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. 2017 Feb 7;8(1):e02073-16.
doi: 10.1128/mBio.02073-16.

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

Magdalena K Bielecka  1 , Liku B Tezera  1 , Robert Zmijan  2 , Francis Drobniewski  3 , Xunli Zhang  2   4 , Suwan Jayasinghe  5 , Paul Elkington  6   4
Affiliations

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

Magdalena K Bielecka et al. mBio. .

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.

Importance: Antimicrobial resistance is a major global threat, and an emerging concept is that infection should be studied in the context of host immune cells. Tuberculosis is a chronic infection that kills over a million people every year and is becoming progressively more resistant to antibiotics. Recent major studies of shorter treatment or new vaccination approaches have not been successful, demonstrating that transformative technologies are required to control tuberculosis. We have developed an entirely new system to study the infection of host cells in a three-dimensional matrix by using bioengineering. We showed that antibiotics that work in patients are effective in this microsphere system but not in standard infection systems. We then combined microspheres with microfluidics to model drug concentration changes in patients and demonstrate the effect of increasing antibiotic concentrations on bacterial survival. This system can be widely applied to address the threat of antimicrobial resistance and develop new treatments.

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Figures

FIG 1
FIG 1
Granulomas form within microspheres, and M. tuberculosis stress genes are upregulated. (A) Cellular distribution within microspheres. Primary human PBMCs were separated and fluorescently stained (monocytes blue [i], T cells green [ii]), recombined, and infected with mCherry-expressing M. tuberculosis (red [iii]). The overlay (iv) shows early granuloma development at day 4. (B) Large multicellular granulomas form at day 14 in infected microspheres (ii), which are not observed in uninfected microspheres (i), imaged by inverted microscopy. Scale bar, 50 μm. (C) M. tuberculosis stress genes are upregulated in the microsphere model compared to 7H9 broth culture. The expression of four stress-related mycobacterial genes was analyzed by RT-qPCR in microspheres at day 14 compared to exponentially growing M. tuberculosis (OD600 = 0.25) in 7H9 broth. The ΔΔCT method was used for relative quantification. Data are presented as fold changes normalized to the sigA gene. Data represent the mean results of three independent experiments ± the standard error of the mean. (D) M. tuberculosis growth in microspheres monitored by bacterial luminescence, demonstrating the typical M. tuberculosis luminescence kinetics of infected PBMCs within microspheres (black). Uninfected PBMCs in the microspheres do not luminesce (gray).
FIG 2
FIG 2
M. tuberculosis proliferation within microspheres is intracellular (A, B). PBMCs were infected with luminescent M. tuberculosis and incorporated into microspheres. Cells were released by decapsulation, and extracellular and cell-associated bacteria were separated by differential centrifugation. Open bars; extracellular mycobacteria; checkered bars; cell-associated mycobacteria. Mycobacterial location determined by luminescence and colony counting on 7H11 agar demonstrated that bacterial proliferation was principally cell associated (C). PBMCs were infected with GFP-expressing M. tuberculosis and incorporated into microspheres. Microspheres were decapsulated, and M. tuberculosis localization was analyzed by flow cytometry (i) Uninfected cells. GFP-expressing M. tuberculosis cells at time zero (ii), day 1 (iii), day 4 (iv), day 7 (v), and day 15 (vi) show progressive intracellular proliferation. Data are from a representative experiment performed on two occasions in triplicate (D). M. tuberculosis infection does not reduce cell viability within microspheres. Cellular survival was measured by the CellTiter-Glo 3D Cell Viability Assay. Data are the mean ± the standard error of the mean of an experiment performed in triplicate on two occasions. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.
FIG 3
FIG 3
Effects of standard antituberculosis antibiotics on M. tuberculosis growth. Antibiotics (rifampin [red, 1 µg/ml], INH [blue, 0.25 µg/ml], and ethambutol [orange, 4 µg/ml]) were added at day 6 to 2D PBMC cultures or the microsphere system, and M. tuberculosis growth was monitored by measuring luminescence. M. tuberculosis growth was inhibited by all of the antibiotics in both 2D cell cultures (A) and the 3D model (B). M. tuberculosis growth was unaffected in the control sample (black) or by the addition of DMSO (gray), which was used as a solvent for rifampin. Symbols: ×, background level of luminescence; black arrows, antibiotic addition. Data are the mean ± the standard error of the mean of an experiment performed in triplicate and are representative of three separate experiments. (C) M. tuberculosis luminescence closely correlates with CFU counts on Middlebrook 7H11 agar. Spearman r value = 0.94; P < 0.0001.
FIG 4
FIG 4
PZA kills M. tuberculosis in the 3D model but not in 7H9 broth or 2D cultures. (A) PZA has no effect on M. tuberculosis growth in 7H9 broth (dark green, 500 µg/ml) compared with an untreated control (black). (B) PZA has a brief effect on M. tuberculosis growth in 2D PBMC cell cultures at 60 µg/ml (light green), 100 µg/ml (medium green), or 500 µg/ml (dark green) in comparison with an untreated control (black), but M. tuberculosis growth rapidly recovers. (C) PZA kills M. tuberculosis in the 3D system at 500 µg/ml (dark green). Minimal killing of M. tuberculosis was observed when 60 µg/ml (light green) or 100 µg/ml (medium green) PZA was added, relative to the control sample (black). Symbols: ×, background level of luminescence; black arrows, antibiotic addition. Data are the mean ± the standard error of the mean of an experiment performed in triplicate and are representative of three separate experiments. (D) Colony counting on 7H11 agar confirms M. tuberculosis killing by PZA. Dilutions of control and 2D PZA plates start from a 1:10 dilution, while all other plates start without dilution. Representative plates are shown.
FIG 5
FIG 5
d-Cycloserine has similar effects on M. tuberculosis growth in 2D and 3D cultures. (A) M. tuberculosis in 7H9 broth. d-Cycloserine at a low concentration (20 µg/ml) had a temporary effect on M. tuberculosis growth (light purple) similar to that of INH at 0.25 µg/ml (blue). d-Cycloserine at 200 µg/ml killed M. tuberculosis more rapidly than (dark purple) and as effectively as moxifloxacin (brown, 5 µg/ml). Linezolid was the most effective second-line antibiotic (magenta, 24 µg/ml). The diluent DMSO (gray) did not affect M. tuberculosis growth relative to that in 7H9 broth only (black). (B) M. tuberculosis growth in 2D PBMC cultures. d-Cycloserine at both concentrations inhibited M. tuberculosis growth (purple) more rapidly than the other antibiotics (moxifloxacin [brown, 5 µg/ml], linezolid [magenta, 24 µg/ml], and INH [blue, 0.25 µg/ml]). Shown is M. tuberculosis growth in control samples (black) and with DMSO (gray). (C) M. tuberculosis growth in a 3D cell culture model. d-Cycloserine, linezolid, and moxifloxacin have an efficacy similar to that in a 2D cell culture (purple), while INH (blue) is more consistently bactericidal. Gray lines indicate background levels of luminescence. Black arrows indicate the day antibiotics were added. Data are the mean ± the standard error of the mean of an experiment performed in triplicate and are representative of three separate experiments.
FIG 6
FIG 6
Modeling of antibiotic pharmacokinetics by integrating microspheres with a microfluidic system. (A) Representation of antibiotic pharmacokinetics in human plasma after daily oral administration during treatment. (B) Microfluidic system with two input channels and one exit channel for a 24-well tissue culture plate. (C, D) Placement of a basal mirror doubles the detection of M. tuberculosis luminescence by the GloMax Discover plate reader. Luminescence from infected PBMCs in microspheres in a single well in the absence (no fill) or presence (stripes) of a basal mirror for 24-well (C) and 96-well (D) tissue culture plates. (E) Modeling of antibiotic concentration profiles with a microfluidic system. From day 5 (black arrow), various peak concentrations of antibiotics were introduced for 6 h via the fluidic system and then washed out to approximate pharmacokinetics in vivo. Increasing rifampin concentrations (0.25 µg/ml [salmon], 1 µg/ml [bright red], and 4 µg/ml [dark red]) progressively accelerated M. tuberculosis killing. The black line represents a control sample to which the carrier DMSO was added and identical washes were performed. Three independent experiments were carried out, and the results of a representative experiment are shown.
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