Abstract
Type I collagen is the most abundant extracellular matrix protein in the human body and is commonly used as a biochemical ligand for hydrogel substrates to support cell adhesion in mechanotransduction studies. Previous protocols for conjugating collagen I have used different solvents; yet, how varying solvent pH and composition impacts the efficiency and distribution of these collagen I coatings remains unknown. Here, we examine the effect of varying solvent pH and type on the efficiency and distribution of collagen I coatings on polyacrylamide hydrogels. We further evaluate the effects of varying solvent on mechanotransduction of human mesenchymal stem cells (MSCs) by characterizing cell spreading and localization of Yes-Associated Protein (YAP), a key transcriptional regulator of mechanotransduction. Increasing solvent pH to 5.2 and above increased the heterogeneity of coating with collagen bundle formation. Collagen I coating highly depends on the solvent type, with acetic acid leading to the highest conjugation efficiency and most homogeneous coating. Compared to HEPES or phosphate-buffered saline buffer, acetic acid-dissolved collagen I coatings substantially enhance MSC adhesion and spreading on both glass and polyacrylamide hydrogel substrates. When acetic acid was used for collagen coatings, even the low collagen concentration (1 μg/ml) induced robust MSC spreading and nuclear YAP localization on both soft (3 kPa) and stiff (38 kPa) substrates. Depending on the solvent type, stiffness-dependent nuclear YAP translocation occurs at a different collagen concentration. Together, the results from this study validate the solvent type as an important parameter to consider when using collagen I as the biochemical ligand to support cell adhesion.
Highlights
IntroductionCells are surrounded by extracellular matrix (ECM) proteins composed of hundreds of proteins and glycoproteins that impart a myriad of physical and biological cues to the cells. Studies of cells ex vivo often require functionalization of surfaces with adhesive ECM proteins in order to permit cell attachment and growth. To enable mechanotransduction studies, polyacrylamide hydrogels with tunable stiffness have been widely used and require coating with ECM proteins to support cell adhesion.4Type I collagen is an important structural component of ECM1 and is formed via self-assembly from tropocollagen units into small fibrils and larger fibers. Given that type I collagen is the most abundant protein in the body and its relatively low cost compared to other ECM proteins, it is one of the most commonly used biochemical ligands for functionalizing hydrogel substrates to support cell adhesion. Previous mechanotransduction studies using collagen I coating have been plagued by varying the efficiency and heterogeneity, with coatings containing a mixture of long thin fibers or thick bundles of collagen aggregates. when interpreting the cell response, the distribution of collagen I coating was often not taken into consideration
We further evaluate the effects of varying solvent on mechanotransduction of human mesenchymal stem cells (MSCs) by characterizing cell spreading and localization of Yes-Associated Protein (YAP), a key transcriptional regulator of mechanotransduction
To corroborate the results from fluorescence image analyses, we used atomic force microscopy (AFM) to characterize the surface topographical cues of glass coated with collagen I dissolved in solvents with varying pH 7.4 or 3.4 [Fig. 1(c)]
Summary
Cells are surrounded by extracellular matrix (ECM) proteins composed of hundreds of proteins and glycoproteins that impart a myriad of physical and biological cues to the cells. Studies of cells ex vivo often require functionalization of surfaces with adhesive ECM proteins in order to permit cell attachment and growth. To enable mechanotransduction studies, polyacrylamide hydrogels with tunable stiffness have been widely used and require coating with ECM proteins to support cell adhesion.4Type I collagen is an important structural component of ECM1 and is formed via self-assembly from tropocollagen units into small fibrils and larger fibers. Given that type I collagen is the most abundant protein in the body and its relatively low cost compared to other ECM proteins, it is one of the most commonly used biochemical ligands for functionalizing hydrogel substrates to support cell adhesion. Previous mechanotransduction studies using collagen I coating have been plagued by varying the efficiency and heterogeneity, with coatings containing a mixture of long thin fibers or thick bundles of collagen aggregates. when interpreting the cell response, the distribution of collagen I coating was often not taken into consideration. Cells are surrounded by extracellular matrix (ECM) proteins composed of hundreds of proteins and glycoproteins that impart a myriad of physical and biological cues to the cells.. Studies of cells ex vivo often require functionalization of surfaces with adhesive ECM proteins in order to permit cell attachment and growth.. Polyacrylamide hydrogels with tunable stiffness have been widely used and require coating with ECM proteins to support cell adhesion.. Type I collagen is an important structural component of ECM1 and is formed via self-assembly from tropocollagen units into small fibrils and larger fibers.. Given that type I collagen is the most abundant protein in the body and its relatively low cost compared to other ECM proteins, it is one of the most commonly used biochemical ligands for functionalizing hydrogel substrates to support cell adhesion..
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