Fig. 3.

Untargeted differential proteomics analysis to investigate the impact of enhanced local cell density after sound patterning of centimeter-scale microcapillary networks. (a) At day 0, Green Fluorescent Protein-Expressing human umbilical vein endothelial cells (GFP-HUVEC) and human pericytes from placenta (hPC-PL) were collected and mixed at a ratio of 10:1 in a single-cell suspension made in fibrin, then dispensed into the sound patterning chamber with a round geometry. Before gelation, Faraday waves were generated at the liquid-air interface upon application of 54 Hz frequency vibration, and sound-induced hydrodynamic forces condensed the cells into reproducible pattern of 4 concentric circles in less than 2 min. At day 5, the concentric circles pattern samples were washed with PBS and incubated at 37 °C with 1 ml of a 2 mg mL⁻¹ Nattokinase solution prepared in 1 mM EDTA PBS. Cellular material was collected in pellets, which underwent protein digestion. The digested peptides were then analyzed by mass spectrometry. (b) Proteomic analysis of the patterned capillaries after 5 days in culture revealed over 900 differentially expressed proteins compared to the stationary condition (random). 643 proteins were up-regulated and 261 down-regulated in the patterned microcapillaries compared to the random condition (n = 3). The heatmap (c) shows the 50 most significant proteins, and (d) the Principal Components Analysis (PCA) of the dataset reduced to two dimensions shows spatial separation between the random and patterned conditions.