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.



Pampaloni F, Stelzer E, Masotti A. 2009. Three-Dimensional Tissue Models for Drug Discovery and Toxicology. BIOT. 3(2):103-117.
Takahashi Y, Hori Y, Yamamoto T, Urashima T, Ohara Y, Tanaka H. 2015. 3D spheroid cultures improve the metabolic gene expression profiles of HepaRG cells. 35(3):
Grabowska I, Szeliga A, Moraczewski J, Czaplicka I, 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(2):125-133.
Fan X, Zou R, Zhao Z, Yang P, Li Y, Song J. 2009. Tensile strain induces integrin ?1 and ILK expression higher and faster in 3D cultured rat skeletal myoblasts than in 2D cultures. Tissue and Cell. 41(4):266-270.
Sainz B, TenCate V, Uprichard SL. 2009. Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol J. 6(1):
Nakamura T, Kato Y, Fuji H, Horiuchi T, Chiba Y, Tanaka K. E-cadherin-dependent intercellular adhesion enhances chemoresistance. Int J Mol Med.
Pruksakorn D, Lirdprapamongkol K, Chokchaichamnankit D, Subhasitanont P, Chiablaem K, Svasti J, Srisomsap C. 2010. Metabolic alteration of HepG2 in scaffold-based 3-D culture: Proteomic approach. Proteomics. 10(21):3896-3904.
Kang X, Xie Y, Kniss DA. 2005. Adipose Tissue Model Using Three-Dimensional Cultivation of Preadipocytes Seeded onto Fibrous Polymer Scaffolds. Tissue Engineering. 11(3-4):458-468.
Stacey DH, Hanson SE, Lahvis G, Gutowski KA, Masters KS. 2009. In vitro Adipogenic Differentiation of Preadipocytes Varies with Differentiation Stimulus, Culture Dimensionality, and Scaffold Composition. Tissue Engineering Part A. 15(11):3389-3399.
Pampaloni F, Stelzer EHK, Leicht S, 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(19):3394-3413.
Shi Z, Abraham G, Tarbell JM. 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(8):e12196.
Debeb BG, Zhang X, Krishnamurthy S, Gao H, Cohen E, Li L, Rodriguez AA, Landis MD, Lucci A, Ueno NT, et al. 2010. Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells. Molecular Cancer. 9(1):180.
Birgersdotter A, Baumforth KRN, Porwit A, Sundblad A, Falk KI, Wei W, Sjöberg J, Murray PG, Björkholm M, Ernberg I. 2007. Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern. Leukemia & Lymphoma. 48(10):2042-2053.
Ki CS, Park SY, Kim HJ, Jung H, Woo KM, Lee JW, Park YH. 2008. Development of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: implications for bone regeneration. Biotechnol Lett. 30(3):405-410.
Hong H, Stegemann JP. 2008. 2D and 3D collagen and fibrin biopolymers promote specific ECM and integrin gene expression by vascular smooth muscle cells. Journal of Biomaterials Science, Polymer Edition. 19(10):1279-1293.
Merwin JR, Anderson JM, Kocher O, Van Itallie CM, Madri JA. 1990. Transforming growth factor beta1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis. J. Cell. Physiol.. 142(1):117-128.
Ceresa CC, Knox AJ, Johnson SR. 2009. Use of a three-dimensional cell culture model to study airway smooth muscle-mast cell interactions in airway remodeling. American Journal of Physiology-Lung Cellular and Molecular Physiology. 296(6):L1059-L1066.
Tian X, Heng B, Ge Z, Lu K, Rufaihah AJ, Fan VT, Yeo J, Cao T. 2008. Comparison of osteogenesis of human embryonic stem cells within 2D and 3D culture systems. Scandinavian Journal of Clinical and Laboratory Investigation. 68(1):58-67.
Hamamura K, Jiang C, Yokota H. 2010. ECM-dependent mRNA expression profiles and phosphorylation patterns of p130Cas, FAK, ERK and p38 MAPK of osteoblast-like cells. 34(10):1005-1012.
Quiros RM, Valianou M, Kwon Y, Brown KM, Godwin AK, Cukierman E. 2008. Ovarian normal and tumor-associated fibroblasts retain in vivo stromal characteristics in a 3-D matrix-dependent manner. Gynecologic Oncology. 110(1):99-109.
Peyton SR, Kim PD, Ghajar CM, Seliktar D, Putnam AJ. 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(17):2597-2607.
Iwan. 2008. Chondrocyte-associated antigen and matrix components in a 2- and 3-dimensional culture of rat chondrocytes. Mol Med Rep.

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