A Robust Longitudinal Co-culture of Obligate Anaerobic Gut Microbiome With Human Intestinal Epithelium in an Anoxic-Oxic Interface-on-a-Chip

Abstract
The majority of human gut microbiome is comprised of obligate anaerobic bacteria that exert essential metabolic functions in the human colon. These anaerobic gut bacteria constantly crosstalk with the colonic epithelium in a mucosal anoxic-oxic interface (AOI). However, in vitro recreation of the metabolically mismatched colonic AOI has been technically challenging. Furthermore, stable co-culture of the obligate anaerobic commensal microbiome and epithelial cells in a mechanically dynamic condition is essential for demonstrating the host-gut microbiome crosstalk. Here, we developed an anoxic-oxic interface-on-a-chip (AOI Chip) by leveraging amodified human gut-on-a-chip to demonstrate a controlled oxygen gradient in the lumen-capillary transepithelial interface by flowing anoxic and oxic culture medium at various physiological milieus. Computational simulation and experimental results revealed that the presence of the epithelial cell layer and the flow-dependent conditioning in the lumen microchannel is necessary and sufficient to create the steady-state vertical oxygen gradient in the AOI Chip. We confirmed that the created AOI does not compromise the viability, barrier function, mucin production, and the expression and localization of tight junction proteins in the 3D ntestinal epithelial layer. Two obligate anaerobic commensal gut microbiome, Bifidobacterium adolescentis and Eubacterium hallii, that exert metabolic cross-feeding in vivo, were independently co-cultured with epithelial cells in the AOI Chip for up to a week without compromising any cell viability. Our new protocol for creating an AOI in a microfluidic gut-on-a-chip may enable to demonstrate the key physiological interactions of obligate anaerobic gut microbiome with the host cells associated with intestinal metabolism, homeostasis, and immune regulation.