Characterization of an engineered mucus microenvironment for in vitro modeling of host–microbe interactions

The human mucus layer plays a vital role in maintaining health by providing a physical barrier to pathogens. This biological hydrogel also provides the microenvironment for commensal bacteria. Common models used to study host–microbe interactions include gnotobiotic animals or mammalian–microbial co-culture platforms. Many of the current in vitro models lack a sufficient mucus layer to host these interactions. In this study, we engineered a mucus-like hydrogel Consisting of a mixed alginate-mucin (ALG-MUC) hydrogel network by using low concentration calcium chloride (CaCl₂) as crosslinker. We demonstrated that the incorporation of ALG-MUC hydrogels into an aqueous two-phase system (ATPS) co-culture platform can support the growth of a mammalian monolayer and pathogenic bacteria. The ALG-MUC hydrogels displayed selective diffusivity against macromolecules and stability with ATPS microbial patterning. Additionally, we showed that the presence of mucin within hydrogels contributed to an increase in antimicrobial resistance in ATPS patterned microbial colonies. By using common laboratory chemicals to generate a mammalian–microbial co-culture system containing a representative mucus microenvironment, this model can be readily adopted by typical life science laboratories to study host–microbe interaction and drug discovery.