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Table 2 Different techniques used for MTS formation and representative applications of the MTS in biomedical research

From: Leveraging and manufacturing in vitro multicellular spheroid-based tumor cell model as a preclinical tool for translating dysregulated tumor metabolism into clinical targets and biomarkers

Methods Concept Advantages Disadvantages Cell types Results References
Scaffold-based Cells adsorb and migrate into solid materials to form micro-tissues. The simplicity of fabrication and the diversity of materials
Appropriate physical support
Resist the outside environment
Harvesting cells from the scaffold is difficult and possibly harmful to the cells.
Suitable for tissue engineering research, not for drug screening.
Lung adenocarcinoma cells (HCC), macrophages and lung fibroblasts Cocultivation promoted the metastatic phenotype due to the excretion of both matrix metalloproteinase-1(MMP-1) and vascular endothelial growth factor (VEGF). (Liu et al. 2016)
Non-small cell lung cancer (NSCLC) Compared with 2D culture model, IC50 value of anticancer drugs in MTSs was significantly increased. (Godugu et al. 2013)
Breast cancer cells (MCF-7) Compared with monolayer cells, 3D MTSs-CCA system was superior to 2D culture system in anticancer drug screening. (Wang et al. 2016)
Hanging drop Cells form a single cluster by dropping a cell suspension onto an inverted glass covering surface by gravity. Mild conditions
Simple materials
Without shear force
Easy to control the number, size and shape of spheroids
Medium changed frequently
Time-consuming and labor-intensive
Hard to scale-up
Limited applicability in drug screening
Limited culture time
Colon cancer cells (HT‑29) Encapsulation of anticancer drugs in liposomes could improve the therapeutic effect. (Galateanu et al. 2016)
Malignant melanoma cells (A375) MTS was an effective tool for investigating the biological effects of oligonucleotides. (Carver et al. 2014)
Pulmonary epithelial cells (EPI), pulmonary vascular endothelial cells (ENDO) and human bone marrow mesenchymal stem cells (MSC) Compared with 2D culture, the expression of ROS and ABCB1 was enhanced and drug resistance was increased. (Lamichhane et al. 2016)
Liquid covering Cells aggregate on non-adherent plates. Easy to operate
Low cost
Large-scale production
High-throughput screening of drugs
Poor consistency
Static culture
Difficult to guarantee cell activity
Lung cancer cell lines, breast cancer cell lines, pancreatic cancer cell lines and fibroblasts (MRC5) After co-culture of tumor cells with fibroblasts, the survival of co-culture cells increased by four times than monoculture cells due to the secretion of soluble factors. (Majety et al. 2015)
Pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC) The complex microenvironment reduced chemotherapy sensitivity. MTSs could be used in drug screening for pancreatic cancer. (Lazzari et al. 2018)
Breast cancer cells (MCF-7), cervical cancer cells (Hela) and primary normal human skin fibroblasts (hFIB) When the cancer cells were co-cultured with hFIB, dense necrotic cores in the tumor spheroids could be observed. (Costa et al. 2014)
Breast cancer cells (MCF-7) and mouse fibroblasts (NIH-3T3) When co-cultured with NIH-3T3, the resistance or IC50 of MCF-7 significantly increased. (Xin and Yang 2019)
U-87 MG glioblastoma and other 40 tumor cell lines 2D and 3D culture models exhibited different sensitivities to targeted drugs. (Vinci et al. 2012)
Colorectal cancer (CRC) cells MTSs with hypoxia and multicellular tumor necrosis areas displayed closer gene expression landscape to tumors in vivo. (Däster et al. 2017)
Ovarian cancer cells (NCI-ADR-RES) Targeted modified drugs were more likely to cross the permeable barrier and accumulate in spheroid. (Perche et al. 2012)
Epithelial ovarian cancer (EOC) cells The response of MTSs to anticancer drugs reduced compared with monolayer culture. (Liao et al. 2014)
Colon cancer cells (Caco-2 and DLD-1) and Peripheral blood mononuclear cell (PBMC) Co-cultured tumor cells were more resistant to 5-FU/Oxaliplatin (FO) than single cultured tumor cells. (Hoffmann et al. 2015)
Micromachining wells Cells cluster in a matrix of micromachining wells. High reproducibility
Uniform size and shape
High-throughput mechanized production
High cost
Specific device
Breast cancer cells (LCC6/Her-2) Compared with traditional monolayer culture, tumor spheroids showed resistance to dose-dependent responses to DOX and anticancer drugs. (Yu et al. 2010)
Breast cancer cells (MCF-7) MTSs retained the physiologic features of solid tumors, thus leading to the understanding of the response of tumor to chemotherapy and radiation therapy. (Markovitz-Bishitz et al. 2010)
Dynamic techniques based on agitation Cells aggregate by continuous stirring in specific reactors. Suitable for long-term culture
Easy to operate
Large-scale industrialization
Controllable condition
Shear force affects cells.
Difficult to control shape, size and quantity
Specific device
Hepatocellular carcinoma cells (Huh7) and endothelial cells (HUVEC) The co-cultured spheroids were more resistant to anticancer drugs (Adriamycin and Sorafenib) than the monolayer cells. (Jung et al. 2017)
Colon cancer cells (HT‑29) Glycolysis, TCA cycle and lipid metabolism-related protein expression from inside MTSs was higher than outside MTSs. (McMahon et al. 2012)