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Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture
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Engineering clinically-relevant human fibroblastic cell-derived extracellular matrices.

Janusz Franco-Barraza1, Kristopher S Raghavan2, Tiffany Luong1

  • 1Cancer Biology, The Martin and Concetta Greenberg Pancreatic Cancer Institute, Fox Chase Cancer Center, Philadelphia, PA, United States.

Methods in Cell Biology
|March 31, 2020
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Summary
This summary is machine-generated.

This study details a method for creating 3D fibroblastic cell-derived matrices (fCDM) to study tissue microenvironments. Protocols enable assessment of fibroblast function and creation of cell-free scaffolds for research.

Keywords:
Cancer-associated fibroblastsCell-derived extracellular matrixCell-matrix interactionsExtracellular matrixPrimary fibroblastsThree-dimensional cell cultureTissue microenvironment

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Area of Science:

  • Cell Biology and Tissue Engineering
  • Extracellular Matrix (ECM) Research
  • Fibroblast Biology and Pathology

Background:

  • Three-dimensional (3D) culturing models, incorporating native extracellular matrix (ECM), are vital for replicating in vivo tissue microenvironments.
  • Fibroblastic cells produce interstitial ECM, providing crucial biochemical and biomechanical cues that regulate cell function, tissue development, and homeostasis.
  • Dysregulation of ECMs and fibroblasts is implicated in pathological events, including developmental defects, fibrosis, chronic inflammation, and cancer.

Purpose of the Study:

  • To provide a step-by-step method for producing multilayered, 3D fibroblastic cell-derived matrices (fCDM).
  • To detail methods for assessing ECM topography and fibroblast functional status (naïve/normal vs. inflammatory/myofibroblastic).
  • To describe protocols for isolating normal and diseased fibroblasts (e.g., cancer-associated fibroblasts, CAFs) and for fCDM decellularization.

Main Methods:

  • Development of protocols for generating 3D fibroblastic cell-derived matrices (fCDM).
  • Implementation of microscopy and semi-quantitative digital imaging analyses to assess ECM topography and fibroblast phenotypes.
  • Establishment of procedures for isolating fibroblasts and for decellularizing fCDM to create in vivo-like scaffolds.

Main Results:

  • A comprehensive methodology for producing and analyzing 3D fCDM is presented.
  • Protocols allow for the characterization of fibroblast functional states and their associated matrix production.
  • Decellularized fCDM serve as effective 3D substrates for subsequent cell culturing, mimicking physiological scaffolds.

Conclusions:

  • The described methods facilitate the study of fibroblast-ECM interactions in a controlled 3D environment.
  • This approach aids in distinguishing between normal and pathological fibroblast phenotypes and their matrix contributions.
  • The generated cell-free scaffolds offer a valuable tool for investigating cell behavior within biomimetic matrices.