Clinical trials and animal studies on the gut microbiota are often limited by the difficult access to the gut, restricted possibility of in vivo monitoring, and ethical issues. An easily accessible and monitorable in vitro model of the gut microbiota represents a valid tool for a wider comprehension of the mechanisms by which microbes interact with the host and with each other. Herein, we present a novel and reliable system for culturing the human gut microbiota in vitro. An electrospun gelatin structure was biofabricated as scaffold for microbial growth. The efficiency of this structure in supporting microbial proliferation and biofilm formation was initially assessed for five microbes commonly inhabiting the human gut. The human fecal microbiota was then cultured on the scaffolds and microbial biofilms monitored by confocal laser and scanning electron microscopy and quantified over time. Metagenomic analyses and Real-Time qPCRs were performed to evaluate the stability of the cultured microbiota in terms of qualitative and quantitative composition. Our results reveal the three-dimensionality of the scaffold-adhered microbial consortia that maintain the bacterial biodiversity and richness found in the original sample. These findings demonstrate the validity of the developed electrospun gelatin-based system for in vitro culturing the human gut microbiota.

The aim of this study is the analysis and characterization of a hydrolyzed keratin-based biomaterial and its processing using electrospinning technology to develop in vitro tissue models. This biomaterial, extracted from poultry feathers, was mixed with type A porcine gelatin and cross-linked with γ-glycidyloxy-propyl-trimethoxy-silane (GPTMS) to be casted initially in the form of film and characterized in terms of swelling, contact angle, mechanical properties, and surface charge density. After these chemical-physical characterizations, electrospun nanofibers structures were manufactured and their mechanical properties were evaluated. Finally, cell response was analyzed by testing the efficacy of keratin-based structures in sustaining cell vitality and proliferation over 4 days of human epithelial, rat neuronal and human primary skin fibroblast cells.

Although the adhesion of bacteria on surfaces is a widely studied process, to date, most of the works focus on a single species of microorganisms and are aimed at evaluating the antimicrobial properties of biomaterials. Here, we describe how a complex microbial community, i.e., the human gut microbiota, adheres to a surface to form stable biofilms. Two electrospun structures made of natural, i.e., gelatin, and synthetic, i.e., polycaprolactone, polymers were used to study their ability to both promote the adhesion of the human gut microbiota and support microbial growth in vitro. Due to the different wettabilities of the two surfaces, a mucin coating was also added to the structures to decouple the effect of bulk and surface properties on microbial adhesion. The developed biofilm was quantified and monitored using live/dead imaging and scanning electron microscopy. The results indicated that the electrospun gelatin structure without the mucin coating was the optimal choice for developing a 3D in vitro model of the human gut microbiota.