(M-495) Single Cell Bacterial Culturing Using Permeable Hydrogel-Shelled Microcapsules

Introduction:: Cell-to-cell heterogeneity in complex microbial communities such as biofilms enables bacteria to survive antibiotic or bactericidal treatment due to subpopulations of cells that may have enhanced fitness under stress. Single cell methods enable the identification and isolation of persistent cells present within the population to provide a deeper understanding into the role of heterogeneity in these complex communities. One such single cell method is drop-based microfluidics, which is a high-throughput method for encapsulating, culturing and assaying growth of individual cells using water-in-oil drops. Some disadvantages of this approach include potential emulsion destabilization during incubation, limited nutrient in nano- and pico-liter drop volumes, and the inability to perform multiple environmental changes over time. The ability to transport nutrient and waste past the drop interfaces would enable observation of varying conditions upon single cell growth. Here, we introduce a method for generating hydrogel-shelled microcapsules using drop-based microfluidics for observing single bacterial cell growth in contained microenvironments that are permeable to the surrounding media. The microscale hydrogel shells are biocompatible, tunable in size and composition, and allow for nutrient and waste exchange. We demonstrate the utility of the capsules by performing single cell growth studies of encapsulated Pseudomonas aeruginosa subjected to doses of the antibiotic, ciprofloxacin, over time. The hydrogel-shelled capsules provide a novel, high-throughput cultivation platform that can expand current methodologies for assaying single cells in complex microbial communities and lend insight into the role of cellular heterogeneity in persistent infections.


Materials and Methods:: Hydrogel-shelled microcapsules were fabricated for single cell microbial isolation and cultivation. A non-planar PDMS microfluidic device enables co-flow of the core and shell fluids. The hydrogel shell solution consisted of 200 mg/mL 10 kDa 4-arm polyethylene glycol –maleimide (PEG4Mal), 5.54 mg/mL Cy5.5-maleimide in DMSO, and 1X PBS at pH 7.4. The core solution consisted of 260 mg/mL 10 kDa dextran suspended in 1X PBS. A 10 kDa, 1 wt% FITC-Dextran solution was added for fluorescence visualization. The oil solution consisted of 3 wt% Span^TM 80 dissolved in light mineral oil while the crosslinking nanoemulsion was a 1:15 aqueous:oil mixture of 50 mg/mL dithiothreitol (DTT) in 0.2 M / pH 8.0 triethanolamine buffer solution and 3 wt% Span80 in light mineral oil. The fluids were driven into the microfluidic device using syringe pumps and varying flow rates controls capsule size and shell thickness. Capsules were washed to replace the mineral oil with aqueous media and facilitate diffusion of dextran out of the cores. Pseudomonas aeruginosa PAO1 constitutively expressing the green fluorescent protein (GFP) from plasmid pMF230 was added to the core solution to create capsules containing single bacterial cells. Time-lapse confocal imaging studies of the capsules in a chambered well slide (Cellvis) was performed at 37 °C in an incubated microscope stage chamber. Growth of single cells in capsules with and without 1 µg/mL ciprofloxacin antibiotic treatment was studied following 4 h, 7 h, and 10 h of incubation. Capsules were imaged every 30 minutes to quantify fluorescence from bacterial growth.


Results, Conclusions, and Discussions:: Hydrogel-shelled capsules for bacterial isolation and cultivation are produced in a non-planar microfluidic device by coflowing dextran and PEG4Mal solutions with mineral oil and DTT. During capsule generation, the non-planar device design creates a dextran-rich core solution in a PEG4Mal shell. The flow intersects with mineral oil solution at a flow-focusing junction, forming drops of the aqueous phases in oil (Fig 1A). The dextran and PEG4Mal are an aqueous two-phase system that phase separate when mixed at high concentrations. Capsules were produced with diameters from 91.8 ± 2.5 µm to 58.9 ± 4.5 µm by changing flow rates while shell thicknesses ranged from 28.1 ± 2.0% to 71.5 ± 0.9% of the total capsule radius.

Single cells of P. aeruginosa (pMF230) were encapsulated into capsules and observed using confocal microscopy for 12 h to create growth curves starting from single cells. In nutrient rich medium, the growth curves of hundreds of capsules had similar growth rates. Compared to growth in water-in-oil drops [1,2], the bacteria aggregate in the capsules, likely due to the ability to attach to the hydrogel surface and form more complex communities (Fig 1B). As the capsules enable diffusion of media through the hydrogel shells (Fig 1C), time-dependent antibiotic treatments can be applied to study their effects on P. aeruginosa growth and aggregation. Cell growth and colony morphology are measured as outcomes of ciprofloxacin timing and dosage.

This is the first application of hydrogel-shelled capsules for the study of single cell bacterial growth. This work will enable future studies into the role of cellular heterogeneity in persistent infections due to the ability to isolate and observe single cell subpopulations under exposure to varying environments.


Acknowledgements (Optional): : Research was supported by NSF-EPSCOR 1736255, NSF-CAREER 1753352 (PI Chang).


References (Optional): : [1] Pratt, S.L., Zath, G.K., Akiyama, T., Williamson, K.S., Franklin, M.J. and Chang, C.B., 2019. DropSOAC: stabilizing microfluidic drops for time-lapse quantification of single-cell bacterial physiology. Frontiers in Microbiology, 10, p.2112.

[2] Akiyama, T., Williamson, K.S., Schaefer, R., Pratt, S., Chang, C.B. and Franklin, M.J., 2017. Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation. Proceedings of the National Academy of Sciences, 114 (12), pp.3204-3209.