Research ArticleHEALTH AND MEDICINE

A model of guided cell self-organization for rapid and spontaneous formation of functional vessels

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Science Advances  12 Jun 2019:
Vol. 5, no. 6, eaau6562
DOI: 10.1126/sciadv.aau6562
  • Fig. 1 3D vascular fabrication process.

    (A) Drawing of the microfluidic platform and picture of the coextrusion device. The three solutions are injected simultaneously by a computer-controlled pump, inside a 3D-printed device soaking in a 100 mM calcium bath; 2.5% AL, IS, and CCS (HUVEC/SMC/ECM). Scale bar, 1 cm. (B) Cell-laden tube after production (day 0) or after 1 day of culture (day 1). Scale bar, 200 μm. (C) Measurements of the alginate wall thickness of alginate tubes produced with a 450-μm nozzle exit coextrusion device for the following injection flow rates (n = 8): 2 ml hour−1 (AL), 1 ml hour−1 (IS), and 1 ml hour−1 (CCS); 2 ml hour−1 (AL), 1.5 ml hour−1 (IS), and 0.5 ml hour−1 (CCS); or 2 ml hour−1 (AL), 0.5 ml hour−1 (IS), and 1.5 ml hour−1 (CCS). (D and E) Tube formation reproducibility: Measurements of external and internal diameters along the same tube, separated by at least 1 mm (n = 16). Photo Credit: Laetitia Andrique (INSERM U1029), Gaelle Recher (CNRS UMR 5298).

  • Fig. 2 Self-organization into artificial mature blood vessels.

    (A and B) Confocal imaging of vesseloid immunostainings at day 1. Nuclei are gray (DAPI), CD31 is blue (endothelial marker), and αSMA or tubulin labeling is orange. Images correspond to a maximal intensity projection along the z axis, except for the half panels indicated as “optical section,” which depict a representative image at the equatorial plane. Scale bars, 50 μm. (C) Fluorescence intensity profiles of αSMA and CD31. The four different measurements were aligned together based on the intersection between both intensity distributions, and this value was taken as the reference in the plot. Negative values indicate distance toward the alginate wall, and positive values indicate distance in the direction of the lumen. (D) Anchoring of SMCs in ECM. Immunostaining of laminin, CD31, and αSMA was performed. Images correspond to a projection of the z axis of each signal. Nuclei are in gray (DAPI), laminin in green, CD31 endothelial marker in blue, and αSMA in orange. Zoom of an equatorial section of the vesseloid. Scale bars, 100 μm. (E) KI67 nuclear signal and histogram representation of proliferation at days 1 and 5. (F) Activated caspase-3 signal and histogram representation of apoptosis at days 1 and 5. Scale bars, 50 μm. AU, arbitrary units; ns, not significant. Photo Credit: Laetitia Andrique (INSERM U1029), Gaelle Recher (CNRS UMR 5298).

  • Fig. 3 Perfusable and liquid tight vesseloids.

    (A) Schematic drawing of the experimental design. The vesseloid is connected to a glass pipette for the perfusion of either dextran solution or culture medium for washing. (B) Photography of the perfusion device. (C) Schematic drawing of the experimental conditions. The left column depicts a longitudinal section alongside the glass pipet and the alginate tubes/vesseloids. The right column depicts the corresponding transverse sections. (D) Bright-field images of the tubes/vesseloids before perfusion (left column). Fluorescent images at t0 and 10 min after FITC-dextran perfusion (second and third columns). (C to E) Row 1: Alginate tube, perfusion of 500-kDa FITC-dextran. Row 2: Alginate tube, perfusion of 20-kDa FITC-dextran. Row 3: Vesseloid with HUVECs only, perfusion of 20-kDa FITC-dextran. Row 4: Vesseloid with HUVECs and vSMCs, perfusion of 20-kDa FITC-dextran. Scale bars, 200 μm. (E) Normalized fluorescence profiles both at the onset of FITC-dextran injection to mark the lumen size (0 min) and after 10 min, superimposed with the bright-field section. Corresponding fluorescence full width at half maximum (FWHM). (F to I) Vesseloids imaged by TEM at high magnification (×25,000 and ×50,000) after 1 day of culture. Corresponding drawing with identification of Weibel-Palade bodies (WPBs), lamellar bodies (LBs), tight junction (TJ), and caveolaes (Cav.). Scale bars, 5 μm. Photo Credit: Laetitia Andrique (INSERM U1029), Gaelle Recher (CNRS UMR 5298).

  • Fig. 4 Functional properties of vesseloids: Contractility and excitability.

    (A) Contraction assay. Vesseloids were exposed to 0.1 μM ET-1 (top) and to 10 μM angiotensin II, and subsequent contraction was measured. Left: Overlay of the first image before vasoconstrictor application (magenta) overlaid with the image at maximal contraction (green). Right: Percentage of contraction measured from the difference of the tube diameter before and after drug application. (B) Calcium imaging analysis subsequent to ET-1 application: Methodology. Top: Color-coded representation of a vesseloid. Cells that exhibit the earliest increase of fluorescence are depicted in red, whereas cells that responded the latest are colored in blue. Scale bars, 200 μm. Four cells are shown as examples (#14, #15, #38, and #34), and their respective fluorescence intensity variation is shown. From these individual traces, parameters were automatically extracted and sorted, notably by PCA with the following parameters: “delay” (time measured between ET-1 application and first fluorescence intensity peak) and “circ” (circularity of the cells, which depicts the cell shape), that allow the segregation of the two cell populations (HUVEC-like and SMC-like) used for further comparisons. (C) Calcium imaging analysis subsequent to ET-1 application: Results. Following segregation of the cell populations according to the previous description, traces were analyzed by comparing relevant parameters such as “cell surface” and time to peak, showing that the cell population identified as HUVECs exhibits a significantly bigger size and a faster response to ET-1 application than vSMCs. AU, arbritary units.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaau6562/DC1

    Fig. S1. Dimension and shape of the coextrusion chip.

    Fig. S2. Uniform laminin coating of the inner alginate wall.

    Fig. S3. Diameter tube measurement using three coextrusion devices of different nozzle sizes.

    Fig. S4. Immunostainings of CD31, fibrillin-1, and VE-cadherin were performed on vesseloids at day 1.

    Fig. S5. Expression measurement of (i) basal and luminal markers, (ii) arterial and venous markers, and (iii) endothelial activation markers in regular culture conditions and in response to inflammatory stimuli.

    Fig. S6. Quiescent cells at day 1 by loading the maximum of cells in the vesseloids.

    Fig. S7. Hypoxia culture stabilizes the vesseloids.

    Fig. S8. Vesseloid imaged by TEM at high magnification (×25,000 and ×50,000) after 1 day of culture.

    Fig. S9. Lumenization with EC lining and formation of an external SMC layer in spherical capsules.

    Movie S1. ECs and SMCs are dynamic after encapsulation.

    Movie S2. The 20-kDa FITC-dextran perfusion.

    Movie S3. ET-1 induces vesseloid contraction.

    Movie S4. ET-1 induces intracellular calcium raise in both ECs and SMCs.

    Data file S1. STL file of the coextrusion chip.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Dimension and shape of the coextrusion chip.
    • Fig. S2. Uniform laminin coating of the inner alginate wall.
    • Fig. S3. Diameter tube measurement using three coextrusion devices of different nozzle sizes.
    • Fig. S4. Immunostainings of CD31, fibrillin-1, and VE-cadherin were performed on vesseloids at day 1.
    • Fig. S5. Expression measurement of (i) basal and luminal markers, (ii) arterial and venous markers, and (iii) endothelial activation markers in regular culture conditions and in response to inflammatory stimuli.
    • Fig. S6. Quiescent cells at day 1 by loading the maximum of cells in the vesseloids.
    • Fig. S7. Hypoxia culture stabilizes the vesseloids.
    • Fig. S8. Vesseloid imaged by TEM at high magnification (×25,000 and ×50,000) after 1 day of culture.
    • Fig. S9. Lumenization with EC lining and formation of an external SMC layer in spherical capsules.
    • Legends for movies S1 to S4
    • Legend for data file S1

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). ECs and SMCs are dynamic after encapsulation.
    • Movie S2 (.avi format). The 20-kDa FITC-dextran perfusion.
    • Movie S3 (.avi format). ET-1 induces vesseloid contraction.
    • Movie S4 (.avi format). ET-1 induces intracellular calcium raise in both ECs and SMCs.
    • Data file S1 (.stl format). STL file of the coextrusion chip.

    Files in this Data Supplement:

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