Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

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, thi...

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Bibliographic Details
Published in:Journal of visualized experiments 2016-01 (107), p.e53691-e53691
Main Authors: Johnson, Paul, Beswick, Ellen J, Chao, Celia, Powell, Don W, Hellmich, Mark R, Pinchuk, Iryna V
Format: Article
Language:English
Subjects:
Actins - analysis
Actins - biosynthesis
Biomarkers - metabolism
Cell Culture Techniques - methods
Colon - cytology
Developmental Biology
Fibroblasts - cytology
Fibroblasts - metabolism
Flow Cytometry - methods
Freezing
Humans
Immunohistochemistry
Intestine, Small - cytology
Myofibroblasts - cytology
Myofibroblasts - metabolism
Phenotype
Stromal Cells - cytology
Stromal Cells - metabolism
Thy-1 Antigens - analysis
Thy-1 Antigens - biosynthesis
Vimentin - analysis
Vimentin - biosynthesis
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Summary: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.
ISSN:1940-087X
1940-087X
DOI:10.3791/53691