Replicating the geometries of infected in vivo vessels using photoablation.

(A) Confocal images of Nm-infected vessels in the human-skin xenograft mouse model. Scale bar: 25 μm. (B) Schematic representation of the development of the Vessel-on-Chip (VoC) device: (i) a collagen-based hydrogel is loaded in the center channel of the microfluidic device, (ii) the focused UV-laser locally carves the chosen geometry within the collagen I matrix, (iii) HUVEC are seeded and attach on the collagen-carved scaffold. (C) Schematic representation of the microfluidic device and zoom of the carved region after cell seeding (F-actin). Scale bar: 250 μm. (D) Confocal images of F-actin and related orthogonal view of the tissue-engineering VoC. Scale bar: 50 μm. (E) Confocal images of VoC in circle-, square- and diamond-shaped structures. Scale bar: 50 μm. (F) Photoablation process to create in vivo-like structures. Definition of the region of interest (ROI) to replicate from intravital microscopy images (left), and the obtained in vivo-like structures in the VoC (right). Scale bars: 20 μm.

The Vessel-on-Chip device provides nuclear morphologies under flow conditions and permeability levels similar to those observed in vivo.

(A) Confocal images of the VE-Cadherin staining and (B) Collagen IV in the VoC. Scale bars: 50 μm (large view) and 7 μm (zoom). (C) Representative confocal images of fluorescent 150 kDa-Dextran (FITC) in the VoC (top, scale bar: 50 μm) and in the human vessel in the mouse model (bottom, scale bar: 30 μm). Fluorescence of the outside and inside regions of the vascular lumen has been measured to determine permeability. (D) Graph representing the permeability to 150 kDa-Dextran. Each dot represents one vessel. For each condition, the mean ± s.d. is represented (VoC, 2.4 mg/ml: 65.12 ± 26.00 cm/s (n=7) – VoC, 4 mg/ml: 3.97 ± 2.49 cm/s (n=5) - Human vessel in vivo: 8.00 ± 2.52 cm/s (n=7)). (E) Representative images of nucleus alignment in the absence and presence of flow (24h). Scale bar: 50 μm. (F-G) Graphs of nucleus orientation (IRQ: interquartile range) and elongation. Each dot corresponds to the mean value of one vessel. For each condition, the mean ± s.d. is represented (No flow: 53.0 ± 30.2° and 1.63 ± 0.11 a.u. (n=34) – 2h of flow: 29.9 ± 11.5° and 1.85 ± 0.25 a.u. (n=22) – 24h of flow: 18.2 ± 7.61° and 1.98 ± 0.20 a.u. (n=25) – in vivo: 12.9 ± 4.6° and 2.01 ± 0.23 a.u. (n=9)). All statistics have been computed with Wilcoxon tests.

Neisseria meningitidis adhesion to the Vessel-on-Chip device.

(A) Schematic of the infected Vessel-on-Chip device. (B) Confocal images of infected vessels in our system 3h post-infection in each condition. Scale bar: 20 μm. (C) Bright-field and fluorescence confocal images of the VoC 3h post-infection with WT (top) and pilD (bottom) Nm strains. Scale bar: 25 μm. (D) Graph representing the number of aggregates 3h post-infection, with WT and pilD Nm strains. 17 μm2 represents the median over the entire population of aggregate sizes. Each dot corresponds to a vessel. For each condition, the mean ± s.d. is represented (WT: 47.5 ± 30.4 (n=2) – pilD: 2.0 ± 2.0 (n=3)). (E) Brighfield (left) and fluorescence (center) images of the infected vessels of different designs (circle, diamond, in vivo-like) and related simulation of shear stress. Scale bar: 50 μm. (F) Normalized histograms of total (blue) and bacteria pixels (orange) depending on the shear stress values for each vessel design (from top to bottom, circle, diamond, in vivo-like). Solid curves represent the mean ± sd (n=3 experiments). Spearman correlation give the correlation between the mean curves.

Flow impacts colony morphology but not growth in both Vessel-on-Chip and animal models, and Neisseria meningitidis infection increases permeability in Vessel-on-Chip.

(A) Normalized surface area of microcolonies over time, in the VoC under flow conditions (left, n=20) and in human vessels of the xenografted mouse model (right, n=33). Solid thin curves, solid thick curves, and dashed thick curves represent individual colony growth, mean, and linear fit (y = ax + 1), respectively. (B) Colony doubling time extracted from the curves (td =1/a). Each dot corresponds to a bacterial microcolony. For each condition, the mean ± s.d. is represented (VoC, Flow−: 16.7 ± 18.5 min (n=21) — VoC, Flow+: 26.2 ± 40.4 min (n=20) — in vivo: 15.2 ± 18.8 min (n=33)). (C) Confocal images of WT Nm microcolonies formed on the vascular wall 3h post-infection, in the absence and presence of flow (VoC), and in the animal model. Scale bar: 10 μm. Circular plots representing the distribution of microcolony orientation. Graph representing the elongation of microcolonies 3h post-infection in each condition. Each dot corresponds to a microcolony. For each condition, the mean ± s.d. is represented (VoC, Flow−: 1.75 ± 0.77 (n=128) — VoC, Flow+: 2.11 ± 1.02 (n=125) — in vivo: 2.07 ± 0.971 (n=61)). (E) Brightfield and fluorescence confocal images of permeability assay (Dextran) in infected (top) and histamine-treated (bottom) Vessel-on-Chips. Scale bar: 25 μm. (F) Relative permeability values of VoC and in vivo, vessels treated with histamine and infected with Nm. Each dot represents a vessel. For each condition, the mean ± s.d. is represented (mouse vessel, Control: 1 ± 0.62 (n=5), Histamine: 3.79 ± 2.25 (n=5) — VoC, Control: 1 ± 0.63 (n=5), Histamine: 5.11 ± 3.92 (n=7), Nm: 11.3 ± 5.48 (n=7)). All statistics have been computed with Wilcoxon tests.

Flow-induced aligned actin stress fibers are reorganized below bacterial microcolonies

(A) Confocal images of the F-actin network in the VoC in the absence and presence of flow (2h and 24h) (top). Scale bar: 30 μm. Corresponding segmented images. The color code shows the alignment of the actin fibers with the direction of the flow (red to blue) (middle). Circular plots of the orientation distribution of actin fibers (bottom). (B) Interquartile range of F-actin fiber orientation. Each dot represents the mean of F-actin fiber orientation per vessel. For each condition, the mean ± s.d. is represented (No Flow: 68.8° ± 31.0° (n=29) — 2h of flow: 33.1° ± 14.3° (n=25) — 24h of flow: 35.5° ± 13.5° (n=19)). (C) Confocal images of honeycomb-shaped cortical plaque formed by Nm microcolonies in the absence (top) and presence of flow (bottom) in the VoC. Scale bar: 10 μm. (D) 3D rendering of a vessel infected with Nm 3h post-infection under flow conditions. Scale bar: 15 μm (main) and 10 μm (zoom). (E) Percentages of colonies forming a cortical plaque (n=5 vessels). (F) F-actin fluorescence intensity under each microcolony and on non-infected regions of the same area. Each dot represents a region of a microcolony. For each condition, the mean ± s.d. is represented (Not infected regions: 0.99 ± 0.33 a.u. (n=74) — Infection, without cortical plaque: 1.30 ± 0.57 a.u. (n=36) — Infection, with cortical plaque: 2.15 ± 0.54 a.u. (n=28)). (G) Coherency of F-actin fibers on non-infected regions and on infection sites. Each dot represents a colony. For each condition, the mean ± s.d. is represented (Not infected regions: 0.40 ± 0.17 a.u. (n=74) — Infection, without cortical plaque: 0.28 ± 0.15 a.u. (n=36) — Infection, with cortical plaque: 0.18 ± 0.09 a.u. (n=28)). All statistics have been computed with Wilcoxon tests.

The Our infection model recapitulates the human neutrophil response to N. meningitidis infection.

(A) Confocal images of E-selectin staining in the VoC for four conditions: without infection nor treatment, after 4h of either TNFε treatment or infection with WT or pilD Nm strains. Scale bar: 50 μm. (B-C) Graphs representing the percentage of E-selectin positive cells and the mean intensity of CD62 in positive cells. For each condition, the mean ± s.d. is represented (Control: 0.32% ± 0.67% (n=10 vessels), 11.4 ± 1.17 (n=2 cells) — TNFε: 72.0% ± 15.0% (n=9 vessels), 245 ± 100 (n=244 cells) — WT: 33.1% ± 8.12% (n=8 vessels), 223 ± 118 (n=126 cells) — pilD: 14.9% ± 8.1% (n=10 vessels), 144 ± 58.4 (n=44 cells)). (D) Schematic representation of the setup. Purified neutrophils were introduced in the microfluidic chip under flow conditions (0.7-1 μl/min). (E) Bright-field and fluorescence images of neutrophils adhering on a non-treated (control), TNFε-treated, or Nm-infected VoC. Scale bar: 25 μm. (F) Graph representing the number of neutrophils adhering to the endothelium for each condition. Each dot represents a vessel. For each condition, the mean ± s.d. is represented (Control: 0.59 ± 1.32 μm−2 (n=5) — TNFε: 33.0 ± 36.1 μm−2 (n=8) — N. meningitidis: 21.5 ± 28.4 μm−2 (n=6)). (G) Representative images of bacteria phagocytosis by neutrophils in infected VoC (left) and in infected human vessels in the grafted mouse model (right). Scale bar: 25 μm. All statistics have been computed with Wilcoxon tests.