Abstract

Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are
structurally and mechanically well-defined and ideal for systematically investigating the influence of
three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like
and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in
DLW-fabricated 3D scaffolds of different mechanics and geometry.

Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phosphopaxillin, phospho-focal adhesion kinase, vinculin, b1-integrin). However, fibroblast-like and epithelial
cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments.

While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments.
Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

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