Superhydrophobicity Effects on Spheroid Formation and Polarization of Macrophages

Abstract

The interaction of biomaterials with the immune system is ruled by the action of macrophages. The surface features of these biomaterials, like wettability, which is an expression of chemical composition, texture, and geometry, can affect macrophages response. Such surface parameters can be then efficiently exploited to improve biocompatibility by lowering undesired immunological reactions and at the same time creating the substrate for positive interactions. In this work, the preparation and physicochemical characterization of highly water-repellent surfaces to develop and characterize 3D spheroids derived from monocyte-macrophages (RAW 264.7 cell line) has been carried out. As a measure of cell viability over time, the obtained aggregates have been transferred under standard 2D cell culture conditions. Significant changes on the morphology-associated polarization of the derived cellular entities have been evaluated at the nanoscale through 3D profilometry. The results suggested that the spheroid formation using highly repellent substrates induced the activation of M2-type cells. This simple and cost-effective approach can be used for preparing M2-based macrophages for regenerative purposes.

Keywords: activation; cell morphology; macrophages; polarization; spheroids; superhydrophobicity; surfaces.

Figure 1

Three-dimensional detail of the superhydrophobic surface (a) and its roughness acquired by interferometric and confocal profilometry at 20× (b). Average roughness, Sa, 170 nm.

Figure 2

Phase contrast images on RAW264.7 cells cultured in TCPS, agarose, and highly water-repellent surfaces (SHS) after 48 h of incubation based on initial cell density (200 or 2000 cel/μL) (a). Circularity and size distribution (b) based on initial cell density (200 or 2000 cel/μL) on agarose and SHS. The results are reported as the average of more than 10 individual spheroids ± standard deviation. * p < 0.05 and ** p < 0.001 indicate significant differences between values as a function of the initial cell density for the same substrate. • p < 0.05 indicates significant differences between values as a function of the substrate for the same initial cell density.

Figure 3

Phase contrast images of the temporal evolution) of cells derived from SHS-induced spheroids. Representative morphology of migrated cells at 168 h after recovery as a function of the initial cell density (200 or 2000 cel/μL). The scale bar represents 200 μm.

Figure 4

Three-dimensional optical profilometry images in confocal mode (100× magnification) for RAW264.7 spheroids formed on SHS surfaces depending on the initial cell density (200 or 2000 cel/μL) for 168 h after recovery and compared to control cells. The scale bar represents 10 μm.