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Modulated Gene Expression in 3D Cell Culture: Implications for Drug Discovery

In-vitro cell models are essential for validating the efficacy of drug candidates. However, large numbers of compounds are discontinued in subsequent phases of clinical studies. The reason for these failures is poor predictability of 2D cell cultures1, which could not mimic physiological conditions. Thus, it is important that the cell model used in testing drug candidates is physiologically relevant and closely mimics in-vivo conditions by expressing appropriate receptors, drug transporters and essential proteins for cell growth and survival.

Cells grown in 3D environment (3D scaffolds, 3D hydrogels, 3D multiwell plates and 3D bioreactors) influences the spatial organization of receptors and interactions with neighboring cells, thereby influencing the gene expression and cellular behavior2. A number of studies demonstrated that behaviors of cells in 3D cultures are similar to the in-vivo conditions; some of the genes and proteins which are modulated in 3D cultures  in comparison with 2D cultures across various cell lines are listed below.

Gene/Protein Name Primary cells/Cell Line Modulation in 3D culture References
Cell adhesion proteins C2C12, Skeletal myoblasts, Huh7, MCF-7 Increase 208367633 192159494 196043765 145329956
Metabolic & Cytoskeletal proteins HepG2, 3T3-L1 Increase 209250557, 158694248, 194027869
Antioxidant proteins MDCK Increase 2071800810
Cytoskeletal proteins Fibroblasts Increase 2080894011
Mesenchymal genes and Pro-metastatic genes 293T Increase 2061523812
Immune response and Apoptosis genes L1236 Increase 1791797213
Kinases MC3T3-E1 Increase 1797308314
Extracellular matrix and cell adhesion proteins Primary smooth muscle cells, Microvascular endothelial cells, Primary-Bronchi smooth muscle cells Increase 1885412215 168885916 1934643117
Osteogenesis proteins H1 (Human embryonic stem cells) Increase 1822455718
Iron Metabolism proteins, Mitochondrial proteins HepG2 Decrease 209250557
Kinases HT1080, MC3T3-E1 Decrease 1670757214 2050728419
Cytoskeletal proteins Smooth muscle cells and Fibroblasts, Primary human ovarian fibroblasts, Primary-Smooth muscle cell (arota) Decrease 2080894011 1844815620 1834236621
Cell division genes L1236 Decrease 1791797213
Extracellular matrix proteins Chondrocytes Decrease 2147950122




  1. Pampaloni, F., Stelzer, E. H. K., and Masotti, A. (2009) Three-dimensional tissue models for drug discovery and toxicology. Recent Pat. Biotechnol. 3, 103–117.
  2. Takahashi, Y., Hori, Y., Yamamoto, T., Urashima, T., Ohara, Y., and Tanaka, H. (2015) 3D spheroid cultures improve the metabolic gene expression profiles of HepaRG cells. Biosci. Rep. 35.
  3. Grabowska, I., Szeliga, A., Moraczewski, J., Czaplicka, I., and Brzóska, E. (2011) Comparison of satellite cell-derived myoblasts and C2C12 differentiation in two- and three-dimensional cultures: changes in adhesion protein expression. Cell Biol. Int. 35, 125–133.
  4. Fan, X., Zou, R., Zhao, Z., Yang, P., Li, Y., and Song, J. (2009) Tensile strain induces integrin beta1 and ILK expression higher and faster in 3D cultured rat skeletal myoblasts than in 2D cultures. Tissue Cell 41, 266–270.
  5. Sainz, B., TenCate, V., and Uprichard, S. L. (2009) Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol. J. 6, 103.
  6. Nakamura, T., Kato, Y., Fuji, H., Horiuchi, T., Chiba, Y., and Tanaka, K. (2003) E-cadherin-dependent intercellular adhesion enhances chemoresistance. Int. J. Mol. Med. 12, 693–700.
  7. Pruksakorn, D., Lirdprapamongkol, K., Chokchaichamnankit, D., Subhasitanont, P., Chiablaem, K., Svasti, J., and Srisomsap, C. (2010) Metabolic alteration of HepG2 in scaffold-based 3-D culture: proteomic approach. Proteomics 10, 3896–3904.
  8. Kang, X., Xie, Y., and Kniss, D. A. (2005) Adipose tissue model using three-dimensional cultivation of preadipocytes seeded onto fibrous polymer scaffolds. Tissue Eng. 11, 458–468.
  9. Stacey, D. H., Hanson, S. E., Lahvis, G., Gutowski, K. A., and Masters, K. S. (2009) In vitro adipogenic differentiation of preadipocytes varies with differentiation stimulus, culture dimensionality, and scaffold composition. Tissue Eng. Part A 15, 3389–3399.
  10. Pampaloni, F., Stelzer, E. H. K., Leicht, S., and Marcello, M. (2010) Madin-Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen-coated surfaces rather than in physiological 3-D cultures. Proteomics 10, 3394–3413.
  11. Shi, Z.-D., Abraham, G., and Tarbell, J. M. (2010) Shear stress modulation of smooth muscle cell marker genes in 2-D and 3-D depends on mechanotransduction by heparan sulfate proteoglycans and ERK1/2. PloS One 5, e12196.
  12. Debeb, B. G., Zhang, X., Krishnamurthy, S., Gao, H., Cohen, E., Li, L., Rodriguez, A. A., Landis, M. D., Lucci, A., Ueno, N. T., Robertson, F., Xu, W., Lacerda, L., Buchholz, T. A., Cristofanilli, M., Reuben, J. M., Lewis, M. T., and Woodward, W. A. (2010) Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells. Mol. Cancer 9, 180.
  13. Birgersdotter, A., Baumforth, K. R. N., Porwit, A., Sundblad, A., Falk, K. I., Wei, W., Sjöberg, J., Murray, P. G., Björkholm, M., and Ernberg, I. (2007) Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern. Leuk. Lymphoma 48, 2042–2053.
  14. Ki, C. S., Park, S. Y., Kim, H. J., Jung, H.-M., Woo, K. M., Lee, J. W., and Park, Y. H. (2008) Development of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: implications for bone regeneration. Biotechnol. Lett. 30, 405–410.
  15. Hong, H., and Stegemann, J. P. (2008) 2D and 3D collagen and fibrin biopolymers promote specific ECM and integrin gene expression by vascular smooth muscle cells. J. Biomater. Sci. Polym. Ed. 19, 1279–1293.
  16. Merwin, J. R., Anderson, J. M., Kocher, O., Van Itallie, C. M., and Madri, J. A. (1990) Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis. J. Cell. Physiol. 142, 117–128.
  17. Ceresa, C. C., Knox, A. J., and Johnson, S. R. (2009) Use of a three-dimensional cell culture model to study airway smooth muscle-mast cell interactions in airway remodeling. Am. J. Physiol. Lung Cell. Mol. Physiol. 296, L1059-1066.
  18. Tian, X.-F., Heng, B.-C., Ge, Z., Lu, K., Rufaihah, A. J., Fan, V. T.-W., Yeo, J.-F., and Cao, T. (2008) Comparison of osteogenesis of human embryonic stem cells within 2D and 3D culture systems. Scand. J. Clin. Lab. Invest. 68, 58–67.
  19. Hamamura, K., Jiang, C., and Yokota, H. (2010) ECM-dependent mRNA expression profiles and phosphorylation patterns of p130Cas, FAK, ERK and p38 MAPK of osteoblast-like cells. Cell Biol. Int. 34, 1005–1012.
  20. Quiros, R. M., Valianou, M., Kwon, Y., Brown, K. M., Godwin, A. K., and Cukierman, E. (2008) Ovarian normal and tumor-associated fibroblasts retain in vivo stromal characteristics in a 3-D matrix-dependent manner. Gynecol. Oncol. 110, 99–109.
  21. Peyton, S. R., Kim, P. D., Ghajar, C. M., Seliktar, D., and Putnam, A. J. (2008) The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system. Biomaterials 29, 2597–2607.
  22. Osiecka-Iwan, A., Hyc, A., Niderla-Bielinska, J., and Moskalewski, S. (2008) Chondrocyte-associated antigen and matrix components in a 2- and 3-dimensional culture of rat chondrocytes. Mol. Med. Rep. 1, 881–887.


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