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Cyborg Organoids: Implantation of Nanoelectronics via Organogenesis for Tissue-Wide Electrophysiology Qiang Li, Kewang Nan, Paul Le Floch, Zuwan Lin, Hao Sheng, Thomas S. Blum, and Jia Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b02512 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 26, 2019

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Cyborg Organoids: Implantation of Nanoelectronics via Organogenesis for Tissue-wide Electrophysiology

Qiang Li1*, Kewang Nan1*, Paul Le Floch1*, Zuwan Lin2, Hao Sheng1, Thomas S. Blum1, Jia Liu1† 1School

of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.

2Department

of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138,

USA.

*These authors contributed equally. †Corresponding author. Email: [email protected]

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ABSTRACT: Tissue-wide electrophysiology with single-cell and millisecond spatiotemporal resolution is critical for heart and brain studies. Issues arise, however, from the invasive, localized implantation of electronics that destroys well-connected cellular networks within matured organs. Here, we report the creation of cyborg organoids: the three-dimensional (3D) assembly of soft, stretchable mesh nanoelectronics across the entire organoid by the cell-cell attraction forces from 2D-to-3D tissue reconfiguration during organogenesis. We demonstrate that stretchable mesh nanoelectronics can migrate with and grow into the initial 2D cell layers to form the 3D organoid structure with minimal impact on tissue growth and differentiation. The intimate contact between the dispersed nanoelectronics and cells enables us to chronically and systematically observe the evolution, propagation and synchronization of the bursting dynamics in human cardiac organoids through their entire organogenesis.

KEYWORDS: Bioelectronics, organogenesis, organoid, three-dimensional culture, stretchable nanoelectronics, electrophysiology


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Complex organs (e.g. heart and brain) rely on spatiotemporally orchestrated communication of heterogenous cells to generate whole-organ functions.1,2 Thus, understanding the development, mechanisms, and diseases of these organs requires system-level mapping of cellular activities with high spatiotemporal resolutions across their entire 3D volumes and over a substantial time window.3-5 Recently, tremendous progress has resulted in the miniaturization of state-of-the-art electrophysiological tools into micro- and nano-scales6, 7 with soft and multiplexed electronic units.8,

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These advances have significantly reduced tissue disruptions from the electronics

implantation while maintaining high spatiotemporal resolution for recording and manipulating.1013

Most of these devices, however, either contact organs only at the surface or penetrate locally

and invasively through micro-needle injections. It remains a key challenge to uniformly implant and distribute a large number of interconnected and individually addressable sensors/stimulators throughout 3D organs with minimal impact for chronic recording. It is noteworthy that in vivo 3D organs evolve from 2D embryonic germ layers during organogenesis. For instance, both the neural tube and heart tube develop from 2D cell layers in embryo via a series of cell proliferation and folding process, the neural tube ultimately forming the brain and spinal cord and heart tube forming the heart. 14, 15 This in vivo 2D-to-3D transition has recently been reproduced in vitro for human organoids culture,16, 17 through the use of human mesenchymal stem cells (hMSCs) to initiate cell condensation and drive human induced pluripotent stems cells (hiPSCs) to fold into 3D organoids for transplantations and drug screening.18, 19 Inspired by normal organogenesis, we propose a new scheme to realize 3D implantation and distribution of nanoelectronics in organoids (Figure. 1A). The first stage consists of transferring and laminating a mesh-like plane of nanoelectronics along with its input/output (I/O) lines onto a


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2D sheet of stem cells or progenitor cells (Stage I). Attraction forces between cells during cell aggregation, proliferation, and migration gradually shrink the cell-sheet into a cell-dense plate, which simultaneously compresses the nanoelectronics into a closely packed architecture and embeds them within the cells (Stage II). This interwoven cell-nanoelectronics structure then contracts and curls as a result of organogenesis-induced self-folding, first into a bowl geometry (Stage III), and then into a 3D spherical morphology (Stage IV). During this process, the mesh nanoelectronics seamlessly reconfigures with the cell-plate due to its soft mechanics while maintaining uniform spatial distributions throughout the tissue leading to a fully-grown 3D organoid with an embedded sensors/stimulators array in a minimally invasive and globally distributed manner (Stage IV) - hence the name “cyborg organoid”. Finally, the stem cells in the as-formed cyborg organoid can further differentiate into targeted types of functional cells such as cardiomyocytes, while their electrophysiological activities can be chronically monitored using the embedded nanoelectronics (Stage V). We underline several important design characteristics of the nanoelectronics which enable the asdescribed seamless, tissue-wide integrations (Figure. 1B). First, the mesh design exploits a serpentine layout with an overall filling ratio of less than 11%, leading to significantly improved in-plane stretchability of up to 30% 20 and out-of-plane compressibility several times smaller than its initial volume due to buckling of the mesh network.21 This design enables the accommodation of drastic volumetric changes (mostly compressive) during organogenesis.16-19 Second, both sets of device dimensions implemented (ribbon width/thickness = 20 m/0.8 m and 10 m/2.8 m) result in minimal device mass (less than 15 µg) and effective bending stiffness of 0.090 n·Nm and 1.9 n·Nm respectively (see Note S1 and Figure. S1 for details) that are essentially imperceptible to the surrounding tissues.7,22 Mechanical properties of the mesh electronics can be compared not


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only to the passive mechanical properties of the cellular media, but also to the amount of force generated during organogenesis. In terms of passive mechanical properties, the elastic modulus of cardiac cells is a few kiloPascals (we chose 6 kPa for calculations).23 For a spherical cardiomyocyte of typical 10 m diameter, the bending stiffness is about 50 nN.m which is more than one order of magnitude higher than our stiffest mesh device. In terms of forces generated during organogenesis, cell-cell, cell-ECM (extracellular matrix), and cell-substrate attraction forces have been quantified and can easily reach magnitudes of 100 nN.24-26 Since the surface area of a unit ribbon of the electronic mesh is about 4x104 μm2, while the typical surface area of a cardiomyocyte cell is 102 μm2, the total attraction force generated by hundreds of cells surrounding the unit ribbon could reach tens of micronewtons, which is strong enough to provide the in-plane force required to buckle a unit ribbon of the mesh electronics (approx. 1-5µN calculated in Supplementary Notes). Both approaches show that the mesh device is mechanically compliant to the surrounding cellular media. Third, sensors with low-impedance and an average diameter of 20 µm, approaching the typical size of an individual cell,22 enable non-invasive, localized, single-cell electrophysiological recordings. Detailed fabrication procedures can be found in the Supplementary Methods and in Figure. S2. Figure 1C shows the mesh nanoelectronics immediately before release from the fabrication substrate while the inset shows an individual platinum electrode electrochemically deposited with poly(3,4-ethylenedioxythiophene) (PEDOT) to further lower the interfacial impedance.28 Folding (up to 180) and stretching (up to 30% biaxially) the released device in a chamber filled with water (Figure. S3) for 100 cycles (Figure. 1D-F and Supplementary videos 1-2) reveals neither visualizable damages (Figure. 1D-E) nor significant changes to the impedance of the electrodes (Figure. 1F). Measurements (Figure. S4) also show >10 times reduction in impedance at 1 kHz


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and a shift of phase towards resistance-dominated behaviors after coating the electrodes with PEDOT.28 Additionally, we determine the device’s longevity using a soaking test in 1x phosphatebuffered saline (PBS) at 37 C, where impedance values of three random channels at 1 kHz remain steady over 30 days (Figure. 1H), showing the potential of such sensory platforms for chronic studies in physiological environments. The mesh nanoelectronics are highly scalable due to the photolithographic nature of the fabrication process. We present a scaled-up design in Figure S5, consisting of 64 independent sensors that span a large area of ~0.5×1 cm2. Notably, the effective bending stiffness and the overall lateral dimensions of the mesh remain unchanged due to the incorporation of multiple (2 in this case) interconnected traces within one mesh ribbon which are stacked on top of each other to maintain a small filling ratio, low device thickness (< 2 m), and suitable lateral dimensions for soft integration with cells using the current scheme. By including more traces (3, 4 or more)29,30 in a single mesh ribbon, as well as adding on-site, flexible multiplexing units27,31, it is possible to further scale up to larger sensor arrays (96, 128 or more) without sacrificing the geometries and mechanics that are essential for integration via organogenesis. Downscaling device dimensions is another approach to increase the density of sensing units integrated into the organoid, such as leveraging the fabrication process through electron beam lithography. To test our concept, we integrated two types of stretchable mesh nanoelectronics (ribbon width/thickness = 10 m/2.8 m and 20 m/ 0.8 m) with hMSCs co-cultured with hiPSC (Figures. S6-S13) or hiPSC-derived cardiac progenitor cells (hiPSC-CPCs) (Figure. 2 and Figures. S14-16). In this culture system, hMSCs stimulate the cellular condensation that triggers the 2Dto-3D reconfiguration, in which Di-phosphorylatio of MII regulatory light chain (ppMRLC) increase the MII ATPase activity to produce a contraction force, and then a actomyosin


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cytoskeletal contraction to direct the cell-cell attraction.18,19 The initial flat cell-sheet sandwiching the nanoelectronics against a layer of Matrigel (Figure. 2A, I) shrunk into a cell-plate via hMSCsdriven condensation, spontaneously packaging the nanoelectronics into a highly compressed structure (Figure. 2A, II). Further 3D re-organization transformed the cell-plate into a 3D organoid (Figure. 2A, III) in ~72 hours. Phase images clearly illustrate initial compression (Figure. 2C, I to II) and subsequent folding (Figure. 2C, III) of the nanoelectronics together with the cell plate at each step. With further growth and expansion, the cyborg organoid fully unfolded and distributed the nanoelectronics throughout its entire 3D structure. Phase and false color images after ~480 hours of organogenesis clearly show uniform distribution of the device over the bottom of the organoid following the seamless folding and assembly into a closed 3D spherical configuration as the organoid grows and expands (Figure. 2D). Importantly, the same tissue morphology from hMSCs/hiPSCs and hMSCs/hiPSC-CPCs cyborg organoids further demonstrates the generality of this method for different types of cells (Figure. 2A-C and Figure. S15; Figure. S11 and Figure. S12). Statistical analysis shows two types of mesh nanoelectronics with different bending stiffness have no statistically significant impacts on the morphology of the organoids during organogenesis (Figure S16). Imaging results show that during organogenesis, up to ~50% compressive and ~20% tensile effective strains, defined as the relative changes in distance between adjacent nodal points on the mesh device, are applied to the nanoelectronics. Simulations by finite element analysis (FEA, see Note S2 for more details) show that the large deformations are accommodated by out-of-plane buckling of the mesh ribbons, leading to a maximum nominal strain of less than 1.2% which is below the fracture limit of both SU-8 and gold (Figure. 2F-G and Figures. S17-S21), indicating


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the robustness of the device design. Notably, the buckling modes in compression and stretching observed in experiments can be qualitatively reproduced by the simulations. In addition, simulations show that the end-to-end force required to deform our device is comparable to those required to deform cells at the same scale,32 indeed indicating a minimally invasive implantation process (Figure. 2H and Figure. S22). Day 40 optical images of tissue development and differentiation show that the nanoelectronics have been completely implanted into organoids and cover the entire tissue area with unfolded mesh ribbons (Figure. 3A). The embedded nanoelectronics’ flexibility and stretchability allow them to beat with the contraction of the cardiac organoids (Supplementary videos 3-6). To further exame the embedding of the nanoelectronics within the 3D organoid, day 40 cyborg cardiac were cleared, stained, and imaged. 3D reconstructed fluorescence images show the

interwoven 3D

nanoelectronics/cellular structure across the entire organoids (Figure. 3B and Figure. S23). The mesh devices are embedded throughout the organoids rather than only at a shallow depth beneath their surface. Cross-sections of 3D reconstructed fluorescence images show the 3D interwoven nanoelectronics/cellular structures across the organoids. The nanoelectronics are sandwiched between the outer layer of the organoid consisting of migrated hMSCs and the matured cardiomyocytes, and statistical analysis show that of the averaged depth of device within organoids is about 250 m (Figure S24). Zoom-in views further reveal that the nanoelectronics are sandwiched between the outer layer of the organoid consisting of migrated hMSCs (Figure. 3C, I) and the core of the organoid consisting of matured cardiomyocytes. Importantly, as the planar nanoelectronics develop together with the 2D cellplate, images show an intimate coupling between differentiated cardiomyocytes and the


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nanoelectronics (Figure. 3C and Figure. S23B, D) throughout the entire organoid, which is rarely observed in other implantation methods involving post-matured living organoids and organs. To investigate the effects of our integrated nanoelectronics on the differentiation of organoids, hiPSC-CPCs/hMSCs cardiac cyborg organoids were fixed, sectioned, and immunostained at different stages (day 7, 20 and 40 of differentiation) for stage-specific marker expressions (Figure. 3D-F and Figures. S25-26) and compared with those from control cardiac organoids. Protein markers including TNT, -actinin, and actin were imaged to quantify the maturation of cardiomyocytes while the marker NKX2.5 was imaged for hiPSC-CPCs (Figure. 3G-J).33 Averaged fluorescence intensity over culture time shows significantly increased fluorescence intensity of TNT and -actinin (Figure. 3K, M), while no statistically significant difference is measured between cyborg and control organoids at any stages. In addition, the fluorescence intensity plot along the axis of individual cells within the organoids shows the same level of increased sarcomere alignments in cyborg and control organoids from day 7 to day 40 of differentiation (Figure. 3L, N). These results demonstrate that the integrated nanoelectronics do not interrupt the 3D differentiation of iPSC-derived cardiac organoids. Furthermore, further immunostaining characterizations on organoids at day 40 of differentiation show that two types of mesh devices have no significant differences in affecting the expression of stage-specific biomarker (TNT) (Figure S27). The strong coupling between stem/progenitor cells and our nanoelectronics offers a unique opportunity to map the evolution of cellular electrophysiology throughout organogenesis. As a proof-of-concept, electrophysiological recordings (Figure. S28) were performed on cardiac cyborg organoids, which typically form in ~48 hours (Figure. 4A-B). Figure 4C shows the voltage trace of a 14-channel (out of 16 channels) electrophysiological recording of a cardiac cyborg organoid


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at day 35 of differentiation. The zoom-in plot of a single spikes (Figure. 4D) shows non-uniform electrophysiological behaviors of the cells distributed across the organoid, as well as a clear time latency revealing tissue-wide propagation of local field potentials (LFP). Chronic tracing of LFP at millisecond temporal resolution during organogenesis (Figure. 4E) reveals changes in the spike dynamics from initially slow waveform through the emergence of repolarization to fast depolarization.36 The averaged amplitude of the fast component associated with depolarization remains undetectable until day 31 of differentiation and then increases monotonically (Figure. 4F). The field potential duration (FPD) is found to remain relatively steady between 0.7 and 0.8 s (Figure. 4G) with a slight increase from day 26 to 35. Activation mapping confirms the gradually synchronized LFP propagation across the cardiac organoid from day 26 to day 35 of differentiation (Figure. 4H and Figure. S29). These recording data suggest that the functional maturation of a cardiac organoid is marked by the synchronization of its bursting phase rather than its bursting frequency or FPD. In summary, we have created the first human cardiac cyborg organoids via organogenetic 2D-to3D tissue reconfiguration, and we have studied the evolution of electrophysiological patterns during organogenesis. While we acknowledge that there are large variations in the generation of organoids leading to low reproducibility and controllability, by combining the implantation through 2D-to-3D reconfiguration process in organogenesis, integration of defined progenitor cells and device mechanical design, this method for the generation of cyborg organoids is highly reproducible and controllable and maintains low variability as characterized by 1) the mesh device’s uniform distribution in the cyborg organoids after >1 month culture, 2) the uniformed diameters of cell sheet, cell plate and organoids during the integration of mesh nanoelectronics


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(Figure 2 and Figure S15) and 3) the low variations of stage-specific biomarker expression (Figure 3, Figure S27). In addition, this method is scalable for integrating a larger number of sensors and stimulators.37-39 Additional work remains to apply cardiac cyborg organoids to study cardiac development, diseases, and therapeutics.40 Further development and generalization of cyborg organoids could serve as a paradigm-shifting platform for spatially resolved, high-fidelity, and chronic electrophysiological recordings for many other types of organoids41, 42 and animal embryos as well as for monitoring and controlling of organoid-enabled cellular therapeutics.43


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ASSOCIATED CONTENT Supporting Information Materials and Methods, Supplementary Notes, Figure S1-S29, Movies S1-S6, and supplementary references. AUTHOR INFORMATION Corresponding Author Email: [email protected] Author Contributions *These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We are grateful for the support of all the members of the Liu group. We thank D. Mélançon for helpful discussions on FEA simulation, and T. Ye and X. Dai for helpful discussions on the electrophysiological recording. We also thank Prof. C. M. Lieber, Prof. Z. Suo, J. C. Salant and D. R. Lilienthal for their valuable contributions to the production of this manuscdript. This research was partially supported by the Harvard Dean’s Fund for Promising Scholarship and the Harvard University Center for Nanoscale Systems supported by

the

National

Science

Foundation.


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(30) Yang, X.; Zhou, T.; Zwang, T. J.; Hong, G.; Zhao, Y.; Viveros, R. D.; Fu, T. M.; Gao, T.; Lieber, C. M. Bioinspired Neuron-like Electronics. Nature Materials 2019, 18, 510-517. (31) Viventi, J.; Kim, D. H.; Vigeland, L.; Frechette, E. S.; Blanco, J. A.; Kim, Y. S.; Avrin, A. E.; Tiruvadi, V. R.; Hwang, S. W.; Vanleer, A. C.; et al. Flexible, Foldable, Actively Multiplexed, High-density Electrode Array for Mapping Brain Activity in vivo. Nature Neuroscience 2011, 14, 1599-1605. (32) Lulevich, V.; Zink, T.; Chen, H. Y.; Liu, F. T.; Liu, G. Y. Cell Mechanics Using Atomic Force Microscopy-Based Single-Cell Compression. Langmuir 2006, 22, 8151–8155. (33) Hoang, P.; Wang, J.; Conklin, B. R.; Healy, K. E.; Ma, Z. Generation of Spatial-Patterned Early Developing Cardiac Organoids Using Human Pluripotent Stem Cells. Nat. Protoc. 2018, 13, 723–737. (34) Yoon, S. J.; Elahi, L. S.; Pașca, A. M.; Marton, R. M.; Gordon, A.; Revah, O.; Miura, Y.; Walczak, E. M.; Holdgate, G. M.; Fan, H. C.; et al. Reliability of Human Cortical Organoid Generation. Nature methods 2019, 16, 75-78. (35) Velasco, S.; Kedaigle, A. J.; Simmons, S. K.; Nash, A.; Rocha, M.; Quadrato, G.; Paulsen, B.; Nguyen, L.; Adiconis, X.; Regev, A.; et al. Individual Brain Organoids Reproducibly form Cell Diversity of the Human Cerebral Cortex. Nature 2019, 570, 523-527. (36) Asakura, K.; Hayashi, S.; Ojima, A.; Taniguchi, T.; Miyamoto, N.; Nakamori, C.; Nagasawa, C.; Kitamura, T.; Osada, T.; Honda, Y.; et al. Improvement of Acquisition and Analysis

Methods

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Multi-Electrode

Array

Experiments

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IPS

Cell-Derived

Cardiomyocytes. J. Pharmacol. Toxicol. Methods 2015, 75, 17–26. (37) Fang, H.; Yu, K. J.; Gloschat, C.; Yang, Z.; Song, E.; Chiang, C. H.; Zhao, J.; Won, S. M.; Xu, S.; Trumpis, M.; et al. Capacitively Coupled Arrays of Multiplexed Flexible Silicon Transistors for Long-Term Cardiac Electrophysiology. Nat. Biomed. Eng. 2017, 1, 0038.


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(38) Jiang, Y.; Li, X.; Liu, B.; Yi, J.; Fang, Y.; Shi, F.; Gao, X.; Sudzilovsky, E.; Parameswaran, R.; Koehler, K.; et al. Rational Design of Silicon Structures for Optically Controlled Multiscale Biointerfaces. Nat. Biomed. Eng. 2018, 2, 508-521. (39) Jiang, Y. and Tian, B., 2018. Inorganic semiconductor biointerfaces. Nat. Rev. Mater. 2018, 3, 473-490. (40) Lancaster, M. A.; Knoblich, J. A. Organogenesis in a Dish: Modeling Development and Disease Using Organoid Technologies. Science 2014, 345, 1247125. (41) Pasca, A. M.; Sloan, S. A.; Clarke, L. E.; Tian, Y.; Makinson, C. D.; Huber, N.; Kim, C. H.; Park, J. Y.; O’Rourke, N. A.; Nguyen, K. D.; et al. Functional Cortical Neurons and Astrocytes from Human Pluripotent Stem Cells in 3D Culture. Nat. Methods 2015, 12, 671–678. (42) Quadrato, G.; Nguyen, T.; Macosko, E. Z.; Sherwood, J. L.; Yang, S. M.; Berger, D. R.; Maria, N.; Scholvin, J.; Goldman, M.; Kinney, J. P.; et al. Cell Diversity and Network Dynamics in Photosensitive Human Brain Organoids. Nature 2017, 545, 48–53. (43) Liu, Y. W.; Chen, B.; Yang, X.; Fugate, J. A.; Kalucki, F. A.; Futakuchi-Tsuchida, A.; Couture, L.; Vogel, K. W.; Astley, C. A.; Baldessari, A.; et al. Human ESC-Derived Cardiomyocytes Restore Function in Infarcted Hearts of Non-Human Primates. Nat. Biotechnol. 2018, 36, 597–605.


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Figure 1. Cyborg organoids: concept and design. (A) Schematics illustrate the stepwise integration of stretchable mesh electronics into organoids through organogenesis. (I) Lamination of stretchable mesh electronics onto a continuous sheet of Matrigel seeded with Human induced pluripotent stem cells (hiPSCs) or hiPSCs-derived progenitor cells. (II) Aggregation of cell-sheet into a cell-dense plate through cell proliferation and migration induced by cell-cell attractive forces, embedding the stretchable mesh electronics into the cell-plate and folding it into a tightly packed

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plate/nanoelectronics hybrid into 3D structure with bowl-like geometry. (IV) Organogenesis unfolds the closely packed nanoelectronics and distributes its structure across the entire 3D organoid. (V) Further development and differentiation of stem/progenitor cells into different cell types with their electrophysiological behaviors continuously monitored by 3D embedded nanoelectronics. (B) Exploded view of stretchable mesh nanoelectronics design consisting of (from top to bottom) a 400 nm-thick top SU-8 encapsulation layer, a 50 nm-thick platinum (Pt) electrode layer coated with poly(3,4-ethylenedioxythiophene) (PEDOT), a 40 nm-thick gold (Au) interconnects layer, and a 400 nm-thick bottom SU-8 encapsulation layer. The inset shows the serpentine layout of the mesh. (C) Optical image of mesh electronics before releasing from the fabrication substrate. The inset shows the zoom-in view of a single Pt electrode coated with PEDOT. (D-E) Optical images of released stretchable mesh electronics in water, folded (D) and stretched (E) by tweezer. (F) Impedance and phase from 0.1 to 10 kHz of representative channels with and without PEDOT. (G) Average impedance (n=5) at 1 kHz for each channel in its original state, after 100 cycles of folding (to the degree as shown in (D)) and 100 cycles of stretching (to ca. 30% biaxially as shown in (E)). (H) Impedance at 1 kHz as a function of incubation time in PBS at 37C for three representative channels.


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Figure 2. 3D implantation and distribution of stretchable mesh nanoelectronics via organogenesis. (A) Optical images show representative stages of the assembly of stretchable mesh nanoelectronics by organoids development corresponding to the schematic in Figure. 1A, including lamination of stretchable mesh nanoelectronics between the cell-sheet on the Matrigel (I), cell-dense cell-plate (II) and 3D re-organized cell-plate (III). hiPSCs-derived cardiac progenitor cells (hiPSCs-CPCs) and human mesenchymal stem cells (hMSCs) are cocultured. The dashed-lines highlight the boundaries of cell-plates at different stages. (B) Zoom-in phase images show the deformation of stretchable mesh nanoelectronics by the mechanical forces from


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organogenesis at different stages corresponding to the blue-dashed-box regions in (A). (C) Further zoom-in phase images with false color highlight the cell-nanoelectronics interaction at different stages of organogenesis. The frame corresponds to the red-dashed-box highlighted regions in (B). Red and blue false color label the SU-8 ribbons from nanoelectronics and stem cells, respectively. White arrows highlight the regions where the ribbons are embedded within the cells. (D) Brightfield phase image shows uniform distribution of the device over the bottom of the organoid following the seamless folding and assembly into a closed 3D spherical configuration as the organoid grows and expands. Inset shows zoom-in phase image with false color from the red dashed box highlight region with the same colors as (C). (E) Representative effective strains of the device calculated through phase image analysis as a function of culture time. Gray dots and lines show the individual data points, while black dots and lines show the averaged results. (Value = mean ± s.e.m., n=10). (F) Two compression modes of a repetitive unit in the mesh electronics obtained from finite element analysis (FEA). Merged simulation and optical images show representative modes in real samples. The color scale indicates nominal strain in the unit. (G) A stretch mode of a repetitive unit of the mesh electronics obtained from FEA. Merged simulation and optical images show the representative modes in real samples. The color scale indicates nominal strain in the unit. (H) Magnitude of force as a function of strain for the three deformation modes in (F) and (G) obtained from FEA.


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Figure 3. Development of human cardiac cyborg organoids. (A) Top (I) and bottom (II) views of the cardiac cyborg organoid at day 40 of differentiation. Red arrows highlight folding and stretching of the mesh device by 3D re-organization of the cell-plate. (B) Top (I) and bottom (II) views of 3D reconstructed fluorescence images of a cleared, immunostained cardiac cyborg organoid at day 40 of differentiation. Green, magenta and blue colors correspond to TNT, CD44 and DAPI, respectively. SU-8 in device was labeled by R6G for imaging in this and the following panels. (C) Zoom-in views of the black-dashed box regions in (B) show migrated hMSCs form the outer layer of the organoid (I) as well as intimate coupling between iPSCs-derived cardiomyocytes and nanoelectronics (white arrows) (I-IV). (D) Schematic of the slicing of the cardiac cyborg organoid for characterization at different stages of organogenesis. (E-F) Projection of 3D reconstructed confocal microscopic fluorescence images of the 30-m-thick cyborg organoid (E) and control organoid (F). Green, red and blue colors correspond to TNT, the device and DAPI, respectively and are denoted at the top of the image panel in this and subsequent images. (G-N), Immunostaining and fluorescence imaging characterize the maturation of cardiomyocyte in the organoid at day 7 (I), 20 (II) and 40 (III-IV) of differentiation. TNT and NKX2.5 (G-H) and actinin and actin (I-J) were used to characterize the level of maturation of organoids with and without embedded nanoelectronics. Zoom-in views (IV) further show subcellular localization of TNT and -actinin at day 40 of differentiation. Averaged fluorescence intensity of TNT (K) and -actinin (M) over culture time. (Value = mean ± s.e.m., n= 6. **, P < 0.01; ***, P < 0.001; ***, P < 0.0001, two-tailed, unpaired t-test.) Fluorescence intensity plot showed the sarcomere alignment (L) and -actinin alignment (N) in control and cyborg organoids at day 7, 20, and 40 of differentiation.


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Figure 4. Chronic, multiplex, tissue-wide electrophysiological mapping of cardiac cyborg organoid during organogenesis. (A) A schematic shows the set-up that connects the cyborg cardiac organoids to external recording equipment for multiplexing electrophysiology during


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organoid development. (B) Optical images show representative processes (0, 24 and 48 hours) of the 3D organization of cardiac cyborg organoids in the culture chamber connected for electrophysiological recording. (C) 14-channel voltage traces recorded from the cardiac cyborg organoid at day 35 of differentiation. (D) Zoom-in views of the blue-box-highlight in (C) shows a single-spiked field potential recording. (E) Zoom-in views of red-dashed-box from (C) on three different culturing days (day 26, 31, and 35 of differentiation). (F-G) Amplitude of fast peak (F) and field potential duration (G) defined in (E) as a function of differentiation time. Gray lines show individual channels. Black line shows the averaged results from 14 channels. (Value = mean ± s.e.m., n= 14). (H) Isochronal mappings at day 26 and day 35 of differentiation show delays of activation time (max. -dV/dt) for three consecutive spike events labeled in Figure. S29.


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