Integration of Mesenchymal Stem Cells into a Novel Micropillar Confinement Assay.

Mechanical cues such as stiffness have been shown to influence cell gene expression, protein expression, and cell behaviors critical for tissue engineering. The mechanical cue of confinement is also a pervasive parameter affecting cells and in tissue-engineered constructs. Despite its prevalence, the mechanical cue of confinement lacks assays that provide precise control over the degree of confinement induced on cells, yield a large sample size, enable long-term culture, and enable easy visualization of cells over time. In this study, we developed a process to systematically confine cells using micropillar arrays. Using photolithography and polydimethylsiloxane (PDMS) molding, we created PDMS arrays of micropillars that were 5, 10, 20, or 50 μm in spacing and between 13 and 17 μm in height. The tops of micropillars were coated with Pluronic F127 to inhibit cell adhesion, and we observed that mesenchymal stem cells (MSCs) robustly infiltrated into the micropillar arrays. MSC and nucleus morphology were altered by narrowing the micropillar spacing, and cytoskeletal elements within MSCs appeared to become more diffuse with increasing confinement. Specifically, MSCs exhibited a ring of actin around their periphery and punctate focal adhesions. MSC migration speed was reduced by narrowing micropillar spacing, and distinct migration behaviors of MSCs emerged in the presence of micropillars. MSCs continued to proliferate within micropillar arrays after 3 weeks in culture, displaying our assay's capability for long-term studies. Our assay also has the capacity to provide adequate cell numbers for quantitative assays to investigate the effect of confinement on gene and protein expression. Through deeper understanding of cell mechanotransduction in the context of confinement, we can modify tissue-engineered constructs to be optimal for a given purpose. Impact Statement In this study, we developed a novel process to systematically confine cells using micropillar arrays. Our assay provides insight into cell behavior in response to mechanical confinement. Through deeper understanding of how cells sense and respond to confinement, we can fine tune tissue-engineered constructs to be optimal for a given purpose. By combining confinement with other physical cues, we can harness mechanical properties to encourage or inhibit cell migration, direct cells down a particular lineage, induce cell secretion of specific cytokines or extracellular vesicles, and ultimately direct cells to behave in a way conducive to tissue engineering.