Isolation of CD 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

Paul Johnson, Ellen J. Beswick, Celia Chao, Don W. Powell, Mark Hellmich, Iryna Pinchuk

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, α-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Original languageEnglish (US)
Article numbere53691
JournalJournal of Visualized Experiments
Volume2016
Issue number107
DOIs
StatePublished - Jan 31 2016

Keywords

  • Developmental biology
  • Fibroblasts/myofibroblasts
  • Frozen
  • Gastrointestinal tract
  • Human
  • Issue 107
  • Mesenchymal stromal cells

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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