Application of Organ-on-chips and micro-physiological systems
Humans have co-evolved with their gut microbiota in a symbiotic relationship essential for health, yet how these thousands of bacterial species influence human biology remains little understood. A better understanding of the interplay between human cells and gut microbiota is required to exploit the complex relationships responsible for the local and distant effects of the microbiome on the human body. Accordingly, significant interest exists in the biotechnology community for improved in vitro models of the human gastrointestinal system, in particular models that support human-microbial co-culture. This feat is complicated by the fact that over 99% of gut bacteria are obligate anaerobes that die in 30-60 min after exposure to room air. Therefore, we have developed an easy-to-use and intuitive platform to replicate the steep O2 gradient across the in vivo colonic epithelium, thus create the appropriate environment required for anaerobes while maintaining viable, healthy epithelial tissue. We have computationally modeled, designed and prototyped the co-culture platform to fit within an SBS standard 12-well plate. The co-culture platform consisted of a basal reservoir and luminal reservoir with a porous polyester membrane and extracellular matrix (ECM) support dividing the two reservoirs. Using our culture methods, colonic epithelial stem cells were expanded on the ECM support and subsequently differentiated into all cell types found in the intestines in a monolayer ideal for compound screens and luminal stimulation/co-culture. Additionally, the ECM could be micromolded to recreate the physical architecture of the colon. Once the colonic epithelial layer was established, the luminal reservoir was sealed with an O2-impermeable barrier which resulted in the auto-generation of an anoxic environment (< 2% O2) in the luminal reservoir within 8 hours by the O2 consumption of the epithelial cells. The basal chamber remained normoxic to supply the epithelial cells with O2 through the porous membrane and ECM support. The generation of a steep O2 gradient was measured and experimentally confirmed. The resulting O2 gradient allowed for anaerobes (lactobacillus rhamnosus GG) to be cultured in the luminal reservoir in contact with an oxygenated colonic epithelial layer. Colonic epithelium and anaerobic bacteria each maintained >90% viability when co-cultured for ≥3 days. Our co-culture platform is simple, robust, self-sustaining and easy-to-use. It does not require any fluidic and gas control systems. It is based on regular standard SBS microplate format that industry and academia use on a daily basis. Thus, it can be adopted in any microbiology laboratory without requiring new equipment.