## 4 Discussion  
To assign and transfer in printable constructs branch-specific information, here we recapitulated an image analysis-based workflow to process small portions of microscopic rodent mesenteric and intestinal tissues images, using the semi-automatic network analysis program VESGEN 2D [18], as we previously showed for human retinal vascular patterns [19]. To materialize this procedure, we printed the patterns in 2D and in the 3D mode with a colored bioink surrogate, with alginate-nanocellulose bioink, as well as with a cell-containing collagen bioink. The logic behind choosing the printable materials to test was to move sequentially from a 'practice level' colored paste used for showing how the 'branch generation' information  
Figure 5: **Cylindrical conversion of the colon image for printing. ****(A)** Flat image from Figure 3 with the three separated branch generations in colors, displayed as prepared for rolling on a cylinder (inclined presentation to provide additional perspective). (B) Cylinder-rolled image of the vascular pattern. (C–E) Rolled monocolor rendering of the.stl files of separated images of each vascular branch generation. Figure 6: **Preparation of the central portion of cylindrical colon pattern for printing. ****(A)** Virtual sectioning of the CAD model in two horizontal semi-cylinders. (B) Oblique view of the model, and selection of a portion for higher magnification to show the decomposition of the continuous lines in short segments (_insert_). can be passed on from VESGEN 2D analysis to an actual print, to biological ones usable for bioprinting. For the latter, we started with a commercial collagenous bioink (\"LifeInk\") as the most suitable support for microvascular cells, which is available in two formulations: (i) LifeInk 240, the acidic and thus more fluid form (suitable for hydrogel-embedded bioprinting where it undergoes jellification by taking the support bath's pH); (ii) LifeInk 200, which has a neutral pH but is more viscous, yet appropriate for direct-write bioprinting of endothelial cells. In our hands, this second formulation led to a loss of printing resolution as compared with the acidic version, and for this reason we also tried another largely used alginate-nanocellulose bioink [31]. However, this did not provide a better printing resolution, with the additional inconvenience of being less transparent than collagenous bioinks, a feature needed for imaging the fluorescently labeled cells. Therefore, we returned to the most physiologic and convenient bioink (neutral collagen), to which we added EC for the final printing. Admittedly, our work did not generate perfusable structures, which is the next phase of our research. However, a hydrogel line-confined self-assembling of microvascular structures from ether primary EC, or from the'stromal vascular fraction' has been previously suggested [15]. Looking forward, it is worth considering the same approach as in the recently described 'Bioprinting-Assisted Tissue Emergence' (BATE) method, which uses very high densities of stem cells that spontaneously self-organize within hydrogels lines [6]. In these, the stem cells-derived tissue constructs are determined to organize according to the printing-imposed geometry, as anticipated elsewhere as well [16; 17]. Another limitation of this study is that the printed images were only topological equivalents, and not exact representations of the original images. In part, this is due to the difficulty to detect and extract the finest microvessels from low-magnification microscopic images, as employed here. In addition, during VESGEN 2D processing one may intentionally reduce the number of vascular generations, as also illustrated here. This simplification is highly desirable in some circumstances, for example when splitting them down into too many groups is counterproductive, considering the additional lengthening of the procedure and of the printing time.