Page 125 - IJB-9-4

International Journal of Bioprinting 3D bioprinted models in pediatric tumors

Use Committee (IACUC) protocol (IACUC-09186) for extruded as a droplet through Cellink’s 22-gauge 1/2inch
PDX maintenance. Once the PDX tumors reached the size blunt tip needle at a pressure of 10 kPa for 1.2 s to create a
dictated by IACUC parameters, they were dissociated into droplet volume of 30 μL onto a 3 μM pore transwell insert
a single cell suspension via the Mouse Tumor Dissociation (Corning Life Sciences, Tewksbury, MA, USA) in a 24-well
Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) plate. Then, 100 μL of 2% calcium chloride was added onto
following the manufacturer’s protocol and utilized for the bioprinted structures for 5 min to achieve crosslinking.
in vitro studies. The prints were then washed with 500 μL of sterile PBS, and
all the liquid was removed from the transwell containing
2.3. 3D bioprinting the bioprint. Finally, 2 mL of the cell line’s respective media
Two different bioprinting methods were employed for this was placed in the well. The prints were incubated at 37°C
study. The initial bioprinting method involved layered in 5% carbon dioxide (CO ) for 5 days to form tumors, and
bioprinting, consisting of (i) a bottom layer of bioink the media were replaced daily.
2
composed of 1% sodium alginate and 6% gelatin (Pronova
UP-LVM, Dupont Nutrition Norge As, Sandvika, Norway); In order to test the bioprints in a high-throughput
(ii) a middle layer of cells (5 × 10 cells in 15 μL); and (iii) a nature, the bioprints created using the mixed method
5
top layer of bioink printed in the same wayas the bottom were scaled down to a 96-well plate. The preparation of
layer into 12-well plates. In order to create the first layer, the the cell-bioink solution followed the same procedure listed
bioink was loaded into a 3 mL printing cartridge (Cellink, above for the mixed method, and the final cell density in
7
Boston, MA, USA) and placed into a 3 mL pneumatic the bioink solution (10 cells per 100 μL of bioink) was
printheadin Cellink’s BIO X printer. The bioink was then the same. The bioink was extruded as a droplet through
extruded as a droplet into the plate through Cellink’s Cellink’s 22-gauge 1/2 inch blunt tip needle at a pressure
20-gauge 1/2 inch blunt tip needle at a pressure of 10 kPa of 10 kPa for 0.4 s to create a droplet volume of 10 μL
for 0.3 s to create a droplet volume of 100 μL. Cells (15 μL) into a 96-well plate. Calcium chloride (2%, 40 μL) was
were then pipetted onto this bottom layer. Next, a second added onto the bioprinted structures for 5 min to achieve
layer of bioink was printed onto this structure following crosslinking. The prints were then washed with 100 μL
the previously used pressure and extrusion time. Diluted of sterile PBS, and all the liquid was removed from the
calcium chloride (100 μL, C1016-500G, Sigma-Aldrich) transwell containing the bioprint. Finally, 200 μL of the cell
to 2% in distilled water was then added on to the bioprint line’s respective media was placed in the well. The prints
for 20 min to achieve crosslinking. The prints were then were used 24 h later for experimentation.
washed with 500 μL of sterile phosphate-buffered saline Many aspects of the bioprinting protocol were adapted
(PBS), and all the liquid was removed from the well from the established bioprinting protocol from Cellink
containing the bioprint. Finally, 2 mL of the cell line’s (https://www.cellink.com/wp-content/uploads/2019/03/
respective media was placed in the well. The prints were Bioprinting-Protocol-CELLINK-Bioink_14-Jun-2021.
used 24 h later for experimentation. pdf), including, but not limited to, the ratio of the bioink to
The second method employed for bioprinting involved cells, the technique for mixing the cells with the bioink, the
mixing the tumor cells directly into the bioink to create printing pressures utilized, the needle size, and the process
a homogeneous bioink solution. A bioink (1,000 μL) for crosslinking with calcium chloride (Supplementary
composed of 1% sodium alginate and 6% gelatin was File, Table S1). The decision to use the bioink composed
prepared and loaded into a BD Plastipak 3 mL syringe of 1% sodium alginate and 6% gelatin (Pronova UP-LVM,
with Luer-Lok tip (Fischer Scientific). A female-female Dupont Nutrition Norge As, Sandvika, Norway) and for
Luer-Lok connector (IMI, Pompano Beach, FL, USA) was the cell density within the bioprints was supported by the
[8]
then connected to the end of the 3 mL syringe. Tumor cells successful bioprints created by Langer et al. .
were prepared in a volume of 100 μL such that the final cell The utilization of 3D bioprinters allows for consistency
density in the bioink solution would be 10 cells per 100 μL and accuracy in the creation of 3D tumor models. Cellink’s
7
of bioink. In order to accomplish the mixing, the prepared BIO X bioprinter has a calibration system that allows for
tumor cells were placed into a separate 3 mL syringe with its bioprints to be placed with precision in the desired
Luer-Lok tip and connected to the free end of the Luer- location. The methods described in this paper utilized the
Lok connector. The bioink and cells were then interspersed droplet feature of the printer, which allows bioink droplets
with one another, creating a homogeneous mixture by to be extruded from the bioprinter and does not require a
repeatedly pushing the materials back and forth across the specified pattern to be programmed into the printer. In the
connector. The cell–bioink mixture was then loaded into a present study, the bioprints were created using the droplet
3 mL printing cartridge and placed into a 3 mL pneumatic approach in view of its simplicity, especially as it applies
print head in Cellink’s BIO X printer. The bioink was then to creating and testing bioprints with a high-throughput

Volume 9 Issue 4 (2023) 117 https://doi.org/10.18063/ijb.723