Bio-printing was carried out using the 3D Discovery bioprinter (Regenhu, Switzerland). The barrel temperature was maintained at 10°C using a temperature controller and the printing chamber temperature was maintained at 20°C. The bio-ink was dispensed through a sterile metal needle (nozzle diameter 0.33 mm), using a pneumatic pressure of 1.8–2.5 Mpa and a deposition speed of 5 mm/s. The deposition speed and pressure were controlled by customized software developed by Regenhu. After the dermal layer constructs were printed, they were crosslinked by sterile 2% (w/v) CaCl2. The crosslinked dermal constructs were washed with sterile saline solution thrice and dipped in DMEM (supplemented with 10% FBS, 1% penicillin-streptomycin) and incubated at 37°C and 5% CO2 .
3d discovery bioprinter
Manufactured by RegenHU
Sourced in Switzerland
The 3D Discovery bioprinter is a desktop-sized device designed for the fabrication of 3D constructs from biocompatible materials. It utilizes an extrusion-based printing technology to deposit biomaterials layer-by-layer, enabling the creation of complex tissue engineering scaffolds and organ constructs. The 3D Discovery provides a controlled and sterile environment for the bioprinting process.
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9 protocols using 3d discovery bioprinter
1
Bioprinting Dermal Constructs with 3D Discovery
2
Bioprinting HDF Cells in Hydrogel Matrix
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HDFN were harvested using 0.05% Trypsin (Gibco), pelleted, and resuspended in prepared DBH at a concentration of 2 million/mL. The molten DBH/cell mixture was loaded into a plunger syringe (regenHU 900013152) and briefly chilled on ice to gel. After attaching a 0.41 mm ID luer lock needle (Nordson 7018263), the syringe was loaded onto a regenHU 3DDiscovery Bioprinter. Volumetric plunger dispensing for the DBH mixture provides an advantage over pneumatic in that the dispense rate is not notably affected by small variation in viscosity with each gel preparation.
3
Bioink Extrusion-based 3D Printing
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To evaluate bioinks shape fidelity, the extrusion-based 3D printing of GelMA or GelMA-LAP (1wt%) bioinks was performed using a 3D Discovery bioprinter (RegenHU, Villaz-Saint-Pierre, Switzerland). Pre-polymer solutions were fabricated at elevated temperatures and loaded into a 3D Discovery bioprinter and protected from light. Computer aided design models were created using the BIOCAD software (3DDiscoverGS, RegenHU, Villaz-Saint-Pierre, Switzerland). The bioinks were extruded using a computer-aided syringe dispenser with a 20-gauge needle, a feed rate of 3 mm/s and pressure of 10 bar. Constructs were printed within a 6 well plate and irradiated for 5 min with visible light using the same Ru/SPS photoinitiator concentrations as described above.
4
3D Bioprinting of Chondrocyte-Laden Hydrogels
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Cell-laden bioinks were prepared by mixing a 5.3% (w/v) alginate solution and a 21.5% (w/v) gelatin solution (Gelatin type B; Sigma-Aldrich, Wicklow, Ireland) with a cell suspension (everything in hgDMEM) to obtain a final solution of 3.5% (w/v) of alginate and 5% (w/v) of gelatin containing 20 × 106 cells/mL. For acellular characterisation, the volume of cell suspension was substituted for hgDMEM to maintain the same polymer concentrations. The different bioinks were loaded into a syringe, and then printed using the 3D Discovery bioprinter (RegenHU, Villaz-St-Pierre, Switzerland) at 16 °C, using a pressure of 0.125 MPa, a translational speed of 4 mm/s and a plastic conical needle of 25G (Adhesive Dispensing Ltd., Buckinghamshire, UK). Cellular constructs were printed following a cylindrical geometry of 4 mm in diameter by 4 mm height, following a grid pattern with strand distance of 0.250 mm and a z distance increase of 0.250 mm. After printing, the different samples were incubated for 30 min in a bath of 45 mM CaCl2 in hgDMEM, and then the cell-laden hydrogel were maintained in XPAN medium for 24 h before switching to chondrogenic medium (CDM) (Table 2 ) at 5% O2. Media exchange was performed twice weekly until the end of the 4-week culture period.
5
3D Bioprinted Scaffold Fabrication
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A multiheaded 3D Discovery
bioprinter (RegenHU, Switzerland) was used to print the scaffolds.
A high precision plunger dispenser was used at a constant volume flow
rate of 1.5 μL/s using a stainless-steel needle with an inner
diameter of 0.41 mm, at 10 mm/s printing speed. The G-code for square
scaffolds was produced using BIOCAD software (RegenHU, Switzerland)
with 0.8 mm spacing. In situ UV-crosslinking was performed after the
deposition of each layer using the light curing cartridge at 365 nm
(500 mW).
bioprinter (RegenHU, Switzerland) was used to print the scaffolds.
A high precision plunger dispenser was used at a constant volume flow
rate of 1.5 μL/s using a stainless-steel needle with an inner
diameter of 0.41 mm, at 10 mm/s printing speed. The G-code for square
scaffolds was produced using BIOCAD software (RegenHU, Switzerland)
with 0.8 mm spacing. In situ UV-crosslinking was performed after the
deposition of each layer using the light curing cartridge at 365 nm
(500 mW).
6
3D Bioprinting of Neural Stem Cells
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RegenHU 3D discovery bioprinter inside a biosafety cabinet at room temperature were used for bioprinting. The printer and the biosafety cabinets were sterilized under UV light for 1 h before printing. Bioink containing NSCs were loaded in a 3 mL sterile syringe and connected to the air pressure supply. A needle with 0.51 mm inner diameter was used for the printing (Needle DD-135N ID=0.51/G21 L=25.4, RegenHU, Switzerland). Print parameters were adjusted to obtain continuous flow rate and smooth hydrogel fibers with minimal spreading. A feed rate of 2 mm/s and pressure of 0.3-0.4 MPa were used, the total print time was under 30 min per one 24-well plate. The printability of the bioink was assessed by switching on the pressure and the filament formation at the tip of the needle. The needle diameter, pneumatic pressure, and nozzle moving speed were optimized to deliver continuous extrusion of the bioink in the designated well of the well plate.
7
3D Bioprinting with Photocurable Hydrogels
8
3D-Printed Wound Dressings with Melt Electrowriting
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CAD (Computer-aided Design) models were designed using a Biomedical Research software BioCAD (REGENHU, Switzerland). The design model was a square shape 20 × 20 mm, distance between fibers of 0.5 mm, angle between layers of 0–90 degrees, and 100 layers. 3DP-MEW wound dressings were manufactured using a 3D Discovery bioprinter (REGENHU) with a MEW printing head. A glass slide (8 × 5 × 1 cm) was used on top of the ceramic collector as a substrate to collect the fibers. The printing parameters were printing-head temperature 70°C (PCL-CC366 5%–10%) and 75°C (PCL), pressure 0.12 MPa, speed of collector 6 mm/s (PCL) and 11 mm/s (PCL-CC366 5%–10%), and offset height “z” 3 (PCL-CC366 5%–10%) and 5 mm (PCL). A gradient applied voltage of 5 to 6.5 kV was applied to maintain a constant electric field.71 (link)
9
3D Printing of Functional Silicone Scaffolds
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Printing was performed using the 3D Discovery bioprinter from RegenHU, Switzerland. Print layouts were developed in the BIOCAD software (RegenHU). The printed scaffolds for the functional fibres were developed with the SE1700 silicone, using a metal conical nozzle with a nominal inner diameter of 100 μm at the tip (Poly Dispersing Systems). The pneumatic pressure was set at 5 bar. The optical silicone OE6520 and the white reflector silicone MS2002 were deposited by the nozzle extrusion method using a nozzle of similar dimensions. The pneumatic pressures were set at 2 and 4 bar, respectively. The platinum suspension was deposited in the printed SE1700 scaffolds by ink-jet or by pipetting. The printed lines were then heated to 120 °C for 5 min to allow evaporation of TGME, leaving behind compacted dry platinum powder inside groove-shaped scaffolds. In all cases, the substrate used for printing was glass treated with 2% sodium dodecyl sulfate (Merck KGaA) to form a debonding layer.
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