Copyright ©The Author(s) 2019.
World J Stem Cells. Dec 26, 2019; 11(12): 1065-1083
Published online Dec 26, 2019. doi: 10.4252/wjsc.v11.i12.1065
Table 1 Differences in two-dimensional vs three-dimensional cell culture models
Type of culture2D3DRef.
In vivo-likeDo not mimic the natural structure of the tissue or tumor massIn vivo tissues and organs are in 3D formTakai et al[102]
ProliferationTumor cells were grown in monolayer faster than in 3D spheroidsSimilar to the situation in vivoLv et al[11]
PolarityPartial polarizationMore accurate depiction of cell polarizationAntoni et al[18]
Cell morphologySheet-like, flat, and stretched cells in monolayerForm aggregate/spheroid structuresBreslin et al[103]
StiffnessHigh stiffness (approximately 3 × 109 Pa)Low stiffness (> 4000 Pa)Krausz et al[104]
Cell-cell interactionLimited cell-cell and cell-extracellular matrix interactions and no “niches”In vivo-like, proper interactions of cell-cell and cell-extracellular matrix, environmental “niches” are createdLv et al[11], Kang et al[105]
Gene/protein expressionChanges in gene expression, mRNA splicing, topology, and biochemistry of cells, often display differential gene/protein levels compared with in vivo modelsExpression of genes and proteins in vivo is relevantly presented in 3D modelsBingel et al[92], Ravi et al[106]
Drug responsesLack of correlation between 2D monolayer cell cultures and human tumors in drug testing.Tumor cells in 3D culture showed drug resistance patterns similar to those observed in patientsLv et al[11], Bingel et al[92]
The culture formationFrom minutes to a few hoursFrom a few hours to a few daysDai et al[33]
Quality of cultureHigh performance, reproducibility, long-term culture, easy to interpret, simplicity of cultureWorse performance and reproducibility, difficult to interpret, cultures are more difficult to carry outHickman et al[107]
Access to essential compoundsUnlimited access to oxygen, nutrients, metabolites, and signaling molecules (in contrast to in vivo)Variable access to oxygen, nutrients, metabolites, and signaling molecules (similar to in vivo)Pampaloni et al[108], Senkowski et al[30]
Cost during maintenance of a cultureCheap, commercially available tests and mediaMore expensive, more time-consuming, fewer commercially available testsFriedrich et al[35]
Table 2 Proposed advantages, disadvantages, and research stage of different three-dimensional cell culture methods
TechniquesAdvantagesDisadvantagesResearch stage
Liquid overlay cultures and Hanging drops(1) Easy-to-use protocol; (2) No added materials; (3) Consistent spheroid formation; control over size Co-culture ability; (4) Transparent; (5) High reproducibility; (6) Inexpensive; (7) Easy to image/harvest samples(1) No support or porosity; (2) Limited flexibility; (3) Limited spheroid size; (4) Heterogeneity of cell lineage; (5) Lack of matrix interaction(1) Basic research; (2) Drug discovery; (3) Personalized medicine
Hydrogel(1) Large variety of natural or synthetic materials; (2) Customizable; (3) Co-culture possible; (4) Inexpensive; (5) High reproducibility(1) Gelling mechanism; (2) Gel-to-gel variation and structural changes over time; (3) Undefined constituents in natural gels; (4) May not be transparent(1) Basic research; (2) Drug discovery
Bioreactors(1) Simple to culture cells; (2) Large-scale production easily achievable; (3) Motion of culture assists nutrient transport; (4) Spheroids produced are easily accessible(1) Specialized equipment required; (2) No control over cell number/size of spheroid; (3) Cells possibly exposed to shear force in spinner flasks (may be problematic for sensitive cells)(1) Basic research; (2) Tissue engineering; (3) Cell expansion
Scaffolds(1) Large variety of materials possible for desired properties; (2) Customizable; (3) Co-cultures possible; (4) Medium cost(1) Possible scaffold-to-scaffold variation; (2) May not be transparent; (3) Cell removal may be difficult(1) Basic research; (2) Drug screening; (3) Drug discovery; (4) Cell expansion
3D bioprinting(1) Custom-made architecture; (2) Chemical, physical gradients; (3) High-throughput production; (4) Co-culture ability(1) Require expensive 3D bioprinting machine; (2) Challenges with cells/materials(1) Cancer pathology; (2) Anticancer drug screening; (3) Cancer treatment; (4) Tissue engineering
Table 3 Examples of three-dimensional research systems utilized for cancer and stem cell cancer studies
Application/platformCells type3D modelCulture systems/matrixResultsRef.
Drug-screeningBreast cancer cells (BT-549, BT-474 and T-47D)Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancerSpheroid formation in 3D-culture platesThree breast cancer cell lines developed dense multicellular spheroids in 3D-culture and showed greater resistance to paclitaxel and doxorubicin compared to the 2D-cultured cellsImamura et al[89]
Metastasis studies and assessing drug sensitivityBreast cancer cells (MDA-MB-231 and MCF-7)Breast cancer bone metastasis3D bioprinting hydrogelBreast cancer cells exhibited spheroid morphology and migratory characteristics, then co-culture of breast tumor cells with bone marrow MSCs increased the formation of spheroid clustersZhu et al[86]
Cancer cell behaviorBreast cancer cells (MCF‑10)Breast cancer progression3D spheroid cultures used U-bottom ultra-low attachment platesGenetic dependencies can be uncovered when cells are grown in 3D conditions similar to in vivoPeela et al[85]
Drug-screeningHuman colon cancer cells (HCT116)Compared gene expression in 2D and 3D systems and identification of context-dependent drug responses3D spheroid cultures used low‐attachment plate (Corning, Amsterdam, The Netherlands)3D spheroids increased expression of genes involved in response to hypoxia and decreased expression of genes involved in cell-cycle progression when compared with monolayer profilesSenkowski et al[30]
GBM biology, anti-GBM drug screeningHuman glioblastoma cells (U87)Compared gene expression in 2D and 3D systems3D PLA porous scaffoldsGBM cells in 3D PLA culture expressed, 8117 and 3060 genes were upregulated and downregulated, respectively, compared to 2D cell culture conditions. Further, KEGG pathway analysis showed the upregulated genes were mainly enriched in PPAR and PI3K-Akt signaling pathways while the downregulated genes were enriched mainly in metabolism, ECM, and TGF-β pathwaysMa et al[87]
Cancer and tumor cell biologyHuman glioblastoma (U-251)Compared gene and protein expression in 2D and 3D systemsESPS scaffolds coated with lamininThe results suggested the influence of 3D context on integrin expression upregulation of the laminin-binding integrins alpha 6 and beta 4Ma et al[91]
Cancer and tumor cell biologyHuman glioblastoma (U-251) cellsCompared drug-sensitivity in 2D and 3D systems3D bioprinting of gelatin/alginate/ fibrinogen hydrogel3D bioprinted glioma stem cells were more resistant to temozolomide than 2D monolayer model at temozolomide concentrations of 400-1600 μg/mLDai et al[33]
Cancer and tumor cell biologyHuman glioblastoma (U-251)Anti-cancer drug screening3D collagen scaffoldGlioma cells in 3D collagen scaffold culture enhanced resistance to chemotherapeutic alkylating agents with a much higher proportion of glioma stem cells and upregulation of MGMTLv et al[11]
Cancer and tumor cell biology, development of new therapies and detection of cardiotoxicityiPSC-derived human cardiomyocytesCardiac microtissuesHanging dropsA 3D culture using iPSC-derived human CMs provided an organoid human-based cellular platform that recapitulated vital cardiac functionalityBeauchamp et al[94]
Tissue engineering and toxicity assessmentHuman hepatoblastoma (HepG2/C3A)A liver-on-a-chip platform for long-term culture of 3D human HepG2/C3A spheroids for drug toxicity assessmentBioprinting of hepatic constructs containing 3D hepatic spheroidsHepatic construct by 3D bioprinting were functional during the 30 d culture period and responded to acetaminophen that induced a toxicBhise et al[98]
Brain diseasesHuman embryonic stem cells (HUES66), C573D neural tissues for use as tractable models of brain diseases3D hydrogels3D cocultures of neuronal and astrocytic cells can change expression patterns so that they correlate with specific brain regions and developmental stagesTekin et al[101]
Cancer and tumor cell biology, drug screeningHuman neuroblastoma cell lines BE(2)-C (ECACC), IMR-32 (DSMZ)Compared gene expression profiles in 2D and 3D systems and tumor tissuePolymeric scaffolds and bioreactor systemsThe autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditionsBingel et al[92]
Cancer and tumor cell biology, neurodegenerative diseasesDPSCsDifferentiation to retinal ganglion-like cells3D fibrin hydrogel3D network can mimic the natural environment of retinal cellsRoozafzoon et al[95]
Cardiovascular diseasehiPSCsCardiomyocytes and endothelial cells, co-differentiated from human pluripotent stem cellsV-bottom 96 well microplatesHuman cardiac microtissues were generated in complex 3D structures and differentiation of human pluripotent stem cells into cardiomyocytes and endothelial cells that expressed cardiac markers also present in primary cardiac microvasculatureGiacomelli et al[110]
Bioartificial liver support devices, drug screening andhiPSCsDifferentiation of hiPSCs into hepatocytesNanofiber hydrogel 3D scaffold3D hydrogel culture conditions promote the differentiation of hiPSCs into hepatocytesLuo et al[100]
Ovarian cancer biology, drug sensitivityOvarian cancer cell lines (A2780 and OVCAR3)Compared drug-sensitivity in 2D and 3D systemsHanging drop3D tumor spheroids demonstrated greater resistance to cisplatin chemotherapy compared to 2D culturesRaghavan et al[90]
Pathogenesis of prostate cancer, prostate cancer therapyProstate cancer cell lines (PC3 and LNCaP)Simulation of prostate cancer bone metastasesCollagen-based scaffoldsThe two cell lines in 3D present increased resistance to docetaxelFitzgerald et al[111]
Radiosensitivity of cancer cellsHuman lung adenocarcinoma cell line (A549)The metabolic response of lung cancer cells to ionizing radiationHydrogels3D model can help regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiationSimon et al[112]
Regenerative medicine, drug screening, and potentially disease modelingHUVECsEndothelial myocardium construction3D bioprintingThis technique could be translated to human cardiomyocytes derived from iPSCs to construct endothelial human myocardiumZhang et al[99]
Cancer cell biology, studying and developing therapies against cancer stem cellsHepatocellular carcinoma (SK-Hep-1), prostate cancer (TRAMP-C2) and breast cancer (MDA-MB-231)Compared cancer morphogenesis and gene expression in 2D and 3D systemsCA scaffoldsThe three cell lines in 3D porous CA scaffolds promote cancer stem-like cell enrichment and increased expression of cancer stem cells genes (CD133 and NANOG)Florczyk et al[28]