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International Journal of Bioprinting 3D bioprinted models in pediatric tumors

1. Introduction of human origin and annually validated via short tandem
repeats as recommended by the American Type Culture
The outcomes for children with hematologic malignancies Collection (ATCC, Manassas, VA, USA). PDX tumors
have substantially improved in the last 30 years, but were also validated via histology. SK-N-AS (MYCN-non-
the same success is not evident in children with solid amplified, CRL-2137) and SK-N-BE(2) (MYCN-amplified,
[1]
tumors . Tumors of neural crest origin, including CRL-2271), were purchased from ATCC. SK-N-AS cells
neuroblastoma, melanoma, peripheral malignant nerve were cultured and tested in Dulbecco’s Modified Eagle’s
sheath tumor, and gastroenteropancreatic neuroendocrine Medium (DMEM; 30-2601, ATCC) containing 10% fetal
tumors (NETs), continue to have dismal prognoses. In the bovine serum (FBS; HyClone, South Logan, UT, USA),
pediatric population, neuroblastoma is the most common 4 mmol/L L-glutamine (Thermo Fisher Scientific Inc.,
neural crest-derived tumor, and children with high-risk Waltham, MA, USA), 1 μmol/L nonessential amino acids,
disease have less than a 50% chance of a 5-year event- and 1 μg/mL penicillin/streptomycin (Sigma Aldrich,
[2]
free survival . Pediatric NETs, which are the rarest, may Burlington, MA, USA). SK-N-BE(2) cells were cultured
[3]
have survival rates as low as 10% . Therefore, discovering and tested in a 1:1 mixture of minimum Eagle’s medium
therapies to improve outcomes in these diseases remains a and Ham’s F-12 medium (30-2004, ATCC) with 10%
necessary endeavor.
FBS (HyClone), 2 mmol/L L-glutamine (Thermo Fisher
Solid tumor modeling has traditionally relied on two- Scientific Inc.), 1 μmol/L nonessential amino acids, and
dimensional (2D) cell culture. However, this method cannot 1 μg/mL penicillin/streptomycin (Sigma Aldrich).
recapitulate the complexities of solid tumors, including Two human neuroblastoma PDXs, COA3 and COA6,
tumor cell heterogeneity and tumor microenvironment. and a gastroenteropancreatic human NETPDX, COA109,
Animal models have also been employed, but they are time- were established and fully characterized as described in
consuming, thus further delaying the clinical translation previous publications [9,10] . The PDXs were passed through
of experimental treatments. Tumor organoids are another animals as these cells cannot be propagated in traditional
cell culture approach, in which these organoid models rely 2D cell culture. For experimentation, dissociated PDX
on tumor cells or tumor stem cells to create their three- tumor cells were maintained and tested in neurobasal
dimensional (3D) structure . All of these approaches fall medium (NB) (Life Technologies, Thermo Fisher Scientific,
[4]
short of 3D bioprinting. 3D bioprinting cancer cells into Waltham, MA)supplemented with B-27 without vitamin A
microtumors allows for the generation of specific tumor (Life Technologies), N2 (Life Technologies), amphotericin
architecture with multiple cell types and provides the B (250 µg/mL, HyClone), gentamicin (50 µg/mL,
opportunity for scalability and more manipulation [5,6] . Millipore), L-glutamine (2 mM, Thermo Fisher Scientific
Investigators have used 3D bioprinting for adult tumor Inc.), epidermal growth factor (EGF; 10 ng/mL, Miltenyi
types, including breast and pancreatic cancers as well Biotec, San Diego, CA, USA), and fibroblast growth factor
as brain tumors, using established cancer cell lines and (FGF; 10 ng/mL, Miltenyi Biotec).
patient-derived tumors [7,8] . These 3D-printed microtumors
demonstrated the tumor phenotype and recapitulated the 2.2. Patient-derived xenografts (PDXs)
tumor microenvironment. The PDXs used in this study included two high-risk
neuroblastomas (COA3 and COA6) and one pediatric,
For the current study, we aimed to create 3D-bioprinted
tumors of pediatric neural crest-derived tumors using high-grade neuroendocrine-like tumor (COA109) that
established cell lines and patient-derived xenograft (PDX) were derived from tumor samples of patients at Children’s
[10,11]
cells. We hypothesized that these 3D bioprint models Hospital of Alabama [10] . The tumors were obtained as
would provide an improved representation of the solid previously described under the University of Alabama
tumor phenotype compared to the traditional 2D culture at Birmingham (UAB) Institutional Review Board (IRB)-
method. Using a combination of approaches, we produced approved protocol (IRB 130627006) and in accordance
tumor models with properties consistent with the solid with the Declaration of Helsinki and the guidelines of the
tumors of origin. Additionally, we were able to scale this National Institutes of Health. Following written informed
3D model for high-throughput drug screening, which consent from parents or guardians and written informed
could be applied to personalized cancer therapy testing. assent from the patients as necessary, fresh tumor
samples were acquired and partitioned for (i) storage at
2. Materials and methods −80°C; (ii) paraffin embedding; and (iii) implantation
into the flanks of athymic nude mice in a mixture of
2.1. Cell lines 25% Matrigel (Corning Inc., Corning, NY, USA) and
Both long-term passage and patient-derived xenograft Roswell Park Memorial Institute (RPMI) 1640 (30-2001,
(PDX) lines were used in this study. Both cell lines were ATCC) following UAB Institutional Animal Care and

Volume 9 Issue 4 (2023) 116 https://doi.org/10.18063/ijb.723