Factors affecting tumor spheroid formation for cancer research

Tumor spheroids are three-dimensional cell culture models that have gained significant attention in cancer research because they provide a more physiologically relevant environment for studying tumor biology compared to traditional two-dimensional cell culture systems.

3D spheroids contain a dense network of cells and deposits of extracellular matrix which allow cell-cell and cell-ECM communication within each dimension. In vitro 3D models recapitulate the dimensionality of cancer microenvironment to understand the initial stages of cancer cell invasion, migration, and signaling pathways (1). Furthermore, since cancer cells in spheroids show more resistance against chemotherapeutic drugs than in 2D cell cultures, spheroids are better suited for drug testing systems.

Spheroids are formed by the aggregation and self-assembly of cells affected by various factors, including nutrient grading, oxygen, and cell line and morphology (2).

This article is an overview of the factors that affect the formation of tumor spheroids for cancer research:

  1. Cell type: Different cell types have different tendencies to form spheroids. For instance, cancer cells form spheroids more readily.
  2. Cell density: Cell density can affect spheroid formation; low cell density results in the formation of small spheroids, while high cell density can result in the formation of larger spheroids. Hence, cell seeding density should be optimized for high reproducibility and reliability (3).
  3. Substrate: The substrate on which cells grow can affect spheroid morphology. Special coating solutions such as the BIOFLOATTM FLEX coating solution can be used by researchers to provide a hydrophilic surface for spontaneous cell aggregation. Irregularly shaped spheroids affect reproducibility, reliability, and high-throughput analysis in cancer research. To avoid it, the use of a BIOFLOATTM coated plate is recommended that allows the formation of a single perfectly round shaped spheroid per well (4,5).
  4. Culture conditions: Temperature, growth factors, sheer stress, medium composition, and cultivation time affect spheroid formation. All these factors influence cell-cell and cell-substrate interactions and determine how cells aggregate and interact with each other (6).


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  2. LaBarbera DV, Reid BG, Yoo BH. The multicellular tumor spheroid model for high-throughput cancer drug discovery. Expert Opin Drug Discov. 2012 Sep;7(9):819-30. doi: 10.1517/17460441.2012.708334. Epub 2012 Jul 12. PMID: 22788761.
  3. Zheng Y, Jiang LI, Yan M, Gosau M, Smeets R, Kluwe L, Friedrich RE. Optimizing Conditions for Spheroid Formation of Dental Pulp Cells in Cell Culture. In Vivo. 2021 Jul-Aug;35(4):1965-1972. doi: 10.21873/invivo.12464. PMID: 34182470; PMCID: PMC8286480.
  4. Cui X, Hartanto Y, Zhang H. Advances in multicellular spheroids formation. J R Soc Interface. 2017 Feb;14(127):20160877. doi: 10.1098/rsif.2016.0877. PMID: 28202590; PMCID: PMC5332573.
  5. Das V, Fürst T, Gurská S, Džubák P, Hajdúch M. Reproducibility of Uniform Spheroid Formation in 384-Well Plates: The Effect of Medium Evaporation. J Biomol Screen. 2016 Oct;21(9):923-30. doi: 10.1177/1087057116651867. Epub 2016 May 25. PMID: 27226477.
  6. Białkowska K, Komorowski P, Bryszewska M, Miłowska K. Spheroids as a Type of Three-Dimensional Cell Cultures-Examples of Methods of Preparation and the Most Important Application. Int J Mol Sci. 2020 Aug 28;21(17):6225. doi: 10.3390/ijms21176225. PMID: 32872135; PMCID: PMC7503223.