Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Table 1 Differences in two-dimensional vs three-dimensional cell culture models

Type of culture	2D	3D	Ref.
In vivo-like	Do not mimic the natural structure of the tissue or tumor mass	In vivo tissues and organs are in 3D form	Takai et al[102]
Proliferation	Tumor cells were grown in monolayer faster than in 3D spheroids	Similar to the situation in vivo	Lv et al[11]
Polarity	Partial polarization	More accurate depiction of cell polarization	Antoni et al[18]
Cell morphology	Sheet-like, flat, and stretched cells in monolayer	Form aggregate/spheroid structures	Breslin et al[103]
Stiffness	High stiffness (approximately 3 × 109 Pa)	Low stiffness (> 4000 Pa)	Krausz et al[104]
Cell-cell interaction	Limited 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 created	Lv et al[11], Kang et al[105]
Gene/protein expression	Changes in gene expression, mRNA splicing, topology, and biochemistry of cells, often display differential gene/protein levels compared with in vivo models	Expression of genes and proteins in vivo is relevantly presented in 3D models	Bingel et al[92], Ravi et al[106]
Drug responses	Lack 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 patients	Lv et al[11], Bingel et al[92]
The culture formation	From minutes to a few hours	From a few hours to a few days	Dai et al[33]
Quality of culture	High performance, reproducibility, long-term culture, easy to interpret, simplicity of culture	Worse performance and reproducibility, difficult to interpret, cultures are more difficult to carry out	Hickman et al[107]
Access to essential compounds	Unlimited 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 culture	Cheap, commercially available tests and media	More expensive, more time-consuming, fewer commercially available tests	Friedrich et al[35]

Table 2 Proposed advantages, disadvantages, and research stage of different three-dimensional cell culture methods

Techniques	Advantages	Disadvantages	Research 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

Modified from Breslin et al[64]; Fang et al[47]; Leong et al[109].

Table 3 Examples of three-dimensional research systems utilized for cancer and stem cell cancer studies

Application/platform	Cells type	3D model	Culture systems/matrix	Results	Ref.
Drug-screening	Breast cancer cells (BT-549, BT-474 and T-47D)	Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer	Spheroid formation in 3D-culture plates	Three breast cancer cell lines developed dense multicellular spheroids in 3D-culture and showed greater resistance to paclitaxel and doxorubicin compared to the 2D-cultured cells	Imamura et al[89]
Metastasis studies and assessing drug sensitivity	Breast cancer cells (MDA-MB-231 and MCF-7)	Breast cancer bone metastasis	3D bioprinting hydrogel	Breast cancer cells exhibited spheroid morphology and migratory characteristics, then co-culture of breast tumor cells with bone marrow MSCs increased the formation of spheroid clusters	Zhu et al[86]
Cancer cell behavior	Breast cancer cells (MCF‑10)	Breast cancer progression	3D spheroid cultures used U-bottom ultra-low attachment plates	Genetic dependencies can be uncovered when cells are grown in 3D conditions similar to in vivo	Peela et al[85]
Drug-screening	Human colon cancer cells (HCT116)	Compared gene expression in 2D and 3D systems and identification of context-dependent drug responses	3D 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 profiles	Senkowski et al[30]
GBM biology, anti-GBM drug screening	Human glioblastoma cells (U87)	Compared gene expression in 2D and 3D systems	3D PLA porous scaffolds	GBM 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 pathways	Ma et al[87]
Cancer and tumor cell biology	Human glioblastoma (U-251)	Compared gene and protein expression in 2D and 3D systems	ESPS scaffolds coated with laminin	The results suggested the influence of 3D context on integrin expression upregulation of the laminin-binding integrins alpha 6 and beta 4	Ma et al[91]
Cancer and tumor cell biology	Human glioblastoma (U-251) cells	Compared drug-sensitivity in 2D and 3D systems	3D bioprinting of gelatin/alginate/ fibrinogen hydrogel	3D bioprinted glioma stem cells were more resistant to temozolomide than 2D monolayer model at temozolomide concentrations of 400-1600 μg/mL	Dai et al[33]
Cancer and tumor cell biology	Human glioblastoma (U-251)	Anti-cancer drug screening	3D collagen scaffold	Glioma cells in 3D collagen scaffold culture enhanced resistance to chemotherapeutic alkylating agents with a much higher proportion of glioma stem cells and upregulation of MGMT	Lv et al[11]
Cancer and tumor cell biology, development of new therapies and detection of cardiotoxicity	iPSC-derived human cardiomyocytes	Cardiac microtissues	Hanging drops	A 3D culture using iPSC-derived human CMs provided an organoid human-based cellular platform that recapitulated vital cardiac functionality	Beauchamp et al[94]
Tissue engineering and toxicity assessment	Human hepatoblastoma (HepG2/C3A)	A liver-on-a-chip platform for long-term culture of 3D human HepG2/C3A spheroids for drug toxicity assessment	Bioprinting of hepatic constructs containing 3D hepatic spheroids	Hepatic construct by 3D bioprinting were functional during the 30 d culture period and responded to acetaminophen that induced a toxic	Bhise et al[98]
Brain diseases	Human embryonic stem cells (HUES66), C57	3D neural tissues for use as tractable models of brain diseases	3D hydrogels	3D cocultures of neuronal and astrocytic cells can change expression patterns so that they correlate with specific brain regions and developmental stages	Tekin et al[101]
Cancer and tumor cell biology, drug screening	Human neuroblastoma cell lines BE(2)-C (ECACC), IMR-32 (DSMZ)	Compared gene expression profiles in 2D and 3D systems and tumor tissue	Polymeric scaffolds and bioreactor systems	The 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 conditions	Bingel et al[92]
Cancer and tumor cell biology, neurodegenerative diseases	DPSCs	Differentiation to retinal ganglion-like cells	3D fibrin hydrogel	3D network can mimic the natural environment of retinal cells	Roozafzoon et al[95]
Cardiovascular disease	hiPSCs	Cardiomyocytes and endothelial cells, co-differentiated from human pluripotent stem cells	V-bottom 96 well microplates	Human 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 microvasculature	Giacomelli et al[110]
Bioartificial liver support devices, drug screening and	hiPSCs	Differentiation of hiPSCs into hepatocytes	Nanofiber hydrogel 3D scaffold	3D hydrogel culture conditions promote the differentiation of hiPSCs into hepatocytes	Luo et al[100]
Ovarian cancer biology, drug sensitivity	Ovarian cancer cell lines (A2780 and OVCAR3)	Compared drug-sensitivity in 2D and 3D systems	Hanging drop	3D tumor spheroids demonstrated greater resistance to cisplatin chemotherapy compared to 2D cultures	Raghavan et al[90]
Pathogenesis of prostate cancer, prostate cancer therapy	Prostate cancer cell lines (PC3 and LNCaP)	Simulation of prostate cancer bone metastases	Collagen-based scaffolds	The two cell lines in 3D present increased resistance to docetaxel	Fitzgerald et al[111]
Radiosensitivity of cancer cells	Human lung adenocarcinoma cell line (A549)	The metabolic response of lung cancer cells to ionizing radiation	Hydrogels	3D model can help regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation	Simon et al[112]
Regenerative medicine, drug screening, and potentially disease modeling	HUVECs	Endothelial myocardium construction	3D bioprinting	This technique could be translated to human cardiomyocytes derived from iPSCs to construct endothelial human myocardium	Zhang et al[99]
Cancer cell biology, studying and developing therapies against cancer stem cells	Hepatocellular carcinoma (SK-Hep-1), prostate cancer (TRAMP-C2) and breast cancer (MDA-MB-231)	Compared cancer morphogenesis and gene expression in 2D and 3D systems	CA scaffolds	The 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]

2D: Two-dimensional; 3D: Three-dimensional; DPSCs: Dental pulp stem cells; iPSC: Induced pluripotent stem cells; hiPSC: Human induced pluripotent stem cells; HUVECs: Human umbilical vein endothelial cells; CA: Chitosan-alginate; MSC: Mesenchymal stem cell; PLA: Polylactic acid; ESPS: Electrospun polystyrene; TGF-β: Transforming growth factor β; KEGG: Kyoto Encyclopedia of Genes and Genomes; GBM: Glioblastoma multiforme; ECM: Extracellular matrix; CM: Cardiomyocyte.