(I-340) Probiotics formulation and delivery using nanocultures

Introduction:: Sub-track: Engineering and the Microbiome


The human gastrointestinal (GI) tract hosts a balance of healthy and pathogenic bacteria collectively called the gut microbiome. Some factors including antibiotic treatment, chemotherapy, and age can cause dysbiosis in the gut flora and increase susceptibility to harmful bacteria such as Clostridium difficile [1]. C. difficile infection of the colon (CDI) causes extreme tissue damage and can lead to sepsis. Given the increasing rate of CDI, causing around 15,000-30,000 deaths annually in the United States alone, it is necessary to improve conventional treatment approaches [2]. Fecal microbiota transplants (FMT) are currently used to restore gut microbiome complexity and suppress CDI by introducing microbes derived from the feces of healthy patients, however, administrative techniques are invasive [3]. New methods for less-invasive delivery of probiotics to the GI tract have been developed by our lab. Nanoliter cultures of probiotic species, known as nanocultures, are generated by encapsulating probiotics such as Escherichia coli Nissle in a protective semi-permeable polydimethylsiloxane (PDMS) microcapsule, and are employed for a targeted delivery into the GI tract. This project specifically aims to study the correlation between E. coli Nissle cell density within microcapsules and their fluorescent intensity.




Materials and Methods:: Routine culturing of E. coli Nissle was performed in lysogeny broth. The cells were collected by centrifugation, washed, and resuspended in 0.1M phosphate buffered saline (PBS) for encapsulation. Cell densities of stock solutions were measured using colony-forming units (CFU) outlined in the protocol by Chen et al. [4]. For encapsulation, flow-focusing microfluidic devices were fabricated using instructions outlined in Niepa et al. [5] and Utada et al. [6] with minor adjustments to form glass capillary needles with inner diameters of 40µm and 180µm. Generation of capsules was performed using the protocol described by Usman et al. [7] with some modifications. The outer phase for encapsulation was 5 wt% poly(vinyl alcohol) aqueous solution (PVA, average Mw = 31,000–50,000), while suspensions of E. coli Nissle in PBS with different cell densities were introduced as the inner phase of the nanocultures. The shell of the nanocultures was prepared with PDMS containing Nile red added at 40µg per 4g of PDMS for staining purposes. Generated nanocultures were collected in PBS and stored at room temperature for two days to complete the curing process, then stored at 4℃ in limited light. Before imaging, 300µl of each sample was stained overnight with 0.45µL of Syto9 to stain the encapsulated cells. Fluorescent images were taken with an Olympus Fluoview FV3000 confocal microscope to visualize the capsule shell (Nile Red) and E. coli Nissle cells (Syto 9)(Figure 1a). The fluorescent images were analyzed with ImageJ software to determine the nanoculture volume and corrected total cell fluorescence (CTCF).


Results, Conclusions, and Discussions:: Results for analyzing colony forming units (CFU) in eight stock solutions used for nanoculture samples were determined fall in a range between 3.5 x 108 - 9.3 x 109 cells/mL, corresponding to 350-9300 cells/nL, when capsule volumes ranged from 1 – 3.8 nL. Corrected total cell fluorescence (CTCF) was calculated for seven different capsules for each of the eight samples. After determining average CTCF, the result was then referenced to the average size of the capsule analyzed to determine CTCF/nL, which helps to better compare results when capsule sizes may vary. Results from this experiment showed that CTCF/nL increased with the cell density of the inoculum. At low density, there is a linear correlation between the cell density and the CTCF/nL (Figure 1b) However, the linear relationship is lost once the cell density of the microcapsules is above 2000 cells/nL, suggesting that the threshold for precisely predicting cell density with CTCF is limited once capsules become too densely packed.

This method for predicting cell density with CTCF can be an efficient technique to analyze the cell capacity of nanocultures. In addition, 3-D projections of confocal images taken can be used as a qualitative way of comparing new samples to a reference of capsules with known cell densities. Future directions for this project include analyzing other species of probiotics to determine the relationship between CTCF and cell density, which can help us predict microcapsule capacity in probiotic cocultures.


Acknowledgements (Optional): : This material is based upon work supported by the National Science Foundation under Grant EEC-2050944.

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