Biox 3d bioprinter

Tinkercad, an online 3D CAD design tool (Fig. 1a), was used to design scaffold structures, which were then divided to smaller implantable systems. The Biox ™ 3D Bioprinter (Cellink, Sweden) with a thermoplastic printhead was employed, using a 22G conical needle (0.41 mm inner diameter) at 1 mm/s speed. PCL only implants were manufactured by adding the powder directly into the thermoplastic printhead without adding any solvent and fill maximum half of the cartridge to ensure the most efficient heating of PCL. Implants containing both PCL and the timolol maleate were added together in thermoplastic cartridge as the powder form and vortexed for 5 min at 60 s intervals. The layer height for printing was set at 0.8 mm and the infill density was set at 0%. Each implant was composed of 2 layers. To achieve good printability, optimisation of the printing pressures and temperatures was required to achieve good flow (data not shown). The PCL extrusion was smooth, and the implant was fully formed at 150 °C and at the pressure of 175 kPa. With the addition of TML, the temperature remained the same; however, the pressure had to be increased to 190 kPa.

For the fabrication of tumor spheroids, a hybrid method consisting of liquid overlay technique and drop-on-demand was utilized. Briefly, U87 were harvested and suspended in α-MEM supplemented with 10% FBS and 1% PS at a cell concentration of (2000 cells/well). 3 mL printing cartridges were then filled with the cell suspension. Using the BIO X™ 3D bioprinter (CELLINK®, Sweden), cell suspensions were then extruded through 25G needles into each well of ultra-low attachment 96 well round-bottomed plates. The extrusion pressure used was 20 kPa, and the extrusion time was set to 1 s. Plates were then centrifuged at 1750 rpm for 10 minutes and first incubated for 6 h in a nitrogen environment and subsequently at 37°C and 5% CO 2 concentration for 24h before further studies.