(K-431) Microfluidic device with continuous oxygen film to study distribution of bacteria

Introduction::

Microfluidic devices have become increasingly important due to their ability to provide precise and controlled environments to mimic different tissues such as the colon. We have previously created a microfluidic device that created a dual-oxygen environment which supported the culture of oxic intestinal epithelial cells simultaneously with anoxic gut bacteria (Wang et al., 2020). An important feature of this device were the in-situ thin film fluorescent oxygen sensors that were used to measure the oxygen levels in the device. A limitation with these sensors was that the thin films were manually placed in discrete locations of the device which increased complexity and lowered reliability. In this study, we have developed a novel process to integrate the oxygen sensor film across the entire device. This strategy was paired with a specific imaging technique to study the spatiotemporal distribution of facultative E. coli Nissle 1917 (ECN) and obligate Bifidobacterium Adolescentis (BFA) anaerobic bacterial species within these devices.

Materials and Methods::

The two-chamber device was fabricated using strandard soft lithography techniques as previously described (Wang et al., 2020). Oxygen film was integrated within the top chamber of the device by dissolving PtTFPP (pentafluorophenylporphine) in toluene and adding this to the PDMS prior to molding. The two bacterial species were cultured using the standard LB broth. The bacteria were mixed in agar and seeded in microfluidic devices and cultured for up to 48-hours after which the devices were sectioned and imaged. Individual bacterial species were identified using specific FISH probes that were designed as well.

Results, Conclusions, and Discussions:: A calibration curve was constructed for the oxygen levels in the device versus the fluorescence of the oxygen sensor film. We found that the individual bacterial species were highly responsive to the oxygen levels within the devices. When anoxic conditions were created in the top layer, the obligate BFA migrated towards the top layer. However, interestingly, ECN was distributed evenly throughout the device. When oxic conditions were created, BFA’s growth was curtailed significantly and there was no significant change in the distribution of ECN.

In this study, we have established a microfluidic device with a continuous layer of oxygen sensitive film that made it simpler to study the distribution of gut bacteria under different oxygen conditions. Instead of placing the oxygen film in discrete locations, we were able to provide a seamless measurement along the entire device, which improved throughput and increased the reproducibility. Further, our results on the migration and distribution of facultative and obligate bacterial species provides the data for the first time on how these species react when subjected together to different oxygenated conditions. We could in the future superimpose conditions with different nutrients or drugs to study how the microbial microenvironment in the colon would be affected under these conditions. This device may also provide information about the quorum sensing, especially the ones associated with cancer.

Acknowledgements (Optional): : We acknowledge funding support from NIH and NSF.

References (Optional): : Wang, C, Dang, T, Baste, J, Anil Joshi, A, Bhushan, A. A novel standalone microfluidic device for local control of oxygen tension for intestinal-bacteria interactions. The FASEB Journal. 2021; 35:e21291. https://doi.org/10.1096/fj.202001600RR