Inkredible bioprinter

To investigate printability, we used the INKREDIBLE bioprinter (Cellink, Gothenburg, Sweden) with the Cellink HeartWare/Repetier Host Software version 2.3.2 model design to fabricate the scaffold in a square shape measuring 20 × 20 × 2 mm³. All samples were printed at room temperature (25 °C) and crosslinked by 365 nm wavelength UV irradiation with an intensity of 10 mW/cm² for 10 min throughout the printing period; 22-gauge (22 G, inner diameter 0.644 mm) conical bioprinting nozzles (Cellink, Sweden) were fixed to 3 mL plastic cartridges for printing, as shown in Figure 1. The minimum pressure at which continuous extrusion occurs was selected, the nozzle speed was set to 5 mm/s, the infill density was set to 100%, and the distance from the needle to the print bed was optimized so that the leading edge of the flow was in line with the needle. Each printed line diameter was 0.41 mm, and the porosity size was 1.1 mm for one layer using a G-code program as a command design. Printability tests were conducted by using a glass Petri dish as the printing surface. We then imaged each print (8-megapixel camera, 1.5 µm pixel size). The ImageJ program (NIH, Bethesda, MD, USA) was used to view the threshold images and measure the printed line size.

The INKREDIBLE bioprinter (CELLINK, Göteborg, Sweden) was employed to produce cell-laden hydrogel strands in a computer-regulated environment. A 10 µm in-plane resolution and 100 µm layer resolution were employed. The equipment contained 2 pneumatic print-points. Their extrusion capability was based on, first, the pressure exerted on the piston, second, the geometry of the extrusion nozzle’s geometry, and third, the bioink’s rheological properties. Air was pressurized and supplied with a compressor, which was set up externally. This compressor was, however, connected to an alternate outlet. The air could be pressurized to up to 250 kPa. Multiple dials could be employed to regulate the pressure transferred to the print leads. In addition, there existed printing nozzles which possessed an inside diameter of 0.6 mm. In general, the printing pressures applied were between 20 kPa and 100 kPa (Figure 1).

In this study, we employed an INKREDIBLE bioprinter (CELLINK, Gothenburg, Sweden) to deliver cell-laden hydrogel strands in a computer-controlled arrangement at 10 µm in-plane resolution, and 100 µm layer resolution. This bioprinter is equipped with two pneumatic print heads, whose extrusion rate depends on the pressure applied to the piston, the geometry of the extrusion nozzle and the rheological properties of the bioink. An external compressor, connected to a different outlet than the bioprinter, provides pressurized air at a pressure of up to 250 kPa. The pressure applied to each print head can be set at a fraction of this pressure via two dials. Using blunt needles of 0.6 mm internal diameter as print nozzles, typical print-head pressures employed in our experiments ranged between 20 kPa and 100 kPa.

In this research, extrusion-based 3D printing technology was utilized using the Inkredible^® bioprinter (CELLINK Corporation, Sweden). The dressings were printed directly onto sterile Petri dishes with the print head temperature adjusted at 25°C and 35°C for hydrogel and hydrogel–BBG bioinks, respectively. The dressings were printed at 2.5 mm/s speed and 100 kPa pressure with a geometry of square (30 × 30 × 3 mm³) and dog bone (30 × 10 × 5 mm³) for different tests. The 3D-printed dressings were immersed in 0.2 M calcium nitride (CaNO₃) solution for 10 min to form crosslinks between alginate chains. After crosslinking, 3D-printed dressings were rinsed with DI water three times and stored at 4°C.

NSCLC PDX (EGFR T790M) cell line was obtained from Dr. Rishi’s Laboratory at Wayne State University and Lung CAFs (AA0022) was obtained from Dr. Noyes Laboratory, Moffit Cancer Center. A total of 10 × 10⁶ cells were mixed homogeneously with the hydrogel using a spatula in 10:1 ratio and printed with INKREDIBLE bioprinter (Cellink, Sweden). For co-culture, 5 × 10⁶  cells of each PDX and CAFs were homogeneously mixed with the hydrogel and printed with print head 1 in INKREDIBLE bioprinter. 22 G needle (CELLINK, Sweden) was used for bio-printing throughout the experiment. After printing, scaffolds were crosslinked with 100mMCaCl₂ crosslinker solution and transferred to 6 well plates. The printed cell laden scaffolds were cultured in DMEM/F12 and RPMI media supplemented with B27 supplement, recombinant human epidermal growth factor (EGF) and recombinant human basic fibroblast growth factor (bFGF).