Fig. 3

Three-dimensional reconstruction, segmentation, and characterization of microvessel morphologies when co-cultured with MSCs for 10 days. The visualization of immunostained, binarized, and segmented vascular structures from confocal laser microscopy and 3D image processing (see Additional file 1: Figs. S2 and S3; Additional file 2: Video S1; Additional file 4: Video S3). HUVECs were double-stained red with UEA I-fluorophore conjugate and immunostaining using Alexa Fluor 555-conjugated secondary antibody against CD31 marker. MSCs were labeled with GFP in green. Nuclei were stained with Hoechst 33,342 in blue. Image processing using IMARIS (Bitplane) allowed the reconstruction of 3D surface models, consisting of triangular meshes with vertices and edges. Surfaces of the 3D microvessel models were classified into “Sprouts” or “Parent vessel” (see “Materials and methods” section). Scale bars: 200 μm. B Segmentation of the entire surface of the 3D microvessel in (A) into “MSC-covered” surfaces (green) or “Uncovered” (red). Panels (i), (ii), and (iii) focus on representative MSC-binding sites as shown in white dashed boxes. Scale bars: 200 μm or 50 μm in pictures of entire or local vascular structures, respectively. C Segmentation of “sprout” surfaces in (A) into “MSC-covered” surfaces (green), “Uncovered” (red), or parent vessel (gray). Scale bars: 200 μm. D Segmentation of “parent vessel” surfaces in (A) into “MSC-covered” surfaces (green), “Uncovered” (red), or sprouts (gray). Scale bars: 200 μm. E Quantitative analyses of microvessel morphology. a Correlation analysis between red intensity (HUVECs) and green intensity (MSCs) using a single 3D image shown in (A). Pearson’s correlation coefficient, r, was calculated as high (> 0.6). b Comparison of sprout ranges between monoculture (n = 4) and co-culture (n = 5). Data are shown as box plots to compare the median values with the Mann–Whitney U test and are recognized as significantly different when the p value < 0.05. c Comparison of surface roughness on the sprouts between monoculture (n = 4) and co-culture (n = 5). The index “surface roughness” was defined as the unit surface area per volume. d Linear regression of MSC coverage ratio and sprout volume using five co-culture samples. P values in (b) and (c) were adjusted using Bonferroni’s multiple corrections