Abstract
Mechanical forces have long been recognized as fundamental drivers in biological processes, such as embryogenesis, tissue formation and disease regulation. The collagen gel contraction (CGC) assay has served as a classic tool in the field of mechanobiology to study cell-induced contraction of extracellular matrix (ECM), which plays an important role in inflammation and wound healing. In a conventional CGC assay, cell-laden collagen is loaded into a cell culture vessel (typically a well plate) and forms a disk-shaped gel adhering to the bottom of the vessel. The decrement in diameter or surface area of the gel is used as a parameter to quantify the degree of cell contractility. In this study, we developed a microscale CGC assay with an engineered well plate insert that uses surface tension forces to load and manipulate small volumes (14 μL) of cell-laden collagen. The system is easily operated with two pipetting steps and the microscale device moves dynamically as a result of cellular forces. We used a straightforward one-dimensional measurement as the gel contraction readout. We adapted a conventional lung fibroblast CGC assay to demonstrate the functionality of the device, observing significantly more gel contraction when human lung fibroblasts were cultured in serum-containing media vs. serum-free media (p ≤ 0.05). We further cocultured eosinophils and fibroblasts in the system, two important cellular components that lead to fibrosis in asthma, and observed that soluble factors from eosinophils significantly increase fibroblast-mediated gel contraction (p ≤ 0.01). Our microscale CGC device provides a new method for studying downstream ECM effects of intercellular cross talk using 7- to 35-fold less cell-laden gel than traditional CGC assays.
Highlights
Fibroblasts are key mesenchymal cells in connective tissue which synthesize extracellular matrix (ECM) components and provide structural support for the extracellular environment (Kendall and Feghali-Bostwick, 2014)
In the fields of regenerative medicine, tissue engineering, and organotypic models, cell-encapsulating microfluidic hydrogels have been widely employed as an effective model to study cell behaviors in ECM environment (Ling et al, 2007; Khetan and Burdick, 2011; Huang et al, 2012, 2013; Bhatia and Ingber, 2014; Duval et al, 2017)
The 3D culture of fibroblasts in native type I collagen gels has enabled researchers to integrate cell behaviors with surrounding matrix components, capturing some key aspects of cell-extracellular matrix interactions that are lost in simple 2D culture on plastic substrates (Bhatia and Ingber, 2014; Duval et al, 2017)
Summary
Fibroblasts are key mesenchymal cells in connective tissue which synthesize extracellular matrix (ECM) components and provide structural support for the extracellular environment (Kendall and Feghali-Bostwick, 2014). As part of the tissue self-repair mechanism, fibroblasts interact with surrounding ECM proteins through a variety of inflammatory mediators and differentiate into a more contractile phenotype known as myofibroblasts (Grinnell, 1994; Jeffery, 2001; Royce et al, 2012). The fibroblast-induced collagen gel contraction (CGC) assay was established by Bell et al to study fibroblast-matrix interactions (Bell et al, 1979) and has been modified and widely used over the past four decades. The traditional CGC assay is performed by embedding fibroblasts into a three-dimensional (3D) gel matrix, such as collagen or fibrin, on the bottom of a well plate, which is manually separated from the well plate surface (for example by scraping a pipette tip around the perimeter of the well) to loosen the gel puck from the well plate walls and enable contraction (Dallon and Ehrlich, 2008; Mikami et al, 2016). As a result, measuring the decrease in size of a gel matrix puck by imaging and subsequent analysis provides a direct way to assess fibroblast contractility
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