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

Magdalena K Bielecka,Francis Drobniewski,Paul Elkington,Liku B Tezera,Suwan Jayasinghe,Robert Zmijan,Xunli Zhang,Carol A Nacy

doi:10.1128/mbio.02073-16

Abstract

ABSTRACTAntimicrobial 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.

Highlights

Antimicrobial resistance presents one of the most significant threats to human health, with the emergence of totally drug-resistant organisms
Considering these concepts together, we concluded that a transformative system to address the threat of antimicrobial resistance requires the following elements: primary host cells infected with fully virulent bacteria, culturing within a 3D structure that incorporates a physiological extracellular matrix, and pharmacokinetic modeling of drug concentrations
Multiple stressrelated genes were upregulated at day 14 in comparison with M. tuberculosis in 7H9 broth culture analyzed by reverse transcription-quantitative PCR (RT-qPCR) (Fig. 1C), including lipF, the acid stress real-time response gene; recA encoding recombinase A, the key mediator of the SOS response to DNA damage; relA, the nutrient stress-related gene; and sodA, the oxidative-stress response gene

Summary

Introduction

Antimicrobial resistance presents one of the most significant threats to human health, with the emergence of totally drug-resistant organisms. The vast majority of in vitro studies are done in the absence of human cells, without an extracellular matrix, and at static antibiotic concentrations Considering these concepts together, we concluded that a transformative system to address the threat of antimicrobial resistance requires the following elements: primary host cells infected with fully virulent bacteria, culturing within a 3D structure that incorporates a physiological extracellular matrix, and pharmacokinetic modeling of drug concentrations. These criteria represent a significant challenge in the context of virulent organisms because of the high biosafety containment level required and the complexity of bacteria being eluted under flow conditions. The host-pathogen interaction is spatially organized [21] and the extracellular matrix influences host cell survival [22], suggesting that a fully humanized system structured in three dimensions with an extracellular matrix is needed to identify novel treatments for TB

Methods

Results

Conclusion