ARTICLES NATURE MATERIALS DOl: 10.1038/NMAT3404 Figure 4 Hybrid macroporous nanoelectronic scaffolds. a, Confocal fluorescence micrograph of a hybrid reticular nanoES/collagen matrix. Green (fluorescein isothiocyanate): collagen type-I; orange(rhodamine 6G): epoxy ribbons. The white arrow marks the position of the nanowire. Scale bar, 10 um b, SEM images of a mesh nanoES/alginate scaffold, top (I) and side(ii)views. The epoxy ribbons from nanoES are false-coloured in brown for clarity. Scale bars, 200um(I)and 100 um(ID). c, A bright-field optical micrograph of the folded scaffold, showing multilayered structures of PLGa and nanoelectronic interconnects The inset shows a photograph of the hybrid sheet before folding. A sheet of PLGA fibres with diameters of 1-3 um was deposited on both sides of the device. No damage or reduction of device yield was observed following this deposition Scale bars, 200 um and 5mm(inset). d, Relative langes in nanowire Fet sensitivity over time in culture (37 C: 5%CO2, supplemented neurobasal medium). n=5: data are means +s d The hybrid nanoES were evaluated in 3D culture, 33 for several Extended studies will be needed to evaluate the nanoES cell types. Embryonic rat hippocampal neurons were cultured in the term implants, although the main component of nano for longer reticular nanoES/Matrigel for 7-21 days(Supplementary Fig S5). has demonstrated long-term chronic biocompatibility suitable for Reconstructed 3D confocal micrographs from a two-week culture in vivo recording" (Fig 5a, b and Supplementary Fig. S6)showed neurons with a high The monitoring capabilities of the nanoES were first demon- density of spatially interconnected neurites that penetrated the strated in a 3D cardiomyocyte mesh construct(Fig. 5g). The output reticular nanoES (Fig 5a), often passing through the ring structures recorded from a single-nanowire FET (Fig. 5g)200 um below the upporting individual nanowire FETs(Fig 5b and Supplementary construct surface showed regularly spaced spikes with a frequency Fig. S6). Notably, the widths of the scaffold elements(passivated of - 1 Hz, a calibrated potential change of -2-3 mv, a signal/noise metal interconnects and structural ribbons) were similar to those >3 and a 2 ms width. The peak amplitude, shape and widt of the neurite projections, demonstrating the combination of are consistent with extracellular recordings from cardiomyocytes. electronics with biological systems at an unprecedented similarity The potential of the nanoES-based 3D cardiac culture to monitor in scale. 3D nanoelectronic cardiac culture was achieved from appropriate pharmacological response was investigated by dosing hybrid mesh nano ES/PLGA scaffolds(Supplementary Figs S7-S9). the 3D cardiomyocyte mesh construct with noradrenaline(al Confocal fluorescence microscopy of a cardiac 3D culture(Fig. 5c) known as norepinephrine), a drug that stimulates cardiac con revealed a high density of cardiomyocytes in close contact with traction through Br-adrenergic receptors. Measurements from nanoES components. Epifluorescence micrographs of cardiac cells the same nanowire Fet device showed a twofold increase in th on the surface of the nanoES cardiac patch showed striations contraction frequency following drug application. Interestingly, characteristic of cardiac tissue,32( Fig. 5d and Supplementary recordings from two FETs from the cardiac patch on Figs S8 and S9). In addition, the in vitro cytotoxicity of nanoES in noradrenaline application showed submillisecond and millise 3D neural and cardiac culture was evaluated( Fig. 5e, f). Differences ond level, heterogeneous cellular responses to the drug (Sup Matrigel over 21 days, assessed with a standard LIvE/DEAd made with a reticular nanoES/neural construct ( Supplementary assay 5e), and between cardiac cells in hybrid mesh Fig. Sl1) showed that the 3D response of glutamate activation nanoES/Matrigel/PLGA and Matrigel/PLGA from 2 to 12 days, could be monitored. Together these experiments suggest nanoES measured with a metabolic activity assay(Fig. 5f), were minimal. constructs can monitor in vitro the response to drugs from 3D These studies show that on the 2-3 week timescale, the nanoes tissue models, and thus have potential as a platform for in vitro component of the scaffolds has little effect on the cell viability, and pharmacological studies Last, simultaneous recordings from thus can be exploited for a number of in vitro studies, including four nanowire FETs with separations up to 6.8 mm in a na drug screening assays with these synthetic neural and cardiac tissues. noES/cardiac construct( Fig. 5h) demonstrated multiplexed sensing NATURE MATERIALS I VOL 11 I NOVEMBER 2012 I G 2012 Macmillan Publishers Limited All rights reservedARTICLES NATURE MATERIALS DOI:10.1038/NMAT3404 a c b 1 23456789 ¬20 20 ¬10 10 0 d ΔS/S (%) Time (week) I II Figure 4 | Hybrid macroporous nanoelectronic scaffolds. a, Confocal fluorescence micrograph of a hybrid reticular nanoES/collagen matrix. Green (fluorescein isothiocyanate): collagen type-I; orange (rhodamine 6G): epoxy ribbons. The white arrow marks the position of the nanowire. Scale bar, 10 µm. b, SEM images of a mesh nanoES/alginate scaffold, top (I) and side (II) views. The epoxy ribbons from nanoES are false-coloured in brown for clarity. Scale bars, 200 µm (I) and 100 µm (II). c, A bright-field optical micrograph of the folded scaffold, showing multilayered structures of PLGA and nanoelectronic interconnects. The inset shows a photograph of the hybrid sheet before folding. A sheet of PLGA fibres with diameters of ∼1–3 µm was deposited on both sides of the device. No damage or reduction of device yield was observed following this deposition. Scale bars, 200 µm and 5 mm (inset). d, Relative changes in nanowire FET sensitivity over time in culture (37 ◦C; 5% CO2, supplemented neurobasal medium). n = 5; data are means ±s.d. The hybrid nanoES were evaluated in 3D culture32,33 for several cell types. Embryonic rat hippocampal neurons were cultured in the reticular nanoES/Matrigel for 7–21 days (Supplementary Fig. S5). Reconstructed 3D confocal micrographs from a two-week culture (Fig. 5a,b and Supplementary Fig. S6) showed neurons with a high density of spatially interconnected neurites that penetrated the reticular nanoES (Fig. 5a), often passing through the ring structures supporting individual nanowire FETs (Fig. 5b and Supplementary Fig. S6). Notably, the widths of the scaffold elements (passivated metal interconnects and structural ribbons) were similar to those of the neurite projections, demonstrating the combination of electronics with biological systems at an unprecedented similarity in scale. 3D nanoelectronic cardiac culture was achieved from hybrid mesh nanoES/PLGA scaffolds (Supplementary Figs S7–S9). Confocal fluorescence microscopy of a cardiac 3D culture (Fig. 5c) revealed a high density of cardiomyocytes in close contact with nanoES components. Epifluorescence micrographs of cardiac cells on the surface of the nanoES cardiac patch showed striations characteristic of cardiac tissue28,32 (Fig. 5d and Supplementary Figs S8 and S9). In addition, the in vitro cytotoxicity of nanoES in 3D neural and cardiac culture was evaluated (Fig. 5e,f). Differences between hippocampal neurons in reticular nanoES/Matrigel versus Matrigel over 21 days, assessed with a standard LIVE/DEAD cell assay33 (Fig. 5e), and between cardiac cells in hybrid mesh nanoES/Matrigel/PLGA and Matrigel/PLGA from 2 to 12 days, measured with a metabolic activity assay (Fig. 5f), were minimal. These studies show that on the 2–3 week timescale, the nanoES component of the scaffolds has little effect on the cell viability, and thus can be exploited for a number of in vitro studies, including drug screening assays with these synthetic neural and cardiac tissues. Extended studies will be needed to evaluate the nanoES for longer￾term implants, although the main component of nanoES, SU-8, has demonstrated long-term chronic biocompatibility suitable for in vivo recording34,35 . The monitoring capabilities of the nanoES were first demon￾strated in a 3D cardiomyocyte mesh construct (Fig. 5g). The output recorded from a single-nanowire FET (Fig. 5g) ∼200 µm below the construct surface showed regularly spaced spikes with a frequency of ∼1 Hz, a calibrated potential change of ∼2–3 mV, a signal/noise ≥3 and a ∼2 ms width. The peak amplitude, shape and width are consistent with extracellular recordings from cardiomyocytes20 . The potential of the nanoES-based 3D cardiac culture to monitor appropriate pharmacological response was investigated by dosing the 3D cardiomyocyte mesh construct with noradrenaline (also known as norepinephrine), a drug that stimulates cardiac con￾traction through β1-adrenergic receptors36. Measurements from the same nanowire FET device showed a twofold increase in the contraction frequency following drug application. Interestingly, recordings from two nanowire FETs from the cardiac patch on noradrenaline application showed submillisecond and millisec￾ond level, heterogeneous cellular responses to the drug (Sup￾plementary Fig. S10). Additionally, multiplexing measurements made with a reticular nanoES/neural construct (Supplementary Fig. S11) showed that the 3D response of glutamate activation could be monitored. Together these experiments suggest nanoES constructs can monitor in vitro the response to drugs from 3D tissue models, and thus have potential as a platform for in vitro pharmacological studies9,10. Last, simultaneous recordings from four nanowire FETs with separations up to 6.8 mm in a na￾noES/cardiac construct (Fig. 5h) demonstrated multiplexed sensing 990 NATURE MATERIALS | VOL 11 | NOVEMBER 2012 | www.nature.com/naturematerials © 2012 Macmillan Publishers Limited. All rights reserved