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Adv Exp Med Biol. 2016;899:167-96. doi: 10.1007/978-3-319-26666-4_10.

Methods: Using Three-Dimensional Culture (Spheroids) as an In Vitro Model of Tumour Hypoxia.

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Nuffield Division of Clinical Laboratory Sciences, Department of Oncology, University of Oxford, Oxford, OX3 9DU, UK.
Gray Laboratory, Cancer Research UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Old Road Campus Research Building, off Roosevelt Drive, Oxford, OX3 7DQ, UK.
Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.
Cancer Biology, Division of Cancer and Stem Cells, University of Nottingham, QMC, D Floor, West Block, W/D/1374, Nottingham, NG7 2UH, UK.


Regions of hypoxia in tumours can be modelled in vitro in 2D cell cultures with a hypoxic chamber or incubator in which oxygen levels can be regulated. Although this system is useful in many respects, it disregards the additional physiological gradients of the hypoxic microenvironment, which result in reduced nutrients and more acidic pH. Another approach to hypoxia modelling is to use three-dimensional spheroid cultures. In spheroids, the physiological gradients of the hypoxic tumour microenvironment can be inexpensively modelled and explored. In addition, spheroids offer the advantage of more representative modelling of tumour therapy responses compared with 2D culture. Here, we review the use of spheroids in hypoxia tumour biology research and highlight the different methodologies for spheroid formation and how to obtain uniformity. We explore the challenge of spheroid analyses and how to determine the effect on the hypoxic versus normoxic components of spheroids. We discuss the use of high-throughput analyses in hypoxia screening of spheroids. Furthermore, we examine the use of mathematical modelling of spheroids to understand more fully the hypoxic tumour microenvironment.


Carbonic anhydrase IX; Hypoxia; Spheroids, 3D culture; pH

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