Since decades cancer cell line has been promptly contributed significant understanding of physiology of heterogeneous tissue environment of cancer. In the recent years, three-dimensional (3D) cell culture technology has received a new dimension in tumor cell biology in identifying the biological characteristics of tumor cells corresponding to drug sensitive area as key point in translational medicine. 3D cell culture technology utilised methods and materials to simulate the in vivo microenvironment of tumor cells by performing culture of tumors cells ex vivo. It provided numerous distinguished characteristics over 2D monolayer or suspension cell culture.
Tumor cell culture as one of the vital technique utilised in tumor cell biology for estimation of potentiality of developed formulation or newly established chemical entity or therapeutics in cancer cell killing. The two dimensional monolayer cell cultures has certain limitation of being identifying the incorrect architect, microenvironment of 2D cultured cells growing in vivo colligated with cell proliferation, morphology, transduction pathway and some other aspects. 3D cell culture technique progressively improved the imitation of cell microenvironment in vivo and thus to considered next generation frontiers in tumor biology research and may be useful in at preclinical development stage in identifying successful prototypes and at early steps. Therefore, the trends of 3D cell culture application increases nowadays by tumor biologist as evidenced in the literature of public domain.
The conventional cell culture system although facilitate in understanding microenvironment of tumor cell, and their complex physiology on how cell functions and respond to stimuli. However, the 3D culture approach is more realistic in dealing the simulation of in vivo condition. Thus, the conspicuous advantage of implenting 3D cell culture system to reduces the gap between cellular function and cell culture system.
Advantage of 3D cell culture technique over conventional technique
Currently, above 80 per cent of tumor biologist rely on 2D cell culture for in vivo investigation due to convenience. Moreover, the in vitro generated data claim on cell line study below or poor performed in clinical investigation in patients bearing tumor may be attributed to the fact that cells grown in 2D cell culture lost the complex 3D tissue architect, cell-to-cell, cell-to-extracellular matrix interaction that significantly influence the cell proliferation, differentiation and other cellular activity. 3D cell culture reveals the more realistic view of tumerogenesis mechanism. The 3D spheroids of A549 cell line have demonstrated constantly high levels of interleukin (IL)-6 and IL-8 secretion over monolayer culture system. The high extracellular matrix deposition for better biomarker expression was reported using 3D culture systems. The cell in 3D system model gives appropriate simulation in vivo conditions and provides novel insight in different ways.
Application of 3D culture system in tumor biology
Cell differentiation: The stem cell research has been widely adopted 3D culture systems for cell differentiation studies. Stem cells are widely used as a cellular source for 3D models to engineer tissue constructs. Apart from providing the information of cell differentiation, it also gives basic insight into research with regards to clinical application. Many researchers understand the mechanism of bone forming cells differentiation i.e. osteoblast to osteocytes and role of oesteocytes on developing bone cancer as well as tissue engineering application.
3D cultures showed 2-fold increased expression of surface-specific markers and Colony-Forming Cell (CFC) assays over their 2D counterparts when mouse embryonic stem cells differentiated into Hematopoietic Precursor Cells (HPC). 3D culture of hepatic cells may tolerate therapeutic treatment same as the tumor shows resistance against therapy in vivo. Breast cancer cell in 3D cell suspension demonstrated higher degree of resistance against temoxifen in endocrine therapy over 2D cell culture. Furthermore, 2D cell culture fails to provide adequate stem cancer cells enrichment culture.
In drug discovery studies: Due to increase in number of new drug entity day by day making animal usage more expensive and unethical. Ultimately 3D cell cultures have greatly improve cell-based drug screening, along with identifying therapeutic, sub-therapeutic and toxic level of drug substances at an earlier stage of the drug discovery. 3D culture could reduce the cost of animal drug testing and a simple, effective tool for drug cytotoxicity for chemotherapeutic agent discovery.
In tumor biology, gene, protein expression : Tumors growing as spheroids could be differentiated into central necrotic layer, outer proliferating part and inner quiescent layer that simulate the microenvironment of human solid tumors. Several features of in vivo grown solid tumors exhibited the features in vitro tumor spheroids. The application of 3D cell cultures in cancer research exactly tells cellular interaction with extracellular materials, cell differential and proliferation pattern. The similar observation could be concluded when compare the gene and protein expressions of cells in 3D cultures over in vivo cancers occurs naturally.
In cell maturation and cell cycle: The investigations based on 3D cell cultures of mammary cells, mouse fibroblasts, vascular cells, and osteoblasts provides ameliorated platform for inferring mechanistic approach involved in cell proliferation. The cell proliferation under controlled compliances of hydrogel from acrylamide, elastic moduli in scaffold and the date generated is used to analyze the cell cycle mitogenesis in tissues.
In Cytoskeleton Studies The cellular cytoskeleton and extracellular matrix composition varies when the cells grown in 2D and 3D cell culture. For example, oral carcinoma of squamous cell cultured as monolayers 2D and scaffold engineered tumors indicated distinguished levels of proteins enable to formed cytoskeleton. The atrophied expression of laminins and raised level of fibronectin 3D cell culture was observed, which is evidently expressed their increased malignancy.
In cell apoptosis: The interaction between ECM and cell cytoskeleton have significant effect on cell apoptosis. Based on a study author suggested that inhibition of β1 integrin unfolds that a novel therapeutic approach refers to ECM based 3D cell cultures a potential culture models to establish the tumor response to specific therapy.
In cellular motility: The movement of cells into or within biomaterial scaffolds is a prerequisite which enables tissue repair and regeneration. The engineered biomaterial is required for 3D migration study. There is two general mode of cell migration through ECM has been studied so far. The stiffness of gel scaffold is deciding factor for migration of cell of whether amoeboid or mesenchymal type migration. On the other hand, soft gels showed amoeboid type of migration through the pores of scaffold because cells lack matrix degradation.
In Drug Response Studies: The malignant cells when they come in contact with chemotherapeutics agent show enhanced resistance than normal cells. Thus drug screening examination requires a veritable in vitro model that can effectively simulate the in vitro tumor tissues and facilitate accurate in vivo drug response. Setting this parameter researcher compared 2D with 3D cell cultures with suitable example of oral carcinoma when with cytotoxic drug PI3-kinase inhibitor LY294002, the 2D model was sensitive to cytotoxic drug while 3D tumor had shown significant resistance.
1. Maddaly Ravi et al., 2014. 3D Cell Culture Systems - Advantages and Applications. DOI 10.1002/jcp.24683.
2. Fischbach C, Chen R., Matsumoto T, Schmelzle T, Brugge JS, Polverini PJ, Mooney DJ.
2007. Engineering tumors with 3D scaffolds. Nature Methods 4:855-860.
3. Park CC, Zhang H, Pallavicini M, Gray WJ, Baehner F, Park JC, Bissell JM. 2006. β1Integrin Inhibitory Antibody Induces Apoptosis of Breast Cancer Cells, Inhibits Growth, and Distinguishes Malignant from Normal Phenotype in Three Dimensional Cultures and in vivo. Cancer Res 66: 1526–1535.
4. Klein EA, Yin L, Kothapalli D, Castagnino P, Byfield FJ, Xu T, Levental I, Hawthorne E,
Janmey PA, Assoian RK. 2009. Cell cycle control by physiological matrix elasticity and in
vivo tissue stiffening. Curr Biol 19:1511-8.
5. Mazzoleni G, Di Lorenzo D, Steimberg N. 2009. Modelling tissues in 3D: the next future of
pharmacotoxicology and food research. Genes Nutr 4:13-22.
6. Liu H, Roy K. 2005. Biomimetic Three-Dimensional Cultures Significantly Increase Hematopoietic Differentiation Efficacy of Embryonic Stem Cells. Tissue Engineering 11:319-330.
7. Pampaloni F, Stelzer EHK, Masotti A. 2009. Three-dimensional tissue models for drug
discovery and toxicology. Recent Patents on Biotechnology 3:103-117.
8. Akhter MH, Amin S. An investigative approach to the treatment modalities of squamous cell carcinoma. 2016. Curr drug deliv. 2017; 14:597-612.
9. Akhter MH, Amin S. Nanocarriers in advanced drug targeting: Setting novel paradigm in cancer therapeutics. Artificial Cells, Nanomedicine, and Biotechnology. 2017; 46:873-884.