Establishing a Three-dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(ε-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.