Matrix-Free Normal Human Epithelial-Fibroblast 3D Spheroid Cultures for In Vitro Lung Modeling

The cellular responses of classical 2D flat monolayer cell culture systems provide only very limited reliable predictions about possible outcomes of corresponding animal experiments and clinical studies, which is due, among other things, to the lack of (bi)directional signaling transmission between different cell types and the lack of a structural microenvironment. To study the interactive communication between different cell types in vitro, two main co-culture methods have emerged as central techniques. In the indirect co-culture method, different cells are cultured physically separately (e.g., using transwell inserts) but can communicate with each other via secreted factors (paracrine mechanism). In the direct co-culture method, the different cells have direct physical contact, which enables direct interactions. Regarding the latter method, the cultivation of cells as spherical cell aggregates, so-called spheroids, embedded in a semi-solid extracellular matrix has been established as an in vivo-related, more complex cell culture model with different functional cell states according to cell-cell and cell-ECM interactions as well as oxygen and nutrient gradients. Here, we present a matrix-free method for direct spheroidal co-cultivation of human bronchial epithelial cells and fibroblasts, which can be considered as an in vivo-approximated cultivation method, especially with regard to the cellular composition of the respective spheroids.