Designing structures to support growth of intestinal biofilms in a colonic in vitro model

The gastrointestinal (GI) tract is host to a complex community of microbes existing within different niches, including free-flowing planktonic microorganisms, and microbial biofilms within the intestinal mucosal layer. Biofilms within the colon are thought to play a key role in triggering inflammatory mechanisms through direct contact with host cells or via microbially produced metabolites. However, , given the difficulties studying these communities in situ, our understanding of colonic biofilms remains limited. Therefore, we propose a platform that can in accurately replicate these complex biofilms in vitro, supporting research of the GI tract sessile populations. Structures to support biofilm growth were designed to integrate a clinically reflective in vitro model of the human colon, called MiGut. The structures were 3D printed in biocompatible resin and featured 12 removable coupons to allow for biofilm sampling. We investigated 2 coupon designs, the first featuring a plain surface and another featuring a 1.3mm honeycomb pattern. Experiments were performed using 4 MiGut reactors, 2 of which contained a plain biofilm structure and 2 with the honeycomb structure. MiGut models were inoculated with human faeces (10% w/v) to replicate the human microbiota and populations were allowed to stabilise/form biofilm for 14 days. Biofilm coupons and planktonic samples were then collected for DNA extraction and real-time quantitative PCR (qPCR) analysis. 2 coupons from each structure were washed and stained with 0.5% (w/v) crystal violet. The biofilm biomass was then determined spectrophotometrically at OD600. Similar DNA concentrations were observed on both coupon types and qPCR showed an increase in Bifidobacterium, Eubacteria, and Prevotella when using the honeycomb design compared to plain. The crystal violet staining revealed that biomass adhered to all coupons, with absorbances of 0.364 AU and 0.855 AU for the plain and honeycomb designs respectively. These results show that the structures are able to support colonic biofilm formation within MiGut, and that specific design choices of the coupons can improve biofilm yield. Future work will focus on incorporating mucin to represent the mucosal environment and to investigate changes in biofilm composition over a period via the use of multiple sampling points.