NATURE MATERIALS DOL: 10.1038/NMAT3404 ARTICLES cardiomyocyte ■ Control noradrenaline Figure 5 3D cell culture and electrical sensing in nanoES. a, b, 3D reconstructed confocal images of rat hippocampal neurons after a two -week culture in Matrigel on reticular nanoES. Red (Alexa Fluor 546): neuronal B-tubulin; yellow (rhodamine 6G): epoxy ribbons. The metal interconnects are false-coloured in blue, and are imaged in the reflected light mode. The white arrow highlights a neurite passing through a ring-like structure supporting a nanowire FET Dimensions in a, x: 317 um; y: 317 um; z: 100 um; in b, x: 127 um; y: 127 um:: z: 68 um. c, Confocal fluorescence micrographs of a synthetic cardiac patch (ll and lD), Zoomed-in view of the upper and lower dashed regions in l, showing metal interconnects, the SU-8 scaffold (arrows in ID)and electrospun PLGA fibres (arrows in IlD) Scale bar, 40 um. d, Epifluorescence micrograph of the surface of the cardiac patch. Green(Alexa Fluor 488) actin; blue(Hoechst 34580): cell nuclei. The position of the source-drain electrodes is outlined with dashed lines. Scale bar, 40 um. e, viable hippocampal neurons cultured in nanoES/Matrigel versus Matrigel. Cell viability was evaluated with a LIVE/DEAD cytotoxicity assay Cells were counted from 3D reconstructed confocal fluorescence micrographs n=6: data are means +s d Differences between groups were very small although statistically significant (p<0.05). f, Metabolic activity of cardiomyocytes evaluated using the MTs assay. n=6: data are means ts.d. Differences between and after(blue)applying noradrenaline. h, Multiplex electrical recording of extracellular field potentials from four nanowire FETs in a mesh nanoES. Dat groups were very small although statistically significant (p< 0.05). g, Conductance versus time traces recorded from a single-nanowire Fet before(black) are conductance versus time traces of a single spike recorded at each nanowire Fet. ith submillisecond time Increases in nanowire FET density, the use of cross-bar circuits and resolution. Our sign yields relatively sparse device implementing multiplexing/demultiplexing for addressing could distribution wi an area of about 3.5 x 1.5 cm. allow the nanoES scaffolds to map cardiac and other synthetic NatureMateriAlsIVol11iNovemBer2012Iwww.nature.com/naturematerial 991 G 2012 Macmillan Publishers Limited. All rights reservedNATURE MATERIALS DOI:10.1038/NMAT3404 ARTICLES x y z Time (ms) 0 10 20 30 40 50 nS 20 nS Time 2 s Conductance Conductance + noradrenaline 100 50 0 7 14 21 2 4 6 8 10 12 1.5 1.0 0.5 0.0 x y z I II III Metal SU-8 PLGA a c d b e g h f Viable cell percentage (%) Neuron Time (day) Metabolic activity (a.u.) Time (day) Cardiomyocyte Control Sample Control Sample Figure 5 | 3D cell culture and electrical sensing in nanoES. a,b, 3D reconstructed confocal images of rat hippocampal neurons after a two-week culture in Matrigel on reticular nanoES. Red (Alexa Fluor 546): neuronal β-tubulin; yellow (rhodamine 6G): epoxy ribbons. The metal interconnects are false-coloured in blue, and are imaged in the reflected light mode. The white arrow highlights a neurite passing through a ring-like structure supporting a nanowire FET. Dimensions in a, x: 317 µm; y: 317 µm; z: 100 µm; in b, x: 127 µm; y: 127 µm; z: 68 µm. c, Confocal fluorescence micrographs of a synthetic cardiac patch. (II and III), Zoomed-in view of the upper and lower dashed regions in I, showing metal interconnects, the SU-8 scaffold (arrows in II) and electrospun PLGA fibres (arrows in III). Scale bar, 40 µm. d, Epifluorescence micrograph of the surface of the cardiac patch. Green (Alexa Fluor 488): α-actin; blue (Hoechst 34580): cell nuclei. The position of the source–drain electrodes is outlined with dashed lines. Scale bar, 40 µm. e, Percentage of viable hippocampal neurons cultured in nanoES/Matrigel versus Matrigel. Cell viability was evaluated with a LIVE/DEAD cytotoxicity assay. Cells were counted from 3D reconstructed confocal fluorescence micrographs. n = 6; data are means ±s.d. Differences between groups were very small although statistically significant (p < 0.05). f, Metabolic activity of cardiomyocytes evaluated using the MTS assay. n = 6; data are means ±s.d. Differences between groups were very small although statistically significant (p < 0.05). g, Conductance versus time traces recorded from a single-nanowire FET before (black) and after (blue) applying noradrenaline. h, Multiplex electrical recording of extracellular field potentials from four nanowire FETs in a mesh nanoES. Data are conductance versus time traces of a single spike recorded at each nanowire FET. of a coherently beating cardiac patch, with submillisecond time resolution. Our current device design yields relatively sparse device distribution with 60 devices over an area of about 3.5 × 1.5 cm2 . Increases in nanowire FET density, the use of cross-bar circuits and implementing multiplexing/demultiplexing for addressing30 could allow the nanoES scaffolds to map cardiac and other synthetic NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials 991 © 2012 Macmillan Publishers Limited. All rights reserved