Figure 5: Evaluation of cell viability and proliferation in the presence of stable radicals.

(a) Bright-field (control) and fluorescence images of 3T3 cells encapsulated in GelMA hydrogels were shown at t=0, 3, 6 days (top to bottom) for control group (no radicals) and for radical group soaked into 30 mg ml⁻¹ for 30 min. Green represented live cells and red represented dead cells. (b) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay results as a function of volume-to-volume ratios (v/v) of added radical suspension volume to cell suspension volume for days 0, 1, 3, 5 and 7. The histograms showed proliferation of cells in the presence of stable radicals. Positive control represents cells without radicals. Negative control is only the MTT reagents. All results were normalized with respect to negative control. Error bars represent s.e.m. (c) After paramagnetizing hydrogels via radicals (30 mg ml⁻¹ for 30 min), exposing them to vitamin E solution resulted in increase of cell proliferation for >50% (v/v) vitamin E solution. Results were given for a range of vitamin E solution volume to cell suspension volume. Error bars represent s.e.m. (d) After paramagnetizing hydrogels via radicals for a range of radical concentration (10, 30, 150 mg ml⁻¹), exposing them to vitamin E solution (100% (v/v)). Labels with ‘r’ in the figure caption, such as ‘10 r’, corresponds to hydrogels radicalized with 10 mg ml⁻¹, and labels with ‘rv’ corresponds to gels exposed to vitamin E following radicalization. Results showed that for large concentration of radical, for example, 150 mg ml⁻¹ (and for 30 min incubation), vitamin E does not significantly help to recover cells. Error bars represent s.e.m. (e) Fluorescent images of square, rod-shaped and L-shaped assemblies of 3T3 encapsulating GelMA hydrogels. Scale bars are 200 μm. (f,g) Cardiomyocyte encapsulating GelMA hydrogels: (f) control (no radicals), (g) × 40 magnification of radical and vitamin E exposed gels. Scale bars are 200 μm (f) and 100 μm (g). Cells were stained with mouse anti-α-actinin (sarcomeric) and anti-GATA-4. Actin cytoskeleton was visualized with phalloidin and DAPI was used as nuclear counter staining. All images were shown on day 10.