“…(Peyron et al 2008) However, many of these in vitro models consist of granulomas grown inside of well plates(Peyron et al 2008; Birkness et al 2007; Kapoor et al 2013; Puissegur et al 2004; Crouser et al 2017), limiting the ability of the researchers to easily manipulate the microenvironment of the granulomas and increase the complexity of their granuloma models through multiculture and introduction of key components of the microenvironment on demand. Recently, more complex, biomimetic models have been developed that have successfully recapitulated important biological phenomena(Venkata Ramanarao Parasa et al 2014; Venkata R. Parasa et al 2017) and examined novel therapeutic approaches to combat TB infection (Tezera et al 2017; Bielecka et al 2017), while simultaneously demonstrating innovative and tractable platforms. However, these models face limitations in studies where users wish to subject granulomas to various microenvironmental cues over time, or in enabling the addition of tissue components after the model is established.…”

Drug permeation and metabolism in Mycobacterium tuberculosis: Prioritising local exposure as essential criterion in new TB drug development

“…Towards this end, a number of factors may have to be considered in ensuring clinical relevance (BOX 2). In this context, it is worth noting the parallel development and application to Mtb infection studies of increasingly sophisticated systems for three-dimensional cell culture (135,136) since these might offer a useful intermediate in bridging the in vitro / in vivo divide.…”