Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Spheroid-based drug screen: considerations and practical approach


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 (≥14 d).

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Tools for routine preparation of agarose-coated 96-well plates.
Figure 2: Routine spheroid analysis includes phase-contrast imaging at days 4 and 7 (before and after drug treatment) and every 48 h thereafter.
Figure 3: Routine monitoring of spheroid formation capacity and establishment of the spheroid initiation protocol for one representative cell line.
Figure 4
Figure 5: Routine monitoring of drug effects on cell survival in HT29 colorectal cancer spheroids following drug treatment as determined through the acid phosphatase (APH) assay.
Figure 6: Routine monitoring of spheroid growth/regrowth of drug-treated HT29 spheroids.


  1. 1

    Abbott, A. Cell culture: biology's new dimension. Nature 424, 870–872 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Kunz-Schughart, L.A., Freyer, J.P., Hofstaedter, F. & Ebner, R. The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J. Biomol. Screen 9, 273–285 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Friedrich, J. et al. A reliable tool to determine cell viability in complex 3-d culture: the Acid phosphatase assay. J. Biomol. Screen. 12, 925–937 (2007).

    CAS  Article  Google Scholar 

  4. 4

    Gudjonsson, T., Ronnov-Jessen, L., Villadsen, R., Bissell, M.J. & Petersen, O.W. To create the correct microenvironment: three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia. Methods 30, 247–255 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    Nelson, C.M. & Bissell, M.J. Modeling dynamic reciprocity: engineering three-dimensional culture models of breast architecture, function, and neoplastic transformation. Semin. Cancer Biol. 15, 342–352 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Lee, G.Y., Kenny, P.A., Lee, E.H. & Bissell, M.J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4, 359–365 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Friedrich, J., Ebner, R. & Kunz-Schughart, L.A. Experimental anti-tumor therapy in 3-D: spheroids—old hat or new challenge? Int. J. Radiat. Biol. 83, 849–871 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Mueller-Klieser, W. Multicellular spheroids. A review on cellular aggregates in cancer research. J. Cancer Res. Clin. Oncol. 113, 101–122 (1987).

    CAS  Article  Google Scholar 

  9. 9

    Kunz-Schughart, L.A., Kreutz, M. & Knuechel, R. Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology. Int. J. Exp. Pathol. 79, 1–23 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    Santini, M.T. & Rainaldi, G. Three-dimensional spheroid model in tumor biology. Pathobiology 67, 148–157 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Khaitan, D., Chandna, S., Arya, M.B. & Dwarakanath, B.S. Establishment and characterization of multicellular spheroids from a human glioma cell line: implications for tumor therapy. J. Transl. Med. 4, 12 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Dubessy, C., Merlin, J.M., Marchal, C. & Guillemin, F. Spheroids in radiobiology and photodynamic therapy. Crit. Rev. Oncol. Hematol. 36, 179–192 (2000).

    CAS  Article  Google Scholar 

  13. 13

    Ballangrud, A.M. et al. Response of LNCaP spheroids after treatment with an alpha-particle emitter (213Bi)-labeled anti-prostate-specific membrane antigen antibody (J591). Cancer Res. 61, 2008–2014 (2001).

    CAS  PubMed  Google Scholar 

  14. 14

    Durand, R.E. & Olive, P.L. Resistance of tumor cells to chemo- and radiotherapy modulated by the three-dimensional architecture of solid tumors and spheroids. Methods Cell Biol. 64, 211–233 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Mueller-Klieser, W. Three-dimensional cell cultures: from molecular mechanisms to clinical applications. Am. J. Physiol. 273, C1109–C1123 (1997).

    CAS  Article  Google Scholar 

  16. 16

    Al-Hajj, M., Becker, M.W., Wicha, M., Weissman, I. & Clarke, M.F. Therapeutic implications of cancer stem cells. Curr. Opin. Genet. Dev. 14, 43–7 (2004).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Vermeulen, L. et al. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc. Natl. Acad. Sci. USA 105, 13427–13432 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Barbone, D., Yang, T.M., Morgan, J.R., Gaudino, G. & Broaddus, V.C. Mammalian target of rapamycin contributes to the acquired apoptotic resistance of human mesothelioma multicellular spheroids. J. Biol. Chem. 283, 13021–13030 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Desoize, B. & Jardillier, J. Multicellular resistance: a paradigm for clinical resistance? Crit. Rev. Oncol. Hematol. 36, 193–207 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Mueller-Klieser, W. Tumor biology and experimental therapeutics. Crit. Rev. Oncol. Hematol. 36, 123–139 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Carlsson, J. & Acker, H. Relations between pH, oxygen partial pressure and growth in cultured cell spheroids. Int. J. Cancer 42, 715–720 (1988).

    CAS  Article  Google Scholar 

  22. 22

    Poland, J. et al. Comparison of protein expression profiles between monolayer and spheroid cell culture of HT-29 cells revealed fragmentation of CK18 in three-dimensional cell culture. Electrophoresis 23, 1174–1184 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Frankel, A., Man, S., Elliott, P., Adams, J. & Kerbel, R.S. Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341. Clin. Cancer Res. 6, 3719–3728 (2000).

    CAS  PubMed  Google Scholar 

  24. 24

    Eshleman, J.S. et al. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res. 62, 7291–7297 (2002).

    CAS  PubMed  Google Scholar 

  25. 25

    Oloumi, A., Lam, W., Banath, J.P. & Olive, P.L. Identification of genes differentially expressed in V79 cells grown as multicell spheroids. Int. J. Radiat. Biol. 78, 483–492 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Jelic, S. Molecular basis of future patients-tailored treatment. Arch. Oncol. 13, 56–58 (2005).

    Google Scholar 

  27. 27

    Liu, M. et al. Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer. Cancer Res. 65, 5325–5336 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Dardousis, K. et al. Identification of differentially expressed genes involved in the formation of multicellular tumor spheroids by HT-29 colon carcinoma cells. Mol. Ther. 15, 94–102 (2007).

    CAS  Article  Google Scholar 

  29. 29

    Howes, A.L. et al. The phosphatidylinositol 3-kinase inhibitor, PX-866, is a potent inhibitor of cancer cell motility and growth in three-dimensional cultures. Mol. Cancer Ther. 6, 2505–2514 (2007).

    CAS  Article  Google Scholar 

  30. 30

    Kunz-Schughart, L.A. Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor. Cell Biol. Int. 23, 157–161 (1999).

    CAS  Article  Google Scholar 

  31. 31

    Furbert-Harris, P.M. et al. Eosinophils in a tri-cell multicellular tumor spheroid (MTS)/endothelium complex. Cell Mol. Biol. 49, 1081–1088 (2003).

    CAS  PubMed  Google Scholar 

  32. 32

    Gottfried, E., Kunz-Schughart, L.A., Andreesen, R. & Kreutz, M. Brave little world: spheroids as an in vitro model to study tumor-immune-cell interactions. Cell Cycle 5, 691–695 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Spoettl, T. et al. Monocyte chemoattractant protein-1 (MCP-1) inhibits the intestinal-like differentiation of monocytes. Clin. Exp. Immunol. 145, 190–199 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34

    Wartenberg, M., Finkensieper, A., Hescheler, J. & Sauer, H. Confrontation cultures of embryonic stem cells with multicellular tumor spheroids to study tumor-induced angiogenesis. Methods Mol. Biol. 331, 313–328 (2006).

    PubMed  Google Scholar 

  35. 35

    Gunther, S. et al. Polyphenols prevent cell shedding from mouse mammary cancer spheroids and inhibit cancer cell invasion in confrontation cultures derived from embryonic stem cells. Cancer Lett. 250, 25–35 (2007).

    Article  CAS  Google Scholar 

  36. 36

    Li, Z.W. & Dalton, W.S. Tumor microenvironment and drug resistance in hematologic malignancies. Blood Rev. 20, 333–342 (2006).

    Article  Google Scholar 

  37. 37

    Kunz-Schughart, L.A. & Mueller-Klieser, W. Three-dimensional culture. In Animal Cell Culture Vol. 3 (Ed. Masters, J.R.W.) 123–148 (Oxford University Press, Oxford, 2000).

    Google Scholar 

  38. 38

    Stark, H.J., Baur, M., Breitkreutz, D., Mirancea, N. & Fusenig, N.E. Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation. J. Invest. Dermatol. 112, 681–691 (1999).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Stark, H.J. et al. Epidermal homeostasis in long-term scaffold-enforced skin equivalents. J. Investig. Dermatol. Symp. Proc. 11, 93–105 (2006).

    CAS  Article  Google Scholar 

  40. 40

    Korff, T. & Augustin, H.G. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J. Cell Biol. 143, 1341–1352 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Ivascu, A. & Kubbies, M. Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J. Biomol. Screen 11, 922–932 (2006).

    CAS  Article  Google Scholar 

  42. 42

    Kosaka, T. et al. Comparison of various methods of assaying the cytotoxic effects of ethanol on human hepatoblastoma cells (HUH-6 line). Acta. Med. Okayama 50, 151–156 (1996).

    CAS  PubMed  Google Scholar 

  43. 43

    Enmon, R. et al. Combination treatment with 17-N-allylamino-17-demethoxy geldanamycin and acute irradiation produces supra-additive growth suppression in human prostate carcinoma spheroids. Cancer Res. 63, 8393–8399 (2003).

    CAS  PubMed  Google Scholar 

  44. 44

    Fehlauer, F. et al. Effects of irradiation and cisplatin on human glioma spheroids: inhibition of cell proliferation and cell migration. J. Cancer Res. Clin. Oncol. 131, 723–732 (2005).

    CAS  Article  Google Scholar 

  45. 45

    Lambert, B. et al. Screening for supra-additive effects of cytotoxic drugs and gamma irradiation in an in vitro model for hepatocellular carcinoma. Can. J. Physiol. Pharmacol. 82, 146–152 (2004).

    CAS  Article  Google Scholar 

  46. 46

    Sutherland, R.M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240, 177–184 (1988).

    CAS  Article  Google Scholar 

  47. 47

    LaRue, K.E., Khalil, M. & Freyer, J.P. Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors. Cancer Res. 64, 1621–1631 (2004).

    CAS  Article  Google Scholar 

  48. 48

    Francia, G., Man, S., Teicher, B., Grasso, L. & Kerbel, R.S. Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents. Mol. Cell. Biol. 24, 6837–6849 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49

    Francia, G. et al. Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids: implications for multicellular resistance to alkylating agents. Mol. Cancer Ther. 4, 1484–1494 (2005).

    CAS  Article  Google Scholar 

  50. 50

    Bindra, R.S., Crosby, M.E. & Glazer, P.M. Regulation of DNA repair in hypoxic cancer cells. Cancer Metastasis Rev 26, 249–260 (2007).

    CAS  Article  Google Scholar 

  51. 51

    Romero, F.J., Zukowski, D. & Mueller-Klieser, W. Glutathione content of V79 cells in two- or three-dimensional culture. Am. J. Physiol. 272, C1507–C1512 (1997).

    CAS  Article  Google Scholar 

  52. 52

    Winters, B.S., Raj, B.K., Robinson, E.E., Foty, R.A. & Corbett, S.A. Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly. Mol. Biol. Cell 17, 3386–3396 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53

    Carlsson, J. & Yuhas, J.M. Liquid-overlay culture of cellular spheroids. Recent Results Cancer Res. 95, 1–23 (1984).

    CAS  Article  Google Scholar 

  54. 54

    Tong, J.Z. et al. Long-term culture of adult rat hepatocyte spheroids. Exp. Cell Res. 200, 326–332 (1992).

    CAS  Article  Google Scholar 

  55. 55

    Hoevel, T., Macek, R., Swisshelm, K. & Kubbies, M. Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids. Int. J. Cancer 108, 374–383 (2004).

    CAS  Article  Google Scholar 

  56. 56

    Wartenberg, M. et al. Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids. Int. J. Cancer 75, 855–863 (1998).

    CAS  Article  Google Scholar 

  57. 57

    Kunz-Schughart, L.A. & Freyer, J.P. Adaptation of an automated selective dissociation procedure to two novel spheroid types. In Vitro Cell. Dev. Biol. Anim. 33, 73–76 (1997).

    CAS  Article  Google Scholar 

  58. 58

    Kerr, D.J., Wheldon, T.E., Kerr, A.M. & Kaye, S.B. In vitro chemosensitivity testing using the multicellular tumor spheroid model. Cancer Drug Deliv. 4, 63–74 (1987).

    CAS  Article  Google Scholar 

  59. 59

    Durand, R.E. Slow penetration of anthracyclines into spheroids and tumors: a therapeutic advantage? Cancer Chemother. Pharmacol. 26, 198–204 (1990).

    CAS  Article  Google Scholar 

  60. 60

    Freyer, J.P. & Schor, P.L. Automated selective dissociation of cells from different regions of multicellular spheroids. In Vitro Cell Dev. Biol. 25, 9–19 (1989).

    CAS  Article  Google Scholar 

  61. 61

    Watanabe, N., Hirayama, R. & Kubota, N. The chemopreventive flavonoid apigenin confers radiosensitizing effect in human tumor cells grown as monolayers and spheroids. J. Radiat. Res. (Tokyo) 48, 45–50 (2007).

    CAS  Article  Google Scholar 

  62. 62

    Essand, M., Nilsson, S. & Carlsson, J. Growth of prostatic cancer cells, DU 145, as multicellular spheroids and effects of estramustine. Anticancer Res. 13, 1261–1268 (1993).

    CAS  PubMed  Google Scholar 

  63. 63

    Russell, J., Wheldon, T.E. & Stanton, P. A radioresistant variant derived from a human neuroblastoma cell line is less prone to radiation-induced apoptosis. Cancer Res. 55, 4915–4921 (1995).

    CAS  PubMed  Google Scholar 

  64. 64

    Marusic, M., Bajzer, Z., Vuk-Pavlovic, S. & Freyer, J.P. Tumor growth in vivo and as multicellular spheroids compared by mathematical models. Bull. Math. Biol. 56, 617–631 (1994).

    CAS  PubMed  Google Scholar 

  65. 65

    Kunz-Schughart, L.A., Groebe, K. & Mueller-Klieser, W. Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation. Int. J. Cancer 66, 578–586 (1996).

    CAS  Article  Google Scholar 

  66. 66

    Tabatabai, M., Williams, D.K. & Bursac, Z. Hyperbolastic growth models: theory and application. Theor. Biol. Med. Model. 2, 14–15 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  67. 67

    Mellor, H.R., Ferguson, D.J. & Callaghan, R. A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs. Br. J. Cancer 93, 302–309 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  68. 68

    Orlandi, P. et al. Idarubicin and idarubicinol effects on breast cancer multicellular spheroids. J. Chemother. 17, 663–667 (2005).

    CAS  Article  Google Scholar 

  69. 69

    Lambert, B. et al. Assessment of supra-additive effects of cytotoxic drugs and low dose rate irradiation in an in vitro model for hepatocellular carcinoma. Can. J. Physiol. Pharmacol. 84, 1021–1028 (2006).

    CAS  Article  Google Scholar 

  70. 70

    Hirschberg, H., Sun, C.H., Krasieva, T. & Madsen, S.J. Effects of ALA-mediated photodynamic therapy on the invasiveness of human glioma cells. Lasers Surg. Med. 38, 939–945 (2006).

    Article  Google Scholar 

  71. 71

    Minchinton, A.I. & Tannock, I.F. Drug penetration in solid tumours. Nat. Rev. Cancer 6, 583–592 (2006).

    CAS  Article  Google Scholar 

  72. 72

    Xiao, Z., Hansen, C.B., Allen, T.M., Miller, G.G. & Moore, R.B. Distribution of photosensitizers in bladder cancer spheroids: implications for intravesical instillation of photosensitizers for photodynamic therapy of bladder cancer. J. Pharm. Pharm. Sci. 8, 536–543 (2005).

    CAS  PubMed  Google Scholar 

  73. 73

    L'Esperance, S., Bachvarova, M., Tetu, B., Mes-Masson, A.M. & Bachvarov, D. Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids. BMC Genomics 9, 99 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. 74

    Shiras, A., Bhosale, A., Patekar, A., Shepal, V. & Shastry, P. Differential expression of CD44(S) and variant isoforms v3, v10 in three-dimensional cultures of mouse melanoma cell lines. Clin. Exp. Metastasis 19, 445–455 (2002).

    CAS  Article  Google Scholar 

  75. 75

    Zietarska, M. et al. Molecular description of a 3D in vitro model for the study of epithelial ovarian cancer (EOC). Mol. Carcinog. 46, 872–885 (2007).

    CAS  Article  Google Scholar 

  76. 76

    Berchner-Pfannschmidt, U. et al. Nuclear oxygen sensing: induction of endogenous prolyl-hydroxylase 2 activity by hypoxia and nitric oxide. J. Biol. Chem. 83, 31745–31753 (2008).

    Article  Google Scholar 

  77. 77

    Leroux, F. et al. Experimental approaches to kinetics of gas diffusion in hydrogenase. Proc. Natl. Acad. Sci. USA 105, 11188–11193 (2008).

    CAS  Article  Google Scholar 

  78. 78

    Nowicki, M.O. et al. Lithium inhibits invasion of glioma cells; possible involvement of glycogen synthase kinase-3. Neuro Oncol. 10, 690–699 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  79. 79

    Wartenberg, M., Hescheler, J. & Sauer, H. Electrical fields enhance growth of cancer spheroids by reactive oxygen species and intracellular Ca2+. Am. J. Physiol. 272, R1677–R1683 (1997).

    CAS  PubMed  Google Scholar 

  80. 80

    Gali-Muhtasib, H. et al. Thymoquinone triggers inactivation of the stress response pathway sensor CHEK1 and contributes to apoptosis in colorectal cancer cells. Cancer Res. 68, 5609–5618 (2008).

    CAS  Article  Google Scholar 

  81. 81

    Wartenberg, M. et al. Reactive oxygen species-linked regulation of the multidrug resistance transporter P-glycoprotein in Nox-1 overexpressing prostate tumor spheroids. FEBS Lett. 579, 4541–4549 (2005).

    CAS  Article  Google Scholar 

  82. 82

    Salmaggi, A. et al. Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia 54, 850–860 (2006).

    Article  Google Scholar 

  83. 83

    Singh, S.K. et al. Identification of human brain tumour initiating cells. Nature 432, 396–401 (2004).

    CAS  Article  Google Scholar 

  84. 84

    Hermann, P.C. et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1, 313–323 (2007).

    CAS  Article  Google Scholar 

  85. 85

    O'Brien, C.A., Pollett, A., Gallinger, S. & Dick, J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445, 106–110 (2007).

    CAS  Article  Google Scholar 

  86. 86

    Ricci-Vitiani, L. et al. Identification and expansion of human colon-cancer-initiating cells. Nature 445, 111–115 (2007).

    CAS  Article  Google Scholar 

  87. 87

    Mizrak, D., Brittan, M. & Alison, M.R. CD133: molecule of the moment. J. Pathol. 214, 3–9 (2008).

    CAS  Article  PubMed  Google Scholar 

  88. 88

    Takaishi, S., Okumura, T. & Wang, T.C. Gastric cancer stem cells. J. Clin. Oncol. 26, 2876–2882 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  89. 89

    Chen, T.R., Dorotinsky, C.S., McGuire, L.J., Macy, M.L. & Hay, R.J. DLD-1 and HCT-15 cell lines derived separately from colorectal carcinomas have totally different chromosome changes but the same genetic origin. Cancer Genet. Cytogenet. 81, 103–108 (1995).

    CAS  Article  Google Scholar 

  90. 90

    Roschke, A.V. et al. Karyotypic 'state' as a potential determinant for anticancer drug discovery. Proc. Natl. Acad. Sci. USA 102, 2964–2969 (2005).

    CAS  Article  Google Scholar 

  91. 91

    Ellison, G. et al. Further evidence to support the melanocytic origin of MDA-MB-435. Mol. Pathol. 55, 294–299 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  92. 92

    Rae, J.M. et al. Common origins of MDA-MB-435 cells from various sources with those shown to have melanoma properties. Clin. Exp. Metastasis 21, 543–552 (2004).

    CAS  Article  Google Scholar 

  93. 93

    Garraway, L.A. et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 436, 117–122 (2005).

    CAS  Article  Google Scholar 

Download references


We gratefully acknowledge the technical assistance of Marit Wondrak, Juana M Castaneda and Tammy Lawrence as well as Frank van Rey and Rupert Feldmeier. The experimental protocols and work documented herein were supported by the Society for Biomolecular Sciences (SBS) and by the German Federal Ministry of Education and Research (BMBF) through grants 0313143 and 01ZZ0502 to L.A.K.-S.

Author information



Corresponding author

Correspondence to Leoni A Kunz-Schughart.

Ethics declarations

Competing interests

R. Ebuer is a minor stockholder C=<1% of Aoalon Pharmaceuticals Inc.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Friedrich, J., Seidel, C., Ebner, R. et al. Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4, 309–324 (2009).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing