Spheroid-based drug screen: considerations and practical approach

Nat Protoc. 2009;4(3):309-24. doi: 10.1038/nprot.2008.226. Epub 2009 Feb 12.


Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein--including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)--is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (> or =14 d).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Cell Line, Tumor
  • Drug Screening Assays, Antitumor / methods
  • Humans
  • Spheroids, Cellular / drug effects*
  • Spheroids, Cellular / metabolism*


  • Antineoplastic Agents