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Copyright ©The Author(s) 2021.
World J Stem Cells. Aug 26, 2021; 13(8): 1112-1126
Published online Aug 26, 2021. doi: 10.4252/wjsc.v13.i8.1112
Table 1 Comparison between two-dimensional and three-dimensional tumor cell culture systems[14-18]

2D tumor cell cultures
3D tumor cell cultures
Time requiredFew daysFew weeks
Physiological relevanceDoes not simulate in vivo tumorSimulates in vivo tumor
Cell-cell and cell-ECM interactionsLow to no interactionsHigh level of interactions
Cell morphologyFlat cells expanding on 2D surfacePreserved in vivo cell shapes and growth patterns; multilayer growth
Oxygen and nutrients perfusionHomogeneousHeterogeneous due to the three-dimensional geometry
Response to drugsMore susceptible to drug actionsMore resistant to drugs with a similar drug penetration profile to in vivo tumor counterparts
Gene expressionMany differences compared to in vivo tumor counterpartsSimilar to in vivo tumor counterparts
DifferentiationPoorWell differentiated cells
CostCheapExpensive
Technique difficultyLowHigh
Table 2 Cancer stem cells biomarkers in brain tumors
Markers
Ref.
CD133Gonçalves et al[12], Singh et al[13], Ogden et al[27], and Li et al[28]
A2B5Ogden et al[27],
CD24Gonçalves et al[12]
Aldehyde dehydrogenase (ALDH)Gonçalves et al[12]
CD15Li et al[28], and Son et al[29]
ABCG2Li et al[28], Bleau et al[29], and Kondo et al[30]
NestinRahman et al[32], and Pollard et al[33]
SOX2Rahman et al[32], and Pollard et al[33]
CD44Rahman et al[32], and Pollard et al[33]
OLIG2Rahman et al[32], and Pollard et al[33]
Table 3 Characteristics of three-dimensional modeling technologies
Modeling technology
Methods of generation
Applications
Limitations
Spheroids[37-39]Static suspension; Hanging drops; Spinner and rotational bioreactor; Magnetic levitation; Microfluidic system; Gel embedding (Matrigel, etc.)Radioresistance through hypoxia and cell-cell contacts; Chemosensitivity and drug screening; Migration and invasion; Propagation and analysis of CSCsTumor heterogeneity; Immune system response; Interaction with normal non-tumor cells; Lack organ-like histology
Organoids[14,17,37,40,41]Culturing done on matrices (Matrigel, collagen I, hyaluronic acid, etc.); Addition of culture supplements including FGF, EGF, Noggin, N2, B27, etc.Disease mechanism; Drug discovery and toxicology; Developmental, stem cell biology and regenerative medicine; Infectious diseaseOxygen and nutrient distribution underdeveloped; Cellular microenvironments are challenging to replicate; Imaging difficulties; Expensive and time consuming assay
Table 4 Organoid and organoid-on-a-chip models
Ref.
Model system
System cell origin
Tumor type
Relevant genes
Results summary
Bian et al[38], 2018neoCORhESCsCNS-PNET; GBMAmplified expression of MYC; Differential expression of GBM associated gene aberrations (GBM-1, GBM-2, and GBM-3)neoCOR used to test gain- and loss of-function mutations, singly or in combination; generation of CNS-PNET or GBM xeno-transplantable tumors
Ogawa et al[58], 2018Human cerebral organoidshESCGBMRAS activation and TP53 deletionGeneration of tumors in cerebral organoids using CRISPR/Cas9 technology; tumors exhibited invasive phenotype and replicated the hallmarks of tumorigenesis in vivo
Ballabio et al[59], 2020Human cerebellar organoidsHuman induced pluripotent stem cell (iPSC)Medulloblastoma (MB) subgroup 3Overexpression of GFI1/c-MYC (GM) and OTX2/c-MYC (OM) gene combinationsOM as a novel driver gene combination required for Group 3 MB tumorigenesis; GM and OM overexpression induces tumor formation in mouse cerebellum; SMARCA4 and Tazemetostat reduces OM tumorigenesis
Linkous et al[60], 2019GLICO hESCs; iPSCsGBM-GLICO recapitulate primary human GBM with in a primitive brain microenvironment; GSCs exhibit high resistance to drug and radiation-inducedgenotoxic stress; GSCs form tumor by relocating to the human cerebral organoid, invasion and proliferation within themicroenvironment of the GLICO
Akay et al[61], 2018Microfluidic chipPatient primary human GBM multiforme specimensGBM-Generation of brain cancer chip that exhibit diffusion prevention mechanism to culture GBM-patient derived 3D spheroids; treatment with TMZ and bevacizumab (Avastin, BEV) in combination enhanced GBM cell death compared to TMZ alone
Ayuso et al[62], 2017Microfluidic chipU-251 MG human GBM cell lineGBM-Generation of microfluidic device to behavior models that simulate blood flow through the tumor; deprivation of nutrients and oxygen induces pseudopalisade formation; pseudopalisading process renders GBM cells to become of more aggressive behavior
Cui et al[63], 2018Microfluidic chipGL261 and CT-2A mouse glioma cell linesGBM-Generation of microfluidic angiogenesis model that simulate GBM tumor angiogenesis and macrophage-associated immunosuppression within GBM tumor microenvironment; GL261 and CT-2A GBM-like tumors promote angiogenesis through driving M2-like macrophage polarization; TGF-b1, and surface integrin (avb3) endothelial-macrophage interactions regulates inflammation-mediated angiogenesis through Src-PI3K-YAP signaling; inhibition of integrin (avb3) and cytokine receptor (TGFb-R1) repress GBM tumor neovascularization
Lin et al[64], 2018Microfluidic chipPatient derived GSCsGBM-Generation of glioma perivascular niches on a chip; Perivascular niches maintain the pluripotent state of GSCs; Stronger chemoresistance of GSCs against TMZ associates with endothelial cell co-culturing, GSCs neurosphere formation and the expression of 6-O-methylguanine and Bmi-1 gene
Yi et al[65], 2019Bio-printed chipPatient primary human GBM specimensGBM-Generation of complex cancerous-tissue constructs constituting brain ECM composition, oxygen gradient-generating system, cancer-stroma structure; exhibited patient-specific response upon the treatment with drug combinations, chemoradiation and TMZ
Table 5 Features and characteristics comparison between spheroids and organoids

Spheroids
Organoids
Cells usedCell lines or CSCsEmbryonic stem cells, induced pluripotent stem cells or CSCs
Physiologic relevanceLowerHigher
Tumor heterogeneityLowerHigher
Technique difficultyLowerHigher
CostLowerHigher
TimeWeeks1-3 mo
Genetic manipulationModerately availableModerately available
BiobanksNot available (cells are difficult to maintain long-term)Available
AdvantagesCost effective; Highly accessible; Good for high throughput drug testingRetains tumor heterogeneity; Better simulation of the physiological environment
DisadvantagesHard to maintain long-term; Not as representative of the physiologic environmentMore complex; Higher failure rate; May give variable results