Table 1

List of utilized in vitro pediatric cancer models, culture conditions, and cell sources

In vitro modelType of modelCulture conditionSource (identifier)
AML
Kasumi-1Cell linesStandard RPMIPrincess Máxima Center (CVCL_0589)
SKNO1RPMI+Glutamax supplemented with 20% FCS, 1% P/S and 10 ng/mL GM-CSF (Miltenyi Biotec).Princess Máxima Center (CVCL_2196)
PDX01PDXPDX material derived from primary t(8;21) pediatric acute myeloid leukemia15 was cocultured with human bone-marrow derived MSCs as feeder layer.
MSCs were cultured in low glucose DMEM (Life Technologies) supplemented with 20% FCS, 8 ng/mL FGF-2 (PeproTech) and 1% P/S and were seeded in 24 well plate at a density of 7500 cell/cm2 1 day prior to PDX culture.
PDX cells were then thawed and resuspended at 1×106 cell/mL in an in-house optimized SFEMII medium (STEMCELL Technologies) supplemented with cytokines including 150 ng/mL SCF, 100 ng/mL TPO, 10 ng/mL FLT3-L, 10 ng/mL IL-3 (all PeproTech), 1350 nM UM729 (STEMCELL Technologies), 750nM SR1 (Biogems), and 10 ng/mL GM-CSF (Princess Máxima Center pharmacy). Cells were kept on MSCs for 3 days before cells were harvested and functional T-cell assays were performed.
Princess Máxima Center
B-ALL
NALM6Cell LinesStandard RPMIPrincess Máxima Center (CVCL_0092)
RCH-ACVPrincess Máxima Center (CVCL_1851)
SUPB-15Princess Máxima Center (CVCL_0103)
697Princess Máxima Center (CVCL_0079)
T-ALL
MOLT16Cell linesStandard RPMIPrincess Máxima Center (CVCL_1424)
CCRF-CEMPrincess Máxima Center (CVCL_0207)
HCC
pHCCOrganoidThe culture medium was based on Saltsman et al.16 with modifications as reported in17 . The medium consisted of the following: Advanced DMEM/F12 supplemented with 1% P/S, 1% GlutaMAX, 10 mM HEPES, 1 x B27 supplement, 1x N2 supplement (all Life Technologies), 0.2% Normocin (Invivogen), 1.25 mM N-acetyl-L-cysteine, 10% (vol/vol) RSPO1 conditioned medium, 10 mM nicotinamide, 10 nM recombinant human (Leu15)-gastrin I (All Sigma), 50 ng/mL recombinant human EGF, 25 ng/mL recombinant human HGF (Both Peprotech), 10 µM forskolin, 5 µM A8301 (Both Tocris Bioscience), 10 µM Y27632 ((Abmole), and 0.5 nM WNT surrogate.18 Cell culture and passing were performed as described by Kluiver et al.19.
Basement membrane extract (BME) (R&D systems) 3D-cultures were dissociated using dispase and TripleE (both Life Technologies). Cells were washed and resuspended in the culture medium above + 2% FCS. Plates were coated with collagen I (Corning) and cells were cultured in 2D-setting before assays were initiated.
Princess Máxima Center
NBL
GIMENCell linesDMEM+Glutamax supplemented with 10% FCS, 1% P/S, 2% non-essential amino acids (Life Technologies).Amsterdam Medical Center (CVCL_1232)
IMR32Princess Máxima Center
(CVCL_0346)
CHP212RPMI+Glutamax supplemented with 10% FCS, 1% P/S, 2% non-essential amino acids.Dana Farber Institute (CVCL_1125)
SMSKANDana Farber Institute (CVCL_7131)
SKNDZDana Farber Institute (CVCL_1701)
NLFDana Farber Institute (CVCL_E217)
691bOrganoidsNBL organoids were established, characterized and cultured as described previously20.
Organoids were maintained in DMEM low glucose, Glutamax-supplemented medium, supplemented with 25% HAM’s F-12 Nutrient Mix, 2% B-27 supplement minus vitamin A, 1% N-2 Supplement, 1% P/S (all Life Technologies), 20 ng/mL Animal-Free Recombinant Human-EGF, 40 ng/mL FGF-basic, 200 ng/mL IGF-I, 10 ng/mL PDGF-AA, and 10 ng/mL PDGF-BB (all Peprotech).
Princess Máxima Center
039
MRT
78T2OrganoidsMRT organoids were established, characterized, and cultured as described previously21 22.
In short, organoids were grown in droplets of growth factor-reduced BME type 2 (R&D Systems), advanced DMEM/F12 containing 1X GlutaMAX, 10 mM HEPES, and 1% P/S, supplemented with 10% R-spondin–conditioned medium, 1.5% B27 supplement, 50 ng/mL EGF, 50 ng/mL FGF-2 (PeproTech), 1.25 mM N-acetylcysteine, 10 mM ROCK inhibitor Y-27632, and 5 mM A83-01. Cells were cultured in 2D-setting in functional T-cell assays.
Princess Máxima Center
JD081T
JD041T
60T2
HBL
HB13FOrganoidsHBL organoids were established, characterized, and cultured as described previously17, see HCC for detailed culture conditions.Princess Máxima Center
HB13E
DMG
JHH-DMG-01NeurospheresNeurospheres were established, characterized, and cultured as previously described23 24.
Cells were maintained in DMEM/F12 and Neurobasal-A medium (1:1), supplemented with, 0.2% Primocin, 10% FCS, 1M HEPES, 1% non-essential amino acids, 1× Glutamax, 1% sodium pyruvate, 2% B-27 supplement (without vitamin A) (All Life Technologies), 20 ng/mL bFGF, 20 ng/mL EGF, 10 ng/mL PDGF-AA, 10 ng/mL PDGF-BB (All Preprotech), and 5 IE/mL Heparin (Pharmacy PMC). Biweekly, a half medium change was performed and once a week Accutase (400–600 units/mL) was used to disrupt spheres to circumvent overcrowding and necrotic core formation.
Princess Máxima Center
HSJD-DMG-07
Adult Cancer Controls
K562Cell linesStandard RPMIUMC Utrecht (CVCL_0004)
JurkatUMC Utrecht (CVCL_0065)
  • Standard RPMI=RPMI + Glutamax (Life Technologies), supplemented with 10% FCS (Sigma)+1% Penicillin/Streptomycin (P/S) (Life Technologies).

  • ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; DMG, diffuse midline glioma; HBL, hepatoblastoma; HCC, hepatocellular carcinoma; MRT, malignant rhabdoid tumor; MSCs, mesenchymal stem cells; NBL, neuroblastoma; PDX, patient-derived xenograft.