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

Summary

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

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.

Introduction

Myofibroblasts/fibroblasts (MFs) represent an abundant population of cells in gastrointestinal (GI) mucosa. These stromal cells are located just beneath the epithelium and form an interconnected network within mucosal lamina propria in the gut. MFs are not only responsible for the deposition of the extracellular matrix, but through paracrine regulation may influence electrolyte transport, restitution, and barrier function of the adjacent epithelium^{1, 2}. Furthermore, MFs have been shown to play a key role in inflammation and tissue remodeling^{3, 4}. Myofibroblasts are a critical part of the tumor microenvironment, where they are also known as cancer....

Protocol

The protocol for obtaining discarded human tissue from surgical patients and the establishment of primary cultures was approved by the University of Texas Medical Branch and University of New Mexico Institutional Review Boards. The general requirements for the procurement of human tissue specimens is described below. Note that since MFs may be isolated from frozen tissue, biobanks and commercial vendors are also viable options.

1. Obtain Human Tissue

1. Submit a protocol to obtain human colon tissue to the institutional review board for approval.
2. Establish collaborations with general surgeons and/or colorectal surgeons in....

Results

Using this enzymatic digestion protocol, we have consistently been able to isolate and grow MF population gut CD90⁺ mucosal stromal cells from GI surgical specimens and biopsies (Figure 1). Visible MF colony proliferation could be observed on day 2 - 5 after seeding mononuclear cell suspensions into 6 well plates (Figure 1A-B). The MF primary cultures reach ~ 50 - 70 % confluence by day 7 - 11 (Figure 1C-D).

Discussion

While this protocol was developed for the research application only, in the light of growing critical importance of stromal cells as a potential cancer prognostic biomarkers and therapeutic target, the ability to freeze "functional" biospecimens for later use offers a significant advantage. This represents a unique advantage for creation of clinical biorepository, which may serve as additional tools serving to advance the development of personalized medicine^{10, 11}. Similar to findings previously report.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MEM, 1x	Corning	MT-10-010-CV
Hanks' Balanced Salt Solution	Sigma-Aldrich	H6648
Sodium pyruvate	Sigma-Aldrich	S8636
Antibiotic-Antimycotic, 100x	Gibco	15240-062
Ciprofloxacin HCl	Corning	61-277-RF
L-Glutamine, 100x	Corning	25-005-CI
MEM Nonessential Amino Acids	Corning	25-025-CI
Dimethyl Sulfoxide (DMSO) Hybri-Max	Sigma-Aldrich	D2650
DL-Dithiothreitol solution (DTT)	Sigma-Aldrich	646563
0.5 M EDTA, pH 8.0	Cellgro	46-034-CI
DNAse	Worthington	LS002139
Collagenase from Clostridium histolyticum, Type I	Sigma-Aldrich	C1639
Collagenase from Clostridium histolyticum, Type II	Sigma-Aldrich	C1764
Collagenase from Clostridium histolyticum, Type IV	Sigma-Aldrich	C5138
Accumax cell dissociation solution	Sigma-Aldrich	A7089
gentleMACS C Tubes	Miltenyi Biotec	130-093-237
gentleMACS Dissociator	Miltenyi Biotec	130-093-235
Countess Automated Cell Counter	Invitrogen	C10227
Cell Strainer (70 µm)	Corning	352350
PE Mouse Anti-Human Vimentin	BD Biosciences	562337	Clone RV202
FITC Monoclonal Anti-Actin, α-Smooth Muscle	Sigma-Aldrich	F3777	Clone 1A4
APC Mouse Anti-Human CD90	BD Biosciences	559869	Clone 5E10