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Protocol for the Generation and 3D Culture of Fluorescently Labeled Multicellular Spheroids
Book chapter

Protocol for the Generation and 3D Culture of Fluorescently Labeled Multicellular Spheroids

Emily C Liu, Amy L Ryan and Sinem Koc-Günel
Methods in molecular biology (Clifton, N.J.)
01/17/2025
DOI: 10.1007/7651_2024_592
PMID: 39821808

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Abstract

Spheroid culture systems have been extensively used to model the three-dimensional (3D) behavior of cells in vitro. Traditionally, spheroids consist of a single cell type, limiting their ability to fully recapitulate the complex inter-cellular interactions observed in vivo. Here we describe a protocol for generating cocultured spheroids composed of two distinct cell types, embedded within a 3D extracellular matrix (ECM) to better study cellular interactions. Fluorescent labeling of each cell type enables clear distinction and visualization, facilitating the analysis of cell invasion, proliferation, and behavior within the matrix. This method is particularly suited for studying matrix invasion, an essential process in cancer metastasis, using both fixed and live cell microscopy. The protocol is versatile and can be adapted for various cell types, providing a robust platform for investigating cell-cell interactions in cancer research, tissue remodeling, and drug screening.
Extracellular Matrix Multicellular interactions Fluorescence imaging Lymphatic endothelium Human lung fibroblasts 3D spheroids Invasion

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