A Unified Protocol to Streamline Molecular and Cellular Analysis for Three-Dimensional Cell Cultures

Lisa M. Kim, Paul Y. Kim, Cheuk T. Leung

Research output: Chapter in Book/Report/Conference proceedingChapter


Three-dimensional (3D) cell cultures based on reconstituted basement membrane materials recapitulate features of extracellular matrix (ECM) and tissue stiffness in vivo and provide a physiologically relevant platform to study complex cellular processes, such as stem cell differentiation and tissue morphogenesis, that are otherwise difficult in animal models. The form and composition of 3D matrices in culture can interfere with and pose challenges for different experimental setups and assays, which necessitate alterations to facilitate analysis. Here, we provide a unified protocol for 3D cell cultures with modular workflows that streamline procedures for compatibility with common molecular and cellular assays such as live-cell imaging, immunofluorescence, qPCR, RNAseq, western blotting, and quantitative mass spectrometry.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages12
StatePublished - 2022

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

Bibliographical note

Funding Information:
The work is supported by a grant award from NIH National Cancer Institute (#R01CA200652). Images were obtained using equipment in the University Imaging Centers at the University of Minnesota.

Publisher Copyright:
© 2022, Springer Science+Business Media, LLC, part of Springer Nature.


  • Extracellular matrix (ECM)
  • Immunofluorescence
  • Live imaging
  • Protein and RNA isolation
  • Three-dimensional (3D) cell culture
  • Extracellular Matrix/metabolism
  • Basement Membrane
  • Cell Culture Techniques/methods
  • Animals
  • Cell Line, Tumor
  • Cell Differentiation

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural


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