Abstract
To study the roles of bacterial secretome in immune cell polarization and gut mucosal homeostasis, we developed a 3-dimensional co-culture model using commercially available inserts in a back-to-back stacked format. We termed this system 3D Flipwells, and successfully co-cultured bacteria, colon epithelial, and immune cells in stratified layers to recapitulate the gut mucosal microenvironment. This co-culture system enables the seeding of colon epithelial cells on one side of the membrane and undifferentiated non-adherent monocytes on the other, allowing for the synchronous progression of colon epithelial polarization, mucus formation, and monocyte-macrophage differentiation and polarization within a single co-culture without the need for detaching and replating pre-differentiated macrophages. This system is easy to construct and only requires a few readily available materials and tools. We tested its utility by analyzing the responses of co-culture components to sepiapterin (SEP), the endogenous precursor of BH4 (tetrahydrobiopterin) -- a cofactor of nitric oxide synthase (NOS). We demonstrated that SEP treatment of 3D Flipwell induced biofilm formation by gut bacteria, mucus production by colon epithelium, and pro-immunogenic polarization of macrophages. This indicated that SEP triggered activation of a mucosal defense mechanism through crosstalk between different cellular components. Further experiments are needed to investigate the role of mucosal communications that lead to immune cell reprogramming and to evaluate the utility of this co-culture system to screen for novel immunomodulatory therapies.