Hybrid Retinal Implant with 3D Micro-Well Electrodes for High-Resolution Visual Restoration via a Single-Cell Neuromodulation
DOI: Pending DOI
Synopsis
Current retinal prostheses are limited by poor electrode-neuron coupling, large electrode-cell separation, and broad activation of neuronal clusters, which disrupt the intrinsic retinal computational circuitry and constrain achievable visual acuity (VA). We developed a Hybrid Retinal Implant (HRI) featuring high-density 3D micro-well Pt electrodes integrated with glutamatergic neurons to promote tight cellular coupling and localized stimulation. The multilayer SU-8 device, biofunctionalized with ECM peptides, enhanced neuronal adhesion and reduced the neural activation threshold by 3-fold, thereby reducing pixel cross-talk. The implant demonstrated stable subretinal integration in RCS rats, with high cell survival up to 30 DIV, no scar formation or immune cell activation, and directed axonal growth toward the bipolar layer. The platform supports single-cell-oriented retinal neuromodulation.
Background
Notwithstanding promising results in evoking visual perception in retinal degeneration diseases, characterized by gradual photoreceptor loss while much of the inner retinal circuitry remains preserved, the VA achieved by current retinal prostheses remains limited. Most deployed systems rely on planar or relatively large electrodes that maintain a large separation from target neurons, resulting in insufficient electrode-neuron coupling. This weak interface increases stimulation thresholds and promotes broad current spread, leading to simultaneous activation of neuronal clusters rather than precise cellular targets. As a result, intrinsic retinal signal processing, which naturally operates at near-single-cell resolution, is not fully preserved, thereby constraining achievable VA[1–3]. Previous efforts have focused on increasing pixel density while reducing electrode size, improving electrode materials to enhance charge storage capacity, and using 3D electrode geometries to increase electrode-neuron proximity [4–6].
Here, in the Hybrid Retinal Implant (HRI) [7], we use a dense 3D micro-well electrode[8] architecture array to locally confine the electric field while incorporating intermediate glutamatergic neurons[9], while the electrodes were coated with ECM-like peptide to promote tight electrode-neuron coupling[10] (See Fig). This strategy reduces the neural activation threshold by >3-fold (as predicted), enabling reduced pixel cross-talk while better preserving intrinsic retinal signal processing and supporting the potential for increased visual acuity through single-cell-oriented stimulation [7].
Methods
Finite-element modeling (COMSOL Multiphysics) was used to analyze the electric-field distribution and predict neural activation thresholds for the 3D micro-well geometry relative to planar controls. The Hybrid Retinal Implant was fabricated by multilayer SU-8 photolithography on high-density microelectrode arrays with Pt working electrodes, optimized for electrode size and high-aspect-ratio well formation. Structural and dimensional fidelity of the electrodes was verified using SEM and FIB cross-sections.
Electrochemical performance was assessed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) to quantify impedance and charge storage capacity. Electrode surfaces were biofunctionalized with ECM-derived peptides (RGD, YIGSR) via thiol self-assembled monolayers. In-vitro adhesion assays used dissociated rat retinal and PRP cells, with gene expression validated by qPCR. Confocal microscopy evaluated focal adhesion formation and neuron-electrode coupling, while TEM assessed ultrastructural cell-electrode proximity.
Functional neurostimulation was evaluated using a custom MEA replicating the implant architecture, combined with patch-clamp recordings and Ca²⁺ imaging to assess activation thresholds. Pharmacological synaptic blockers distinguished direct from network-mediated responses. Integration and survival studies used human embryonic stem cell-derived photoreceptor precursor cells (hESC-PRPs) [9] seeded within the HRI micro-wells. In vivo feasibility was evaluated after subretinal implantation in RCS rats using fundus imaging, OCT, and histological immunohistochemistry.
Results
The fabricated 1-mm Hybrid Retinal Implant comprised 3,172 high-density 3D micro-well Pt electrodes (10-15 µm diameter, 15-20 µm depth, 13-18 µm pitch), demonstrating high structural and dimensional fidelity across the array. SEM and FIB analyses confirmed well-defined micro-well geometry, uniform electrode formation, and consistent high-aspect-ratio wells throughout the device, validating the robustness of the multilayer SU-8 fabrication process [8].
Electrochemical characterization using EIS and CV demonstrated stable impedance profiles and increased charge storage capacity of Pt electrodes compared with gold controls, supporting their suitability for neural stimulation [8].
Surface biofunctionalization with cyclic-YIGSR ECM molecules significantly enhanced neuronal adhesion relative to non-functionalized controls, with pronounced focal adhesion formation observed by confocal imaging[10]. Confocal analysis further identified an optimal micro-well diameter of ~10 µm, enabling single-cell insertion, tight membrane contact with the electrode and sidewalls, and formation of a fibronectin adhesion ring consistent with effective electrode sealing. TEM imaging revealed tight ultrastructural proximity between neuronal membranes and electrode sidewalls within the wells, confirming enhanced physical coupling at the cell-electrode interface [7].
Finite-element modeling compared planar, partially sealed, and fully sealed configurations, predicting strong electric-field confinement and an approximately three-fold reduction in activation thresholds in the sealed geometry. These predictions were experimentally validated. Functional testing with a custom MEA demonstrated spatially confined stimulation that persisted under synaptic-blocking conditions [7].
Patch-clamp recordings and Ca²⁺ imaging in HEK293 cells stably expressing the NaV1.2 sodium channel confirmed an approximately three-fold reduction in activation threshold in the seal configuration compared with distance-mimicking electrodes, reaching the pico-coulomb regime and supporting geometry-dependent activation as predicted by FEM modeling. TTX abolished responses, confirming voltage-gated Na⁺ channel-mediated activation, while synaptic blockers distinguished direct from network-mediated activity, consistent with localized single-cell stimulation [7].
Following in-vivo subretinal implantation in RCS rats, fundus imaging confirmed correct subretinal placement and stable implant positioning over time. OCT cross-sections demonstrated preservation of retinal lamination above the implant with no evidence of retinal detachment or major structural disruption, scar formation, or inflammatory cell infiltration [8]. Histological immunohistochemistry revealed high PRP survival up to 30 DIV [7,9]. Notably, directed neurite extension toward the bipolar cell layer was observed adjacent to the micro-well structures, supporting favorable anatomical integration and targeted cellular interaction with the implant [7].
Together, these results demonstrate that the HRI architecture improves neuron-electrode coupling, reduces activation thresholds, and enables spatially confined retinal stimulation, paving the way for high-resolution vision restoration.
Discussion
This work presents the Hybrid Retinal Implant (HRI), a dense 3D micro-well electrode array combined with intermediate glutamatergic photoreceptor precursors, designed to address key limitations of existing retinal prostheses. Current systems are often constrained by large electrode-neuron separation and broad current spread that disrupt intrinsic retinal signal processing and limit achievable visual acuity (VA). The HRI was engineered to promote tight cell-electrode coupling and enable spatially confined, single-cell-oriented stimulation through its high-density micro-well architecture and biofunctionalized surfaces.
In vitro, the implant demonstrated high structural fidelity of the micro-well array, tight cell-electrode coupling at the single-cell level, and an approximately three-fold reduction in activation thresholds, reaching the pico-coulomb stimulation range and preventing electrode cross-talk. These findings are consistent with direct cellular activation that is geometry-dependent. In vivo, subretinal implantation showed good anatomical integration within the subretinal space, together with directed neurite growth toward the bipolar layer, indicating productive interaction with the host retina.
Clinically, by enabling tight neuron-electrode coupling and spatially confined activation, the HRI strategy may support high-resolution prosthetic vision while better preserving the intrinsic retinal signal-processing. Together, these findings position the platform as a promising pathway toward next-generation retinal prostheses aimed at high-acuity vision restoration.
Disclosures
Funded by the European Research Council (ERC) starter grant #755748, Israeli Ministry of Science and Technology grant # 1001817837, and Israeli Ministry of Defense grant # 4441401256. The authors declare no conflict of interest.
References
[1] S. Picaud, J.-A. Sahel, Retinal prostheses: clinical results and future challenges., C. R. Biol. 337 (2014) 214–222. https://doi.org/10.1016/j.crvi.2014.01.001.
[2] D. Palanker, Electronic Retinal Prostheses, Cold Spring Harb. Perspect. Med. 13 (2023). https://doi.org/10.1101/cshperspect.a041525.
[3] K.A. Ramirez, L.E. Drew-Bear, M. Vega-Garces, H. Betancourt-Belandria, J.F. Arevalo, An update on visual prosthesis, Int. J. Retina Vitreous 9 (2023) 73. https://doi.org/10.1186/s40942-023-00498-1.
[4] X.Y. Kang, J.Q. Liu, H.C. Tian, B. Yang, Y. Nuli, C.S. Yang, Fabrication and electrochemical comparison of SIROF-AIROF-EIROF microelectrodes for neural interfaces, 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBC 2014 1 (2014) 478–481. https://doi.org/10.1109/EMBC.2014.6943632.
[5] A. Corna, T. Herrmann, G. Zeck, Electrode-size dependent thresholds in subretinal neuroprosthetic stimulation, J. Neural Eng. 15 (2018). https://doi.org/10.1088/1741-2552/aac1c8.
[6] T. Flores, T. Huang, M. Bhuckory, E. Ho, Z. Chen, R. Dalal, L. Galambos, T. Kamins, K. Mathieson, D. Palanker, Honeycomb-shaped electro-neural interface enables cellular-scale pixels in subretinal prosthesis, Sci. Rep. 9 (2019) 1–12. https://doi.org/10.1038/s41598-019-47082-y.
[7] N. Farah, A. Markus, G. Shpun, Y. Chemla, E. Lasnoy, T. Azrad-Leibovitch, F. Fentahon, Z. Zalvesky, Y. Mandel, Towards High-Acuity Vision Restoration: Hybrid Retinal Prosthesis Composed of a High-Density Multielectrode Array Integrated With Glutamatergic Cells, Adv. Funct. Mater. 35 (2025). https://doi.org/10.1002/adfm.202512621.
[8] G. Shpun, N. Farah, Y. Chemla, A. Markus, T.A. Leibovitch, E. Lasnoy, D. Gerber, Z. Zalevsky, Y. Mandel, Optimizing the fabrication of a 3D high-resolution implant for neural stimulation, J. Biol. Eng. 17 (2023). https://doi.org/10.1186/s13036-023-00370-8.
[9] R.S.G. and Y.M. Amos Markus, Astar Shamul, Yoav Chemla, Nairouz Farah, Lital Shaham, An optimized protocol for generating labeled and transplantable photoreceptor precursors from human embryonic stem cells, Exp. Eye Res. 180 (2019) 29–38. https://doi.org/10.1016/j.exer.2018.11.013.
[10] G. Shpun, A. Markus, N. Farah, Z. Zalevsky, Y. Mandel, Adhesion of retinal cells to gold surfaces by biomimetic molecules, Front. Cell Dev. Biol. 12 (2024). https://doi.org/10.3389/fcell.2024.1438716.