Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Authors

  • Jens M. Kelm,

    1. Laboratory for Biological Engineering, Department of Chemical Engineering, University of Queensland, 4072 Brisbane, Australia; telephone: +617-3365-4682; fax: +617-3365-4199
    2. Institute of Biotechnology, Swiss Federal Institute of Technology, Zurich, Switzerland
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  • Nicholas E. Timmins,

    1. Laboratory for Biological Engineering, Department of Chemical Engineering, University of Queensland, 4072 Brisbane, Australia; telephone: +617-3365-4682; fax: +617-3365-4199
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  • Catherine J. Brown,

    1. Laboratory for Biological Engineering, Department of Chemical Engineering, University of Queensland, 4072 Brisbane, Australia; telephone: +617-3365-4682; fax: +617-3365-4199
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  • Martin Fussenegger,

    1. Institute of Biotechnology, Swiss Federal Institute of Technology, Zurich, Switzerland
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  • Lars K. Nielsen

    Corresponding author
    1. Laboratory for Biological Engineering, Department of Chemical Engineering, University of Queensland, 4072 Brisbane, Australia; telephone: +617-3365-4682; fax: +617-3365-4199
    • Laboratory for Biological Engineering, Department of Chemical Engineering, University of Queensland, 4072 Brisbane, Australia; telephone: +617-3365-4682; fax: +617-3365-4199
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Abstract

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 173–180, 2003.

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