Fabrication and Characterization of an Artificial Mucus Layer for Mammalian-Microbial Co-Culturing Applications

Abstract

The human mucus layer plays a vital role in maintaining health by acting as a selectively diffusive barrier to opportunistic pathogens and foreign particles that enter the body. In areas such as the respiratory and gastrointestinal tract, the mucus hydrogel lining provides a unique microenvironment inhabited by both commensal and pathogenic bacteria, where the bacteria use mucin, the glycoprotein that makes up the mucus hydrogel network, as an attachment site. To model the host-microbe interactions, in vitro, in both health and disease, it is crucial to provide a proper microenvironment that can support the growth of both mammalian and microbial cells in a controlled manner. Recent advances in modelling host-microbe interactions include compartmentalized microfluidic devices or transwell co-cultures. However, these techniques lack sufficient control of the mucus microenvironment and require highly specialized skills to fabricate and operate, and therefore are difficult for the average life science laboratory to adopt. In this study, we aim to address these concerns by fabricating an artificial mucus layer using a simple ionic gelation technique with calcium chloride to generate an alginate-mucin (ALG-MUC) semi-interpenetrating polymer network hydrogel to be incorporated into a polyethylene glycol-dextran (PEG-DEX) aqueous two-phase system (ATPS) co-culture platform. We demonstrated the utility of ALG-MUC hydrogels using two sets of mammalian-microbial co-cultures. First, we used a human bronchial epithelial cell line, 16-HBE, co-cultured with a common airway pathogen, Pseudomonas aeruginosa. The second was a colorectal adenocarcinoma cell line, Caco-2, co-cultured with a gut pathogen, Shigella flexneri. Additionally, we characterized the viscosity and diffusivity of the ALG-MUC hydrogels. The findings showed that the ALG-MUC hydrogels were compatible with a PEG-DEX ATPS by reducing PEG-mediated cytotoxicity when cells were overlaid with a hydrogel layer. It was also found that diffusion of biomolecules (IgG and LL-37) was more affected by hydrogel composition (presence of mucin) rather than differences in viscosity. Moreover, the concentration of mucin as well as ATPS formulation affected the spatial distribution and antibiotic resistance of bacteria within this multiphase co-culture system. The ALG-MUC hydrogel was shown to have similar diffusive characteristics to natural mucus as well as support the simultaneous culture of pathogenic bacteria with mammalian cells, in vitro. With the ability to readily form a mucus-like hydrogel directly on top of mammalian cells, we provide a controlled co-culture platform that has the potential for assessing host-pathogen interaction and antibiotic testing in a realistic microenvironment. Future studies will further characterize the bacterial colony formation within the hydrogel layer and explore different ATPS formulation with modified ALG-MUC compositions to model complex bacterial infections at the mucosal interface.