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

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

doi:10.1007/978-1-0716-1979-7_27

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 proceeding › Chapter

Abstract

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 language	English (US)
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	405-416
Number of pages	12
Volume	2429
DOIs	https://doi.org/10.1007/978-1-0716-1979-7_27
State	Published - 2022

Publication series

Name	Methods in molecular biology (Clifton, N.J.)
Publisher	Humana 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.

Keywords

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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10.1007/978-1-0716-1979-7_27

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abstract = "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.",

keywords = "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",

author = "Kim, {Lisa M.} and Kim, {Paul Y.} and Leung, {Cheuk T.}",

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: {\textcopyright} 2022, Springer Science+Business Media, LLC, part of Springer Nature.",

year = "2022",

doi = "10.1007/978-1-0716-1979-7_27",

language = "English (US)",

volume = "2429",

series = "Methods in molecular biology (Clifton, N.J.)",

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AU - Kim, Lisa M.

AU - Kim, Paul Y.

AU - Leung, Cheuk T.

N1 - 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.

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - Extracellular matrix (ECM)

KW - Immunofluorescence

KW - Live imaging

KW - Protein and RNA isolation

KW - Three-dimensional (3D) cell culture

KW - Extracellular Matrix/metabolism

KW - Basement Membrane

KW - Cell Culture Techniques/methods

KW - Animals

KW - Cell Line, Tumor

KW - Cell Differentiation

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U2 - 10.1007/978-1-0716-1979-7_27

DO - 10.1007/978-1-0716-1979-7_27

M3 - Chapter

C2 - 35507177

AN - SCOPUS:85129383349

VL - 2429

T3 - Methods in molecular biology (Clifton, N.J.)

SP - 405

EP - 416

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

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

Abstract

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

Abstract

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Bibliographical note

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PubMed: MeSH publication types

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Nikon A1RHD Multiphoton Upright Confocal

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