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International Journal of Bioprinting 3D bioprinted models in pediatric tumors

approach. CellTiter Glo Luminescent Cell Viability Assay alamarBlue dye (10 µL, Thermo Fisher Scientific) was
(Promega, Madison, WI, USA)was performed to ensure added, and absorbance was measured using a microplate
that the selected printing pressure did not negatively impact reader (Epoch Microplate Spectrophotometer, BioTek
the viability of cells (Supplementary File, Figure S1). Instruments, Winooski, VT, USA) at 570 nm and 600 nm.
The viability of 2D-cultured cells was also assessed with
2.4. Reagents alamarBlue,and the cells (1.5 × 10 ) were plated in 96-well
5
CalceinAM (C1430),a cell-permeant dye used to stain live plates and treated with the same conditions. The viability
tumor cells, and SYTOX Orange nucleic acid stain (S11368) of cells from at least three biologic replicates was reported
used to stain dead tumor cells were purchased from Thermo as fold change ± standard error of the mean (SEM).
Fischer Scientific. The antibodies used included rabbit
antichromogranin A (ab15160) from Abcam (Cambridge, 2.7. In vivo studies
MA, USA) and rabbit antineuron-specific enolase (NSE) SK-N-AS and COA109 cells were used to print mixed
(AB951-I).Anti-rabbit immunoglobulin (Ig)G (NI0-1) was 3D bioprints. The resulting bioprints were then minced
purchased from Millipore Sigma. Cisplatin was obtained and combined with 25% Matrigel/RPMI for injection
from Cayman Chemical Company (13119, Ann Arbor, into the right flank of athymic nude mice. This study was
MI, USA), and trametinib, a small molecule inhibitor of conducted under UAB IACUC approval (IACUC-09064)
MEK1/2, was purchased from Selleckchem (Houston, and in accordance with national, international, and NIH
TX, USA). guidelines. The animals were maintained in pathogen free
environment with free access to food and water. The tumor
2.5. Immunohistochemistry (IHC) size was measured once a week, while the overall body
In vivo tumor samples were formalin-fixed, paraffin- condition was monitored daily. Per the National Cancer
embedded, cut into 4 µm sections, and processed as Institute’s guidelines, the formula (width × length)/2 was
2
previously described . The 3D-bioprinted tumors were used to calculate tumor volume, with width being the
[12]
immediately fixed in 80% ethanol for at least 24 h following smaller value .The animals were humanely euthanized
[14]
experimentation. The fixed bioprinted tumors were then and tumors were harvested once IACUC parameters were
processed and embedded as noted for in vivo tumors. met. The tumor specimens were divided for storage at
For both in vivo and 3D-bioprinted tumors, standard −80°C and paraffin-embedded for IHC studies.
hematoxylin and eosin (H&E) staining methods were
utilized. Chromogranin A (1:100) and NSE (1:25) staining 2.8. Ex vivo studies
was performed by adding primary antibody to slides SK-N-AS cells were printed as layered 3D bioprints on the
for overnight incubation in a humidity chamber at 4ºC. upper rowof a 12-well plate, while additional SK-N-AS cells
The slides were washed with PBS, and rabbit secondary were cultured under 2D conditions at the lower row(5 ×
antibody (R.T.U. biotinylated universal antibody, Vector 10 in 2 mL of media). The bioprints and cultured cells
5
Laboratories, Burlingame, CA, USA) was added for 30 min were preliminarily incubated overnight under normoxic
at 22ºC. The reaction was completed with VECTASTAIN conditions at 37°C in 5% CO and then transferred to a
2
Elite ABC reagent (PK-7100, Vector Laboratories) at hypoxia chamber at 1% oxygen (O ) for 120 h. The media
2
room temperature for 30 min, and Metal-Enhanced DAB were replaced at the midpoint of the experiment.
Substrate (Thermo Fisher Scientific) was applied for 2 min.
All slides with primary antibody were counterstained with After 120 h, the tumors were stained with Calcein AM
hematoxylin and had negative controls stained with anti- for viable cells and counterstained with SYTOX Orange for
IgG at 1 µg/mL. dead tumor cells at 1:1000 for 30 min. Following staining,
a minimum of nine images were taken using EVOS
2.6. Viability studies (AMG EVOS FL Digital Inverted Imaging System) at 10×
The viability of homogenous mixed 3D-bioprinted tumors magnification. The images were then individually analyzed
(24-well plates) was assessed via Calcein AM staining and with ImageJ software (Version 1.49, http://imagej.nih.
ImageJ analysis (Version 1.49, http://imagej.nih.gov/ij, gov/ij, accessed on September 10, 2022), where the total
accessed on July 17, 2022) as described in a previous fluorescence area positive for Calcein AM was compared
publication . The viability of the 96-well plate mixed to the total fluorescence area positive for SYTOX Orange
[13]
tumors was determined with alamarBlue colorimetric to determine the tumor viability. In order to determine
assays (Thermo Fisher Scientific). In 96-well plates, the viability of 2D-cultured tumor cells under hypoxic
3D-bioprinted tumors were printed as described and conditions, the surrounding media were collected, and
treated with cisplatin or trametinib and an equivalent 0.5 mL of Trypsin-EDTA (0.05%, with phenol red; Thermo
concentration of vehicle (dimethyl sulfoxide, DMSO), Fisher Scientific) was added to detach cells from the well
as a control, at increasing concentrations. After 24 h, plate. The detached cells were then pelleted, resuspended

Volume 9 Issue 4 (2023) 118 https://doi.org/10.18063/ijb.723