Page 392 - IJB-10-2

International Journal of Bioprinting Automated bioink mixer improves bioprinting

from GenTarget (GenTarget Inc., San Diego, CA, USA). the program executed the predetermined settings, causing
These cells were cultured in low-glucose Dulbecco’s the sliding platform to move the syringe barrels back and
Modified Eagle Medium (DMEM; Biowest, Nuaillé, forth in a highly controllable manner. The mixing process
France) containing 10% fetal calf serum (FCS; c.c.pro, was terminated according to the specified parameters.
Oberdorla, Germany), 4.5 mg/mL glucose (Sigma), 2 mM For comparison, the mixing process was also carried out
L-glutamine (Biowest), and 1× non-essential amino acid manually by human operators.
(NEAA; Biowest). The HepaRG cell line was purchased
from Biopredic International (Saint Gregoiré, France) 2.5. Bioink characterization
and cultured in William’s E medium (Gibco, Paisley, UK) Following the mixing process, the bioink was transferred
supplemented with 10% FCS, 2 mM L-glutamine, 5 µg/ into a cartridge for further evaluation of cell distribution
mL Insulin (Sigma), and 50 µM Hydrocortisone (Sigma). and viability. A sample of 0.5 mL bioink was taken from
The human non-small lung cancer cell line A549 (ACC three different positions (start, middle, and end) within
107; DSMZ, Braunschweig, Germany) was cultured in the cartridge and placed onto a glass slide. As illustrated in
high-glucose DMEM (Biowest) with 10% FCS and 2 Figure S1A (Supplementary File), the bioink sample was
mM L-glutamine (Biowest). All cell lines were cultured compressed into a thin layer by a cover slide held up by
in a 37°C incubator with 5% CO2. Upon reaching 75% two 100-µm-thick spacers. The resulting bioink layer was
confluence, the cells were washed with phosphate-buffered observed under a fluorescence microscope (Observer Z1,
saline (PBS; Biowest) prior to harvesting using trypsin- Zeiss, Jena, Germany) to assess the distribution of cells
EDTA (Biowest) for either passaging or bioink mixing. within the bioink.
Metabolic activity of the cells was also quantified
2.4. Bioink mixing process after bioprinting using the tetrazolium hydroxide salt
Three different hydrogels were prepared according to the (XTT, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-
specifications outlined in Table 1: Alginate and the blend 2H-Tetrazolium-5-Carboxanilide) assay according to
of gelatin/alginate are widely used natural hydrogels for the manufacturer’s instructions (Thermo Fisher, Kandel,
bioink preparation. Gelatin (G2500-500G) used in this Germany). The mixed bioink from the aforementioned
experiment was obtained from Sigma (St. Louis, MO, positions was printed as single-layer disc constructs
USA). Poly(acrylic acid) (PAA; Carbopol 940, Acros into a 48-well plate using an extrusion-based printhead
Organics, Belgium) is a synthetic component with high equipped with a 22G nozzle on a regenHU bioprinter (3D
biocompatibility. The respective powder components discovery, regenHU, Switzerland). After printing, 300 µL
were dissolved in complete medium and stirred overnight of complete medium supplemented with 20 mM CaCl2
at 37°C. Subsequently, 1 mL of the resulting hydrogel (Roth, Karlsruhe, Germany) were added to each well.
was loaded into a syringe, while 0.9 mL cell suspension The XTT assay was performed 1 day after bioprinting by
(2 × 10 cells) and 0.1 mL 1 M CaSO4 suspension were adding 150 µL of XTT working solution, which is a mixture
7
combined in a separate syringe. In the case of PAA bioink of 1 mg/mL XTT solution and 3.83 mg/mL phenazine
mixing, 0.1 mL CaSO4 suspension was replaced by 0.1 mL methosulfate (PMS, AppliChem, Darmstadt, Germany)
complete medium.
at a volume ratio of 500:1. Following a 4-h incubation
For mixing of the bioink, two syringes were loaded period, 100 µL supernatant from each well was transferred
with materials and carefully connected using a female Luer to a 96-well plate for absorbance measurement at 450 nm
thread style coupler (Masterflex, Gelsenkirchen, Germany) (with 620 nm as the reference wavelength) in a microplate
without introducing air bubbles. The connected syringes reader (Sunrise, Tecan, Männedorf, Switzerland). Cell-
were then placed on the sliding platform with the syringe free constructs were included as background control, and
barrels secured, while the thumb rests were appropriately all absorbance values were corrected by subtracting the
positioned at both ends. Once the parameters were set, corresponding background value.

Table 1. Formula of hydrogels for bioink mixing in this study
Hydrogel Solute Solvent Note
6% alginate 3 g sodium alginate 50 mL complete medium N/A
6% gelatin + 4% alginate 3 g gelatin 50 mL complete medium N/A
2 g sodium alginate
2% PAA 1 g PAA 50 mL complete medium pH neutralized using 10 M NaOH
solution after dissolving

Volume 10 Issue 2 (2024) 384 doi: 10.36922/ijb.1974