Journal: Scientific Reports

Article Title: Templated Assembly of Collagen Fibers Directs Cell Growth in 2D and 3D

doi: 10.1038/s41598-017-10182-8

Figure Lengend Snippet: Incorporation of collagen networks in 3D hydrogel scaffolds supports cell alignment and differentiation. ( a ) Dry fabrics can be readily incorporated into commonly-used non-cell-adherent hydrogels such as agarose. ( b ) Low magnification epifluorescence images of actin-phalloidin and Hoechst nuclear staining demonstrate that collagen-doped fabrics promote large-scale alignment of agarose-encapsulated C2C12 cells. Cell alignment and growth is not observed for control fabrics that do not contain collagen. Arrows indicate direction of fabric alignment. ( c ) High-resolution confocal maximum intensity projections show details of aligned fascicle-like structures. Positioning of cell nuclei and interconnected cytoskeleton structures are indicative of myotube formation. Myotubes and fascicle-like structures are not observed for cells cultured with control fabrics. C2C12 cells growing along collagen networks within the hydrogels display immunofluorescence for skeletal muscle cell differentiation markers including myosin heavy chain, parvalbumin and NOS-I. All scale bars, 50 µm. ( d ) Representative Western blots for myosin heavy chain, parvalbumin and GAPDH for cells grown in 2D on tissue culture plastic, within agarose containing 3D control fabrics (3D (−)) and within agarose containing 3D collagen fabrics (3D (+)). Images are cropped from the original full blots with adjustment of brightness and contrast to ensure that faint bands are visible. Unprocessed Western blot images are available in Supplementary Figure 7 . Levels of myosin heavy chain ( e ) and parvalbumin ( f ) relative to GAPDH (AU; Arbitrary Units) were compared across culture conditions. Significant differences of p

Article Snippet: The following primary antibodies were used to assess C2C12 differentiation in 3D culture conditions by immunofluorescence: mouse-anti-myosin-hc (1:100; Novus Biologicals), mouse-anti-parvalbumin (1:200; Chemicon), rabbit-anti-parvalbumin (1:100; Novus Biologicals), rabbit-anti-NOS-I (1:200; Chemicon) and goat-anti-nNOS (NOS-I) (1:100; Novus Biologicals).

Techniques: Staining, Cell Culture, Immunofluorescence, Cell Differentiation, Western Blot