Establishment of Two Dimensional (2D) and Three-Dimensional (3D) Melanoma Primary Cultures as a Tool for In Vitro Drug Resistance Studies

  • Nataly Cruz Rodríguez
  • Johanna Lineros
  • Carol Stefany Rodríguez
  • Lina María Martínez
  • Josefa Antonia RodríguezEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1913)


Characteristics of melanoma cells have been deciphered by studies carried out in two dimensional cell cultures growing as adherent monolayers on the bottom of plastic flasks. Melanoma cells can be cultured with a considerable degree of success, and, depending on the further use of the cells obtained in the culture, methodologies have to be adjusted to obtain reliable results. Although there are many melanoma continuous cell lines, in vitro 2D and 3D melanoma primary cell culture may be a more useful model to investigate interactions between cancer cells and immune system, as well as the effect of cytotoxic treatments and personalized medicine in environments more similar to the physiological conditions.

Here, we described a protocol which employs many strategies to obtain primary 2D and 3D melanoma cultures as a model to study cell–cell and cell–microenvironment interactions that must be considered to properly design personalized cancer treatments, as well as for testing novel anticancer drugs and drug delivery vehicles.

Key words

Melanoma Primary cell culture Two dimensional cell culture Three-dimensional cell spheroids Cancer model Drug resistance 


  1. 1.
    Yamamoto Y, Ochiya T (2017) Epithelial stem cell culture: modeling human disease and applications for regenerative medicine. Inflamm Regen 37:3–10CrossRefGoogle Scholar
  2. 2.
    Mouriaux F, Zaniolo K, Bergeron MA et al (2016) Effects of long-term serial passaging on the characteristics and properties of cell lines derived from uveal melanoma primary tumors. Invest Ophthalmol Vis Sci 57(13):5288–5301CrossRefGoogle Scholar
  3. 3.
    Freshney RI (2010) Culture of animal cells: a manual of basic technique and specialized applications, 6th edn. John Wiley & Sons, Inc., Hoboken, New JerseyCrossRefGoogle Scholar
  4. 4.
    Virgone-Carlotta A, Lemasson M, Mertani HC, Diaz JJ et al (2017) In-depth phenotypic characterization of multicellular tumor spheroids: effects of 5-fluorouracil. PLoS One 12(11):e0188100CrossRefGoogle Scholar
  5. 5.
    Grill J, Lamfers ML, van Beusechem VW et al (2002) The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro. Mol Ther 6(5):609–614PubMedGoogle Scholar
  6. 6.
    Raghavan S, Mehta P, Horst EN et al (2016) Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity. Oncotarget 7(13):16948–16961CrossRefGoogle Scholar
  7. 7.
    Huanga B, Gao JQ (2018) Application of 3D cultured multicellular spheroid tumor models in tumor-targeted drug delivery system research. Bull Cancer 270:246–259Google Scholar
  8. 8.
    Vorsmann H, Groeber F, Walles H et al (2013) Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing. Cell Death Dis 4:e719CrossRefGoogle Scholar
  9. 9.
    Foty R (2011) A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp 51:2720Google Scholar
  10. 10.
    Hirschhaeuser F, Menne H, Dittfeld C et al (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148(1):3–15CrossRefGoogle Scholar
  11. 11.
    Coit DG, Thompson JA, Algazi A et al (2016) Melanoma, version 2 NCCN clinical practice guidelines in oncology. J Natl Compr Cancer Netw 14(4):450–473CrossRefGoogle Scholar
  12. 12.
    Edge SB, Compton CC (2010) The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 17(6):1471–1474CrossRefGoogle Scholar
  13. 13.
    Halaban R, Rubin JS, Funasaka Y (1992) Met and hepatocyte growth factor/scatter factor signal transduction in normal melanocytes and melanoma cells. Oncogene 7(11):2195–2206PubMedGoogle Scholar
  14. 14.
    Matsumoto K, Tajima H, Nakamura T (1991) Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth. Biochem Biophys Res Commun 176(1):45–51CrossRefGoogle Scholar
  15. 15.
    Yada Y, Higuchi K, Imokawa G (1991) Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem 266(27):18352–18357PubMedGoogle Scholar
  16. 16.
    Godwin LS, Castle JT, Kohli JS (2014) Isolation, culture, and transfection of melanocytes. Curr Protoc Cell Biol 63:1–20PubMedGoogle Scholar
  17. 17.
    Anitua E, Andia I, Sanchez M et al (2005) Autologous preparations rich in growth factors promote proliferation and induce VEGF and HGF production by human tendon cells in culture. J Orthop Res 23(2):281–286CrossRefGoogle Scholar
  18. 18.
    Anitua E, Sanchez M, Nurden AT et al (2007) Reciprocal actions of platelet-secreted TGF-beta1 on the production of VEGF and HGF by human tendon cells. Plast Reconstr Surg 119(3):950–959CrossRefGoogle Scholar
  19. 19.
    Gassling VL, Acil Y, Springer IN et al (2009) Platelet-rich plasma and platelet-rich fibrin in human cell culture. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108(1):48–55CrossRefGoogle Scholar
  20. 20.
    Li H, Usas A, Poddar M (2013) Platelet-rich plasma promotes the proliferation of human muscle derived progenitor cells and maintains their stemness. PLoS One 8(6):e64923CrossRefGoogle Scholar
  21. 21.
    Eisinger M, Marko O, Ogata S et al (1985) Growth regulation of human melanocytes: mitogenic factors in extracts of melanoma, astrocytoma, and fibroblast cell lines. Science 229(4717):984–986CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Nataly Cruz Rodríguez
    • 1
  • Johanna Lineros
    • 1
  • Carol Stefany Rodríguez
    • 2
  • Lina María Martínez
    • 1
  • Josefa Antonia Rodríguez
    • 1
    • 2
    Email author
  1. 1.Cancer Biology Research GroupNational Cancer Institute of ColombiaBogotáColombia
  2. 2.Bachelor Project in BiologyFrancisco José de Caldas District UniversityBogotáColombia

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