*2.4. Fabrication Method*

### 2.4.1. Fabrication of Mold System

The methods employed for the fabrication of the outermost layer and middle layer of the TEVG were realized via a mold system. The mold system consists of five separate molds. They were made of photosensitive resin (Clear Resin, Formlabs, Somerville, MA, USA) and printed directly by a 3D printer (Form 2, Formlabs) from stereolithography (STL) files (SolidWorks 2018, Dassault Systèmes, Vélizy-Villacoublay, France). The printing parameters were set following the general configurations in the program. Briefly, the layer thickness was set to be 0.025 mm and the resin temperature was 35 ◦C. In the settings of the supporting constructions, the density of the supports, the contact point size and the thickness of the basement were set to be 1.00, 0.7 mm and 2 mm respectively. The profiles of the five molds are shown in Figure 4. The cross section of each mold's track is semicircular. The center trajectories of each mold's track are identical. The curvature radiuses of the fitting surfaces of the five molds are also the same (60 mm), which corresponds to the curved structure of the bioengineered TEVG. The diameter of the groove/convex on each mold's curved surface is determined by the dimensions of the designed TEVG.

**Figure 4.** Mold system: (**A**) mold 1; (**B**) mold 2; (**C**) mold 3; (**D**) mold 4; and (**E**) mold 5.

### 2.4.2. Fabrication of Multilayered Bifurcated TEVG with Curved Structure

A schematic representation of the step-by-step process for the fabrication of multilayered bifurcated TEVGs with a curved structure is presented in Figure 5a. At first, mold 1 and mold 2 were fitted together, with their center trajectories coincident, and three plastic tubes were inserted into the flank hole of mold 1 as the mold side gate to compose the feed system. Next, a freshly prepared gelatin/mTG solution was housed in a syringe and pipetted into the channel formed by mold 1 and mold 2 (Figure 5b). The solution delivery rate was maintained at 6 mL/min by a micro-pump attached to the syringe. After cooling at room temperature for 15 min and then 30 min at 10 ◦C to induce the gelation of the gelatin injected in the molds, mold 2 was gently removed, leaving the hydrogel structure in the channel of mold 1, which corresponds to half of the outermost layer of the TEVG. In the same manner, mold 3 was fitted together with mold 1, and the gelatin/mTG solution was pipetted into the channel from the gate, gelatinating to form half of the middle layer of the structure. After the gelation of the gelatin, we removed mold 3. Then, the fugitive ink Pluronic F127 was loaded in a syringe and printed into the groove of the hydrogel on mold 1. The printing process was executed by a custom-built three-axle linkage platform (Figure 5c). The diameter of the printed F127 ink could be regulated by adjusting the moving speed of the platform and the delivery rate of the micro-pump online. After the printing process, the rest was done in the same manner as mentioned above. Briefly, mold 4 and mold 5 were subsequently fitted together with mold 1, and the gelatin/mTG solution was pipetted into the inner channel to shape the left half of the middle layer and outermost layer of the TEVG respectively. After being crosslinked at room temperature for 15 min and then at 10 ◦C for 30 min, the upper mold was gently removed; then, we carefully removed mold 1, and a multilayered vessel-like structure was achieved. We placed the construct at 4 ◦C for 20 min, and during this process, fugitive ink Pluronic F127 liquefied and flowed away, forming the inner channel of the TEVG. The obtained construct was incubated at 37 ◦C for 5 h to ge<sup>t</sup> fully crosslinked. Then, the construct was immersed in 65 ◦C distilled water for 5 h to heat-inactivate the residual enzyme.

**Figure 5.** Fabrication process of the TEVG: (**a**) procedure of the step-by-step fabrication process. The molds 2–5 were gathered after mold 1 to compose the injection mold systems to form the different layers of the TEVG; (**b**) the gelatin/mTG perfusion system; (**c**) the fugitive ink printing system.

### *2.5. In Vitro Cytocompatibility of the TEVG*

Human umbilical cord derived endothelial cells (HUVECs) were trypsinized, centrifuged and suspended in a culture medium at a cell density of 1.0 × 10<sup>6</sup> cells/mL. The cells in the third passage after defrosting were used for the experiments. The TEVG was soaked in 75% ethanol for 1 h and then washed 3 times with phosphate buffer saline (PBS) to remove the ethanol. After that, the sample was exposed to ultraviolet light for 30 min. The cell suspension was injected into the channel. After 4 h of cell attachment, the cellular construct was placed in Dulbecco's modified Eagle medium containing high glucose and sodium pyruvate (DMEM) (HyClone, GE), supplemented with 10% fetal bovine serum (FBS) (HyClone, GE), and cultured in a humidified incubator at 37 ◦C. It was statically cultured with medium that was changed each day. At 4 h and 72 h after culturing, the TEVG was washed in sterile PBS (HyClone, GE) three times and stained using Live-DyeTM ("live"; 1 μL/mL; BioVision) and propidium iodide ("dead"; 1 μL/mL; BioVision) for 30 min. After that, the cellular morphology and fluorescent images were observed with an inversed fluorescent microscope (Eclipse Ti-U, Nikon Instruments Inc., Tokyo, Japan).

### *2.6. Uniaxial Compressive Testing*

The uniaxial compressive testing was operated on a material testing machine (Z2.5, ZWICK, Ulm, Germany). To ensure stability during the test, the diameter of the cross-section was scaled up by three times its original diameter. The uniaxial compressive tests were performed at the rate of 1 mm/min at an ambient temperature of 23 ◦C and 37 ◦C respectively. The environmental humidity was about 65%. To investigate the effect of the addition of mTG on the mechanical properties, samples with and without crosslinking were fabricated to carry out the tests.

### *2.7. Thermostability of the TEVG*

To examine the thermostability of the bioengineered TEVG, samples with and without mTG were both submerged in PBS (pH 7.4) and stored in the incubator at 37 ◦C for different time periods (2, 4, 6, 8 and 10 days). After each time period, the samples were washed and subsequently dried in a vacuum oven at room temperature for 6 h. Their weights were recorded during this period.
