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Human gut microbiota on gut-on-a-chip

Periodic Reporting for period 1 - Goc-MM (Human gut microbiota on gut-on-a-chip)

Reporting period: 2019-05-06 to 2021-05-05

Goc-MM consists of minimal functional portion of gut cultured in a microfluidic device, gut-on-a-chip, that mimics the environment of intestine. This gut-on-chip was designed to host gut-microbiota derived by stool samples from patients with metabolic conditions. The purpose was to study the gut microbiota and its metabolites in presence of oxygen and in anoxic conditions provided by a new perfusion system, CubiX produced at Cherry Biotech. The gut microbiota provides nutrients and energy for the host through the products of substrate fermentation in Short Chain Fatty Acids (SCFA). Understanding how the changes in the gut microbiota composition may influence the production of the SCFA is of interest to find diets and treatments for obese and anorexic patients. Previous studies are limited and imprecise with a lack of information in detailed dietary intake, environmental exposure, and mediators. Understanding the role of the trillions of bacteria, viruses, and fungi that comprise the human microbiota is critical to understand a variety of human disease. Dysbiosis of the microbiota had been found critical not only for intestinal disease but also in the immunological responses and in other human pathologies. Criticalities on studying the microbiota in vitro are mainly represented by the variety of the gut microbiota presences and their interactions. Microfluidic organ-on-a-chip models offer new opportunities to analyse the single contribute of individual cellular, chemicals and physical parameters on-at-a-time.
The scientific goal of Goc-MM was to design, test and bring to a TRL (technological readiness level) 5 a gut-on-chip containing two channels: one for the endothelium and the other one for the intestinal epithelium to host the gut microbiota. The main motivation that leads us to get involved in this project was to provide to scientific community and industries a convenient and standardized methodology of isolation and cultivation of microbiota from human faecal microbiota transplantation. The faecal transplantation is used in the management of recurrent Clostridioides difficile infection because of its clear advantages over antibiotics. Benefits from faecal transplantation have been suggested for other conditions but studying in vitro the mechanism of action has been a bottleneck for two main reasons: (1) reproducing the peristaltic movements in vitro; and (2) reproducing the thermic and gas environment which is partially produced by the fermentative reactions of the gut microbiota. In this view, the Gut-on-a-Chip is an interesting technology to understand gut microbiota.
In the Goc-MM project, a new gut-on-chip that mimics the environment of intestine was produced. We used biological and FDA approved membranes that are stretched by the liquid flow to simulate the physiological intestinal peristaltic movements. The architecture of this device was designed to prevent clogs due to stool samples and the overgrowth of bacteria. The right microenvironmental temperature as well as O2 and CO2 content are provided by Cherry Biotech CubiX. This new versatile platform is the first commercially available device dedicated to generating programmed flows, temperature, and gas compositions to microfluidic devices without using an incubator. Unfortunately, the impact of the pandemic emergency and lockdown measures caused delays and difficulties in the finalisation of the project as it was designed initially. However, studies from the literature and the experience achieved with this project have been used for dissemination activities through publications (one submitted and one in preparation) and invited speeches to conferences. A technology, products and patents analysis on the commercially available gut-on-a-chip devices as well as on those developed in research institutes and academia has been performed. This market study has been resumed and collected in a review article that has been uploaded in the Open Research Europe portal and that will be made accessible to the scientific community and citizens. A Twitter page has also been created as well as a page in the Cherry Biotech website dedicated to the project.
The Goc-MM project goes beyond the state-of-art by the integration on a human-derived and FDA approved membrane in a gut-on-a-chip (GOC) model also able to simulate the peristalsis movement of lower intestinal tract. The media perfused are enriched with different gasses simulating the rather anoxic gut environment as well as the oxygen rich microvascular flow. The different gasses concentrations are possible thanks to CubiX, a perfusion system developed in Cherry. We have performed an in-depth search of suitable membranes and we chose a decellularized amniotic membrane given its suitable specifications: transparency, thickness, elasticity, resistance, and ability to support cells grown. This membrane separates the upper hypoxic channel containing Caco-2 cell line and the lower chamber, coated with endothelial cells. The chip has been designed specifically to tackle the challenge of having a realistic gut microbiota inside the chip. The upper channel is indeed designed to host faecal transplantation and avoid the bacteria overgrown that may produce clogging into the chip. Despite the plan to test the chip in a relevant environment with patient-derived stool samples, due to the concomitant pandemic of COVID-19 we were prevented the access to the samples. The proof of concept as well as the chips characterizations/iterations have been performed by using a decellularized porcine bowel. Fluid simulations as well as real tested allowed us to optimize the design of the chip. Specific efforts in the usability of the chip were included since the beginning in order to reduce to the minimum the use of robust connectors. The socio-economic impact of the Goc-MM rely on the possibility to grow the anaerobic bacteria under physiological flow by using the microfluidic perfusion, simulating not only the peristalsis but also controlling the transit time. The microfluidic systems, as such as Goc-MM, aims to replace, reduce, and refine the animal experimentation demolishing the ethical barrier, reducing the pre-clinical costs and being closed to human physiology for the use of human-derived cell lines. In parallel to the GOC designing phase we also performed an in-depth market assessment as well as a freedom to operate in order to have a clear overview of current strategies and bottlenecks. The results of this survey was presented as a publication submitted to Open Research Europe. The ER had the possibility to learn-by-doing microfluidic and microphysiological systems. Moreover during the IF the ER could explore aspects that are more related to the non-academic sectors. During the period of the project marketing/business development actions were undertaken to explore the potential market segment of the project outcomes. The rotation and the responsibilities taken in different positions inside the start-up enabled the ER to open the vision of unexpected new career prospective.
Roadmap of the translation in preclinical studies of gastrointestinal (GI) model
Goc-MM prototype