Characterizing ECM production by cells encapsulated in hydrogels.
Hydrogels composed of hydrophilic polymers such as polyethylene glycol and alginate have been used as scaffolds for various tissue engineering applications. This chapter describes procedures for encapsulation of cells in hydrogels and subsequently characterizing the extracellular matrix (ECM) production by those cells using biochemical assays, gene expression analysis, and histology. In particular, the biochemical assays described here are used to quantify collagen, glycosaminoglycan (GAG), and DNA content in each scaffold. The methods for analyzing the level of gene expression of specific ECM molecules such as collagen I, collagen II, and aggrecan are also described. Finally, included are protocols for histological methods used to analyze overall matrix production and GAG synthesis via hematoxylin and eosin staining and Safranin-O, respectively. These methods can be modified so that other scaffolds apart from hydrogels can be used.
Strehin
Iossif A
IA
Department of Biomedical Engineering, John Hopkins University, Baltimore, MD, USA.
Elisseeff
Jennifer H
JH
D001483:Base Sequence; D017931:DNA Primers; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis*; D020100:Hydrogels
D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D020100:Hydrogels