Effects of extracellular matrix analogues on primary human fibroblast behavior

Abstract

In vitro cell culture is a vital research tool for cell biology, pharmacology, toxicology, protein production, systems biology and drug discovery. Traditional culturing methods on plastic surfaces do not accurately represent the in vivo environment, and a paradigm shift from two-dimensional to three-dimensional (3-D) experimental techniques is underway. To enable this change, a variety of natural, synthetic and semi-synthetic extracellular matrix (ECM) equivalents have been developed to provide an appropriate cellular microenvironment. We describe herein an investigation of the properties of four commercially available ECM equivalents on the growth and proliferation of primary human tracheal scar fibroblast behavior, both in 3-D and pseudo-3-D conditions. We also compare subcutaneous tissue growth of 3-D encapsulated fibroblasts in vivo in two of these materials, Matrigel and Extracel. The latter shows increased cell proliferation and remodeling of the ECM equivalent. The results provide researchers with a rational basis for selection of a given ECM equivalent based on its biological performance in vitro and in vivo, as well as the practicality of the experimental protocols. Biomaterials that use a customizable glycosaminoglycan-based hydrogel appear to offer the most convenient and flexible system for conducting in vitro research that accurately translates to in vivo physiology needed for tissue engineering.

Fig. 1

Morphology of T31 human tracheal scar fibroblasts on thin layers of various sECMs after 5 days in culture. (A) Tissue culture plate (TCP); (B) Matrigel^™; (C) PuraMatrix^™; (D) PureCol^™; (E) Extracel^™.

Fig. 2

Pseudo-3-D proliferation of T31 human tracheal scar fibroblasts on wells coated with a thin layer of biomaterials. Values represented are mean ± SD, n = 6. Cell proliferation rates on TCP (control), PuraMatrix^™ (p > 0.05), PureCol^™ (p < 0.001), Matrigel^™ (p < 0.001) and Extracel^™ (p < 0.001). All reported statistical data are relative to TCP on day 7 data points.

Fig. 3

Schematic representation of 3-D cell encapsulation into hydrogels (original drawing by Xiaoyu Chen).

Fig. 4

Proliferation rates of fibroblasts encapsulated within various ECM analogues. Columns represent mean ± SD, n = 4. p > 0.05 relative to Matrigel^™.

Fig. 5

Live/dead staining of 3-D encapsulated cells. (A) Matrigel^™; (B) PuraMatrix^™; (C) PureCol^™; (D) Extracel^™. Green = live cells, red = dead cells.

Fig. 6

H&E staining of hydrogel-embedded cells. Nuclei = purple to dark purple; cytoplasm = pink to light purple. Cells or cell clusters are indicated with arrows. (A) Matrigel^™; (B) PuraMatrix^™; (C) PureCol^™; (D) Extracel^™.

Fig. 7

H&E staining of newly formed fibrous tissues after the injection of T31 fibroblasts in either Matrigel^™ (A) or in Extracel^™ (B). The arrowheads indicate selected regions of undegraded hydrogel.