Emerging and re-emerging infectious diseases are threats to the military day to day operations and training. A recent surveillance study in Iraq found that enterotoxigenic Escherichia coli (E. coli) 23% and enteroaggregative E. Coli (12.5%) were the most common pathogens to cause gastrointestinal tract (GI) infections. Now many pathogens pose high resistance in multidrug or antibiotics. These kinds of bacterial virulence and their effect on cellular dynamics and cognition are essential to study in the human model to determine emerging pathogens with potential future treatment. Toward the solution, NeyroblastGX LLC (NGL), proposed to develop âhOPSenseâ, a human pluripotent stem cell (hPSC)-derived 3D GI tract organoid (GIO) system compatible to high content electroconductance microfluidics-based sensing array for detecting physiologically relevant pathogen response. In our study, we have selected GI tract since various military surveys revealed that diarrhea is the most frequent complaint in deployed military personnel from the United States (U.S). In this project, NGL will generate GIO from hPSC derived 3D spheroid culture. Our in-house 3D spheroids are highly enriched in endodermal marker expression with self-renewal properties like a stem cell. These spheroids are highly stable and the ability to regenerate even after the freeze-thaw cycle. We will use these spheroids to generate GIO in combination within house biocompatible gel matrix plate to speed up the organoid generation process (~21 days), where traditional GIO generation takes 60 days. Thus, NGL GI organoid generation will be fast, cost-effective, and will have the ability to scale up easily. NGL also will introduce microbiome culture to these organoids to produce a fully functional GI tract system to study pathogenic versus non-pathogenic responses. To study host-pathogen interaction, NGL will treat GIO with E. coli and Salmonella, and both were reported pathogenic bacterial species in various military outbreaks. After the treatment of GIO with bacteria, we will collect organoids and run for organoid paracellular barrier function, cytokines array profile, and shotgun metagenomic for these samples. We anticipate observing correlation with intestinal disruption with increasing cytokine releasing signal and alteration of metagenomic pattern with pathogenic bacteria compared to non-pathogenic. We will also explore GIO electroconductance activity by using an electrical interface combined microfluidics chip. All these results will be quantifiable and can be able to be scaling up to using high-content microfluidics assay platform, further refining to use in Phase II to study pathogen on a large scale. Thus, NGL GIO system technology will able to replicate the physiologically relevant response of host-pathogen interaction with higher sensitivity and specificity to improve warfighter heal
