Skip to main content

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

  • Protocol
  • First Online:
Immune Checkpoint Blockade

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Yamamoto Y, Ochiya T (2017) Epithelial stem cell culture: modeling human disease and applications for regenerative medicine. Inflamm Regen 37:3–10

    Article  Google Scholar 

  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):5288–5301

    Article  CAS  Google Scholar 

  3. Freshney RI (2010) Culture of animal cells: a manual of basic technique and specialized applications, 6th edn. John Wiley & Sons, Inc., Hoboken, New Jersey

    Book  Google Scholar 

  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):e0188100

    Article  Google Scholar 

  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):609–614

    CAS  PubMed  Google Scholar 

  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):16948–16961

    Article  Google Scholar 

  7. Huanga B, Gao JQ (2018) Application of 3D cultured multicellular spheroid tumor models in tumor-targeted drug delivery system research. Bull Cancer 270:246–259

    Google Scholar 

  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:e719

    Article  CAS  Google Scholar 

  9. Foty R (2011) A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp 51:2720

    Google Scholar 

  10. Hirschhaeuser F, Menne H, Dittfeld C et al (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148(1):3–15

    Article  CAS  Google Scholar 

  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):450–473

    Article  Google Scholar 

  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):1471–1474

    Article  Google Scholar 

  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):2195–2206

    CAS  PubMed  Google Scholar 

  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):45–51

    Article  CAS  Google Scholar 

  15. Yada Y, Higuchi K, Imokawa G (1991) Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem 266(27):18352–18357

    CAS  PubMed  Google Scholar 

  16. Godwin LS, Castle JT, Kohli JS (2014) Isolation, culture, and transfection of melanocytes. Curr Protoc Cell Biol 63:1–20

    PubMed  Google Scholar 

  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):281–286

    Article  CAS  Google Scholar 

  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):950–959

    Article  CAS  Google Scholar 

  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):48–55

    Article  Google Scholar 

  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):e64923

    Article  CAS  Google Scholar 

  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):984–986

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Josefa Antonia Rodríguez .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Cruz Rodríguez, N., Lineros, J., Rodríguez, C.S., Martínez, L.M., Rodríguez, J.A. (2019). Establishment of Two Dimensional (2D) and Three-Dimensional (3D) Melanoma Primary Cultures as a Tool for In Vitro Drug Resistance Studies. In: Pico de Coaña, Y. (eds) Immune Checkpoint Blockade. Methods in Molecular Biology, vol 1913. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8979-9_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8979-9_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8978-2

  • Online ISBN: 978-1-4939-8979-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics