The design of a coextrusion microfluidic device dedicated for spherical capsules formation has been previously described (52). Here, we slightly modified the design. The stereolithographic file (STL) file used for 3D printing is provided as data file S1. Using this device, we produced alginate hollow tubes filled with both ECs and SMCs. Briefly, cells were injected into the central zone isolated from the 2.5% (w/v) AL by a 300 mM IS. Cell loading of the CCS varied from 33% (v/v) (routine conditions) to 38% (v/v) (maximum loading). Cells were dispersed in Matrigel (phenol red free: 354262, Corning). The cell mixture was composed of 2:1 HUVEC/vSMC (for example, 14 million ECs/7 million SMCs). Practically, for each encapsulation, we started from 300 cm2 of HUVECs and 150 cm2 of vSMCs. Fifty-five microliters of cell pellet was resuspended directly with 110 or 90 μl of Matrigel (for the 33 and 38% cell loadings, respectively). These conditions ensure that the final Matrigel concentration in the tubes is about 35% (v/v), which is sufficient to create a continuous Matrigel layer anchored to the inner alginate wall (16). Most experiments were performed using the following injection flow rates: 2 ml hour−1 (AL), 1 ml hour−1 (IS), and 1 ml hour−1 (CCS). The impact of flow rates variation on the homogeneity of the tubes was investigated in Fig. 1C. A 100 mM calcium bath at 37°C was used to trigger rapid gelation of the alginate gel while avoiding osmotic shock. Immediately after formation (within less than 10 min), the tubes were transferred in HUVEC and vSMC growth medium mixture (1:1 EGM2/SMCGM2) and incubated at 37°C. This procedure developed for vesseloid preparation was patented (please see details in Acknowledgments).

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