The Floor‐Ceiling‐Chip, or 2 × 2D = Pseudo‐3D—Approaching 3D Cell Morphology and Organization between Two Opposing 2D Substrates with Cell‐Adhesive Protein Micropatterns

In this proof-of-concept study, we develop a novel 3D cell patterning and culture platform. The "Floor-Ceiling-Chip" (FC-Chip) simply consists of two opposing 2D substrates in the form of ion track-etched membranes, creating a pseudo-3D microenvironment for the cells between them. This allows the stimulation of both the dorsal and ventral sides of cells, thereby also eliminating the artificial polarization, for example, of stromal cells, observed in standard culture dishes and inserts. By providing the membranes with micropatterned cell-adhesive islands of varying geometries and sizes, the FC-Chip enables control over cell shape and alignment in a 3D environment. Analysis of fluorescence microscopic images reveals distinct cellular and nuclear morphology, along with perinuclear actin organization, in the on-chip cultures compared to cultures on traditional 2D substrates. Cells in the FC-Chip exhibit fewer focal adhesions, lower expression of lamin A/C, and less nuclear localization of the yes-associated protein 1. The chip demonstrates compatibility with standard biochemical assays and supports long-term cultures up to 10 days, expanding its potential applications. Overall, the early version of the FC-Chip presented here confirms the feasibility of a straightforward, accessible, and versatile future culture platform for the manipulation and modeling of cell morphology and organization in 3D.