Protocols

Human Ear

Protocol

Design of Blueprint

Software used to draw 3D model

Name of software

A 3D model of a human ear was reconstructed from an MRI scan, as described by Nimeskern et al. The STL file of the auricular model was processed in the software Slic3r (v1.2.9) and scaled to 35% of the original size.

Software used to convert 3D model to bioprinting protocol and toolpath

Spacing between lines

Layer Height = 0.40mm

Porosity of model

External perimeters extrusion width = 0.45mm

Layer thickness

Perimeters = 1

Bioinks

Biomaterial used

CELLINK Bioink

Company supplying biomaterial

Cellink

Composition

Formulation of bioink

Nanocellulose and sodium alginate-based bioink

References

3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration. Héctor Martínez Ávila, Silke Schwarz, Nicole Rotter, Paul Gatenholm. Bioprinting 2016, Volumes 1–2, 22-35.
dx.doi.org/10.1016/j.bprint.2016.08.003

3D Bioprinting Human Chondrocytes with Nanocellulose–Alginate Bioink for Cartilage Tissue Engineering Applications. Kajsa Markstedt, Athanasios Mantas, Ivan Tournier, Héctor Martínez Ávila, Daniel Hägg, and Paul Gatenholm. Biomacromolecules 2015 16 (5), 1489-1496.
dx.doi.org/10.1021/acs.biomac.5b00188

Cells

Cell type(s)

Human primary nasal chondrocytes (hNC)

Cell concentration

20x106 cells/ml
A suspension of 72x106 hNCs in a volume of 600µL were mixed with 3mL of CELLINK bioink using the CELLMIXER to obtain a final concentration of 20x106 cells/ml.

Culture media composition

DMEM/Ham's F-12 (1:1, Biochrom), supplemented with 10% fetal bovine serum (Biochrom) and 1% penicillin–streptomycin

Differentiation medium: StemMACS ChondroDiff media (Miltenyi Biotec GmbH) supplemented with 1% penicillin–streptomycin.

Bioprinter

Model and Manufacturer

INKRIBLE+ 3D Bioprinter by CELLINK

Bioprinting Technology & parameters

Pneumatic-driven microextrusion

Pressure

15-17 kPa

Printing speed

600 mm/min

Printhead temperature

Room temperature (22°C)

Printbed temperature

Room temperature (22°C)

Crosslinking process of bioink

After the bioprinting process, the cell-laden ear constructs were crosslinked by submerging in an ionic solution of 100mM CaCl2 for 10 minutes. The constructs were then rinsed and incubated in culture medium in standard culture conditions (37°C, 5% CO2 and 95% relative humidity).

Crosslinking method

Other

Bioprinting metrics

Time for bioprinting and crosslinking process per construct

5 minutes and 51 seconds per construct

Volume of bioink per construct

1.2 mL

Post-bioprinting

Time of 3D culture in vitro

28 days

Stability of constructs in vitro?

Macroscopic evaluation of constructs showed no change in shape during in vitro 3D culture.

Results

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