Functional retinal organoids recorded by a flexible multielectrode array sandwich platform

Functional retinal organoids recorded by a flexible multielectrode array sandwich platform | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Functional retinal organoids recorded by a flexible multielectrode array sandwich platform Yagmur Demircan-Yalcin, Hosseinzadeh Zohreh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8768713/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Stem cell-derived retinal organoids provide a powerful in vitro platform for studying retinal development, degeneration, and therapeutic strategies. However, methods for assessing their electrophysiological function remain limited by the organoids’ 3D structure and fragility. Here, we demonstrate that a flexible multielectrode array (flexMEA) enables stable and flexible dimension recording of activity from retinal organoids. Using a sandwich approach that combines a polyimide-based flexMEA with a standard 60-electrode MEA, we achieved high-quality extracellular recordings and mapped neuronal activity across the organoid surface. In this configuration, the standard MEA was used for electrical stimulation, while neural signals were recorded with the flexMEA. This work is proof-of-the-concept of a flexMEA integrated technology as a non-invasive and conformal approach for functional phenotyping of retinal organoids for the future functional interventions, such as drug testing. Figures Figure 1 Figure 2 Introduction Retinal organoids, derived from human induced pluripotent stem cells (hiPSCs), recapitulate key structural and molecular features of the developing human retina. They contain laminated neural layers, photoreceptors capable of outer segment formation, and synaptic connections crucial for retinal circuitry. While molecular and morphological assessment of organoids has advanced rapidly, functional evaluation remains challenging [ 1 – 3 ]. Traditional 2-dimensional (2D) multielectrode arrays (MEAs) are often very rigid to interface well with 3D organoid tissues, leading to incomplete contact and low recording yield. 2D MEAs, originally developed for planar cell cultures like retina [ 4 ], are poorly suited for probing the electrophysiological activity of 3D tissues, such as organoids. Their lack of conformal contact often necessitates tissue slicing or mechanical compression and restricts recordings to a single surface. To overcome these constraints, a range of advanced MEA technologies has been introduced, including microneedle-based electrodes [ 5 ] and mesh MEAs [ 6 ], supporting 3D tissue growth and becoming internalized over time. More recently, conformal MEA platforms have further improved tissue-electrode interfaces, exemplified by flexible 3D MEAs designed for organoid studies, such as kirigami-inspired electronics with basket-like spiral or honeycomb architectures [ 7 ]. In parallel, microelectrode-tissue-microelectrode sandwich platforms have been used for developing electrical stimulation for a subretinal retinal implant, in which ex vivo mouse retinal tissue is interfaced between two opposing high-density MEAs and flexible MEA (flexMEA) for recording and stimulating, respectively [ 8 ]. While powerful for designing electrical stimulations, this configuration used planar mouse retina rather than 3D organoid tissues. Flexible MEAs (flexMEAs) represent a particularly promising class of bioelectronic interfaces, as their thin, compliant substrates enable intimate contact with curved tissues, thereby enhancing recording stability and signal fidelity. Although flexible electronics have been successfully applied to brain organoids and engineered cardiac tissues, their use in retinal organoids, especially at advanced stages of differentiation, remains limited [ 9 ]. Here, we introduce a flexMEA platform for electrophysiological recordings from mouse retinal organoids. Using this system, we reliably capture spontaneous neural activity from intact organoids. Unlike existing conformal MEA approaches in which stimulation and recording are performed using a single device, our strategy decouples these functions. This configuration allows electrical stimulation to be delivered from a standard MEA and neural responses to be simultaneously recorded from a second MEA (flexMEA) positioned on a different surface. This modular and spatially flexible architecture enables independent optimization of stimulation and recording interfaces, enhances experimental control, and provides a versatile framework for functional interrogation of complex 3D retinal tissues. Materials and Methods Mouse Retinal Organoid Culture Mouse retinal organoids were generated from mouse embryonic stem cells (mESCs) following a modified 3D differentiation protocol based on optic vesicle self-organization [ 10 ] (Fig. 2 A). Briefly, mESCs were maintained under feeder-free conditions and dissociated into single cells. Cells were aggregated in low-attachment plates to form embryoid bodies and cultured in retinal induction medium supplemented with knockout serum replacement (KSR), (96.4% Glasgow minimum essential medium, 1% nonessential aminoacids, 1% sodium pyruvate, 0.1% 2-mercaptoethanol, 1.5% KSR). Additionally, Matrigel (2%) was added on top of embryoid bodies on day 1. Retinal specification was initiated by inhibition of Wnt and Nodal signaling, followed by gradual neural differentiation. Optic vesicle-like structures emerged at day 7 and were manually isolated and transferred to suspension culture to promote optic cup formation in Tom’s medium supplemented with N2 and B27 (96% DMEM/F12, 1% nonessential aminoacids, 1% sodium pyruvate, 1% N2, 2% B27) till day 10. Organoids were subsequently maintained in a maturation medium containing retinoic acid and taurine to support photoreceptor differentiation (94% DMEM/F12, 1% nonessential aminoacids, 1% sodium pyruvate, 1% N2, 2% B27, 0.5µM retinoic acid, 1mM taurine) between day 10 and 15. Starting on day 16, organoids were cultured for up to 30–45 days in a plain medium (98% DMEM/F12, 1% nonessential aminoacids, 1% sodium pyruvate), with medium changes every 2–3 days. MEA Sandwich Configuration Electrophysiological recordings were performed using a sandwich configuration, combining a flexMEA for neural signal recording and a standard rigid MEA for electrical stimulation. Retinal organoids were positioned between these two arrays (Fig. 1 A-C). Electrophysiological Recording Extracellular neuronal activity of retinal organoids was recorded using a flexMEA, compatible with the ME2100-HS32 headstage (Multichannel Systems MCS GmbH, Reutlingen, Germany). Recordings were performed at 30–34°C in carbogenated artificial cerebrospinal fluid. The array was fabricated on a flexible polyimide 2611 foil substrate, allowing close conformal contact with the retinal organoid. Recording electrodes were made of titanium nitride (TiN) to ensure low impedance and high signal-to-noise ratio performance, while gold tracks were used for signal conduction. The electrode layout consisted of a 6 × 6 grid, comprising 32 recording electrodes, supplemented by two reference electrodes and two ground electrodes. Individual electrodes had a diameter of 30 µm and were spaced at 300 µm intervals. The impedance of the electrodes was 500 kΩ at 1 kHz. This flexible MEA configuration permitted stable recordings while preserving tissue viability during electrophysiological measurements. Here, the flexMEA was connected to preamplifiers (Multichannel Systems MCS GmbH, Reutlingen, Germany) via an omni connector, and the recording was performed using MC Rack software. The recording sampling rate was 50 kHz with an amplifier input range of ± 400 mV and occasionally of ± 4 V. The raw data was sampled at a rate of 50 kHz/channel, using a filter with a bandwidth of 1 Hz-3 kHz and a gain of 1100. Electrical stimulation For the stimulation, a planar MEA with standard MEA containing 59 circular TiN electrodes (diameter: 30 µm, interelectrode spacing: 200 µm, 60MEA200/30iR-ITO, Multichannel Systems MCS GmbH, Reutlingen, Germany) with electrode tracks insulated by Si 3 N 4 on a glass substrate was used. The electrodes had impedances of approximately 250 kΩ at 1 kHz. A stimulus generator (STG 2008, Multichannel Systems MCS GmbH, Reutlingen, Germany) was used to generate electrical stimulus pulses via only one of the 59 electrodes. The stimulus consisted of monophasic cathodic rectangular voltage pulses of 1000 µs duration with the following amplitudes 100, 300, 500, 1000 mV. Results FlexMEA platform for recording retinal organoids To enable non-invasive and conformal electrophysiological recordings from retinal organoids, we developed a sandwich recording configuration, integrating a polyimide-based flexMEA with a conventional 60-electrode MEA. The experimental setup is shown in representative photograph (Fig. 1 A). In this configuration, retinal organoids were placed on the standard 2D MEA (Fig. 1 B), while the flexMEA was mounted on a micromanipulator and gently positioned onto the curved surface of the organoid, ensuring conformal contact without mechanical compression (Fig. 1 C). Upon placement of the organoid on the MEA and contact with the flexMEA, stable extracellular recordings were consistently obtained. The flexible array adapted to the organoid surface geometry, resulting in reliable electrode-tissue coupling across multiple recording sites. Representative raw traces demonstrate clear extracellular spike activity with low noise levels across channels (Fig. 2 B). Overlay of spike waveforms further confirmed consistent signal and reproducibility across channels (Fig. 2 B). Simultaneous recordings from multiple electrodes revealed temporally correlated spiking, indicating network-level activity within the retinal organoids (Fig. 2 C). Together, these data establish the flexMEA platform as a robust interface for recording spontaneous neural activity from retinal organoids. Tracking stimulus-evoked responses using a dual-MEA sandwich configuration To assess the ability of the platform to capture stimulus-evoked activity, electrical stimulation was delivered through the standard 60-electrode MEA while neural responses were recorded from the flexMEA positioned on the opposite surface of the organoid. Stimulation amplitudes ranging from 100 to 1000 (mV) reliably elicited extracellular responses across multiple recording channels. Raster plots illustrate spatially distributed spiking activity detected by the flexMEA following stimulation, demonstrating that evoked signals can be tracked across the organoid surface (Fig. 2 D). Discussion This study demonstrates that flexMEAs provide an effective platform for functional interrogation of retinal organoids. The conformal contact, achieved by flexMEAs, significantly improves signal quality compared to conventional rigid arrays, enabling stable recordings of both spontaneous and stimulus-evoked activity. Key advantages of flexMEA technology. First, these devices offer enhanced organoid compatibility due to their minimal mechanical stress and adaptable geometry, allowing intimate contact with curved tissue surfaces. Second, flexMEAs provide improved signal yield, with a greater number of active channels and higher signal-to-noise ratios compared with conventional approaches. Third, their mechanical flexibility enables detection of spontaneous network activity across multiple surfaces of organoids [11, 12]. Finally, our design allows electrical stimulation to be delivered through one array while recording from a different surface with a second array, providing greater spatial control and experimental versatility. Limitations. The used flexMEAs are constrained by electrode density and coverage, as the limited number of recording sites can restrict spatial resolution and hinder the mapping of large-scale network activity. In addition, the thin, compliant substrates that provide mechanical flexibility are inherently fragile and can be prone to tearing, delamination, or damage during handling or repetitive use [12]. Future impact and direction. These capabilities make flexMEAs a valuable tool for future studies of retinal development, disease modeling, such as retinitis pigmentosa, drug screening, and the evaluation of cell- or gene-based therapeutic interventions. Further advances in flexMEA design, including curved architectures and increased electrode density, may enable more comprehensive coverage of organoid surfaces, higher spatial resolution, and improved mapping of retinal network dynamics. Declarations Author Contribution ZH conducted the experiments and performed the data analysis. ZH and YDY interpreted the data and wrote the manuscript Acknowledge ERC to ZH (101039764). References Lee S, Chung WG, Jeong H, Cui G, Kim E, Lim JA, et al. Electrophysiological Analysis of Retinal Organoid Development Using 3D Microelectrodes of Liquid Metals. Adv Mater 2024;36(35):e2404428. Onyak JR, Vergara MN, Renna JM. Retinal organoid light responsivity: current status and future opportunities. Transl Res 2022;250:98-111. Fathi M, Ross CT, Hosseinzadeh Z. Functional 3-Dimensional Retinal Organoids: Technological Progress and Existing Challenges. Front Neurosci 2021;15:668857. Shabani H, Zrenner E, Rathbun DL, Hosseinzadeh Z. Electrical Input Filters of Ganglion Cells in Wild Type and Degenerating rd10 Mouse Retina as a Template for Selective Electrical Stimulation. IEEE Transactions on Neural Systems and Rehabilitation Engineering 2024;32:850-64. Lee SH, Thunemann M, Lee K, Cleary DR, Tonsfeldt KJ, Oh H, et al. Scalable Thousand Channel Penetrating Microneedle Arrays on Flex for Multimodal and Large Area Coverage BrainMachine Interfaces. Adv Funct Mater 2022;32(25). McDonald M, Sebinger D, Brauns L, Gonzalez-Cano L, Menuchin-Lasowski Y, Mierzejewski M, et al. A mesh microelectrode array for non-invasive electrophysiology within neural organoids. Biosensors and Bioelectronics 2023;228:115223. Yang X, Forro C, Li TL, Miura Y, Zaluska TJ, Tsai CT, et al. Kirigami electronics for long-term electrophysiological recording of human neural organoids and assembloids. Nat Biotechnol 2024;42(12):1836-43. Yang F, Yang C-H, Wang F-M, Cheng Y-T, Teng C-C, Lee L-J, et al. A high-density microelectrode-tissue-microelectrode sandwich platform for application of retinal circuit study. BioMedical Engineering OnLine 2015;14(1):109. Lee J, Liu J. Flexible and stretchable bioelectronics for organoids. Med-X 2025;3(1):5. La Torre A. Retinal Differentiation of Mouse Embryonic Stem Cells. Bio-protocol 2016;6(13):e1851. Siwakoti U, Jones SA, Kumbhare D, Cui XT, Castagnola E. Recent Progress in Flexible Microelectrode Arrays for Combined Electrophysiological and Electrochemical Sensing. Biosensors 2025;15(2):100. Liu X, Gong Y, Jiang Z, Stevens T, Li W. Flexible high-density microelectrode arrays for closed-loop brain–machine interfaces: a review. Frontiers in Neuroscience 2024;Volume 18 - 2024. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8768713","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":587458401,"identity":"e9f2ce2c-4362-473b-8ded-5c0a13a1ae67","order_by":0,"name":"Yagmur Demircan-Yalcin","email":"data:image/png;base64,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","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Yagmur","middleName":"","lastName":"Demircan-Yalcin","suffix":""},{"id":587458402,"identity":"1faf9cc3-1932-4a89-9c66-95c35466d9bd","order_by":1,"name":"Hosseinzadeh Zohreh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hosseinzadeh","middleName":"","lastName":"Zohreh","suffix":""}],"badges":[],"createdAt":"2026-02-02 20:24:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8768713/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8768713/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102194483,"identity":"8f0553c4-6995-467a-ae48-69c8625a4529","added_by":"auto","created_at":"2026-02-09 09:46:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":662794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlexible MEA sandwich platform for retinal organoid recordings. (A)\u003c/strong\u003e The flexMEA mounted on a holder and micromanipulator and directly connected to an omni connector, interfaced with the recording amplifiers \u003cstrong\u003e(B)\u003c/strong\u003e The retinal organoid is positioned on a standard MEA, and the flexMEA is placed on top to form the sandwich configuration. \u003cstrong\u003e(C)\u003c/strong\u003eSchematic illustration of the sandwich configuration, enabling simultaneous electrical stimulation through the standard MEA and extracellular recording from the flexMEA.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8768713/v1/f6ee3667a9ed9955550123a6.png"},{"id":102194484,"identity":"f59636de-1aab-4217-87c2-841ce2b770be","added_by":"auto","created_at":"2026-02-09 09:46:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":193279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrophysiological activity recorded from retinal organoids by sandwich MEA platform. (A) \u003c/strong\u003eSchematic overview of the stepwise differentiation protocol used to generate retinal organoids, from mouse embryonic stem cells to optic vesicle/optic cup–like structures and 3D retinal organoids. Representative bright-field image of a retinal organoid at a mature stage of differentiation and fluorescence image of a cryosection of a retinal organoid stained with DAPI (blue), showing nuclear labeling and the layered cellular organization of the organoid. \u003cstrong\u003e(B)\u003c/strong\u003e Spontaneous activity recorded from a single electrode in three retinal organoids (organoid 1 to 3) on days 20, 25, and 30 of differentiation. Individual colored overlay of traces of organoid 1 correspond to signals acquired from different electrodes (green and orange), demonstrating simultaneous detection of evoked neural activity across multiple sites.\u003cstrong\u003e(C)\u003c/strong\u003e Network activity in organoid 1, showing synchronous firing across seven recording channels.\u003cstrong\u003e (D)\u003c/strong\u003e Electrical stimulation delivered through a standard 60-electrode MEA at increasing amplitudes (100, 300, 500 and 1000 mV) evokes measurable responses recorded by the flexMEA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8768713/v1/a460421807bf4238251d1313.png"},{"id":102842383,"identity":"3f08d0e1-1d3b-4518-bb98-dc4cf8086382","added_by":"auto","created_at":"2026-02-17 12:27:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1502015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8768713/v1/6235a35e-8117-4c00-bbc3-1268af8f7039.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional retinal organoids recorded by a flexible multielectrode array sandwich platform","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRetinal organoids, derived from human induced pluripotent stem cells (hiPSCs), recapitulate key structural and molecular features of the developing human retina. They contain laminated neural layers, photoreceptors capable of outer segment formation, and synaptic connections crucial for retinal circuitry. While molecular and morphological assessment of organoids has advanced rapidly, functional evaluation remains challenging [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Traditional 2-dimensional (2D) multielectrode arrays (MEAs) are often very rigid to interface well with 3D organoid tissues, leading to incomplete contact and low recording yield. 2D MEAs, originally developed for planar cell cultures like retina [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], are poorly suited for probing the electrophysiological activity of 3D tissues, such as organoids. Their lack of conformal contact often necessitates tissue slicing or mechanical compression and restricts recordings to a single surface. To overcome these constraints, a range of advanced MEA technologies has been introduced, including microneedle-based electrodes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and mesh MEAs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], supporting 3D tissue growth and becoming internalized over time. More recently, conformal MEA platforms have further improved tissue-electrode interfaces, exemplified by flexible 3D MEAs designed for organoid studies, such as kirigami-inspired electronics with basket-like spiral or honeycomb architectures [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In parallel, microelectrode-tissue-microelectrode sandwich platforms have been used for developing electrical stimulation for a subretinal retinal implant, in which \u003cem\u003eex vivo\u003c/em\u003e mouse retinal tissue is interfaced between two opposing high-density MEAs and flexible MEA (flexMEA) for recording and stimulating, respectively [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While powerful for designing electrical stimulations, this configuration used planar mouse retina rather than 3D organoid tissues.\u003c/p\u003e \u003cp\u003eFlexible MEAs (flexMEAs) represent a particularly promising class of bioelectronic interfaces, as their thin, compliant substrates enable intimate contact with curved tissues, thereby enhancing recording stability and signal fidelity. Although flexible electronics have been successfully applied to brain organoids and engineered cardiac tissues, their use in retinal organoids, especially at advanced stages of differentiation, remains limited [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we introduce a flexMEA platform for electrophysiological recordings from mouse retinal organoids. Using this system, we reliably capture spontaneous neural activity from intact organoids. Unlike existing conformal MEA approaches in which stimulation and recording are performed using a single device, our strategy decouples these functions. This configuration allows electrical stimulation to be delivered from a standard MEA and neural responses to be simultaneously recorded from a second MEA (flexMEA) positioned on a different surface. This modular and spatially flexible architecture enables independent optimization of stimulation and recording interfaces, enhances experimental control, and provides a versatile framework for functional interrogation of complex 3D retinal tissues.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMouse Retinal Organoid Culture\u003c/h2\u003e \u003cp\u003eMouse retinal organoids were generated from mouse embryonic stem cells (mESCs) following a modified 3D differentiation protocol based on optic vesicle self-organization [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Briefly, mESCs were maintained under feeder-free conditions and dissociated into single cells. Cells were aggregated in low-attachment plates to form embryoid bodies and cultured in retinal induction medium supplemented with knockout serum replacement (KSR), (96.4% Glasgow minimum essential medium, 1% nonessential aminoacids, 1% sodium pyruvate, 0.1% 2-mercaptoethanol, 1.5% KSR). Additionally, Matrigel (2%) was added on top of embryoid bodies on day 1. Retinal specification was initiated by inhibition of Wnt and Nodal signaling, followed by gradual neural differentiation. Optic vesicle-like structures emerged at day 7 and were manually isolated and transferred to suspension culture to promote optic cup formation in Tom\u0026rsquo;s medium supplemented with N2 and B27 (96% DMEM/F12, 1% nonessential aminoacids, 1% sodium pyruvate, 1% N2, 2% B27) till day 10. Organoids were subsequently maintained in a maturation medium containing retinoic acid and taurine to support photoreceptor differentiation (94% DMEM/F12, 1% nonessential aminoacids, 1% sodium pyruvate, 1% N2, 2% B27, 0.5\u0026micro;M retinoic acid, 1mM taurine) between day 10 and 15. Starting on day 16, organoids were cultured for up to 30\u0026ndash;45 days in a plain medium (98% DMEM/F12, 1% nonessential aminoacids, 1% sodium pyruvate), with medium changes every 2\u0026ndash;3 days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMEA Sandwich Configuration\u003c/h3\u003e\n\u003cp\u003eElectrophysiological recordings were performed using a sandwich configuration, combining a flexMEA for neural signal recording and a standard rigid MEA for electrical stimulation. Retinal organoids were positioned between these two arrays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C).\u003c/p\u003e\n\u003ch3\u003eElectrophysiological Recording\u003c/h3\u003e\n\u003cp\u003eExtracellular neuronal activity of retinal organoids was recorded using a flexMEA, compatible with the ME2100-HS32 headstage (Multichannel Systems MCS GmbH, Reutlingen, Germany). Recordings were performed at 30\u0026ndash;34\u0026deg;C in carbogenated artificial cerebrospinal fluid. The array was fabricated on a flexible polyimide 2611 foil substrate, allowing close conformal contact with the retinal organoid. Recording electrodes were made of titanium nitride (TiN) to ensure low impedance and high signal-to-noise ratio performance, while gold tracks were used for signal conduction. The electrode layout consisted of a 6 \u0026times; 6 grid, comprising 32 recording electrodes, supplemented by two reference electrodes and two ground electrodes. Individual electrodes had a diameter of 30 \u0026micro;m and were spaced at 300 \u0026micro;m intervals. The impedance of the electrodes was 500 kΩ at 1 kHz. This flexible MEA configuration permitted stable recordings while preserving tissue viability during electrophysiological measurements.\u003c/p\u003e \u003cp\u003eHere, the flexMEA was connected to preamplifiers (Multichannel Systems MCS GmbH, Reutlingen, Germany) via an omni connector, and the recording was performed using MC Rack software. The recording sampling rate was 50 kHz with an amplifier input range of \u0026plusmn;\u0026thinsp;400 mV and occasionally of \u0026plusmn;\u0026thinsp;4 V. The raw data was sampled at a rate of 50 kHz/channel, using a filter with a bandwidth of 1 Hz-3 kHz and a gain of 1100.\u003c/p\u003e\n\u003ch3\u003eElectrical stimulation\u003c/h3\u003e\n\u003cp\u003eFor the stimulation, a planar MEA with standard MEA containing 59 circular TiN electrodes (diameter: 30 \u0026micro;m, interelectrode spacing: 200 \u0026micro;m, 60MEA200/30iR-ITO, Multichannel Systems MCS GmbH, Reutlingen, Germany) with electrode tracks insulated by Si\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e on a glass substrate was used. The electrodes had impedances of approximately 250 kΩ at 1 kHz. A stimulus generator (STG 2008, Multichannel Systems MCS GmbH, Reutlingen, Germany) was used to generate electrical stimulus pulses via only one of the 59 electrodes. The stimulus consisted of monophasic cathodic rectangular voltage pulses of 1000 \u0026micro;s duration with the following amplitudes 100, 300, 500, 1000 mV.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlexMEA platform for recording retinal organoids\u003c/h2\u003e \u003cp\u003eTo enable non-invasive and conformal electrophysiological recordings from retinal organoids, we developed a sandwich recording configuration, integrating a polyimide-based flexMEA with a conventional 60-electrode MEA. The experimental setup is shown in representative photograph (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In this configuration, retinal organoids were placed on the standard 2D MEA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), while the flexMEA was mounted on a micromanipulator and gently positioned onto the curved surface of the organoid, ensuring conformal contact without mechanical compression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eUpon placement of the organoid on the MEA and contact with the flexMEA, stable extracellular recordings were consistently obtained. The flexible array adapted to the organoid surface geometry, resulting in reliable electrode-tissue coupling across multiple recording sites. Representative raw traces demonstrate clear extracellular spike activity with low noise levels across channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Overlay of spike waveforms further confirmed consistent signal and reproducibility across channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Simultaneous recordings from multiple electrodes revealed temporally correlated spiking, indicating network-level activity within the retinal organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Together, these data establish the flexMEA platform as a robust interface for recording spontaneous neural activity from retinal organoids.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTracking stimulus-evoked responses using a dual-MEA sandwich configuration\u003c/h3\u003e\n\u003cp\u003eTo assess the ability of the platform to capture stimulus-evoked activity, electrical stimulation was delivered through the standard 60-electrode MEA while neural responses were recorded from the flexMEA positioned on the opposite surface of the organoid. Stimulation amplitudes ranging from 100 to 1000 (mV) reliably elicited extracellular responses across multiple recording channels. Raster plots illustrate spatially distributed spiking activity detected by the flexMEA following stimulation, demonstrating that evoked signals can be tracked across the organoid surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that flexMEAs provide an effective platform for functional interrogation of retinal organoids. The conformal contact, achieved by flexMEAs, significantly improves signal quality compared to conventional rigid arrays, enabling stable recordings of both spontaneous and stimulus-evoked activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eKey advantages of flexMEA technology.\u003c/em\u003e\u003c/strong\u003e First, these devices offer enhanced organoid compatibility due to their minimal mechanical stress and adaptable geometry, allowing intimate contact with curved tissue surfaces. Second, flexMEAs provide improved signal yield, with a greater number of active channels and higher signal-to-noise ratios compared with conventional approaches. Third, their mechanical flexibility enables detection of spontaneous network activity across multiple surfaces of organoids [11, 12]. Finally, our design allows electrical stimulation to be delivered through one array while recording from a different surface with a second array, providing greater spatial control and experimental versatility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLimitations.\u003c/em\u003e\u003c/strong\u003e The used flexMEAs are constrained by electrode density and coverage, as the limited number of recording sites can restrict spatial resolution and hinder the mapping of large-scale network activity. In addition, the thin, compliant substrates that provide mechanical flexibility are inherently fragile and can be prone to tearing, delamination, or damage during handling or repetitive use [12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFuture impact and direction.\u003c/em\u003e\u003c/strong\u003e These capabilities make flexMEAs a valuable tool for future studies of retinal development, disease modeling, such as retinitis pigmentosa, drug screening, and the evaluation of cell- or gene-based therapeutic interventions. Further advances in flexMEA design, including curved architectures and increased electrode density, may enable more comprehensive coverage of organoid surfaces, higher spatial resolution, and improved mapping of retinal network dynamics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZH conducted the experiments and performed the data analysis. ZH and YDY interpreted the data and wrote the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledge\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eERC to ZH (101039764).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLee S, Chung WG, Jeong H, Cui G, Kim E, Lim JA, et al. Electrophysiological Analysis of Retinal Organoid Development Using 3D Microelectrodes of Liquid Metals. Adv Mater 2024;36(35):e2404428.\u003c/li\u003e\n \u003cli\u003eOnyak JR, Vergara MN, Renna JM. Retinal organoid light responsivity: current status and future opportunities. Transl Res 2022;250:98-111.\u003c/li\u003e\n \u003cli\u003eFathi M, Ross CT, Hosseinzadeh Z. Functional 3-Dimensional Retinal Organoids: Technological Progress and Existing Challenges. Front Neurosci 2021;15:668857.\u003c/li\u003e\n \u003cli\u003eShabani H, Zrenner E, Rathbun DL, Hosseinzadeh Z. Electrical Input Filters of Ganglion Cells in Wild Type and Degenerating rd10 Mouse Retina as a Template for Selective Electrical Stimulation. IEEE Transactions on Neural Systems and Rehabilitation Engineering 2024;32:850-64.\u003c/li\u003e\n \u003cli\u003eLee SH, Thunemann M, Lee K, Cleary DR, Tonsfeldt KJ, Oh H, et al. Scalable Thousand Channel Penetrating Microneedle Arrays on Flex for Multimodal and Large Area Coverage BrainMachine Interfaces. Adv Funct Mater 2022;32(25).\u003c/li\u003e\n \u003cli\u003eMcDonald M, Sebinger D, Brauns L, Gonzalez-Cano L, Menuchin-Lasowski Y, Mierzejewski M, et al. A mesh microelectrode array for non-invasive electrophysiology within neural organoids. Biosensors and Bioelectronics 2023;228:115223.\u003c/li\u003e\n \u003cli\u003eYang X, Forro C, Li TL, Miura Y, Zaluska TJ, Tsai CT, et al. Kirigami electronics for long-term electrophysiological recording of human neural organoids and assembloids. Nat Biotechnol 2024;42(12):1836-43.\u003c/li\u003e\n \u003cli\u003eYang F, Yang C-H, Wang F-M, Cheng Y-T, Teng C-C, Lee L-J, et al. A high-density microelectrode-tissue-microelectrode sandwich platform for application of retinal circuit study. BioMedical Engineering OnLine 2015;14(1):109.\u003c/li\u003e\n \u003cli\u003eLee J, Liu J. Flexible and stretchable bioelectronics for organoids. Med-X 2025;3(1):5.\u003c/li\u003e\n \u003cli\u003eLa Torre A. Retinal Differentiation of Mouse Embryonic Stem Cells. Bio-protocol 2016;6(13):e1851.\u003c/li\u003e\n \u003cli\u003eSiwakoti U, Jones SA, Kumbhare D, Cui XT, Castagnola E. Recent Progress in Flexible Microelectrode Arrays for Combined Electrophysiological and Electrochemical Sensing. Biosensors 2025;15(2):100.\u003c/li\u003e\n \u003cli\u003eLiu X, Gong Y, Jiang Z, Stevens T, Li W. Flexible high-density microelectrode arrays for closed-loop brain\u0026ndash;machine interfaces: a review. Frontiers in Neuroscience 2024;Volume 18 - 2024.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8768713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8768713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStem cell-derived retinal organoids provide a powerful in vitro platform for studying retinal development, degeneration, and therapeutic strategies. However, methods for assessing their electrophysiological function remain limited by the organoids\u0026rsquo; 3D structure and fragility. Here, we demonstrate that a flexible multielectrode array (flexMEA) enables stable and flexible dimension recording of activity from retinal organoids. Using a sandwich approach that combines a polyimide-based flexMEA with a standard 60-electrode MEA, we achieved high-quality extracellular recordings and mapped neuronal activity across the organoid surface. In this configuration, the standard MEA was used for electrical stimulation, while neural signals were recorded with the flexMEA. This work is proof-of-the-concept of a flexMEA integrated technology as a non-invasive and conformal approach for functional phenotyping of retinal organoids for the future functional interventions, such as drug testing.\u003c/p\u003e","manuscriptTitle":"Functional retinal organoids recorded by a flexible multielectrode array sandwich platform","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 09:46:21","doi":"10.21203/rs.3.rs-8768713/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4fc9e356-b137-441a-aac5-b5f40db29741","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T12:26:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 09:46:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8768713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8768713","identity":"rs-8768713","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}