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A method for reproducible high-resolution imaging of 3D cancer cell spheroids

Research output: Contribution to journalArticlepeer-review

Original languageEnglish
JournalJournal of Microscopy
Early online date13 Jan 2023
Accepted/In press9 Jan 2023
E-pub ahead of print13 Jan 2023

Bibliographical note

Funding Information: NA was funded by a Leverhulme Trust Doctoral Scholarship Grant. TP was funded by the London Interdisciplinary Doctoral Training Programme and BBSRC. SC acknowledges support from a Royal Society University Research Fellowship. TS was funded by the Medical Research Council (MR/K019732/1). CC and VC received funding from the European Research Council through ERC Starting Grant EnBiON (759577). Y.M. was funded by MRC awards MR/L009056/1, MR/T031646/1, MR/W027437/1, a Lister Institute Research Prize and EMBO Young Investigator Programme. R.T. was funded by an MRC award MR/T031646/1. Publisher Copyright: © 2023 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

King's Authors


Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell–ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

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