## 2 Materials and Methods  
### Modification of a Commercial Kentstrapper Verve 3D Printer in a BioVERVE Bioprinter  
The aim of this work was to modify a commercial 3D printer (Kentstrapper Verve, Kentstrapper S.r.l, Florence, Italy) to create a bioprinter (BioVERVE) by replacing the default extruder used to print plastic filaments with a custom-made syringe extruder suitable for printing bioinks. The original syringe extruder was designed by Kentstrapper S.p.A. to accommodate a huge 60 mL luer-lock syringe and the ensemble of a step motor,gears, transmission belt, bearingss and helical screw used to control the movement of the syringe plunger (Figure 1a).  
The 60 mL syringe extruder was used to verify the correct operation of the 3D printer in executing the G-code containing the print commands. Moreover, a first tuning of the syringe plunger movements was performed. The commands for the printer were provided as a G-code file. In particular, the correct parameter value for the M92 G-code (set axis step per millimeter) that determines how many turns of the step motor are needed to advance the syringe plunger of 1 mm was established. A value of 50 was found to be imposed as M92 parameter to print commercial silicone (i.e., 50 motor turns for 1 mm plunger advance).  
The original Kentstrapper extruder was later modified to allocate a smaller 1 mL luer-lock syringe (more suitable for cell bioprinting purposes) and to reduce the overall size of the syringe extruder above the printer (Figure 1b,c).  
The new extruder design was largely inspired by the Kentstrapper's design. The main modifications were related to the reduced size of a 1 mL syringe and to the reduction in the extruder size above the printer. In consideration of the small dimensions of the cell construct to be printed, the syringe was positioned below the rods sustaining the extruder for the \(x\)-\(y\) motion, leaving just the step motor above the printer (Figure 2).  
Figure 1: (**a**) Original Kentstrapper Verve 3D printer syringe adapter. (**b**) Isometric view of the 1 mL syringe adapter designed by FreeCAD; (**c**) 3D printed parts of the 1 mL syringe adapter.  
Figure 2: (**a**) BioVERVE 3D bioprinter, (**b**) VERVE bioprinter extruder and syringe adapter and (**c**) professional bioprinter (3D-Discovery, REGEN-HU, CH) of MAIA Center at ENEA.  
Such syringe arrangement was chosen to realize an upper cover for the printer in order to protect the printing environment from outer contaminants.  
The new adapter was entirely designed using FreeCAD ([[https://www.freecadweb.org](https://www.freecadweb.org)]([https://www.freecadweb.org](https://www.freecadweb.org)), (accessed on 20 September 2021)), a freeware and open-source parametric 3D modeler. The resulting model was subsequently sliced using Ultimaker Cura (V4.13, Ultimaker, Utrecht, the Netherlands--[[https://github.com/Ultimaker/Cura/releases](https://github.com/Ultimaker/Cura/releases)]([https://github.com/Ultimaker/Cura/releases](https://github.com/Ultimaker/Cura/releases)) (accessed on 20 September 2021)), a freeware and open-source slicing application for 3D printers. The resulting G-code file was printed with a Kentstrapper Mavis 3D printer using polylactic acid (PLA) filaments. In order to print bioinks, the correct M92 G-code parameter value, which largely depends on the viscosity of the specific bioink used, the syringe nozzle diameter and, ultimately, the desired spatial resolution, was determined.  
Several attempts were performed to establish the correct M92 value to be used in the bioprinting experiments, taking into account the ink viscosity. To this aim, conical 25 G nozzles were used to print a simple ring (5 mm diameter and 1 mm height), and several inks were tested (i.e., Cellink Xplore, Cellink Start and Cellink Laminink 411, Cellink Life Sciences, Goteborg, Sweden).