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7 physiological characteristics that distinguish spheroids from 2D cell cultures

A spheroid is a three-dimensional (3D) cell culture model that mimics the in vivo environment more closely than traditional two-dimensional (2D) monolayer cultures. Physiologically, spheroids exhibit several distinct characteristics compared to 2D cell cultures.

This article is an overview of the key physiological aspects of spheroid cell cultures (1,2,3):

  1. Cell-Cell Interactions: Cells in a spheroid culture are in close proximity to each other which allows for extensive cell-cell interactions. These interactions also mimic the natural cell signaling and communication in tissues and organs.
  2. Oxygen and Nutrient Gradients: The outer layer of cells in a spheroid has better access to oxygen and nutrients from the culture medium, while the inner layers experience lower availability of these elements. As spheroids grow, the diffusion of oxygen and nutrients becomes limited, resulting in the formation of concentration gradients and further variations in cell metabolism and gene expression.
  3. Morphology: Spheroids typically have a spherical or an ovoid morphology, with cells arranged in concentric layers. The outermost layer is in direct contact with the surrounding medium, while the inner layers are exposed to decreasing nutrient and oxygen concentrations.
  4. Hypoxia: Due to limited oxygen diffusion, cells in the core of large spheroids may experience hypoxia (low oxygen conditions). Hypoxia can induce cellular responses, such as the upregulation of hypoxia-inducible factors (HIFs) that regulate various cellular processes, such as angiogenesis and metabolism.
  5. Apoptosis and Necrosis: As spheroids grow, the limited nutrient and oxygen supply to the inner layers can lead to cellular stress and subsequent cell death. It can result in the formation of necrotic cores in larger spheroids.
  6. Cell Polarization: In spheroids, cells often exhibit polarization, with distinct apical and basal regions. This polarization can be observed in epithelial spheroids, where the outer cells form a tightly packed layer, reminiscent of an epithelial cell sheet.
  7. Extracellular Matrix (ECM): Some spheroid models develop a specialized ECM to provide structural support and mimic the in vivo microenvironment of tissues and tumors. This matrix can influence cell behavior, including proliferation, migration, and differentiation (4).


  1. Kapałczyńska M, Kolenda T, Przybyła W, Zajączkowska M, Teresiak A, Filas V, Ibbs M, Bliźniak R, Łuczewski Ł, Lamperska K. 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci. 2018 Jun;14(4):910-919. doi: 10.5114/aoms.2016.63743. Epub 2016 Nov 18. PMID: 30002710; PMCID: PMC6040128.
  2. Yamada KM, Cukierman E. Modeling tissue morphogenesis and cancer in 3D. Cell. 2007 Aug 24;130(4):601-10. doi: 10.1016/j.cell.2007.08.006. PMID: 17719539.
  3. Pampaloni F, Reynaud EG, Stelzer EH. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007 Oct;8(10):839-45. doi: 10.1038/nrm2236. PMID: 17684528.
  4. Baker BM, Chen CS. Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J Cell Sci. 2012 Jul 1;125(Pt 13):3015-24. doi: 10.1242/jcs.079509. Epub 2012 Jul 13. PMID: 22797912; PMCID: PMC3434846.