Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Summary

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr period.

Abstract

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Introduction

An important tool for studying cell-matrix interactions is the cell populated collagen gel^1,2. The gel provides a 3D environment that is closer to the in vivo character of the tissue and better suited for understanding cell behavior than is offered by traditional 2D cultures³. Early studies in which fibroblasts were homogenously distributed within a collagen gel found that the cells rapidly consolidate the collagen fibers and compact the gel^4,5. The contractile fibroblasts in free floating gels then transition into a quiescent state soon after the gel has fully reached compaction^1,6,7. The fibroblasts in gels that a....

Protocol

1. Stencil Preparation

Prepare a stencil on Parafilm to layout the location of each explant, following the desired geometry (Figures 1A and 1B). Space each explant approximately 1-2 mm apart. This distance corresponds to an ideal spacing for generating fiber alignment between explants. Attach the stencil underneath the area of the coverglass where the sample will be prepared with tape.

2. Sterilization

Thoroughly clean all components of the bioreactor using 70% ethanol and sterilize for 2-3 hr under UV light prior to experimentation. If an....

Results

Tissue remodeling is a complex process that is driven in part by reciprocal physical interactions between cells and the surrounding matrix. The cells reorganize the surrounding fibers and generate tension in the fiber network. The alignment of fibers and mechanical environment in turn controls cell behavior, so that both cells and matrix globally reorganize to produce remodeled tissue. In this experiment, the cells of the explants, initially round in morphology, began to extend into the gel and adhere to the fibrin fiber.......

Discussion

This protocol was developed for the purpose of observing and quantifying the mechanics involved in cell-mediated ECM remodeling. Such processes underlie a number of biological phenomena and have important implications for engineering tissues^2,22, reducing scar^1,23, and understanding pathological tissue remodeling^12,24. The use of time-lapse DIC microscopy allows one to resolve and quantify the displacement and alignment of fibrin fibers that occurs as a result of cell traction forces. The.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sigma-Aldrich	F8630
Sigma-Aldrich	T4648
Gibco	11965-092
Gibco	15140-122
Sigma-Aldrich	A2942
Sigma-Aldrich	H0887
Sigma-Aldrich	223506
Gibco	25200-056
Invitrogen	3000
Lonza	DE14-701F
Molecular Probes	F8858
GIBCO	A10483-01
GIBCO	11430-030
Fisher-Scientific	SS264-1
Sigma-Aldrich	A3428-25MG
Biotense Bioreactor	ADMET
Ti-Eclipe Microscope	Nikon
# 0 35 mm Glass Bottom Petri Dish	MatTek	P35G-0-20-C
# 0 35 mm Glass Top Petri Dish	MatTek	P35GTOP-0-20-C
Plastic Luer fittings, PVC tubing with Luer ends	Cole-Parmer	30600-65