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

Abstract

Most achievements to engineer blood vessels are based on multiple-step manipulations such as manual sheet rolling or sequential cell seeding followed by scaffold degradation. Here, we propose a one-step strategy using a microfluidic coextrusion device to produce mature functional blood vessels. A hollow alginate hydrogel tube is internally coated with extracellular matrix to direct the self-assembly of a mixture of endothelial cells (ECs) and smooth muscle cells (SMCs). The resulting vascular structure has the correct configuration of lumen, an inner lining of ECs, and outer sheath of SMCs. These "vesseloids" reach homeostasis within a day and exhibit the following properties expected for functional vessels (i) quiescence, (ii) perfusability, and (iii) contractility in response to vasoconstrictor agents. Together, these findings provide an original and simple strategy to generate functional artificial vessels and pave the way for further developments in vascular graft and tissue engineering and for deciphering the angiogenesis process.

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.