WO2026028201A1 - Hybrid retinal neuroprosthesis and methods of using the same - Google Patents

Hybrid retinal neuroprosthesis and methods of using the same

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Publication number
WO2026028201A1
WO2026028201A1 PCT/IL2025/050652 IL2025050652W WO2026028201A1 WO 2026028201 A1 WO2026028201 A1 WO 2026028201A1 IL 2025050652 W IL2025050652 W IL 2025050652W WO 2026028201 A1 WO2026028201 A1 WO 2026028201A1
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cells
retinal
cell
prosthesis according
micro
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French (fr)
Inventor
Yossi MANDEL
Nairouz Farah
Amos MARKUS
Gal SHPUN
Zeev Zalevsky
Yoav CHEMLA
Tamar AZRAD-LEIBOVITCH
Aviad SLOTKY
Erel LASNOY
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Bar Ilan University
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Bar Ilan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/05—Electrodes for implantation or insertion into the body, e.g. heart electrode
    • A61N1/0526—Head electrodes
    • A61N1/0543—Retinal electrodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea

Definitions

  • the invention relates to retinal prosthetics. More specifically, it concerns hybrid neuro- electronic retinal implants integrating micro-well electrode arrays, biomimetic surface coatings, glutamatergic neurons, and neurotrophic modulation for at least partial vision restoration in retinal dysfunctional conditions.
  • Degenerative diseases of the outer retina are among the most common causes of blindness in developed countries. These disorders are marked by degeneration of the lightsensitive cells of the retina (photoreceptors), whereas the inner retinal neurons are relatively spared.
  • Cell replacement therapy involving photoreceptor transplantation for example, transplantation of human pluripotent cell-derived retinal pigment epithelium (RPE) or photoreceptor precursors (PRP)
  • RPE retinal pigment epithelium
  • PRP photoreceptor precursors
  • Another vision restoration approach in such conditions is electronic retinal prosthetics, wherein viable cells, either the ganglion or the bipolar cells, are electrically stimulated in a patterned manner.
  • the visual acuity obtained with the currently available technologies is still relatively low (e.g., visual acuity of 20/550-20/1200 in most patients).
  • Some of the underlying causes for this low acuity in current retinal prostheses include, for example, low pixel resolution, electrode crosstalk, and axonal stimulation.
  • improving the spatial resolution, and thus increasing the obtained acuity can be theoretically achieved by reducing the electrodes and pixels size. More specifically, reducing the electrode spacing to 5 pm and increasing the pixel density could theoretically increase the obtained visual acuity to 20/20.
  • the ability to increase the electrode density is limited by the electrode-neuron distance, since the electric field drops quadratically as the distance increases.
  • electrodes should be located no farther than one electrode diameter (i.e., a few micrometers) away from the target cells.
  • the bipolar cells which are the target cells in this case, are separated from the implant surface by about 40pm. This limitation is further exacerbated in the epiretinal prosthesis approach, where the estimated distance of the electrodes from the target cells is around 200pm. To increase the proximity, three-dimensional pillar electrode arrays were suggested, however, the layered structure of the retina does not allow proximity between neurons and electrodes.
  • the second challenge with current retinal prosthetic approaches is their inability to replicate the selective and distinct opposite responses of the ON and OFF and other pathways during natural vision. This arises because the electrical activation elicits non-selective depolarization of all types of bipolar cells (BPC) (e.g., ON, OFF) or all types of retinal ganglion cells (RGCs).
  • BPC bipolar cells
  • RGC retinal ganglion cells
  • Several groups have addressed this issue by optimizing pulse parameters, to selectively activate specific retinal cells or layers (e.g., BPC, RGC, photoreceptors, and nerve fiber layer) or specific circuits.
  • a third limitation of current prosthesis technologies is that they stimulate retinal neurons in a pulsed rather than in a continuous graded potential manner, which is the mode of operation that provides the natural visual system with unrivaled sensitivity over a wide dynamic range.
  • This non-natural mode of activation could be the underlying mechanism of the low contrast sensitivity found in pre-clinical (12% in a behavioral study in Royal College of Surgeons (RCS) rats) and clinical work.
  • Cell adhesion to electrode surfaces is important for improving neural prosthetic interfaces, particularly in retinal implants.
  • Traditional coatings poly-L-lysine, laminin, fibronectin) enhance general cell adhesion but lack cell-type specificity.
  • the present invention provides, in embodiments thereof, relates to retinal prosthetics, incorporating hybrid neuro-electronic retinal implants integrating micro-well electrode arrays, glutamatergic neurons (e.g. photoreceptor precursor cells), with optional biomimetic coating and neurotrophic modulation for at least partially allowing vision restoration in various dysfunctional conditions, including, retinal degenerative diseases.
  • retinal prosthetics incorporating hybrid neuro-electronic retinal implants integrating micro-well electrode arrays, glutamatergic neurons (e.g. photoreceptor precursor cells), with optional biomimetic coating and neurotrophic modulation for at least partially allowing vision restoration in various dysfunctional conditions, including, retinal degenerative diseases.
  • advantageous hybrid retinal implants which include high-density electrode array integrated with glutamatergic neurons.
  • the electrodes are designed to create a tight neuron-electrode coupling by a 3D micro-well geometry, thereby enabling the activation of the neurons by small-sized electrodes at high density arrays, allowing both a significant reduction in the activation threshold, reduced electrodes cross-talk and an increase in spatial resolution.
  • the neurons can synapse with the retinal neural circuits of the host bipolar cells and other cells such as the horizontal cells.
  • Upon patterned electrical stimulation of the integrated glutamatergic neurons by the electrodes such neurons can activate the host bipolar cells while mimicking and preserving natural visual pathways.
  • the disclosed micro-well configuration facilitated the confinement and amplification of the electrical field around the glutamatergic neurons. Further, the simulation shows a significant increase in spatial resolution, down to at least about 10pm, while eliminating electrode crosstalk. Furthermore, as exemplified herein, advantageously, the array allows three-order-of-magnitude reduction in the activation threshold in neurons and photoreceptors, reaching picocoulomb (pC) level.
  • pC picocoulomb
  • a hybrid implant incorporating glutamatergic neurons (hESC-derived photoreceptor precursors) implanted, in-vivo, in the subretinal space of retinal degenerated rats confirmed the survival of the seeded cells within the micro-wells, axonal sprouting toward the host bipolar cells and plausible synapses.
  • a hybrid retinal prosthesis including: a micro-well electrode array having wells with diameters between about 5 pm and about 50 pm for confining neurons; and a biomimetic peptidomimetic coating comprising adhesion motifs associated with the electrode surfaces.
  • the wells have diameters in the range of about 5 gm- 15 gm.
  • the peptidomimetic coating includes thiol- functionalized adhesion motifs.
  • the peptidomimetic coating may include thiol- functionalized adhesion motifs selected from YIGSR (SEQ ID NO:6), IKVAV (SEQ ID NO: 7), and cyclic RGD (SEQ ID NO: 1).
  • the peptidomimetic coating includes a peptide having an amino acid sequence CGG-YIGSR (SEQ ID NO: 4) or C-PolyProline(10)-YIGSR (SEQ ID NO: 5).
  • the peptide density on the electrode surface is at a density sufficient to promote integrin-mediated adhesion of photoreceptors while minimizing non-specific binding of non-retinal cells, as measured by differential adhesion assay.
  • the micro-well electrode array may include a conductive material electrode at the well base, said electrode made of or coated with gold, platinum, indium tin oxide (ITO), activated iridium oxide film (AIROF), Sputtered iridium oxide films (SIROF), Titanium, Titanium nitride (TiN), Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate(PEDOT:PSS), or any combinations thereof.
  • ITO indium tin oxide
  • AIROF activated iridium oxide film
  • SIROF Sputtered iridium oxide films
  • TiN Titanium nitride
  • PEDOT:PSS Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate
  • the micro-well height is in the range of about 10 gm to 20 gm.
  • the prosthesis may further include glutamatergic neuronal cells, horizontal cells, amacrine cells, or other neural cells that can communicate with the bipolar cells and/or retinal ganglion cells and other retinal circuitry within said micro-wells.
  • glutamatergic neuronal cells horizontal cells
  • amacrine cells or other neural cells that can communicate with the bipolar cells and/or retinal ganglion cells and other retinal circuitry within said micro-wells.
  • the cells may include photoreceptor precursor cells (PRPs).
  • PRPs photoreceptor precursor cells
  • the PRPs may be human embryonic stem cell-derived photoreceptor precursors (hESC-PRPs), induced pluripotent stem cells (iPSCs), fetal retinal progenitors, or primary photoreceptor cells.
  • the prosthesis may further include a composition including small molecule substances and/or growth factors, configured to enhance neurite elongation of cells (such as, PRPs).
  • the small molecule substances may include a ROCK- inhibitor and/or a BMP4-activator.
  • the small molecule substances may include Y-2763, fasudil, or hydroxyfasudil, taurine, retinal-conditioned medium (RCM), or any combinations thereof.
  • the prosthesis may further include collagen, Neuronal growth factor (NGF) and/or extracellular matrix (ECM) hydrogel scaffold to enhance cells (e.g. PRP) survival and axonal guidance.
  • NGF Neuronal growth factor
  • ECM extracellular matrix
  • the micro wells may be pretreated with plasma, prior to adding cells to the wells.
  • the prosthesis is configured for implantation into a target area of a subject having retinal dysfunction, to thereby at least partially restore vision in the subject.
  • the target area may include subretinal, epiretinal, or suprachoroidal space.
  • the prosthesis is for use in at least partially restoring scotopic, photopic, mesopic, color vision, improving contrast sensitivity, enhancing visual acuity, or any combinations thereof.
  • the subject is having or afflicted with retinitis pigmentosa, age-related macular degeneration, or photoreceptor degeneration.
  • a method for at least partially restoring vision to a subject having retinal disfunction includes implanting into a target area of the subject the prosthesis as disclosed herein.
  • the hybrid retinal implantable prosthesis includes: a high-density micro-well electrode array having wells of about 5-50pm diameter; and glutamatergic neurons seeded within said wells; wherein geometry of the microwells provides tight sealing to the cells, to thereby amplify local electric fields, and reduce activation thresholds to about 950pC or less.
  • the activation thresholds is reduced to less than about 500pC.
  • a pixel pitch of the micro-well array may be about 10 pm or less, thereby enabling a visual acuity of at least 20/40.
  • electrical stimulation of the sealed neurons may modulate intracellular calcium, potassium and/or sodium dynamics, leading to neurotransmitter release, resulting in selective ON/OFF bipolar pathway activation and/or activation of other retinal circuitry
  • the electrical stimulation of the sealed neurons may facilitate controlled calcium influx and release of glutamate, resulting in selective ON/OFF bipolar pathway activation and/or activation of other retinal circuitry.
  • the sealing may provide an activation contrast ratio of about 1:400 or more, between a target micro-well and adjacent micro-wells, thereby reducing crosstalk between adjacent cells, thereby enhancing resolution and contrast of the restored vision.
  • the wells may be pretreated with plasma prior to seeding the cells.
  • the wells may be made of or coated with photoresists or epoxy-based polymers (e.g., SU-8).
  • a method for surface modification for retinal cell-specific adhesion to a substrate includes:
  • a substrate comprising an electrode; and coating said electrode with a biomimetic peptide comprising an amino acid sequence YIGSR (SEQ ID NO: 6), an anchor (such as, a thiol anchor) and a spacer, wherein the coating promotes preferential adhesion of retinal cells over non-retinal cells by enhancing cell density, spreading, and/or focal adhesion formation.
  • a biomimetic peptide comprising an amino acid sequence YIGSR (SEQ ID NO: 6), an anchor (such as, a thiol anchor) and a spacer, wherein the coating promotes preferential adhesion of retinal cells over non-retinal cells by enhancing cell density, spreading, and/or focal adhesion formation.
  • the surface modification method may further include seeding cells on a surface of the coated substrate.
  • a biomimetic coating composition for promoting retinal cell adhesion to a substrate the composition includes a peptide having an amino acid sequence CGG-YIGSR (SEQ ID NO: 4) or C-PolyProline(10)-YIGSR (SEQ ID NO: 5), wherein said peptide selectively promotes retinal cell adhesion to a substrate.
  • the substrate is or comprises electrodes.
  • the electrode comprises gold, platinum, indium tin oxide (ITO), activated iridium oxide fdm (AIROF), Sputtered iridium oxide fdms (SIROF), Titanium, Titanium nitride (TiN), (PEDOT:PSS) coated electrodes, or any combinations thereof.
  • a method of promoting neurite outgrowth in photoreceptor precursor cells includes contacting said PRPs with a ROCK inhibitor for a period of time, under suitable conditions, to thereby induce increase in neurite length by at least 5 -fold compared to untreated PRPs.
  • the ROCK inhibitor is Y -27632, provided at a concentration in the range of about 50 pM and 100 pM.
  • Certain embodiments of the present disclosure may include some, all, or none of the above advantages.
  • One or more other technical advantages may be readily apparent to those skilled in the art from the figures, descriptions, and claims included herein.
  • specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.
  • FIG. 1 - a schematic illustration of a hybrid retinal prosthesis, according to some embodiments
  • Figs. 2A-2C show the effect of sealing wells, on electrical Field confinement.
  • Fig. 2A shows an illustration of a 2D cross section of the electrical field in a flat (non-sealed), partially sealed (1pm cell-microwell wall spacing) and a sealed (0.05 m spacing) configuration.
  • White extracellular space, gray: SU-8 microwell.
  • Blow up Zoom in on the cell membrane- extracellular domain interface;
  • Fig. 2B shows bar graphs of the electrical field at the interface of the cell membrane for the different configurations at 10pm (the height marked with a dashed line in a);
  • Fig. 2A shows an illustration of a 2D cross section of the electrical field in a flat (non-sealed), partially sealed (1pm cell-microwell wall spacing) and a sealed (0.05 m spacing) configuration.
  • White extracellular space
  • gray SU-8 microwell.
  • Blow up Zoom in on the cell membrane- extracellular domain interface
  • Fig. 2B shows bar graph
  • 2C shows a graphs of the electrical field at the cell membrane-microwell interface for varying cell-microwell spacing (ranging from the sealed to the partially sealed configuration), demonstrating the electrical field decrease (circles).
  • the simulations results are fitted to a 1/Spacing power function (Solid line);
  • Figs. 3A-B show graphs demonstrating the effect of sealing on cell membrane potential.
  • Fig. 3A- The induced membrane potential deflection (AVm) for three tested configurations (Flat, partially sealed and sealed), as a function of the charge (C) injection are presented;
  • Fig. 3B- The charge threshold for a 1 msec pulse for three tested configurations, compared to thresholds obtained in intracellular stimulation experiments, h is electrode-cell distance, S is cell-well spacing. All experimental data bars represent the mean ⁇ SD;
  • Figs. 4A-4B show Spatial Resolution testing -
  • Fig. 4A shows an illustration of the simulation set-up. The ratio between the activation threshold of the target cell and the neighboring cell upon current injection in the target cell is tested;
  • Fig. 4B- graph presenting he ratio of the activation threshold of the target cell to the activation threshold of the adjacent cell during electrical stimulation of the target cell, as a function of target cell microwell coverage;
  • Figs. 5A-5D Calcium imaging of voltage driven activity in PRP using the calcium indicator OGB.
  • Fig. 5A shows PRP cells stained with the calcium indicator OGB. The patched cell is demarcated with a purple circle and the tested neighboring cell with green;
  • Fig. 5B shows the calcium change observed in a cell (green) in proximity to the patched one (purple), revealing a synaptic mediated response. Black arrows indicate the voltage step onset;
  • Fig. 5C shows representative calcium change in a cell that is in proximity to a patched one, before (grey solid line) and after the application of synaptic blockers (red solid line) highlighting the synaptic mediated response;
  • Figs. 6A-6E show imaging of a 1 mm diameter implant.
  • Figs. 6A-C show SEM images of a 10-micron microwell size implant.
  • Fig. 6A shows a low magnification image where the implant’s center has a dense area of microwells (Scale bar 400 pm).
  • Figs. 6B-6C provide magnified views of the implant with a 10pm microwell diameter.
  • Figs. 6D-6E show images of another implant with 15 pm microwell diameter, where an electrode is positioned in the center of each well.
  • Fig. 6E shows cross-sectional FIB/SEM images, highlighting a single microwell with an electrode at the bottom (indicated by an arrow). Scale bar in Figs. 6B-E- 10 pm;
  • Fig. 7 shows Confocal imaging of cell-well interface for microwells with varying diameters showing the confinement of the PRP within the micro-well geometry.
  • Panels a-c show (a) 20pm well (b) 15pm well (c) 10pm well. Imaging shows cells within the wells (wells in red, cells in green).
  • Panels a', b', c' shows magnified view of a cell within a well, illustrating how the cells assume the well shape.
  • Panels d-f show Visualization of the contact area between the wells and the cells (yellow), (d) 20pm well (e) 15pm well (f) 10pm well.
  • Panels d', e', f show magnified representative view of the contact area for a cell within a well.
  • Panels g-I show Imaging of the cell nuclei within the wells (nuclei shown in blue), (g) 20pm well (h) 15pm well (i) 10pm well.
  • Panels d', e', f’ show magnified representative view of a cell inside a well. Each cell nucleus is represented by a different color.
  • Panels j, k, 1 - show top view of phalloidin staining of actin filaments (j) 20pm well (k) 15pm well (1) 10pm well.
  • Panels j’, k’, 1’ show side view for actin filaments. Scale bar :(panels a-i) 50pm, (panels a'-i’): 10pm. (panels j, j ’, k, k’, 1, 1’) -10pm;
  • Figs. 8A-8C show Quantitative analysis of the cell-well interface for the various microwell diameters.
  • Fig. 8A- shows percentage of the internal surface area of the microwell that is in contact with the cells;
  • Fig. 8B shows Percentage of sealed microwells occupied by cells;
  • Fig. 8C shows Average number of nuclei per well (the mean ⁇ SD). ** p ⁇ 0.01, ** p ⁇ 0.001;
  • Figs. 9A-9C show TEM imaging of a PRP cell inside a SU8 well showing the sealing of the PRP by the micro-well wall.
  • Fig. 9A- Few cells can be seen within the well indicated by pound sign.
  • the SU8 is indicated by an asterisk.
  • Figs. 9B-9C show magnified views of the region demarcated by the dashed squares in Fig. 9A, namely a’ and a”, respectively. Arrows point to the contact points between the cell membrane and the SU8;
  • Fig. 10A-D show In-vitro studies demonstrating activation threshold reduction.
  • Fig. 10A shows illustration of three different experimental conditions: Distant (mimicking subretinal implants), flat, and sealed; Fig.
  • FIG. 10B shows A fluorescence image demonstrating sealed cell (indicated by the perfectly round fluorescence around the well rim, yellow arrow) and the patch-clamp pipette (white arrow).
  • Fig. IOC shows Strengthduration curves for all experimental configurations showing a significant reduction in the activation threshold for cells sealed in a well;
  • Fig. 10D shows Charge activation thresholds for a 100 psec pulse for flat and microwell-type electrodes and for distant conditions. All bars present the mean ⁇ SD.
  • Figs. 11A-11D show in-vivo testing of seeded PRP viability following device implantation in the subretinal space of an RCS rat;
  • Fig. 11A Confocal scanning ophthalmoscopy (cSLO) imaging (left) and OCT of the cross section marked by the green line, highlighting the localization of the implant within the subretinal space;
  • Fig. 11B Left-IR cSLO fundus imaging; right - fluorescence fundus imaging revealing the survival of seeded cells 7 days post implantation;
  • Fig. 11C Fundus imaging of a transplanted animal at various time points following transplantation (Top) and Fluorescence imaging of the same region (bottom);
  • Fig. 11D Cell viability at various time points post transplantation on implant or by bolus injection;
  • Figs. 12A-12D show Confocal imaging of a whole mount retina, 30 days following the subretinal transplantation of the hybrid implant.
  • Fig. 12A - The implant (yellow arrow) is located in the subretinal space below the INL.
  • red- implant green - GFP labeled PRPs cells
  • Fig. 12B Confocal imaging of the whole mount retina in Fig. 12A - focusing on the GFP labeled PRPs plane
  • Fig. 12C Retinal cryosection of the whole mount, demonstrating the location of the PRPs in the implant's wells, and the outgrowth of GFP labeled neurites (white arrows).
  • FIG. 2D shows enlarged area of cells showing the presumed synapse between transplanted cells and host bipolar cells, based on the colocalization of GFP PRP cells (green) and ribeye synapses (red), in proximity with bipolar cells (magenta, stained with PKC alpha). Arrows denote the areas of co-localization. Scale bar: 5pm;
  • Figs. 13A-13B Assembly of coating biomolecules to gold surfaces. Shown are STED Fluorescent images of untreated (Fig. 13A) and fluorescent RGD-NBD-coated glass-gold paterned surfaces (Fig. 13B). RGD assembly is revealed mainly in the gold areas. Scale bar 300pm;
  • Figs. 15A-15B- Effect of coating biomolecules on cell density.
  • Fig. 15A- shows results relating to HEK 293 cells and
  • Fig. 15B shows results relating to rat retinal cells.
  • Cells were seeded on gold surfaces coated with various biomolecules at several concentrations.
  • Cell density was evaluated using a Leica LMD7 microscope and a binarization Image J algorithm. For each biomolecule, the cell density was normalized to a cell density of DDW.
  • *Red star denotes significance compared to control, p «0.05.
  • **Black star denotes significance compared to a long cyclo-RGD [Poly-Pro-c(RGD)] -coated surface, p «0.05.
  • Figs. 16A-16B Effect of coating molecules on the cell surface area.
  • Fig. 16A shows results relating to HEK293 cells and
  • Fig. 16B shows results relating to rat retinal cells.
  • the cells were incubated for 72h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/ml) followed by fixation and staining. The average surface area from each biomolecule-coated surface has been normalized to that of DDW. *p ⁇ 0.05, ***p ⁇ 0.001, compared to untreated (bare) gold;
  • Fig. 17A shows results relating to HEK293 cells;
  • Fig. 17B shows results relating to retinal cells. The cells were incubated for 72h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/ml) followed by fixation and staining. The number of bright Vinculin spots was counted manually. *p ⁇ 0.03, **p ⁇ 0.001;
  • Figs. 18A-18B show bar graphs presenting relative normalized gene expression levels of adhesion integrins and focal adhesion proteins as affected by coating biomolecules as measured by real-time qPCR.
  • Fig. 18A shows results relating to HEK293 cells;
  • Fig. 18B shows results relating to Rat-dissociated retinal cells. Cells were incubated for 72h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/ml) before RNA extraction. Expression levels were normalized to the expression level of the GAPDH gene, used as a reference gene. *p ⁇ 0.05 compared to DDW.
  • Figs. 19A-19F Quantification of neurite extension following ROCK-I inhibitor administration (Y-27632) in-vitro.
  • Fig. 19A- untreated cells Fig. 19B- cells treated with ROCK inhibitor, Y-27632 (Y -27) 50pM
  • Fig. 19C- cells treated with lOOpM Y-27
  • Fig. 19D- cells treated with 200pM Y-27. nuclei (blue), CRX (green), actin (magenta).
  • White arrows point to neurite extents from PRP cells.
  • Scale bar 50pm
  • Fig. 19E- Quantification of Y-276332 effect on neurites longer than 5pm under the various treatments N>200 cells, mean ⁇ SEM
  • Figs. 20A-20J show the effect of various molecules (Taurine, RCM, and BMP4 inhibitors) on neurite extension in PRP. Shows are Confocal images of untreated PRP (Fig. 20A), PRP treated with ImM Taurine (Tau, Fig. 20B), ImM Taurine + 50pM Y-27632 (Tau+Y-27, Fig. 20C), retinal condition media (RCM, Fig. 20D), RCM + 50pM Y-27632 (RCM+Y-27, Fig. 20E).
  • Noggin BMP4 inhibitor
  • Noggin with 50pM Y-27632 Y-27 + Nog, Fig. 20F
  • Noggin with Tau Tau + Nog, Fig.
  • Figs. 21A-21D- BMP4 mediates neurite extension in PRP.
  • Figs. 21A-D show Confocal images of neurite extension in-vitro of untreated PRP (Fig. 21A) and cells treated with 40ng/mL BPM (Fig. 21B). Nuclei (blue), CRX (green), actin (magenta). White arrows point to neurite extents from CRX positive cells. Scale bar: 50pm;
  • Figs. 22A-22E- show Ex-vivo neurite extension of retinal cells seeded on 3m-old RCS retina following 24h treatments of 50pM Y-27632, ImM Taurine, collagen. Confocal top view images of: Fig. 22A- control sample (ctrl); Fig. 22B- 50pM Y-27632 treated culture (Y -27); Fig. 22C- ImM Taurine treated culture (Tau); Fig. 22D- cells and collagen mixture (Clgn). Nuclei (blue) seeded cells (green). Panels A’, B’, C’, D’- only green channel. In each image a characteristic single neurite is indicated by purple and orange arrows.
  • Panels A”, B”, C”, D depict cross section of the selected neurite path. Scale bar: 20pm; Fig. 22E- Quantitative analysis of retinal cells neurite extension for the various treatments. N ⁇ 85, mean ⁇ SEM, ***P ⁇ 0.00I. INL - Inner nuclear layer.
  • hybrid neuro-electronic retinal implants integrating micro-well electrode arrays having photoreceptor precursor cells seeded/grown thereon, optional biomimetic surface coatings, while optionally inducing neurotrophic modulation (for example, using ROCK inhibitor and/or BMP activation), for vision restoration in various conditions, including, retinal degenerative diseases.
  • neurotrophic modulation for example, using ROCK inhibitor and/or BMP activation
  • Micro-well refers to a conical, concaved or cylindrical, microfabricated depression or cavity in a substrate, typically having lateral dimensions between about 2 pm and 50 pm and a height between about 5 pm and 50 pm, capable of confining a single neuron, photoreceptor precursor, or small cell cluster.
  • Electrode array refers to a patterned assembly of conductive elements (electrodes) configured for electrical stimulation or recording.
  • the electrodes may include, be made of or coated with gold, platinum, titanium, titanium-nitride, iridium oxide, Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), conductive polymers, carbon nanotubes, graphene, or any equivalent conductive material. Each possibility is a separate embodiment.
  • the term “Sealing resistance” relates to electrical resistance created by tight neuron-micro-well contact configured to improve stimulation efficiency.
  • the term “Pixel pitch” refers to the center-to-center spacing between adjacent micro-wells or electrode sites.
  • a pixel pitch ⁇ 10 pm denotes ultra-high-density electrode configurations enabling improved spatial resolution of stimulation.
  • Biomimetic coating refers to a surface modification mimicking, for example, extracellular matrix (ECM) cues to promote selective retinal cell adhesion.
  • ECM extracellular matrix
  • the term refers to a synthetic or semi-synthetic surface modification comprising peptides, proteins, polymers, or combinations thereof, designed to mimic native extracellular matrix (ECM) signals and promote selective cell adhesion
  • Cell-adhesion motif refers to a minimal amino acid sequence derived from ECM proteins (e.g., laminin, fibronectin, vitronectin) that binds integrin or other cell-surface receptors, including but not limited to YIGSR, IKVAV, RGD, cyclic RGD, and their functional analogs.
  • Spacer relates to a flexible linker (e.g., 1-50 amino acids, PEG) between a surface anchor and adhesion motif, optimizing receptor accessibility.
  • the spacer is configured to separate the adhesion motif from a surface anchor, allowing optimal receptor engagement and conformational flexibility.
  • anchor refers to a moiety (e.g., thiol, such as cysteine residue, mercapto-silane) capable of forming covalent bonds with conductive electrodes, such as, metal electrodes, including, for example, gold or platinum.
  • Photoreceptor precursor cells refers to progenitor cells capable of differentiating into rod/cone photoreceptors.
  • the cells may be derived from embryonic stem cells, induced pluripotent stem cells (iPSCs), fetal retinal progenitors, primary retina, and the like. Each possibility is a separate embodiment.
  • Neurotrophic composition refers to a formulation which includes small molecules, peptides, growth factors, or other bioactive agents that enhance neuronal survival, neurite outgrowth, synaptic integration, or neuroprotection.
  • examples include ROCK inhibitors (Y-27632, fasudil), BMP pathway modulators, cAMP analogs, taurine, retinal-conditioned medium (RCM), BDNF, CNTF, or combinations thereof. Each possibility is a separate embodiment.
  • ECM hydrogel scaffold refers to a three-dimensional matrix including natural (e.g., collagen, laminin, fibrin) or synthetic polymers configured to support PRP survival, adhesion, and guided neurite extension.
  • Activation threshold refers to minimal electric charge required to elicit an action potential/ or neurotransmitter release from a neuron confined within a microwell.
  • Activation contrast ratio refers to the ratio of stimulation- induced activation between a target micro-well and adjacent non-target wells (unintended activation), where higher ratios indicate reduced crosstalk.
  • the term refers to the ratio between the activation threshold of the “target” cell and the activation threshold of the “adjacent” cell when the stimulation is given in the “target” cell electrode, were studied.
  • the prosthesis includes a high density multi -we 11 electrode array (for example having about 1 Omicron pitch) .
  • Each of the wells of the array is capable of holding photoreceptor cells, such as, Glutamatergic neurons (such as human embryonic stem cells PRPs).
  • the prosthesis can be implanted into target tissue (e.g., host retina), whereby the implant cells can form synapses with host cells of the retina (e.g., bipolar cells, horizontal cells).
  • the neurons synapse with the retinal neural circuits of the host bipolar cells. Upon patterned electrical stimulation of the integrated glutamatergic neurons by the electrodes, these neurons can activate the host retinal circuits while mimicking and preserving natural visual pathways.
  • the size, shape, and/or geometry of the wells, as well as optional coating thereof using biomimetic molecules, and/or use of various compositions enhancing neurogenesis further enhance the efficiency of the prosthesis, allowing the prosthesis to restore high visual acuity.
  • Examples 9-13 presented herein below demonstrate biomimetic compounds that can enhance cells attachment to metal electrodes.
  • the electrodes may be coated with short biomolecules to mimic the retinal ECM and to elicit the adhesion of retinal cells to metal electrodes.
  • various molecular biomimetic compounds having various head group sequence (e.g., RGD or YIGSR), having different spatial conformation (linear or cyclic), and spacer length (short or long), were used.
  • the various biomolecules were attached to electrodes via a semi-covalent bond through a thiol group (SH); a cell adhesion assay and a cell spreading surface were used to estimate the efficiency of the biomolecules.
  • SH thiol group
  • PRP photoreceptor precursors
  • RhoA kinase inhibitor namely Y -27632
  • Y -27632 significantly increased both the percentage of cells with neurites and the extent of neurite elongation.
  • the amino acid taurine and retinal conditioned media RCM
  • BMP4 Bone morphogenic protein 4
  • BMP4 inhibitor reduced neurite extension in cultures treated with taurine and RCM, supporting the involvement of these pathways in the neurite extension process. Additionally, neurite outgrowth in PRP seeded on organotypic explants of degenerated retinae was used to determine the influence of the 3D microenvironment on neurite extension. Neurite extension was significantly upregulated when PRPs were seeded on the retinal explant; this effect was enhanced by the addition of a collagen matrix that mimics the outer plexiform layer. Accordingly is has been surprisingly shown that ROCK, BMP4 and the phototransduction pathways are involved in PRP neurite elongation and further underscore the importance of small molecules and the 3D environment in this process.
  • engineered micro-well electrode array having a geometry that enables tight neuron-electrode coupling by creating a sealed interface.
  • Such geometry may include, for example, cylindrical and/or conus-like geometry of the wells.
  • such geometry may elicit electrical field confinement, thereby reducing the charge activation threshold to the pC level (e.g., about l-1000pC (picocoulombs), about 10-800pC, about 20-700pC, about 300-600pC, less than about 950pC, less than about 500pC, less than a bout 250pC, less than about 150pC, less than about lOOpC), and practically eliminates electrode crosstalk, thereby enabling high-resolution and high-contrast retinal stimulation.
  • the charge activation threshold e.g., about l-1000pC (picocoulombs), about 10-800pC, about 20-700pC, about 300-600pC, less than about 950pC, less than about 500pC, less than a bout 250pC, less than about 150pC, less than about lOOpC
  • Such activation threshold is significantly lower compared to the few nanocoulombs (nC) reported for the subretinal activation threshold.
  • the hybrid retinal prosthesis is configured to stimulate neurons at ultra-low charge thresholds, including, less than about 100 pC per stimulation pulse, less than about 50 pC, less than about 10 pC, less than about 5 pC, thereby reducing energy consumption and avoiding electrode crosstalk and enabling the analog mode of stimulation (rather than pulsed mode), while achieving high-resolution neuronal activation.
  • the sealing of cells and the spatial confinement of the electrical field through the insolating micro-wells can eliminate the crosstalk, one of the main limitations of currently available retinal prostheses.
  • an activation threshold ratio of about 1:400 between the targeted cell and a cell in an adjacent pixel may be obtained, for a pixel pitch of 5, 10 pm, which can provide a visual acuity of about 20/20 and 20/40, respectively.
  • the disclosed array enables close cell-well proximity, with a large contact area between the cell membrane and the micro-well wall, whereby, the higher the seal resistance (Rseal), the lower the required activation current.
  • the sealing of the cells may be further strengthened by the presence of actin rings, which facilitate the mechano-electrical coupling between the micro-wells and the cells, leading to enhanced electrical coupling with the electrode and increasing the Rseal.
  • the hybrid retinal implant may advantageously utilize glutamatergic neurons for translating the electrical stimulation into neurotransmitter release.
  • glutamatergic neurons for translating the electrical stimulation into neurotransmitter release.
  • the transplanted PRPs create glutamatergic synapses that could modify the postsynaptic cells’ calcium level.
  • the glutamatergic cells in the hybrid implant can selectively activate the distinct retina circuitry (e.g., ON /OFF, color, horizontal cell circuitry) by glutamatergic release and not essentially by direct electrical activation. This capability is needed for mimicking normal vision and processing visual information before relaying it to the visual cortex.
  • the distinct retina circuitry e.g., ON /OFF, color, horizontal cell circuitry
  • the survival rate of the transplanted cells in the microwell, serving as a scaffold and support for the cells may be at least about 50%, at least 60 A %, at least about 70%, at least about 80%, over a period of time (e.g., 2 days, 5 days, 14 days, 30 days, etc.).
  • oxygen and metabolic supply to the PRPs in micro-wells may be provided in-vivo, by the inner retina through diffusion.
  • the transplanted cells may integrate with the host retina, whereby at least some of the cells may extend axon-like structures toward inner retinal layers.
  • patterned electrical activation and energy transfer should be provided.
  • photovoltaic approach can be used to convert light into electric current.
  • neural stimulation can be obtained by light irradiance at ambient light levels, or by using a customized projector system with light levels closer to ambient light levels, compared with currently available photovoltaic retinal prostheses.
  • a hybrid retinal prosthesis device which includes a high-density multi-electrode array configured as micro-wells with diameters of between about 5 pm and 50 pm, or any subranges thereof (for example about 5 pm, about 10 pm, about 15 pm, about 5 pm, about 5-15 pm, about 5 pm -20 pm).
  • the wells may be similar, identical or different with respect of size, (e.g., diameter, height), composition, geometrical shape, and/or type of electrode.
  • the array may include wells of different sizes.
  • the array may be patterned. In some embodiments, various regions of the array, may have a different distribution of wells.
  • different regions of the array may each have a different type of wells (i.e., different with respect of size, composition, shape, etc.).
  • the array may include two extreme regions having a same distribution of wells, while the central region, disposed between the two extreme regions, may have a different distribution of wells.
  • the micro-well array is spatially patterned such that different regions exhibit distinct well characteristics. These characteristics may include variations in well size, shape, composition, or spatial distribution. For example, a rectangular array may comprise two peripheral regions (at opposite ends) that share the same well configuration, while a central region, positioned between them, contains a different distribution or type of wells. This design allows the array to be tailored for region-specific retinal architecture or stimulation needs.
  • the wells may be made of suitable photoresist polymers.
  • SU-8 may be used as a negative photoresist for the microfabrication of the wells, while allowing high aspect ratio, optical transparency, and biocompatibility.
  • SU-8 is used to define micro-well walls or scaffolding layers.
  • functionally equivalent photoresists or epoxy-based polymers may be used, including but not limited to KMPR, JSR THB, Ormocomp, EpoCore, or mr-DWL resists, provided they enable comparable resolution, thermal stability, and biocompatibility for retinal implant fabrication.
  • the wells of the device may further include electrodes.
  • the electrodes may be made of any suitable material conductive material, including, for example, but not limited to: gold, platinum, iridium oxide, indium tin oxide (ITO), activated iridium oxide fdm (AIROF), Sputtered iridium oxide fdms (SIROF), Titanium, Titanium nitride (TiN), conductive polymers, (PEDOT:PSS) coated electrodes, and the like, or any combinations thereof.
  • conductive material including, for example, but not limited to: gold, platinum, iridium oxide, indium tin oxide (ITO), activated iridium oxide fdm (AIROF), Sputtered iridium oxide fdms (SIROF), Titanium, Titanium nitride (TiN), conductive polymers, (PEDOT:PSS) coated electrodes, and the like, or any combinations thereof.
  • ITO indium tin oxide
  • the electrodes may be coated with biomimetic compounds, facilitating the attachment of cells to the electrodes.
  • the cells may be treated with compositions designed to enhance differentiation and/or growth, thereby enhancing, for example neurite outgrowth, and/or extend cells viability.
  • the wells may be associated with cells.
  • the cells may be seeded.
  • the cells may include any retinal or retinal-compatible neuronal cell type that is capable of forming functional synaptic connections with surviving host retinal circuitry.
  • the cells may be engineered cells (retinal compatible cells or other types of cells).
  • such cells may include, for example, but not limited to: Photoreceptor lineage cells (for example, Human embryonic stem cell-derived photoreceptor precursors (hESC- PRPs), Induced pluripotent stem cell-derived photoreceptor precursors (iPSC-PRPs), Fetal or neonatal photoreceptor progenitor cells, Rod or cone photoreceptor precursor cells differentiated from retinal organoids); Retinal progenitor cells (RPCs) (for example, Multipotent progenitor cells isolated from fetal or neonatal retina capable of differentiating into photoreceptors, bipolar cells, or Muller glia, Muller glia-derived progenitors reprogrammed to a photoreceptor lineage); Inner retinal interneuron precursors (for example, Bipolar cell precursors, which can directly relay signals to ganglion cells, Amacrine or horizontal cell precursors for alternative network integration); Other excitatory or inhibitory neurons (for example, Cortical glutamatergic
  • PRP genetically modified to express synaptic adhesion molecules favoring connectivity with bipolar cells
  • Neuronal cells e.g. PRP
  • PRP neuronal cells
  • Genetically engineered or optogenetically modified cells for example, Neurons expressing light-sensitive opsins (e.g., channel rhodopsins) to allow hybrid electrical-optical stimulation, neurons engineered to release glutamate or other excitatory neurotransmitters in response to electrical stimulation, and the like, or any combinations thereof.
  • the cells may include glutamatergic neurons, photoreceptor precursors, human embryonic stem cell-derived photoreceptor precursors (hESC-PRPs).
  • the micro-well system may be configured to accommodate multiple types of cells, including but not limited to neuronal cells, glial cells, photoreceptor precursors, stem cell-derived neurons, or other electrically excitable or support cells.
  • each well is configured to accept a single cell.
  • each well is capable of holding a single cell.
  • the microwell geometry provides tight sealing between the neuron membrane and micro-well wall, resulting in amplification of the electric field, elimination of electrode crosstalk, and reduction of neuron activation thresholds to picocoulomb levels, thereby mimicking graded retinal potentials.
  • the well may be essentially cylindrical.
  • the well may be essentially conical (e.g., having a conus-like shape, with the narrow portion being closer to the electrode region).
  • the wells may be pre-treated with plasma (e.g., N2- or 02- plasma) prior to seeding the cells.
  • plasma e.g., N2- or 02- plasma
  • each micro-well has a conical or cylindrical geometry configured to confine a single photoreceptor precursor cell (PRP), and wherein said confinement promotes the formation of an actin cytoskeletal ring at the cell-substrate interface, thereby achieving tight sealing of the cell within the micro-well, amplifying local electric fields, and reducing cross-talk between adjacent wells.
  • PRP photoreceptor precursor cell
  • the pixel pitch of the micro-well array may be in the range of about 5-50 pm, or any subranges thereof. In some embodiments, the pixel pitch of the micro-well array may be less than about 40 pm, less than about 30 pm, less than about 20 pm, less than about 15 pm, less than about 10 pm. In some embodiments, the array may have a pattemed/spatial distribution of pixel pitches.
  • the hybrid retinal prosthesis is configured to achieve graded levels of visual acuity restoration depending on the pixel pitch of the micro-well electrode array, the degree of neuronal integration, and the disease stage of the host retina.
  • the hybrid prosthesis may enable a visual acuity of at least 20/20, 20/40, 20/50, 20/100. Each possibility is a separate embodiment.
  • the prosthesis may have a pixel pitch of about 10 pm or more, enabling visual acuity of at least 20/40, sufficient for reading standard text and performing daily tasks.
  • an intermediate -density array with 10-20 pm pitch may provide moderate acuity of 20/50 to 20/100, enabling mobility and large-print reading.
  • lower-density arrays with over 20 pm pitch may achieve functional vision over 20/400, improving orientation and light perception for advanced retinal degeneration.
  • ultra-high-density micro-wells with less than about 10 pm (e.g., 5 pm) pitch may achieve near-normal visual acuity (20/20 or better). According to some embodiments, such a range allows the prosthesis to be adapted for varying disease severities and clinical needs, from basic orientation vision to high-resolution central vision restoration.
  • the neuron-micro-well interface may form an actin cytoskeletal ring that enhances seal resistance and neuron-electrode coupling.
  • the glutamatergic neurons may be differentiated photoreceptor precursors expressing CRX marker and capable of synaptogenesis with bipolar cells.
  • the electrical stimulation of the sealed PRPs may trigger a controlled calcium influx and release of glutamate, resulting in selective ON/OFF, color vision, and other bipolar pathway activation.
  • the sealing may provide an activation contrast ratio of over about 1:400 between a target micro-well and adjacent micro-wells, thereby reducing or eliminating crosstalk.
  • the hybrid retinal prosthesis is configured to provide a stimulation contrast ratio between a target micro-well and adjacent micro-wells of at least 1:50, at least 1: 100, at least 1:400, and in some embodiments at least 1: 1,000.
  • This high contrast ratio may be achieved by sealing resistance created by the close neuron-micro-well contact reducing current leakage, optimized micro-well geometry confining the electric field, selective cell adhesion (using, for example coatings minimizing non-target cell adhesion, as detailed below). According to some embodiments, such contrast ratios effectively reduce or eliminate crosstalk, allowing selective activation of single neurons for high-resolution stimulation.
  • a micro-well electrode configuration for neural stimulation which includes an insulating micro-well with a neuron-contacting wall having a cell-membrane spacing of less than about 50nm (e.g., less than about 40nm, less than about 40 nm, less than about 30nm, less than about 20nm, less than about 15nm, less than about lOnm); an electrode disposed at the bottom of the micro-well, wherein said sealed configuration yields a charge activation threshold of less than about 500pC (e.g., less than about 400pC, less than about 300pC, less than about 200pC, less than about lOOpC, less that about 75pC, less than about 50pC, less than about 30pC, less than about 20pC, less than about 15 pC, less than about lOpC).
  • the charge activation threshold may be at least two, three, or more orders of magnitude lower than flat electrode configurations at less than about 40 pm neuron-
  • the array may include multi-layer electrodes with distinct conductive materials to enable multiplexed stimulation.
  • the prosthesis may further include an integrated wireless telemetry system for power delivery, bidirectional data transfer, and real-time monitoring.
  • an integrated wireless telemetry system for power delivery, bidirectional data transfer, and real-time monitoring.
  • the prosthesis may be fabricated using lithographic, nanoimprint, or 3D printing technologies, enabling customizable well geometries. Each possibility is a separate embodiment.
  • the wells in particular, the electrodes
  • the electrodes may be coated prior to seeding the cells, in order to enhance the attachment of the cells to the electrodes.
  • the coating disclosed herein can promote retinal cell adhesion by the self-assembly monolayer (SAM) of bio-peptidomimetics, which imitates the ECM adhesion motifs and promotes FA (Focal adhesion) formation, to thereby improve neuron-electrode interfaces in the electronic retinal prosthesis or for devising a scaffold integrated with retinal cells.
  • SAM self-assembly monolayer
  • FA Fecal adhesion
  • cell adhesion occurs through focal adhesion formation, which is regulated via the integrin mechanism, specifically IntasPi and IntavPs, involving the overexpression of adhesion integrins and focal adhesion protein genes.
  • the cell adhesion over the biomolecules was determined by measuring the cell density, the cell area (spreading), the number of focal adhesion sites, and adhesion-related gene expression compared to untreated gold surfaces. According to some embodiments, in order to determine optimal biomolecule type and concentration, the cell density was evaluated after 24h postseeding.
  • retinal cells were specifically attracted to surfaces coated with the YIGSR molecules (e.g., molecules having an amino acid sequence as denoted by SEQ ID NOs 4-6), whereas the HEK293 cells were mainly attracted to the RGD-type molecules (GG- RGD and poly-pro-cRGD, e.g., molecules having an amino acid sequence as denoted by SEQ ID NOs 1-3).
  • the YIGSR-type molecules significantly increased the retinal cell surface area, whereas the RGD- type molecules did not.
  • the adhesion biomolecule-coated electrode surfaces can stimulate the recruitment of cytoplasmic proteins (e.g., Vinculin) to form focal adhesion complexes.
  • cytoplasmic proteins e.g., Vinculin
  • YISGR containing molecules may increase the expression of adhesion integrins IntaV, Inta5, Intpi, and Intp3 as well as the focal adhesion proteins (Vinculin and PTK-2); According to some embodiments, YIGSR-type molecules elicit the cellular adhesion mechanism, leading to an increase in cellular density, cell spreading, and FA expression of neurite cells.
  • the adhesion peptide has an amino acid sequence of SEQ ID NO: 6 (YIGSR) or a functionally equivalent variant thereof, wherein one or more conservative substitutions may be made without abolishing retinal cell adhesion activity.
  • YIGSR amino acid sequence of SEQ ID NO: 6
  • the adhesion peptide may have an amino acid sequence of any one of SEQ ID NOs: 4-6, or a functionally equivalent variant thereof.
  • the biomimetic adhesion peptide has an amino acid sequence having at least 70%, 80%, 90%, or 95% sequence identity to SEQ ID NO: 6 (YIGSR), SEQ ID NO: 5 (CGG-YIGSR), or SEQ ID NO: 4 (C-(Pro)lO-YIGSR), and retains integrin- mediated retinal neuron adhesion activity.
  • the biomimetic adhesion peptide has an amino acid sequence having at least 70%, 80%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 1-3.
  • the amount of biomimetic peptide coating applied to the electrode surface may be selected to achieve optimal retinal neuron adhesion while minimizing nonspecific cell attachment.
  • the peptide coating may be applied at a concentration of about 0.05-0.3 mg/mL, for example, about 0.08-0.15 mg/mL, to achieve a surface density of about 0.5-5 pmol/cm 2 , for example, about l-2 pmol/cm 2 .
  • the electrode substrate may be incubated for at least 10 minutes, 30 minutes, 1 hour in the peptide solution to form a self-assembled monolayer, after which excess peptide may be removed.
  • the electrode substrate may optionally be cleaned by oxygen or nitrogen plasma, then immersed in a solution of thiol-fiinctionalized YIGSR peptide (SEQ ID NO: 4-6) at a concentration of about 0.05-0.3 mg/mL for about 1-3 hours at room temperature.
  • the thiol anchor may bind covalently to the metal of the electrodes, to form a selfassembled monolayer, optionally combined with PEG-thiols to control peptide spacing.
  • the resulting coated electrode may exhibit a reduced water contact angle of less than about 40°, e.g., 22°, and enhanced retinal neuron adhesion.
  • the biomimetic coating may include controlled-release nanoparticles delivering neuroprotective or anti-inflammatory drugs.
  • the modification may include any thiol-anchor, silane coupling agent, or click-chemistry linker enabling stable peptide immobilization.
  • the spacer length may be in the range of about 1-50 amino acids, or a polyethylene glycol (PEG) linker.
  • PEG polyethylene glycol
  • a surface modification for retinal cellspecific adhesion including: an electrode-like substrate (e.g., conductive electrode); a biomimetic peptide selected from YIGSR-based molecules; wherein the peptide comprises a thiol anchor, a short or long spacer, and a ligand head group, such that the coating promotes preferential adhesion of retinal cells over non-retinal cells by enhancing cell density, spreading, and/or focal adhesion formation.
  • an electrode-like substrate e.g., conductive electrode
  • a biomimetic peptide selected from YIGSR-based molecules wherein the peptide comprises a thiol anchor, a short or long spacer, and a ligand head group, such that the coating promotes preferential adhesion of retinal cells over non-retinal cells by enhancing cell density, spreading, and/or focal adhesion formation.
  • a method of enhancing retinal cell adhesion to an electrode surface including: coating the electrode surface with a YIGSR peptide sequence anchored via a cysteine thiol group forming a self-assembled monolayer (SAM), seeding retinal cells on the coated surface, wherein the coating increases retinal cell adhesion density and focal adhesion (Vinculin) formation compared to uncoated or RGD-coated controls.
  • SAM self-assembled monolayer
  • a biomimetic coating composition for retinal neuroprosthesis including a peptide sequence CGG-YIGSR (SEQ ID NO: 4) or C- PolyProline( 10) -YIGSR (SEQ ID NO: 5), wherein the spacer length (short GG or long PolyProline) may have lower impact on retinal cell spreading, and the peptide selectively promotes retinal cell adhesion without significantly affecting non-retinal HEK293 cells.
  • the coating may be applied by immersion in a peptide solution (e.g., 1/12 to 1/3 mg/ml) for at least 1-2 hours to achieve optimal surface coverage and hydrophilicity.
  • a peptide solution e.g., 1/12 to 1/3 mg/ml
  • the coated surface may reduce water contact angle to at least 22°, indicating increased hydrophilicity compared to uncoated electrode.
  • the peptide may further increase cell spreading area at least 2-fold, compared to control surfaces.
  • the coating is configured to provide a biomimetic ECM-mimicking interface for retinal neuroprostheses.
  • axonal elongation of the transplanted photoreceptors has a key role in their integration and creation of functional synapse with the host retina.
  • Neurite elongation is affected by various intrinsic or extrinsic factors, including signals from neighboring cells and the cellular microenvironment, which trigger pathways within the cell.
  • enhancing PRP axon elongation may be facilitated by the ROCK pathway.
  • ROCK inhibitors such as Y-27632 can significantly increase PRP neurite length (for example, up to 27.22 pm ⁇ 3.19).
  • such inhibitor can also increase the percentage of photoreceptor cells extending axons with more than half of the cells extended neurons following treatment.
  • RCM treatment of PRP cells may also enhance neurite elongation. Surprisingly it was demonstrated herein that the medium containing Muller cell-derived factors, rather than the cells themselves, is sufficient to induce photoreceptor cell extension, without the direct contact between retinal neurons and glial cells.
  • taurine can extend axons in retinal cells.
  • RNA-seq was carried out on Y- 27632 treated cultures.
  • Signal pathway analysis revealed augmentation in several pathways associated with neurite elongation, encompassing pathways involving BMP4, CREB, and the phototransduction process.
  • the gene expression analysis did not reveal alterations in the levels of genes directly associated with the Rho pathway, such as ROCK, LIMK, or Cofilin (CFL1).
  • BMP4 may be upregulated in cultures treated with Y- 27632, taurine, and RCM.
  • adding BMP4 protein to PRP cells can results in elongation of the cell’s extensions.
  • a 3D organotypic retinal explant model (harvested from three-month-old RCS animals, devoid of photoreceptors), may be used to seed PRP cells and monitor their outgrowth formation following different treatments.
  • ROCKi and taurine significantly enhance neurite extension compared with the control group.
  • seeding the PRP cells onto a 3D retinal explant in a collagen matrix to mimic the ECM microenvironment can cause a significantly longer neurite when compared to an explant condition (no collagen) after treatment with Y-27632.
  • three-dimensional environment is important in influencing the elongation of the PRP extensions.
  • a method of promoting neurite outgrowth in photoreceptor precursor cells includes contacting the PRPs with a ROCK inhibitor, wherein the ROCK inhibitor may be, for example, Y-27632 at a concentration between about 50 pM and 100 pM, thereby increasing neurite length by at least two-fold compared to untreated PRPs and optionally enhancing expression of BMP4 and phototransduction genes.
  • the photoreceptor precursor cells may be treated with a ROCK inhibitor, (such as, Y-27632), at a concentration of about 20-500 pM, e.g., about 50- 100 pM for a time period of about 2-96 hours (e.g., 12-72 hours), prior to and/or after seeding on the biomimetic micro-well prosthesis.
  • a ROCK inhibitor such as, Y-27632
  • the ROCK inhibitor may reduce actin cytoskeletal tension, inducing neurite elongation independently of BMP4 signaling.
  • the ROCK inhibitor may be combined with taurine or retinal conditioned medium (RCM) to provide additive neurite outgrowth effects, while BMP4 pathway inhibitors do not block Y-27632-induced elongation.
  • RCM retinal conditioned medium
  • the ROCK inhibitor may be delivered post-implantation to enhance PRP survival, enhance neurite extension and/or reduce glial scarring at the implant site.
  • the ROCK-inhibitor may be provided to the eye, after implantation for a period of time of, for example, 1-28 days, 2-4 weeks, 1-3 months, etc.
  • photoreceptor precursor cells may be treated with at least one neurite-promoting compound selected from: ROCK inhibitors (e.g., Y-27632), taurine, retinal conditioned medium, neurotrophic growth factors (e.g., BDNF, CNTF, NGF, GDNF), ECM- derived peptides (e.g., IKVAV), cAMP elevators (e.g., forskolin), mTOR pathway activators (e.g., IGF-1), and small molecule neurogenic compounds (e.g., HDAC inhibitors).
  • ROCK inhibitors e.g., Y-27632
  • taurine e.g., BDNF, CNTF, NGF, GDNF
  • ECM- derived peptides e.g., IKVAV
  • cAMP elevators e.g., forskolin
  • mTOR pathway activators e.g., IGF-1
  • small molecule neurogenic compounds e.g., HDAC
  • a method for enhancing neurite extension in photoreceptor precursors including: culturing PRPs in a medium containing taurine and/or retinal conditioned medium (RCM) enriched with retinal and Muller glial cell secretome, wherein neurite extension is mediated via BMP4 signalling pathway activation, and wherein inhibition of BMP4 signalling reduces said neurite extension.
  • RCM retinal conditioned medium
  • a three-dimensional (3D) collagen hydrogel or other bio-compatible matrix wherein the 3D microenvironment provides synergistic mechanical and neurotrophic cues resulting in enhanced neurite outgrowth compared to without the 3D matrix.
  • a method of stimulating neurite outgrowth in PRPs via BMP4 signaling including: contacting the PRPs with a BMP4 protein or inducing BMP4 gene expression, wherein the neurite outgrowth effect is blocked by a BMP4 inhibitor such as Noggin, thereby confirming BMP4 pathway mediation in taurine- and RCM- induced axonogenesis.
  • a 3D bio-compatible matrix for promoting photoreceptor precursor axonal elongation comprising PRPs cells embedded in a collagen hydrogel matrix.
  • the 3D matrix may further include a small molecule substance including a ROCK inhibitor, Y-2763, taurine, retinal-conditioned medium (RCM), or any combinations thereof.
  • ROCK inhibitor Y-2763
  • taurine taurine
  • RCM retinal-conditioned medium
  • a method for promoting neurite outgrowth including contacting PRPs with a neurite-promoting agent under conditions sufficient to increase neurite length by at least 2-, 5-, or 10-fold relative to untreated PRPs.
  • an ex vivo organoid models wherein the prosthesis can be applied in retinal organoid cultures for disease modelling or drug screening.
  • treating refers to means of obtaining a desired physiological effect (e.g., by implanting the hybrid retinal implant as disclosed herein).
  • the effect may be therapeutic in terms of partially or completely restoring retinal function, e.g., at least partially restoring vision.
  • a method for at least partially restoring retinal function or vision in a subject in need thereof includes: implanting the hybrid retinal prosthesis as disclosed herein into a target region; electrically stimulating the glutamatergic neurons within the sealed micro-wells; thereby inducing glutamatergic synaptic signalling to host bipolar cells and restoring graded retinal neurotransmission with high spatial resolution.
  • electrical stimulation can induce formation of synapses.
  • a method for at least partially restoring visual function including: seeding photoreceptor precursor cells into a structured electrode array configured for implantation; promoting axonal outgrowth using a combination of ROCK inhibitor and trophic factors; and implanting the array into a subject’s subretinal space to achieve retinal circuit activation.
  • a method of selectively activating retinal cells or inducing selective retinal stimulation includes applying electrical stimuli through the micro-well electrode array at an amplitude of less than about InC (e.g., less than about lOOpC) to sealed glutamatergic neurons, thereby inducing glutamate or other neurotransmitters release.
  • the hybrid retinal prosthesis or co-cultured neuronal cells can release neurotransmitters instead of or in addition to glutamate. These may include one or more of GABA, acetylcholine, dopamine, serotonin, nitric oxide, or neuropeptides, either to replicate native retinal processing or to modulate host-graft integration.
  • neurotransmitter release is patterned spatially or temporally to reproduce natural retinal computations such as directional selectivity or lateral inhibition.
  • the hybrid retinal prosthesis may be used for treating retinal degenerative diseases characterized, for example, by photoreceptor cell loss with at least partial preservation of inner retinal neurons.
  • the hybrid retinal prosthesis may be used for treating retinal degenerative diseases that include, but not limited to: age- related macular degeneration, retinitis pigmentosa (rod-cone dysterophies), Leber congenital amaurosis, cone-rod dystrophies, Stargardt macular dystrophy, myopic macular degeneration, traumatic or toxic retinopathies, phototoxic injury, and the like, or any combinations thereof. Each possibility is a separate embodiment.
  • the prosthesis in more advanced stages of retinal diseases where bipolar cells are also compromised, may be seeded with alternative interneurons or optogenetically modified neurons to relay signals directly to retinal ganglion cells.
  • the term retinal degenerative condition is interchangeable with the term retinal dysfunction condition.
  • the hybrid retinal prosthesis may be implanted in various anatomical locations within or adjacent to the retina depending on the stage of degeneration and surgical accessibility.
  • the hybrid retinal prosthesis may be implanted into a site selected from the subretinal space, epiretinal space, suprachoroidal space, intraretinal layers, or any combinations thereof, depending on the underlying retinal disease and surgical requirements.
  • subretinal implantation is preferred for diseases with preserved bipolar cells, while epiretinal implantation may be used in advanced degeneration where direct ganglion cell activation is necessary; however in a separate embodiments, epiretinal implantation is used for enabling synapse between the device cells and the bipolar cells.
  • suprachoroidal placement may be advantageous for minimally invasive procedures.
  • the implantation is facilitated such that the electrode surface of the array is facing the retinal inner nuclear layer, optionally anchored via hydrogel adhesion or suture-less geometric fitting.
  • the hybrid retinal prosthesis as disclosed herein, for at least partially restoring retinal function or vision in a subject in need thereof.
  • the implant may be used in retinal organoids or ex vivo retinal explants for disease modeling, drug screening, or testing neuroprotective compounds.
  • the implant may be used for simultaneously restoring photoreceptor function and monitoring electrophysiological responses of the host retina.
  • the methods may include combining prosthesis implantation with gene therapy or optogenetics.
  • the prosthesis may be used in conjunction with viral delivery of opsins, CRISPR-based gene editing, or pharmacological modulation to enhance vision restoration.
  • a method for modifying a substrate for retinal cell-specific adhesion includes providing a substrate including an electrode; and coating said substrate with a biomimetic peptide having a cell-adhesion motif selected from YIGSR, IKVAV, RGD, or equivalents, a thiol or silane anchor, and a flexible spacer, thereby promoting selective retinal cell adhesion over non-retinal cells.
  • a method for promoting neurite outgrowth in photoreceptor precursor cells includes contacting the PRPs with a neurite-promoting agent selected from ROCK inhibitors (e.g., Y-27632, fasudil), BMP modulators, or combinations thereof at 10-100 pM, under conditions sufficient to induce at least a 2- to 10-fold increase in neurite length compared to untreated PRPs.
  • ROCK inhibitors e.g., Y-27632, fasudil
  • BMP modulators e.g., BMP modulators, or combinations thereof at 10-100 pM
  • kits for at least partially restoring retinal function in a subject in need thereof includes the multi -array electrode as disclosed herein, and instructions for using the same.
  • the kit may further include a container including cells (e.g., PRP cells), for seeding on the array.
  • cells e.g., PRP cells
  • the array in the kit is pre-seeded with the cells (e.g., PRP cells).
  • the wells and or electrodes of the array are pre-coated with a biomimetic coating, as disclosed herein.
  • the wells of the array and/or the cells are treated or incubated in the presence of a neurite-growth enhancing composition as disclosed herein.
  • the kit may further include a surgical tool for enhancing implantation.
  • the words “include” and “have”, and forms thereof, are not limited to members in a list with which the words may be associated.
  • PRPs photoreceptor precursors
  • PRPs photoreceptor precursors differentiated from hESCs were selected, due to their putative ability to naturally connect with the host retina upon transplantation.
  • PRPs were generated as previously described (Markus, A. et al. An optimized protocol for generating labeled and transplantable photoreceptor precursors from human embryonic stem cells. Exp Eye Res 180, 29-38 (2019)). Briefly, GFP-labelled hESCs (U.S. National Stem Cell Bank [WA09]) were grown on mitomycin C-inactivated STO cells (a murine line derived from embryonic fibroblasts) in NutriStemR hPSC XF Culture Medium.
  • hESCs were trypsinized to single cells and seeded in differentiation medium in uniform-sized agarose micro-wells (9000 cells per well) prepared using silicone micro-molds.
  • a differentiation medium - GMEM Gibco, 11710035, Life Technologies, Warrington, UK
  • 20% knockout serum replacement Gibco, 10828028, Life Technologies, Warrington, UK
  • O.lmM nonessential amino acids Biological Industries, 01-340-1B, Israel
  • 1 mM pyruvate Biological Industries, 03-042-1B, Israel
  • 0.1 mM 2-mercaptoethanol Sigma, M7522, Israel
  • lOOU/ml penicillin lOOpg/ml streptomycin and 0.25pg/ml amphotericin
  • Bio Industries, 03-033- 1B, Israel lOOU/ml penicillin, lOOpg/ml streptomycin and 0.25pg/ml amphotericin
  • the embryoid bodies were trypsinized on day 24 and filtered through a 40-pm strainer (Coming, Cx-431750, NC, USA). Using this protocol, 70-80% of cells expressing CRX, a PRP marker, can be achieved following 30 days of differentiation.
  • Dye solution was then added to the cell culture to a final concentration of 7.92pM (20pl/ml).
  • the coverslip was then placed in the recording chamber of an upright microscope (Slicescope 6000, Scientifica) equipped with a CCD camera (EXI-Blue QIMAGING) and filters to visualize the cells for calcium imaging. Images were captured at 10 frames per second.
  • the chamber contained an extracellular solution consisting of (mM): NaCl (119); KC1 (2.5); MgC12 (2); HEPES (25); CaC12, (2); and D-glucose (30).
  • the OGB/Rhod2 was washed with 1ml of the extracellular solution, after which 3ml of the extracellular solution were added.
  • the induced calcium currents through the simultaneous intracellular stimulation of a cell through patch clamp were studied. Under the voltage clamp configuration, the cell was held at -60mV and voltage steps of 30m V were applied every lOsec. To test the formation of glutamatergic synapses in the cell, the glutamate receptor blockers APV (80pM), CNQX (lOpM), and L-AP4 (30pM) were applied to block the synaptic transmission between the cells and then intracellular stimulations were performed. As control, the simulations without the blockers were repeated under the same conditions.
  • the micro-well array (1mm diameter) was fabricated using a sequence of conventional photolithography steps in a clean room (Shpun, G. et al. Optimizing the fabrication of a 3D high-resolution implant for neural stimulation. J Biol Eng 17, (2023)). Briefly, the device includes a 3pm-thick flat base layer and a 3D micro-well layer fabricated onto it (10pm, 20pm or 10pm in diameter).
  • an SU-8 negative photoresist polymerized under UV light was spin coated on a pre-cleaned soda lime glass coated with a sacrificial layer (LORI OB, MCC, USA), soft baked and patterned by UV radiation (395nm) using Maskless aligner (Heidelberg MLA150 maskless aligner, Germany) or by mask aligner with a photomask (MA6 Karl Suss, Germany).
  • Maskless aligner Heidelberg MLA150 maskless aligner, Germany
  • MA6 Karl Suss, Germany Karl Suss, Germany
  • the polymeric array was stripped from the glass by dissolving the sacrificial layer in organic solution (DMSO) overnight and then rinsed in DDW. Implant characterization was then performed using both confocal and SEM (E-SEM 326, Quanta FEG 250 by FEI) imaging.
  • the devices were N2-plasma-treated several hours before seeding. Then, PRPs (day 24) were trypsinized and separated to single cells using a 10pm strainer (Pluriselect #43-10010-40), and seeded onto SU8 micro-wells in 50pL PRP medium. After two hours of incubation at 37°C, additional PRP medium was added, and the implant was centrifuged (100rcf/4min) and incubated for at least another 24 hours.
  • the inherent fluorescence of the GFP-PRP cells was used, in conjunction with actin staining, using Phalloidin (Cat. #PHDN1-A) to visualize the cell cytoskeleton.
  • Phalloidin Cat. #PHDN1-A
  • Three-dimensional images of the cells with implants were acquired using a Leica Stelaris confocal microscope and processed with IMARIS software.
  • the GFP fluorescence channel was merged with the actin staining fluorescence channel, combining both to define the cell volume and edge and create a "surface” .
  • the implant “surface” was obtained using the red fluorescence of the implant.
  • TEM imaging was used (Henn, I. et al. SEM/FIB Imaging for Studying Neural Interfaces. Dev Neurobiol (2019) doi: 10.1002/dneu.22707). Briefly, implants were seeded with PRP cells as detailed above. After 5 days of incubation, cells were fixed with a fixation buffer (2.5% (wt/vol) paraformaldehyde, 2.5% glutaraldehyde, 0.1M cacodylate buffer, PH 7) for Ih at 24°C and then left overnight at 4°C.
  • a fixation buffer (2.5% (wt/vol) paraformaldehyde, 2.5% glutaraldehyde, 0.1M cacodylate buffer, PH 7
  • TEM sample fixation was performed: 1 hour of 1% osmium in a buffer containing CaCO 0.1M, 5mM CaC12, 0.5% potassium dichromate K2Cr2O7, 0.5% potassium hexacyonaferrate K4[Fe(CN)6], followed by three washes with 0.1M cacodylate buffer, and two washes with DDW. Next, the samples were stained with 2% aqueous uranyl acetate for Ih, followed by dehydration in ethanol solutions of 50% 70% and 95% and three times 100%, for ten minutes.
  • samples were embedded using the Epon technique carried out in 5 steps: 30% for 3 hours, 50% overnight, 75% for 3 hours, 100% resin overnight, followed by 100% repeated twice.
  • samples underwent a series of washes with 10 splashes of 5 ml 100% ethanol. Then, samples were placed in an oven for 24 hours at 60°C. Finally, the samples were cut with an ultra-microtome (Leica UC7) and sections of 50-70nm were viewed under an electron microscope (Tecnai G2 Spirit).
  • a device prototype fabricated using the same optimized process, including high-density electrodes (an electrode pitch of 50pm, well diameter of 10 pm and height of 5pm) was used.
  • an excitable HEK293 cell line were rendered excitable through the expression of the voltage-gated sodium channel NaV1.2 (as detailed below).
  • 1,500,000 NaV 1.2-positive cells were seeded onto polyethylenimine (PEI, Sigma Aldrich, 408727)-coated devices, followed by centrifugation (150rcf/4mins) to ensure cell entrance into wells.
  • Electrode array current injector MAA 2001, Multi-Channel System, Germany.
  • Cathodic pulses 0.04ms-lms, IpA-lOOpA, were delivered at a rate of 0.2Hz.
  • the induced responses were recorded using the patch clamp technique, similarly to the description above.
  • the threshold was defined as the first current step inducing an inward membrane current.
  • HEK239 cell line expressing the voltage-gated sodium channel NaV1.2 was made, by transfecting cells with the plasmids’ CD-splice variant_pcDNA3.1(+) IRES GFP corrected and SCN I B-SCN2B_pcDNA3. 1 (+) IRES (GenScript, NJ, USA), using polyethyleneimine (PEI, Sigma Aldrich, 408727) reagent dissolved in DDW (lOmg/lml). The transfection solution contained 0.5pg DNA, lOpl PEI, 70pl DMEM per well, with the transfection.
  • PEI polyethyleneimine
  • the HEK293-GFP cells were then incubated in a medium containing: MEM-eagle (Biological Industries), 1% PSA (Biological Industries), 1% glutamine (Sigma-Aldrich), and 10% fetal bovine serum (Danyel biotech, Rehovot, Israel). Cells were incubated at 37°C with 5% CO 2 .
  • a binary mask was applied over the image (thresholding) to highlight regions that are GFP positive.
  • the average fluorescence signal at each time point for a region of interest was then normalized to the average fluorescence of the same region evaluated on the first day post transplantation.
  • the rats were sacrificed and the eyes incubated with paraformaldehyde 4% for 24 hours. The eyes were then rinsed using PBS, and the corneas removed to create eyecups. A 5mm sample containing the device was cut out of the eyecups. The nuclei were stained overnight using Hoechst (Hoechst 14533). The samples were then rinsed using PBS and flat-mounted on slides, and imaging performed using an 1X81 Olympus microscope and confocal microscope (Leica TCS SP8).
  • the samples were cryo-sectioned. To this end, the samples were incubated in increasing concentrations of sucrose (5%, 15%, 30%) (Millipore, 573113- IKg) at room temperature for 5-30 min, according to the sucrose concentration. They were then incubated with PBS containing 30% sucrose for 24 hours at 4°C. At the end of the process, the samples were frozen in OCT medium (Tissue-Plus, OCT compound embedding matrix Tissue- Tek Scigen) and sectioned into 10pm slices with a cryostat (CM1800 LEICA). Retinal cryosections were rinsed with PBS and then twice with PBST.
  • sucrose 5%, 15%, 30%
  • PBS containing 30% sucrose for 24 hours at 4°C.
  • OCT medium Tissue-Plus, OCT compound embedding matrix Tissue- Tek Scigen
  • Sections were then incubated in a blocker solution containing 1% bovine serum albumin (MP Biomedicals, 160069) for 60 min. Following overnight incubation in primary antibodies PKCa (Sigma- Aldrich, P4334), Gluthamine Synthetase (Abeam ab49873), ribeye (BD biosciences 612044), and Mitochondria Monoclonal Antibody (Thermo Fisher MTC02), sections were rinsed with PBS and incubated with secondary antibodies Alexa 594 anti-mouse (ENCO, 711-545-152) and Alexa 647 antiRabbit (ENCO, 711-545-152 ) for 1 hour at room temperature. Samples were imaged using confocal microscopy (Leica TCS SP8).
  • the hybrid retina concept was modelled using COMSOL Multiphysics 5.2 with MATLAB R2019a.
  • the electrical potential and electric field for each spatial point in the model were calculated using COMSOL’s electrical current physics model by solving the electric field equation:
  • V(o V0) 0
  • a the electrical conductivity
  • 0 the potential at a specific location.
  • the outer boundaries of the model were set to an insulating condition in which
  • n the unit’s outward normal vector and J represents the current density; a continuity condition was applied to all other boundaries.
  • Hodgkin- Huxley equations state that where Cm is the membrane capacitance, E na is the sodium reversal potential, G na is the maximal sodium channel conductance, m and h are the rate functions governing the activation and inactivation of the sodium channel, respectively, and G k is the maximal potassium channel conductance.
  • the m, n and h rate functions were obtained by solving the following exponential rate functions with a time constant: where the equation for each a and is empirically adapted.
  • the simulations were performed under a 2D axis symmetry geometry to reduce both the computational complexity and the calculation time.
  • the cell was modelled as a 5pm wide, 15pm high rectangle, a shape chosen following the observation that a sealed cell assumed the shape of the well.
  • the microwell was then modelled as a 15 pm high, 1 pm wide rectangle. These parameters were chosen to resemble those of the actual fabricated device.
  • the electrode was then modelled as a 2.5pm wide, 1pm high rectangle on top of the microwell base. An additional rectangle (55pm wide, 1pm high) served as a remote ground, placed far from the cell). Finally, these four domains were surrounded by a large 200pmx200pm rectangle.
  • the electrode was given the properties TiN (electrical conductivity: 5.998e7[S/m]).
  • a similar modelling excluding the SU8 domains, was performed to simulate the non-sealed configuration, which represents the standard cell-flat electrode interface.
  • the rate functions a and P to were fit to PRPs by iteratively adjusting the rate function based on previous patch-clamp data acquired from PRPs.
  • the patched cell was held at -60mV and voltage steps ranging from 120mV to 70mV, with a step size of lOmV and width of 200msec, were applied to test ionic currents.
  • the observed electrophysiological signals were amplified (xl) and low pass filtered (Bessel 10kHz) (Multiclamp 700b AXON), sampled at 10kHz and saved (Digidata, Axon).
  • the cell membrane potential was set to steps identical to those used in the electrophysiological experiments, and various parameters employed in the Hodgkin- Huxley equations were iteratively adjusted until the computer modelling data fit the measured parameters.
  • a gold layer deposited on glass was used.
  • microscope glass slides #7105, BOJACK, China
  • piranha solution 3: 1, Ammonia solution: Hydrogen peroxide
  • DDW double-distilled water
  • Cr/Au (lOnm/lOOnm) layers were spatter deposited (Bestec Berlin, Germany) after Ch+Ar (3min, 100W) plasma attaching (Dainer electronics, Pico, Germany) and Ar ion milling (lOsec).
  • p-slides (12 wells (#81201, GmbH, Grafelfing, Germany)) were mounted on the gold-coated slides and sealed with SYLGARD®-184 (#761028-5EA, Merck, New Jersey, USA), followed by curing at 80°C overnight. Finally, the samples were rinsed twice in DDW, and uprooted by boiling in DDW for 20 min, followed by a 70% Ethanol wash and exposure to UV radiation for 30min in a biological hood. For the gene expression studies, the slides were uprooted without mounting the p-slides.
  • the design of the biomolecules used in this study was based on the following:
  • the thiol group (SH) was used, which is found in Cysteine amino acid, and spontaneously forms a self-assembly monolayer (SAM) of a semi-covalent bond with gold.
  • SAM self-assembly monolayer
  • GG short amino acid spacer
  • PPPPPPPPPP long spacer polyproline
  • the amino acid type and sequence e.g., RGDxx, where xx represents any amino acid or YIGSR (SEQ ID NO: 6), as well as their conformational structure (e.g., linear, cyclic, or branched), greatly influence the molecule’s affinity to the integrin subtype and especially to the adhesion integrins.
  • the RGD motif is an adhesion motif for adhesive basal cells and neurons, YIGSR and IKVAV (SEQ ID NO: 7) are known to specifically promote the adhesion of neuronal cells.
  • biomolecules were used: a) Short linear RGD; Cys-Gly-Gly-Arg-Gly-Asp-D-Phe-Lys (CGGRGDfK). (SEQ ID NO: 1) b) Short cyclic RGD; Cys-Gly-Gly-cyclo(Arg-Gly-Asp-D-Phe-Ly), (CGG-c(RGDfK)). (SEQ ID NO: 2) c) Long -cyclic RGD; Cys-PolyProline(10)-cyclo(Arg-Gly-Asp-D-Phe-Lys), (C-PolyPro9- c(RGDfK)).
  • the peptides were dissolved in aqueous solution 1: 1 (DDW: Acetonitrile (AN), #75058) at a concentration of 1/3 mg/ml and stored until use at -20°C after being divided into several test tubes. Before use, the solutions were thawed and diluted to various concentrations (1/3, 1/6, 1/12, and 1/24 mg/ml).
  • the biomolecule-gold electrode coating was obtained through a self-assembly monolayer (SAM) facilitated by semi-covalent bonds spontaneously forming between the gold and the thiol group (SH) present in the biomolecule, which can be found at cysteine (C) amino acid.
  • SAM self-assembly monolayer
  • SH thiol group
  • gold surfaces were coated by immersion for 2 hours in the solutions, followed by rinsing with PBS. For control purposes, untreated gold surfaces were soaked only in DDW. All samples were soaked for 2h at RT and rinsed three times in PBS before cell seeding.
  • XPS X-ray photoelectron spectroscopy
  • the contact angle between a water droplet and the surface was measured using a Contact Angle Goniometer (System OCA, model OCA20, Data Physics Instruments GmbH, Filderstadt, Germany). Briefly, gold-coated (lOOnm) cover glasses were coated by 2 hours of immersion in Acetonitrile-DDW (1: 1) solutions for each biomolecule at various concentrations. A drop of 5pL of DDW was placed in the center of each sample. The measurements were performed at 25°C and 55% moisture; Laplace-Young curve fitting was used to determine the static water contact angle values.
  • N2 B27, N2, and EGF (lOng / pl, Peprotech, Israel), NGF (lOng / pl, Peprotech), NT3(10ng / pl, Peprotech), BDNF (lOng / pl, Peprotech), and FGF (lOng / pl, Peprotech) were added to the medium.
  • the medium was replaced after 24h with fresh medium containing ROCK inhibitor (5pl/lml, #1254/10, Biotest, Dreieich, Germany) and antimetabolite cytosine -D-arabinofuranoside hydrochloride (Ara-c, 0.5pl/lml, #C6645, Merck, USA), aiming to decrease the number of glia cells.
  • ROCK inhibitor 5pl/lml, #1254/10, Biotest, Dreieich, Germany
  • Antimetabolite cytosine -D-arabinofuranoside hydrochloride Ara-c, 0.5pl/lml, #C
  • HEK293 cells were transfected with polyethyleneimine (PEI, Sigma Aldrich, 408727) reagent dissolved in DDW (lOmg/lml) cells that were seeded on a 24-well plate.
  • the transfection solution contained 0.5pg DNA, lOpl PEI, 70 pl DMEM per well, with the transfection plasmids’ CD-splice variant_pcDNA3.1(+) IRES GFP_corrected and SCN1B- SCN2B_pcDNA3.1(+) IRES (GenScript, NJ, USA).
  • Human HEK293-GFP cell medium contains MEM-eagle (Biological Industries), 1% PSA (Biological Industries), 1% glutamine (Sigma- Aldrich), and 10% fatal bovine serum (Danyel biotech, Rehovot, Israel). Cells were incubated at 37°C with 5% CO2.
  • Biomolecule effect on cell density Aiming to assess the optimal biomolecule and concentration, the five types of biomolecules were diluted into four concentrations (1/3, 1/6, 1/12, and 1/24 mg/ml) in aqueous solutions DDW: Acetonitrile (1: 1). The gold surfaces were coated as mentioned above in two micro-wells for each concentration (in duplicate).
  • HEK-293-GFP and rat-dissociated retinal cells were seeded at an initial concentration of 0.8M cells/mm 2 , and incubated for 24h. Then, the cells were gently rinsed with PBS to remove the unattached cells and fixed with 4% paraformaldehyde (#BN15711, Bar Naor, Israel) for 15 min at room temperature.
  • Nuclear staining was performed with Hoechst (#14533, Sigma- Aldrich). Stained samples were rinsed (PBS) and mounted on slides in 90% glycerol (#G9012, Sigma- Aldrich)/ 10% PBS/1% n- propyl-gallate (#P3130, Sigma-Aldrich) and sealed with nail polish. The samples were imaged using a Leica LMD7 Microscope (Leica-microsystems, Germany) at a magnification of x20. The navigation and stitching applications were used to extend the field of view by a factor of 9; three regions of interest (ROI) were imaged from the center of each micro-well, with two duplicates for each concentration, overall, with four repetitions.
  • ROI regions of interest
  • the cell area was measured as a function of the coated biomolecule.
  • gold surfaces were coated by aqueous solutions (1/6 mg/ml, DDW: Acetonitrile, 1: 1) of the biomolecule and the control as mentioned above with two micro-wells for each molecule (duplicates) and three repetitions.
  • HEK-293-GFP and rat-dissociated retinal cells were seeded at an initial concentration of 0.15M cells/mm 2 to allow single-cell spreading and incubated for 72h.
  • lOpM Ara-c and 50pM ROCK inhibitors were added after 24h, aiming to restrict the glial cell growth.
  • rat-dissociated retinal cells were stained for cytoplasmic staining using ViaFluor® 488 (#BTM-30086, 1:4000, Biotium, Fremont, CA, USA) as the supplier protocol, fixed with 4% paraformaldehyde (#BN 15711 , Bar Naor, Israel) for 15 min at RT, and nuclear staining was performed by Hoechst (#14533, 1: 1000, Sigma- Aldrich).
  • the average cell surface area was measured by counting the fluorescence pixels and dividing them by the number of nuclei in the frame; this was further normalized to the average surface area of the “DDW coated” images.
  • Statistical analysis was determined using both multivariate one-way ANOVA analysis and a two-tailed t-test, provided by the MATLAB statistics toolbox.
  • HEK-293-GFP and rat-dissociated-retinal cells were seeded at an initial concentration of 0.8K cells/mm 2 to allow single cell spreading and incubated for 72h (for rat-dissociated retinal cells lOpM Ara-c and 50pM ROCK inhibitor were added after 24h).
  • cells were immuno-stained for cytoskeletal markers as follows: Surfaces were rinsed gently with PBS to remove unattached cells; then the cells were fixed with 4% paraformaldehyde (Bar Naor, BN15711) for 15 min at room temperature. Next, the cells were rinsed in PBS with 0.5% Triton-XlOO (Amersham, 22686) and 1% Tween (Amersham, 20605) (PBST). Blocking was performed for 30 min in a blocker solution containing 1% bovine serum albumin (MP Biomedicals, 160069).
  • F-actin staining was performed with #P5282, Sigma-Aldrich, 1:500 and nuclear staining was performed with Hoechst (Sigma-Aldrich, 100MG-14533). Stained samples were rinsed in PBS, mounted on slides in 90% glycerol (Sigma- Aldrich, G9012)/10% PBS/1% n-propyl-gallate (Sigma- Aldrich, P3130), and sealed with nail polish. Cells were imaged using a Leica-Stellaris-5 microscope (Leica-microsystems, Germany) at xlOO oil immersion objective. Aiming to estimate the focal adhesion (FA) spots, the Vinculin channel was binarized using ImageJ and the bright spots for each cell were counted. A DDW- normalized average amount of FA for each molecule was calculated, and the statistical significance was determined using both multi-variate one-way ANOVA analysis and a two- tailed t-test, provided by the MATLAB statistics toolbox.
  • FA focal
  • RT-PCR was performed on RNA extracted from the cells, which was synthesized to cDNA using M-MLV reverse transcriptase (#M1701, Promega, Madison, WI, USA). Quantitative PCR analysis was then performed using PerfeCTa SYBR Green FastMix (#95074-250, Quantabio, Beverly, MA, USA).
  • the primers were designed according to the gene sequences database of the National Library of Medicine website, and the specificity of each primer was assessed using the BLAST software and UCSC In-Silico PCR. Primers were manufactured by Sigma Aldrich, checked via gel electrophoresis, and melting curves at 60°C; the efficiency and specificity were assessed using linear calibration curves at various concentrations. The corresponding primers of the genes of interest are listed in Table Supp-Tl in the Supplementary Material.
  • SD Standard Deviation
  • the retinas of Pl Sprague Dawley rats were isolated and dissociated using a papain dissociation kit (Worthington #LK003150) following the manufacturer’s protocol with minor adaptations.
  • 1 x 105 cells were seeded on coverslips coated with poly-lysine (Sigma, #P4707) and laminin (Sigma, #L2020).
  • PRP medium contains DMEM/F12 (Gibco) and Neurobasal (Gibco) in a 1: 1 ratio, 1% glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids, and 2% horse serum.
  • the medium was supplemented with B27, N2, EGF (10 ng/pL, PeproTech), NGF (10 ng/pL, PeproTech), NT3 (10 ng/pL, PeproTech), BDNF (10 ng/pL, PeproTech), and FGF (10 ng/pL, PeproTech).
  • EGF 10 ng/pL, PeproTech
  • NGF 10 ng/pL, PeproTech
  • NT3 10 ng/pL, PeproTech
  • BDNF 10 ng/pL, PeproTech
  • FGF 10 ng/pL, PeproTech
  • Dissociated retinal cells were labeled with AAV1-GFP virus (Charles River Laboratories, USA) and treated with ara-C (10 pM) for 5 days before being seeded on the retinal explant.
  • AAV1-GFP virus Charles River Laboratories, USA
  • ara-C 10 pM
  • 1 x 105 retinal cells were seeded in collagen I (pH 7-8), incubated for 15 minutes and then added PRP media for 3 days as described above.
  • Dissociated rat retinal culture were fixed with 4% PFA (Bar Naor, #BN15711) for 15 minutes, then rinsed twice in PBS (Biological Industries, 02-020-1A) for 5 minutes each, followed by rinsing in PBS with 0.5% Triton X-100 (Amersham, 22686) (PBST) for 5 minutes, and then rinsed again twice in PBS for 5 minutes each.
  • the cells were incubated in a blocker solution containing 1% bovine serum albumin (BSA, MP Biomedicals, 160069) for 45-60 minutes, then incubated overnight with a primary antibody for Rabbit anti-CRX (Biotest, NBP2-15964).
  • BSA bovine serum albumin
  • the retinas were fixed with 4% PFA (Bar Naor, #BN15711) for 15 minutes, followed by two rinses with PBS (Biological Industries, 02-020-1 A) for 20 minutes each.
  • PFA Bar Naor, #BN15711
  • PBS Biological Industries, 02-020-1 A
  • Hoechst (1.8pM, Sigma-Aldrich, 100MG-14533) was used, followed by one additional rinse with PBS.
  • the retinas were mounted on slides in a solution consisting of 90% glycerol (Sigma-Aldrich, G9012), 10% PBS, and 1% n-propyl -gallate (Sigma-Aldrich, P3130), and then sealed with nail polish.
  • RT-PCR was performed using MLV reverse transcriptase (Promega, M1701).
  • qPCR analysis was performed using PerfeCTa SYBR® Green FastMix (Quanta, 95074-250).
  • Illumina Stranded mRNA library preparation kit (#20040534) was used from 1000 ng of total RNA starting material. All eight RNA samples underwent PolyA selection following the manufacturers protocols, with a 10 PCR cycles in the amplification step. Quantification and quality control of the libraries were done using Denovix fluorimeter and Agilent 4200 TapeStation.
  • N indicates the number of cells or fields of view tested.
  • SEM standard error of the mean
  • N indicates the number of cells or fields of view tested.
  • neurite extension imaging and quantification Cells and retinal imaging were performed using a confocal microscope (STELLARIS, LEICA), equipped with a 63X oil-immersion objective. The length of neurites was measured manually, and quantification was carried out in a blinded manner. Cells with neurites whose total length equaled or exceeded 5 pm were taken into account. For comparisons between the different groups, a two-tailed Student’s t test was utilized, assuming Gaussian distribution.
  • RT-qPCR data were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and analyzed using the 2-AACt method to compare relative gene. Significant levels are denoted as P ⁇ 0.05, '**' P ⁇ 0.01, '***' P ⁇ 0.001.
  • GPDH Glyceraldehyde 3-phosphate dehydrogenase
  • the effect of the microwell electrode geometry on the electrode-neuron coupling was tested. More specifically, the electric field around the neurons and the lateral spread of the electric field toward adjacent electrodes (as a measure of potential crosstalk, which can reduce the obtained resolution, visual acuity, and contrast sensitivity) was tested.
  • Fig. 2A show that the induced electrical field around the cell (in response to a 2nA, 5ms pulse) in the sealed configuration is highly amplified compared to the partially sealed and flat configurations.
  • a cross-section at 10pm above the electrode (Fig. 2B) revealed that for a sealed configuration, the electrical field (and the current density) at the cell membrane-well interface (2001.73 V/m) is more than three- orders-of-magnitude higher than that of the flat configuration (1.54V/m) and almost two- orders-of-magnitude higher than that of the partially sealed (56V/m) configuration.
  • the Hodgkin-Huxley ion channels’ rate functions and dynamics were incorporated as partial differential equations applicable to the boundary representing the cell membrane.
  • the glutamatergic cells human embryonic-stem-cell-derived photoreceptor precursors, hESC- PRPs
  • both ionic currents under the voltage clamp configuration
  • membrane potential under the current clamp configuration
  • the charge activation threshold required to trigger an action potential in neurons was tested. Although photoreceptors operate through graded potentials rather than action potentials, it was decided to simulate the charge activation threshold for inducing an action potential since this metric, rather than the graded potential, is commonly used to evaluate electrode-neuron coupling. This approach allows to more effectively compare this novel geometry with previous data. To this aim, the same micro-well cell geometry modeled with various neuron-wall spacings and neuron-electrode distances was used. The injected cathodic current with a pulse width of 1msec from the electrode situated at the bottom of the well was increased until an action potential was induced.
  • the potential crosstalk between pixels which is a main determinant of the resolution of patterned retinal activation, was modelled.
  • two neighbouring pixels (10pm pixel diameter and 5pm diameter electrode) were modelled using the same electrical and biophysical properties used for the single-pixel activation threshold study.
  • the effect of activating a “target” cell on the “adjacent” cell was studied. More specifically, the ratio between the activation threshold of the “target” cell and the activation threshold of the “adjacent” cell when the stimulation is given in the “target” cell electrode, were studied. Further, the effect of various coverage percentages of the cells by the micro-well were studied.
  • activation contrast ratio is defined as the ratio of the activation threshold of the target pixel to the activation thresholds of the adjacent pixels during electrical stimulation of the target pixel.
  • Figs. 4A-B show that in the sealed configuration (complete coverage case) there is a difference of more than two orders of magnitude (1 : 430) between the activation charge threshold of the target cell and the adjacent cell (Fig. 4A), suggesting an excellent contrast of patterned neural activation. This ratio decreased as the cell coverage declined, reaching a difference of 5 -fold for the case of almost no coverage (0.1% coverage). It was further found that when the target cell was stimulated by 9.8pC to elicit a lOmV change in the Vm, the change in the adjacent cell was only 0.09mV, corresponding to a ratio that is higher than 100.
  • PRPs partially differentiated photoreceptors
  • Example 5- The PRP cells exhibit glutamatergic synapses
  • the fabricated in- vivo implant features a central section containing a multi-electrode array of 3,172 micro-well electrode complexes with a pixel size of 10pm.
  • Each micro-well is 10pm in height (Figs 6A-C) and has an electrode with a diameter of 6pm at the center of its bottom ( Figures 6D-E, b', b").
  • a handle is attached to facilitate its placement beneath the retina and for electrical connectors (Fig. 6A).
  • the experimental implant enables the stimulation of the cells at a single row resolution.
  • implant features enhanced cell -micro-well coupling.
  • PRPs glutamatergic neurons
  • three implant prototypes were fabricated using the same optimized lithography process with different micro-well diameters (10, 15, and 20pm) (Fig. 7, panes a-c).
  • the photolithography substance SU8 was mixed with rhodamine- fluorophore to enhance the visibility of the micro-well walls.
  • GFP -labelled PRP cells were then seeded onto these devices; cells were fixed 24 hours after the culture and stained for nuclei (Hoechst) and actin (phalloidin).
  • the percentage of the micro-well wall area that was in contact with the cell membrane was calculated by colocalization analysis of the cell fluorescence (GFP and actin staining) and the SU8 implant fluorescence (rhodamine B, red) (Fig. 7, panels d-f).
  • a micro-well was defined as sealed by the neuron for wells in which the contact area formed a complete closed circle without any gaps or breaks (e.g., Fig. 7, panels e’, f ) and non-sealed if there was a gap (e.g., Fig. 7 panel d’)
  • micro-wells seeded with PRP were fixed and embedded using Epon, cut using an ultramicrotome into 50-70nm sections and then imaged with transmission electron microscopy (TEM; JEM-1400 Flash Electron Microscope).
  • TEM of PRP cells seeded in a 20pm diameter SU8 well revealed several cells inside the well, as inferred by the nuclei in the image (Fig. 9A).
  • a zoom-in on the cell-well interface revealed a close contact between the cells and the well walls on both sides (Figs. 9B-C), with a visible gap present in only a few locations.
  • Example 7 The micro-well electrode configuration significantly reduces the activation charge
  • a stimulating current with pulse durations ranging from 40ps to 1msec and with increasing amplitude was injected (through the electrode at the bottom of the well), while the cell membrane potential was recorded via the intracellular patch-clamp electrode.
  • the activation threshold was defined as the lowest current required to induce an action potential or an inward Na + current.
  • relatively larger cells namely, HeK239 cells engineered to express the voltage -sensitive Na + channel Na2.1 were used.
  • 5pm-high micro-wells were used, rather than ones with the full height, 15pm.
  • micro-well sealed the micro-well electrode configuration was used and only cells in which the fluorescence staining pattern suggested complete sealing with the ring of the micro-well were studied (Fig. 10B, yellow arrow).
  • flat electrode a similar setup was used, but here the electrodes were not encircled by micro-well structures.
  • distant condition the effect of the cell-electrode distance on activation thresholds was further tested by coating the implant prototype with a uniform layer of collagen (30-50pm, using 10% collagen gel). This layer thickness was selected to reflect the distance between the implanted subretinal electrodes and the bipolar cells (estimated at about 40pm).
  • Figs. IOC and 10D demonstrate the strong effect of the micro-well sealing and the cell-electrode distance on the activation threshold.
  • the threshold for cell activation was pulses of 5ms and 1.5mA. Most cells were not activated by the maximum current amplitude and duration parameters of the system (10ms, 1.5mA).
  • cells in the flat condition which were cultured directly on flat electrodes (no microwells), exhibited a significantly lower activation current and charges (Fig. 10C-D).
  • micro-well sealed configuration which decreased the threshold even further, to the picocoulomb range (for 40ps pulses, a charge threshold of 420 ⁇ 170pC and 2.6 ⁇ 2.8nC (average ⁇ st.dev.) for the sealed and flat configuration, respectively, p ⁇ 0.01). More importantly, since in this experiment micro-wells with a height of 5pm (rather than 15-20pm) were used, and it is estimated that the cell-micro-well wall coverage is only 25%, based on the simulation results, the expected threshold of a completely sealed configuration is 1.5-orders- of-magnitude lower and is, therefore, expected to be around 13.28pC. Thus, the measured reduction in the activation threshold by the tight-sealing micro-well configuration is within the pC range and fits well with the computer simulation.
  • Example 8 Implantation of the retinal prosthesis into the sub-retinal space and integration of the glutamatergic cells with the host retina
  • the in-vivo survival of the PRPs in an implanted hybrid device and their axonal sprouting and synaptic connection with the host retina was tested.
  • the device was seeded with GFP- labeled PRPs before implantation into the sub-retinal space of immunosuppressed RCS rats (a known retinal degeneration model).
  • Fundus imaging and optical coherence tomography revealed the anatomical position of the implant in the subretinal space, in close proximity with the inner nuclear layer (INL), where the target bipolar cells are located (Figs. 11A-B).
  • biomolecules are hydrophilic peptides containing amid and carboxylic groups on their chain side, they elicited a decrease in the contact angle compared with the untreated (bare) gold surface, as is shown in Figs 14A-C. All the biomolecules used showed a similar decrease in contact angle compared with the bare gold surface (p ⁇ 0.05); the lowest contact angle was measured for linear short RGD at 22.2° (Fig. 14D). The contact angle decreased with increasing biomolecule concentration, reaching a plateau at l/12mg/ml (Fig 4E).
  • Example 10- Surface modification using biomimetic peptides - Retinal Cells are Attracted by YIGSR biomolecules
  • HEK293 and retinal cells were cultured on gold surfaces coated with the tested biomolecules at a concentration of 1/3 [mg/ml]. Quantification of the results, which are presented in Figs. 15A-B suggest that HEK293 cells were mainly attracted to the RGD-type biomolecules, whereas the retinal cells were mainly affected by the YIGSR-type molecules.
  • HEK293 cells Fig. 15A
  • all biomolecules elicited a higher cell density compared with DDW (one way ANOVA p ⁇ 0.001 for all biomolecules).
  • the highest cell density was observed for the long-spacer cRGD of 6.2 ⁇ 1.3 (p ⁇ 0.005 for a comparison with all other molecules), followed by the short spacer non-cyclic RGD (GG-RGD, (5.02 ⁇ 2.3).
  • GG-RGD short spacer non-cyclic RGD
  • the retinal cells (Fig. 15B) were mainly attracted to the YIGSR ligand.
  • the highest cell density was found for the long spacer YIGSR (Poly-Pro- YIGSR) at the lowest concentration (3.6 ⁇ 1.6), p ⁇ 0.001 compared with DDW).
  • FA focal adhesion
  • the RGD molecules affected the HEK293 surface attachment, showing developed filopodia and cell spreading.
  • YIGSR affected the rat- dissociated-retinal cells more than the RGD-type molecules.
  • Quantitative analysis of the average cellular number of FA spots is presented in Figs 17A-B, for HEK293 (Fig 17A) and retinal cells (Fig. 17B).
  • the cyclic RGD molecules elicited significantly more FA spots compared with the YIGSR molecules (p «0.01 for both cyclic RGD vs both YIGSR and DDW).
  • a Y-27632 on neurite extension in primary retinal rat dissociated cells was tested. Following dissociation, cells were treated with araC to eliminate all non-neural cells, including Muller glial cells, that can introduce confounding effects and hinder the interpretation of results. To this aim, a dose-dependent effect assay was conducted, comparing axonal length following 72h treatment with Y-27632 at concentrations of 50 pM, 100 pM, and 200 pM (Figs. 19A-D), with untreated samples serving as controls. Staining for CRX, a PRP-specific marker, was carried out to ensure the quantification of PRP neurite length exclusively. Actin staining was used to visualize the cell neurites (Figs.
  • Y-27632 at 200 pM could be due to the molecule's toxicity, as it was reported that ROCK inhibitors at elevated concentrations affect other kinases, such as protein kinases A and C and myosin light chain kinase, potentially leading to unintended side effects.
  • Example 15- ROCK inhibitor enhances axonal guidance and phototransduction gene expression in retinal cells.
  • BMP signaling including BMP4 and MAPK12 genes
  • phototransduction genes such as rhodopsin (RHO) and transducin (GNAT1)
  • CREB signaling such as DRD4, OXTR and SSTR3
  • RNA-seq PCR analysis was conducted.
  • Example 17 - BMP4 inhibition reduces neurite extension in taurine and RCM but not Y- 27632-treated cells.
  • Example 18- Y-27632 enhanced neurite elongation in an ex-vivo model of Degenerated Retina
  • Neuronal cells cultivated in a two-dimensional culture are exposed to different microenvironments and biological signals compared to those grown in a three-dimensional culture or within intact tissue. Therefore, when studying neurite extension, it is important to replicate the natural neural and retinal tissue environment.
  • retinal explants from three- month-old RCS rats a model of outer retinal degeneration disease, where the photoreceptor cells degenerate, leaving the bipolar and ganglion cell layers relatively preserved, were used (Figs. 22A-D).
  • PRP cells labeled with AAV1-GFP virus were seeded onto the retinal explant, with the bipolar cell layer facing upward. The samples were treated with Y-27632 50pM or Taurine ImM.
  • an artificial photoreceptor outer nuclear layer was generated by applying photoreceptor cells incorporated within a collagen hydrogel mixture and seeded them above the inner nuclear layer (INL). It was found that the length of cell extensions within the collagen was longer compared to cells treated with Y -27632 (22.01 ⁇ 0.71 vs 17.87i0.79, p ⁇ 0.001, Fig. 22E).

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Abstract

Provided herein are hybrid neuro-electronic retinal implants integrating micro-well electrode arrays, biomimetic surface coatings, glutamatergic neurons, and neurotrophic modulation for at least partial vision restoration in retinal dysfunctional conditions.

Description

HYBRID RETINAL NEUROPROSTHESIS AND METHODS OF USING THE SAME
FIELD OF THE INVENTION
The invention relates to retinal prosthetics. More specifically, it concerns hybrid neuro- electronic retinal implants integrating micro-well electrode arrays, biomimetic surface coatings, glutamatergic neurons, and neurotrophic modulation for at least partial vision restoration in retinal dysfunctional conditions.
BACKGROUND
Degenerative diseases of the outer retina are among the most common causes of blindness in developed countries. These disorders are marked by degeneration of the lightsensitive cells of the retina (photoreceptors), whereas the inner retinal neurons are relatively spared. Cell replacement therapy involving photoreceptor transplantation (for example, transplantation of human pluripotent cell-derived retinal pigment epithelium (RPE) or photoreceptor precursors (PRP)), faces challenges such as survival, axonal elongation and synapse formation between donor cells and the host retina. Another vision restoration approach in such conditions is electronic retinal prosthetics, wherein viable cells, either the ganglion or the bipolar cells, are electrically stimulated in a patterned manner. However, the visual acuity obtained with the currently available technologies is still relatively low (e.g., visual acuity of 20/550-20/1200 in most patients).
Some of the underlying causes for this low acuity in current retinal prostheses include, for example, low pixel resolution, electrode crosstalk, and axonal stimulation. However, improving the spatial resolution, and thus increasing the obtained acuity, can be theoretically achieved by reducing the electrodes and pixels size. More specifically, reducing the electrode spacing to 5 pm and increasing the pixel density could theoretically increase the obtained visual acuity to 20/20. However, the ability to increase the electrode density is limited by the electrode-neuron distance, since the electric field drops quadratically as the distance increases. Optimally, electrodes should be located no farther than one electrode diameter (i.e., a few micrometers) away from the target cells. Although a subretinal approach appears to provide consistent proximity to the retina along the implant, even with this approach, the bipolar cells, which are the target cells in this case, are separated from the implant surface by about 40pm. This limitation is further exacerbated in the epiretinal prosthesis approach, where the estimated distance of the electrodes from the target cells is around 200pm. To increase the proximity, three-dimensional pillar electrode arrays were suggested, however, the layered structure of the retina does not allow proximity between neurons and electrodes.
The second challenge with current retinal prosthetic approaches is their inability to replicate the selective and distinct opposite responses of the ON and OFF and other pathways during natural vision. This arises because the electrical activation elicits non-selective depolarization of all types of bipolar cells (BPC) (e.g., ON, OFF) or all types of retinal ganglion cells (RGCs). Several groups have addressed this issue by optimizing pulse parameters, to selectively activate specific retinal cells or layers (e.g., BPC, RGC, photoreceptors, and nerve fiber layer) or specific circuits. Several groups have demonstrated the selective activation of ON and OFF RGCs (not BPCs) by modulating the amplitude of short high-frequency biphasic pulses, as well as through other pulse configuration manipulations. However, this method does not allow for selective network-mediated activation of retinal circuits. Consequently, this leads to significant differences in the network-mediated RGC responses compared to those induced by natural light activation.
A third limitation of current prosthesis technologies is that they stimulate retinal neurons in a pulsed rather than in a continuous graded potential manner, which is the mode of operation that provides the natural visual system with unrivaled sensitivity over a wide dynamic range. This non-natural mode of activation could be the underlying mechanism of the low contrast sensitivity found in pre-clinical (12% in a behavioral study in Royal College of Surgeons (RCS) rats) and clinical work.
Cell adhesion to electrode surfaces is important for improving neural prosthetic interfaces, particularly in retinal implants. Traditional coatings (poly-L-lysine, laminin, fibronectin) enhance general cell adhesion but lack cell-type specificity.
Accordingly, there is a need for hybrid retinal implants that can overcome such challenges of low pixel density and electrode crosstalk, non-selective activation and pulsatile stimulation, and which can provide retinal implants with a tight neuron-electrode coupling, with reduced activation thresholds and increased spatial resolution, which are safe and efficient and which can provide enhanced visual acuity and which can provide at least partial vision restoration in subjects in need thereof. SUMMARY OF THE INVENTION
The present invention provides, in embodiments thereof, relates to retinal prosthetics, incorporating hybrid neuro-electronic retinal implants integrating micro-well electrode arrays, glutamatergic neurons (e.g. photoreceptor precursor cells), with optional biomimetic coating and neurotrophic modulation for at least partially allowing vision restoration in various dysfunctional conditions, including, retinal degenerative diseases.
According to some embodiments, there are provided herein advantageous hybrid retinal implants, which include high-density electrode array integrated with glutamatergic neurons. The electrodes are designed to create a tight neuron-electrode coupling by a 3D micro-well geometry, thereby enabling the activation of the neurons by small-sized electrodes at high density arrays, allowing both a significant reduction in the activation threshold, reduced electrodes cross-talk and an increase in spatial resolution. Following implantation of the hybrid prosthesis, the neurons can synapse with the retinal neural circuits of the host bipolar cells and other cells such as the horizontal cells. Upon patterned electrical stimulation of the integrated glutamatergic neurons by the electrodes, such neurons can activate the host bipolar cells while mimicking and preserving natural visual pathways.
According to some embodiments, as exemplified herein below, the disclosed micro-well configuration facilitated the confinement and amplification of the electrical field around the glutamatergic neurons. Further, the simulation shows a significant increase in spatial resolution, down to at least about 10pm, while eliminating electrode crosstalk. Furthermore, as exemplified herein, advantageously, the array allows three-order-of-magnitude reduction in the activation threshold in neurons and photoreceptors, reaching picocoulomb (pC) level. Further, advantageously, a hybrid implant incorporating glutamatergic neurons (hESC-derived photoreceptor precursors) implanted, in-vivo, in the subretinal space of retinal degenerated rats, confirmed the survival of the seeded cells within the micro-wells, axonal sprouting toward the host bipolar cells and plausible synapses.
According to some embodiments, there is provided a hybrid retinal prosthesis including: a micro-well electrode array having wells with diameters between about 5 pm and about 50 pm for confining neurons; and a biomimetic peptidomimetic coating comprising adhesion motifs associated with the electrode surfaces. According to some embodiments, the wells have diameters in the range of about 5 gm- 15 gm.
According to some embodiments, the peptidomimetic coating includes thiol- functionalized adhesion motifs.
According to some embodiments, the peptidomimetic coating may include thiol- functionalized adhesion motifs selected from YIGSR (SEQ ID NO:6), IKVAV (SEQ ID NO: 7), and cyclic RGD (SEQ ID NO: 1).
According to some embodiments, the peptidomimetic coating includes a peptide having an amino acid sequence CGG-YIGSR (SEQ ID NO: 4) or C-PolyProline(10)-YIGSR (SEQ ID NO: 5).
According to some embodiments, the peptide density on the electrode surface is at a density sufficient to promote integrin-mediated adhesion of photoreceptors while minimizing non-specific binding of non-retinal cells, as measured by differential adhesion assay.
According to some embodiments, the micro-well electrode array may include a conductive material electrode at the well base, said electrode made of or coated with gold, platinum, indium tin oxide (ITO), activated iridium oxide film (AIROF), Sputtered iridium oxide films (SIROF), Titanium, Titanium nitride (TiN), Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate(PEDOT:PSS), or any combinations thereof. Each possibility is a separate embodiment.
According to some embodiments, the micro-well height is in the range of about 10 gm to 20 gm.
According to some embodiments, the prosthesis may further include glutamatergic neuronal cells, horizontal cells, amacrine cells, or other neural cells that can communicate with the bipolar cells and/or retinal ganglion cells and other retinal circuitry within said micro-wells. Each possibility is a separate embodiment.
According to some embodiments, the cells may include photoreceptor precursor cells (PRPs).
According to some embodiments, the PRPs may be human embryonic stem cell-derived photoreceptor precursors (hESC-PRPs), induced pluripotent stem cells (iPSCs), fetal retinal progenitors, or primary photoreceptor cells. According to some embodiments, the prosthesis may further include a composition including small molecule substances and/or growth factors, configured to enhance neurite elongation of cells (such as, PRPs).
According to some embodiments, the small molecule substances may include a ROCK- inhibitor and/or a BMP4-activator.
According to some embodiments, the small molecule substances may include Y-2763, fasudil, or hydroxyfasudil, taurine, retinal-conditioned medium (RCM), or any combinations thereof.
According to some embodiments, the prosthesis may further include collagen, Neuronal growth factor (NGF) and/or extracellular matrix (ECM) hydrogel scaffold to enhance cells (e.g. PRP) survival and axonal guidance.
According to some embodiments, the micro wells may be pretreated with plasma, prior to adding cells to the wells.
According to some embodiments, the prosthesis is configured for implantation into a target area of a subject having retinal dysfunction, to thereby at least partially restore vision in the subject.
According to some embodiments, the target area may include subretinal, epiretinal, or suprachoroidal space.
According to some embodiments, the prosthesis is for use in at least partially restoring scotopic, photopic, mesopic, color vision, improving contrast sensitivity, enhancing visual acuity, or any combinations thereof.
According to some embodiments, the subject is having or afflicted with retinitis pigmentosa, age-related macular degeneration, or photoreceptor degeneration.
According to some embodiments, there is provided a method for at least partially restoring vision to a subject having retinal disfunction, the method includes implanting into a target area of the subject the prosthesis as disclosed herein.
According to some embodiments, the hybrid retinal implantable prosthesis includes: a high-density micro-well electrode array having wells of about 5-50pm diameter; and glutamatergic neurons seeded within said wells; wherein geometry of the microwells provides tight sealing to the cells, to thereby amplify local electric fields, and reduce activation thresholds to about 950pC or less.
According to some embodiments, the activation thresholds is reduced to less than about 500pC.
According to some embodiments, a pixel pitch of the micro-well array may be about 10 pm or less, thereby enabling a visual acuity of at least 20/40.
According to some embodiments, electrical stimulation of the sealed neurons may modulate intracellular calcium, potassium and/or sodium dynamics, leading to neurotransmitter release, resulting in selective ON/OFF bipolar pathway activation and/or activation of other retinal circuitry
According to some embodiments, the electrical stimulation of the sealed neurons may facilitate controlled calcium influx and release of glutamate, resulting in selective ON/OFF bipolar pathway activation and/or activation of other retinal circuitry.
According to some embodiments, the sealing may provide an activation contrast ratio of about 1:400 or more, between a target micro-well and adjacent micro-wells, thereby reducing crosstalk between adjacent cells, thereby enhancing resolution and contrast of the restored vision.
According to some embodiments, the wells may be pretreated with plasma prior to seeding the cells.
According to some embodiments, the wells may be made of or coated with photoresists or epoxy-based polymers (e.g., SU-8).
According to some embodiments, there is provided a method for surface modification for retinal cell-specific adhesion to a substrate, the method includes:
Providing or obtaining a substrate comprising an electrode; and coating said electrode with a biomimetic peptide comprising an amino acid sequence YIGSR (SEQ ID NO: 6), an anchor (such as, a thiol anchor) and a spacer, wherein the coating promotes preferential adhesion of retinal cells over non-retinal cells by enhancing cell density, spreading, and/or focal adhesion formation.
According to some embodiments, the surface modification method may further include seeding cells on a surface of the coated substrate. According to some embodiments, there is provided a biomimetic coating composition for promoting retinal cell adhesion to a substrate, the composition includes a peptide having an amino acid sequence CGG-YIGSR (SEQ ID NO: 4) or C-PolyProline(10)-YIGSR (SEQ ID NO: 5), wherein said peptide selectively promotes retinal cell adhesion to a substrate.
According to some embodiments, the substrate is or comprises electrodes. According to some embodiments, the electrode comprises gold, platinum, indium tin oxide (ITO), activated iridium oxide fdm (AIROF), Sputtered iridium oxide fdms (SIROF), Titanium, Titanium nitride (TiN), (PEDOT:PSS) coated electrodes, or any combinations thereof.
According to some embodiments, there is provided a method of promoting neurite outgrowth in photoreceptor precursor cells (PRPs), the method includes contacting said PRPs with a ROCK inhibitor for a period of time, under suitable conditions, to thereby induce increase in neurite length by at least 5 -fold compared to untreated PRPs.
According to some embodiments, the ROCK inhibitor is Y -27632, provided at a concentration in the range of about 50 pM and 100 pM.
Other objects, features and advantages of the present invention will become clear from the following description, examples and drawings.
Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more other technical advantages may be readily apparent to those skilled in the art from the figures, descriptions, and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.
BRIEF DESCRIPTION OF THE DRAWINGS
Some embodiments of the disclosure are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments may be practiced. The figures are for the purpose of illustrative description and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the disclosure. For the sake of clarity, some objects depicted in the figures are not to scale.
In the Figures: Fig. 1 - a schematic illustration of a hybrid retinal prosthesis, according to some embodiments;
Figs. 2A-2C show the effect of sealing wells, on electrical Field confinement. Fig. 2A shows an illustration of a 2D cross section of the electrical field in a flat (non-sealed), partially sealed (1pm cell-microwell wall spacing) and a sealed (0.05 m spacing) configuration. White: extracellular space, gray: SU-8 microwell. Blow up: Zoom in on the cell membrane- extracellular domain interface; Fig. 2B shows bar graphs of the electrical field at the interface of the cell membrane for the different configurations at 10pm (the height marked with a dashed line in a); Fig. 2C shows a graphs of the electrical field at the cell membrane-microwell interface for varying cell-microwell spacing (ranging from the sealed to the partially sealed configuration), demonstrating the electrical field decrease (circles). The simulations results are fitted to a 1/Spacing power function (Solid line);
Figs. 3A-B show graphs demonstrating the effect of sealing on cell membrane potential. Fig. 3A- The induced membrane potential deflection (AVm) for three tested configurations (Flat, partially sealed and sealed), as a function of the charge (C) injection are presented; Fig. 3B- The charge threshold for a 1 msec pulse for three tested configurations, compared to thresholds obtained in intracellular stimulation experiments, h is electrode-cell distance, S is cell-well spacing. All experimental data bars represent the mean±SD;
Figs. 4A-4B show Spatial Resolution testing - Fig. 4A shows an illustration of the simulation set-up. The ratio between the activation threshold of the target cell and the neighboring cell upon current injection in the target cell is tested; Fig. 4B- graph presenting he ratio of the activation threshold of the target cell to the activation threshold of the adjacent cell during electrical stimulation of the target cell, as a function of target cell microwell coverage;
Figs. 5A-5D - Calcium imaging of voltage driven activity in PRP using the calcium indicator OGB. Fig. 5A shows PRP cells stained with the calcium indicator OGB. The patched cell is demarcated with a purple circle and the tested neighboring cell with green; Fig. 5B shows the calcium change observed in a cell (green) in proximity to the patched one (purple), revealing a synaptic mediated response. Black arrows indicate the voltage step onset; Fig. 5C shows representative calcium change in a cell that is in proximity to a patched one, before (grey solid line) and after the application of synaptic blockers (red solid line) highlighting the synaptic mediated response; Fig. 5D shows the maximal calcium change in in a cell in proximity to a patched one before and after the addition of blockers (n=6, p< 0.001 paired t-test). Scale bar=50pm;
Figs. 6A-6E show imaging of a 1 mm diameter implant. Figs. 6A-C show SEM images of a 10-micron microwell size implant. Fig. 6A shows a low magnification image where the implant’s center has a dense area of microwells (Scale bar 400 pm). Figs. 6B-6C provide magnified views of the implant with a 10pm microwell diameter. Figs. 6D-6E show images of another implant with 15 pm microwell diameter, where an electrode is positioned in the center of each well. Fig. 6E shows cross-sectional FIB/SEM images, highlighting a single microwell with an electrode at the bottom (indicated by an arrow). Scale bar in Figs. 6B-E- 10 pm;
Fig. 7 shows Confocal imaging of cell-well interface for microwells with varying diameters showing the confinement of the PRP within the micro-well geometry. Panels a-c show (a) 20pm well (b) 15pm well (c) 10pm well. Imaging shows cells within the wells (wells in red, cells in green). Panels a', b', c', shows magnified view of a cell within a well, illustrating how the cells assume the well shape. Panels d-f show Visualization of the contact area between the wells and the cells (yellow), (d) 20pm well (e) 15pm well (f) 10pm well. Panels d', e', f show magnified representative view of the contact area for a cell within a well. Panels g-I show Imaging of the cell nuclei within the wells (nuclei shown in blue), (g) 20pm well (h) 15pm well (i) 10pm well. Panels d', e', f’ show magnified representative view of a cell inside a well. Each cell nucleus is represented by a different color. Panels j, k, 1 - show top view of phalloidin staining of actin filaments (j) 20pm well (k) 15pm well (1) 10pm well. Panels j’, k’, 1’ show side view for actin filaments. Scale bar :(panels a-i) 50pm, (panels a'-i’): 10pm. (panels j, j ’, k, k’, 1, 1’) -10pm;
Figs. 8A-8C show Quantitative analysis of the cell-well interface for the various microwell diameters. Fig. 8A- shows percentage of the internal surface area of the microwell that is in contact with the cells; Fig. 8B shows Percentage of sealed microwells occupied by cells; Fig. 8C shows Average number of nuclei per well (the mean±SD). ** p<0.01, ** p<0.001;
Figs. 9A-9C show TEM imaging of a PRP cell inside a SU8 well showing the sealing of the PRP by the micro-well wall. Fig. 9A- Few cells can be seen within the well indicated by pound sign. The SU8 is indicated by an asterisk. Figs. 9B-9C show magnified views of the region demarcated by the dashed squares in Fig. 9A, namely a’ and a”, respectively. Arrows point to the contact points between the cell membrane and the SU8; Fig. 10A-D show In-vitro studies demonstrating activation threshold reduction. Fig. 10A shows illustration of three different experimental conditions: Distant (mimicking subretinal implants), flat, and sealed; Fig. 10B shows A fluorescence image demonstrating sealed cell (indicated by the perfectly round fluorescence around the well rim, yellow arrow) and the patch-clamp pipette (white arrow). Asterisks: electrode leads; Fig. IOC shows Strengthduration curves for all experimental configurations showing a significant reduction in the activation threshold for cells sealed in a well; Fig. 10D shows Charge activation thresholds for a 100 psec pulse for flat and microwell-type electrodes and for distant conditions. All bars present the mean±SD. Flat n=l 1, sealed n=6, p=0.025;
Figs. 11A-11D show in-vivo testing of seeded PRP viability following device implantation in the subretinal space of an RCS rat; Fig. 11A Confocal scanning ophthalmoscopy (cSLO) imaging (left) and OCT of the cross section marked by the green line, highlighting the localization of the implant within the subretinal space; Fig. 11B - Left-IR cSLO fundus imaging; right - fluorescence fundus imaging revealing the survival of seeded cells 7 days post implantation; Fig. 11C Fundus imaging of a transplanted animal at various time points following transplantation (Top) and Fluorescence imaging of the same region (bottom); Fig. 11D Cell viability at various time points post transplantation on implant or by bolus injection;
Figs. 12A-12D show Confocal imaging of a whole mount retina, 30 days following the subretinal transplantation of the hybrid implant. Fig. 12A - The implant (yellow arrow) is located in the subretinal space below the INL. In blue- cell nucleus, red- implant, green - GFP labeled PRPs cells; Fig. 12B- Confocal imaging of the whole mount retina in Fig. 12A - focusing on the GFP labeled PRPs plane; Fig. 12C- Retinal cryosection of the whole mount, demonstrating the location of the PRPs in the implant's wells, and the outgrowth of GFP labeled neurites (white arrows). Red- implant green- GFP-labeled PRPs, blue- nuclei. INL- inner nuclear layer, GCL- ganglion cell layer; Fig. 2D shows enlarged area of cells showing the presumed synapse between transplanted cells and host bipolar cells, based on the colocalization of GFP PRP cells (green) and ribeye synapses (red), in proximity with bipolar cells (magenta, stained with PKC alpha). Arrows denote the areas of co-localization. Scale bar: 5pm;
Figs. 13A-13B Assembly of coating biomolecules to gold surfaces. Shown are STED Fluorescent images of untreated (Fig. 13A) and fluorescent RGD-NBD-coated glass-gold paterned surfaces (Fig. 13B). RGD assembly is revealed mainly in the gold areas. Scale bar 300pm;
Figs. 14A-14E show contact angle measurements presenting gold surface modifications: Fig. 14A- Contact angle images of untreated surfaces; Fig. 14B- short linear RGD coated surfaces; Fig. 14C- short linear YIGSR coated gold surfaces. Fig. 14D- water contact angle of gold surfaces coated with the various molecules (N=3), p<0.05; Fig. 14E- effect of the biomolecule concentration on the contact angle (N=3), p<0.05;
Figs. 15A-15B- Effect of coating biomolecules on cell density. Fig. 15A- shows results relating to HEK 293 cells and Fig. 15B shows results relating to rat retinal cells. Cells were seeded on gold surfaces coated with various biomolecules at several concentrations. Cell density was evaluated using a Leica LMD7 microscope and a binarization Image J algorithm. For each biomolecule, the cell density was normalized to a cell density of DDW. *Red star denotes significance compared to control, p«0.05. **Black star denotes significance compared to a long cyclo-RGD [Poly-Pro-c(RGD)] -coated surface, p«0.05.
Figs. 16A-16B- Effect of coating molecules on the cell surface area. Fig. 16A shows results relating to HEK293 cells and Fig. 16B shows results relating to rat retinal cells. The cells were incubated for 72h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/ml) followed by fixation and staining. The average surface area from each biomolecule-coated surface has been normalized to that of DDW. *p< 0.05, ***p< 0.001, compared to untreated (bare) gold;
Figs. 17A-17B- Effect of coating molecules on the focal adhesion spots. Fig. 17A shows results relating to HEK293 cells; Fig. 17B shows results relating to retinal cells. The cells were incubated for 72h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/ml) followed by fixation and staining. The number of bright Vinculin spots was counted manually. *p < 0.03, **p < 0.001;
Figs. 18A-18B show bar graphs presenting relative normalized gene expression levels of adhesion integrins and focal adhesion proteins as affected by coating biomolecules as measured by real-time qPCR. Fig. 18A shows results relating to HEK293 cells; Fig. 18B shows results relating to Rat-dissociated retinal cells. Cells were incubated for 72h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/ml) before RNA extraction. Expression levels were normalized to the expression level of the GAPDH gene, used as a reference gene. *p< 0.05 compared to DDW. Figs. 19A-19F - Quantification of neurite extension following ROCK-I inhibitor administration (Y-27632) in-vitro. Confocal images of rPRP culture seeded for 72h on coverslips are shown: Fig. 19A- untreated cells; Fig. 19B- cells treated with ROCK inhibitor, Y-27632 (Y -27) 50pM, Fig. 19C- cells treated with lOOpM Y-27; Fig. 19D- cells treated with 200pM Y-27. nuclei (blue), CRX (green), actin (magenta). White arrows point to neurite extents from PRP cells. Scale bar: 50pm; Fig. 19E- Quantification of Y-276332 effect on neurites longer than 5pm under the various treatments (N>200 cells, mean ± SEM); Fig. 19F- percentage of PRP cells with neurites for the various treatments: N>3 cells, mean ± SEM. *P<0.05, **P<0.01;
Figs. 20A-20J show the effect of various molecules (Taurine, RCM, and BMP4 inhibitors) on neurite extension in PRP. Shows are Confocal images of untreated PRP (Fig. 20A), PRP treated with ImM Taurine (Tau, Fig. 20B), ImM Taurine + 50pM Y-27632 (Tau+Y-27, Fig. 20C), retinal condition media (RCM, Fig. 20D), RCM + 50pM Y-27632 (RCM+Y-27, Fig. 20E). Noggin (BMP4 inhibitor) with 50pM Y-27632 (Y-27 + Nog, Fig. 20F), Noggin with Tau (Tau + Nog, Fig. 20G), Noggin with RCM (RCM + Nog, Fig. 20H). Nuclei (blue), CRX (green), actin (magenta). White arrows point to neurite protrusions from PRP (CRX stained cells). Scale bar: 50 pm. Quantification of average neurite length for the various treatments for extensions longer than 5pm are shown in Fig. 201 (N>200 cells, mean ± SEM); the percentage of PRP cells with neurites per treatment are presented in Fig. 20J (N>3 fields of view, mean ± SEM). *P<0.01, **P<0.01, ***P<0.001;
Figs. 21A-21D- BMP4 mediates neurite extension in PRP. Figs. 21A-D show Confocal images of neurite extension in-vitro of untreated PRP (Fig. 21A) and cells treated with 40ng/mL BPM (Fig. 21B). Nuclei (blue), CRX (green), actin (magenta). White arrows point to neurite extents from CRX positive cells. Scale bar: 50pm; Figs. 21C-21D show bar graphs of quantification of the effect of BMP4 treatment on average neurite length in extensions longer than 5pm (Fig. 21C) and percentage PRP with neurites per treatment (Fig. 21D); N=12 fields of view, mean ± SEM. *P<0.05; and
Figs. 22A-22E- show Ex-vivo neurite extension of retinal cells seeded on 3m-old RCS retina following 24h treatments of 50pM Y-27632, ImM Taurine, collagen. Confocal top view images of: Fig. 22A- control sample (ctrl); Fig. 22B- 50pM Y-27632 treated culture (Y -27); Fig. 22C- ImM Taurine treated culture (Tau); Fig. 22D- cells and collagen mixture (Clgn). Nuclei (blue) seeded cells (green). Panels A’, B’, C’, D’- only green channel. In each image a characteristic single neurite is indicated by purple and orange arrows. Panels A”, B”, C”, D” depict cross section of the selected neurite path. Scale bar: 20pm; Fig. 22E- Quantitative analysis of retinal cells neurite extension for the various treatments. N<85, mean ± SEM, ***P<0.00I. INL - Inner nuclear layer.
DETAILED DESCRIPTION
The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art will be able to implement the teachings herein without undue effort or experimentation. In the figures, same reference numerals refer to same parts throughout. In the figures, same reference numerals refer to same parts throughout.
In some embodiments, there are provided herein hybrid neuro-electronic retinal implants integrating micro-well electrode arrays having photoreceptor precursor cells seeded/grown thereon, optional biomimetic surface coatings, while optionally inducing neurotrophic modulation (for example, using ROCK inhibitor and/or BMP activation), for vision restoration in various conditions, including, retinal degenerative diseases.
As used herein, the term “Micro-well” refers to a conical, concaved or cylindrical, microfabricated depression or cavity in a substrate, typically having lateral dimensions between about 2 pm and 50 pm and a height between about 5 pm and 50 pm, capable of confining a single neuron, photoreceptor precursor, or small cell cluster.
As used herein, the term “Electrode array” refers to a patterned assembly of conductive elements (electrodes) configured for electrical stimulation or recording. The electrodes may include, be made of or coated with gold, platinum, titanium, titanium-nitride, iridium oxide, Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), conductive polymers, carbon nanotubes, graphene, or any equivalent conductive material. Each possibility is a separate embodiment.
As used herein, the term “Sealing resistance” relates to electrical resistance created by tight neuron-micro-well contact configured to improve stimulation efficiency.
As used herein, the term “Pixel pitch” refers to the center-to-center spacing between adjacent micro-wells or electrode sites. A pixel pitch <10 pm denotes ultra-high-density electrode configurations enabling improved spatial resolution of stimulation. As used herein, the term “Biomimetic coating” refers to a surface modification mimicking, for example, extracellular matrix (ECM) cues to promote selective retinal cell adhesion. The term refers to a synthetic or semi-synthetic surface modification comprising peptides, proteins, polymers, or combinations thereof, designed to mimic native extracellular matrix (ECM) signals and promote selective cell adhesion
As used herein, the term “Cell-adhesion motif’ refers to a minimal amino acid sequence derived from ECM proteins (e.g., laminin, fibronectin, vitronectin) that binds integrin or other cell-surface receptors, including but not limited to YIGSR, IKVAV, RGD, cyclic RGD, and their functional analogs.
As used herein, the term “Spacer” relates to a flexible linker (e.g., 1-50 amino acids, PEG) between a surface anchor and adhesion motif, optimizing receptor accessibility. The spacer is configured to separate the adhesion motif from a surface anchor, allowing optimal receptor engagement and conformational flexibility.
As used herein, the term “anchor” refers to a moiety (e.g., thiol, such as cysteine residue, mercapto-silane) capable of forming covalent bonds with conductive electrodes, such as, metal electrodes, including, for example, gold or platinum.
As used herein, the term “Photoreceptor precursor cells (PRPs)” refers to progenitor cells capable of differentiating into rod/cone photoreceptors. In some embodiments, the cells may be derived from embryonic stem cells, induced pluripotent stem cells (iPSCs), fetal retinal progenitors, primary retina, and the like. Each possibility is a separate embodiment.
As used herein, the term “Neurotrophic composition” refers to a formulation which includes small molecules, peptides, growth factors, or other bioactive agents that enhance neuronal survival, neurite outgrowth, synaptic integration, or neuroprotection. Examples include ROCK inhibitors (Y-27632, fasudil), BMP pathway modulators, cAMP analogs, taurine, retinal-conditioned medium (RCM), BDNF, CNTF, or combinations thereof. Each possibility is a separate embodiment.
As used herein, the term “ECM hydrogel scaffold” refers to a three-dimensional matrix including natural (e.g., collagen, laminin, fibrin) or synthetic polymers configured to support PRP survival, adhesion, and guided neurite extension. As used herein, the term “Activation threshold” refers to minimal electric charge required to elicit an action potential/ or neurotransmitter release from a neuron confined within a microwell.
As used herein, the term “Activation contrast ratio” refers to the ratio of stimulation- induced activation between a target micro-well and adjacent non-target wells (unintended activation), where higher ratios indicate reduced crosstalk. The term refers to the ratio between the activation threshold of the “target” cell and the activation threshold of the “adjacent” cell when the stimulation is given in the “target” cell electrode, were studied.
Reference is now made to Fig. 1, showing an exemplary illustration of a hybrid retinal prosthesis, according to some embodiments. As shown in Fig. 1, the prosthesis includes a high density multi -we 11 electrode array (for example having about 1 Omicron pitch) . Each of the wells of the array is capable of holding photoreceptor cells, such as, Glutamatergic neurons (such as human embryonic stem cells PRPs). The prosthesis can be implanted into target tissue (e.g., host retina), whereby the implant cells can form synapses with host cells of the retina (e.g., bipolar cells, horizontal cells). This allows activation of the host cells (via the electrode array and the implanted cells that can release neurotransmitters to mimic natural retinal signaling), to facilitate at least partial restoration of vision to the host. In other words, following implantation of the hybrid prosthesis, the neurons synapse with the retinal neural circuits of the host bipolar cells. Upon patterned electrical stimulation of the integrated glutamatergic neurons by the electrodes, these neurons can activate the host retinal circuits while mimicking and preserving natural visual pathways.
According to some embodiments, as further detailed hereinbelow, the size, shape, and/or geometry of the wells, as well as optional coating thereof using biomimetic molecules, and/or use of various compositions enhancing neurogenesis, further enhance the efficiency of the prosthesis, allowing the prosthesis to restore high visual acuity.
Examples 1-8 presented herein below, demonstrate the simulation, fabrication and in- vivo functioning of an hybrid-multi-well electrode array prosthesis.
Examples 9-13 presented herein below, demonstrate biomimetic compounds that can enhance cells attachment to metal electrodes. According to some embodiments, in order to enhance the attachment of the cells to the electrodes, the electrodes may be coated with short biomolecules to mimic the retinal ECM and to elicit the adhesion of retinal cells to metal electrodes. To this aim, various molecular biomimetic compounds having various head group sequence (e.g., RGD or YIGSR), having different spatial conformation (linear or cyclic), and spacer length (short or long), were used. As exemplified herein, the various biomolecules were attached to electrodes via a semi-covalent bond through a thiol group (SH); a cell adhesion assay and a cell spreading surface were used to estimate the efficiency of the biomolecules. The generation of focal adhesion complex creation and gene regulation, which were associated with various biomolecule surface coatings were tested.
Examples 14-18 presented herein below, demonstrate the effect of various molecules and/or microenvironment on neurite extension of photoreceptor precursors (PRP). As exemplified herein, notable effect of the RhoA kinase inhibitor (ROCKi), namely Y -27632, significantly increased both the percentage of cells with neurites and the extent of neurite elongation. Additionally, the amino acid taurine and retinal conditioned media (RCM) significantly augmented neurite outgrowth. RNA-seq analysis conducted on PRP cells treated with 50pM Y-27632 revealed an upregulation in genes associated with Bone morphogenic protein 4 (BMP4) and phototransduction pathways. BMP4 inhibitor reduced neurite extension in cultures treated with taurine and RCM, supporting the involvement of these pathways in the neurite extension process. Additionally, neurite outgrowth in PRP seeded on organotypic explants of degenerated retinae was used to determine the influence of the 3D microenvironment on neurite extension. Neurite extension was significantly upregulated when PRPs were seeded on the retinal explant; this effect was enhanced by the addition of a collagen matrix that mimics the outer plexiform layer. Accordingly is has been surprisingly shown that ROCK, BMP4 and the phototransduction pathways are involved in PRP neurite elongation and further underscore the importance of small molecules and the 3D environment in this process.
According to some embodiments, there are provided herein engineered micro-well electrode array having a geometry that enables tight neuron-electrode coupling by creating a sealed interface. Such geometry may include, for example, cylindrical and/or conus-like geometry of the wells. According to some embodiments, such geometry may elicit electrical field confinement, thereby reducing the charge activation threshold to the pC level (e.g., about l-1000pC (picocoulombs), about 10-800pC, about 20-700pC, about 300-600pC, less than about 950pC, less than about 500pC, less than a bout 250pC, less than about 150pC, less than about lOOpC), and practically eliminates electrode crosstalk, thereby enabling high-resolution and high-contrast retinal stimulation. Such activation threshold is significantly lower compared to the few nanocoulombs (nC) reported for the subretinal activation threshold. In some embodiments, the hybrid retinal prosthesis is configured to stimulate neurons at ultra-low charge thresholds, including, less than about 100 pC per stimulation pulse, less than about 50 pC, less than about 10 pC, less than about 5 pC, thereby reducing energy consumption and avoiding electrode crosstalk and enabling the analog mode of stimulation (rather than pulsed mode), while achieving high-resolution neuronal activation.
According to some embodiments, the sealing of cells and the spatial confinement of the electrical field through the insolating micro-wells can eliminate the crosstalk, one of the main limitations of currently available retinal prostheses. According to some exemplary embodiments, an activation threshold ratio of about 1:400 between the targeted cell and a cell in an adjacent pixel may be obtained, for a pixel pitch of 5, 10 pm, which can provide a visual acuity of about 20/20 and 20/40, respectively.
According to some embodiments, the disclosed array enables close cell-well proximity, with a large contact area between the cell membrane and the micro-well wall, whereby, the higher the seal resistance (Rseal), the lower the required activation current. According to some embodiments, the sealing of the cells may be further strengthened by the presence of actin rings, which facilitate the mechano-electrical coupling between the micro-wells and the cells, leading to enhanced electrical coupling with the electrode and increasing the Rseal.
According to some embodiments, in order to overcome the non-specific stimulation of retinal circuitry, the hybrid retinal implant may advantageously utilize glutamatergic neurons for translating the electrical stimulation into neurotransmitter release. According to some embodiments, with the hybrid retinal implant, there is no essential need for a functional RPE layer. This feature is of particular interest for treating outer retinal degenerative diseases with both RPE and PR loss.
According to some embodiments, as exemplified herein, the transplanted PRPs create glutamatergic synapses that could modify the postsynaptic cells’ calcium level.
According to some embodiments, advantageously, the glutamatergic cells in the hybrid implant can selectively activate the distinct retina circuitry (e.g., ON /OFF, color, horizontal cell circuitry) by glutamatergic release and not essentially by direct electrical activation. This capability is needed for mimicking normal vision and processing visual information before relaying it to the visual cortex.
According to some embodiments, the survival rate of the transplanted cells in the microwell, serving as a scaffold and support for the cells, may be at least about 50%, at least 60A%, at least about 70%, at least about 80%, over a period of time (e.g., 2 days, 5 days, 14 days, 30 days, etc.). According to some embodiments, oxygen and metabolic supply to the PRPs in micro-wells may be provided in-vivo, by the inner retina through diffusion.
According to some embodiments, as exemplified herein below, the transplanted cells may integrate with the host retina, whereby at least some of the cells may extend axon-like structures toward inner retinal layers.
According to some embodiments, in order to facilitate at least partial vision restoration, patterned electrical activation and energy transfer should be provided. In order to enable the current injection photovoltaic approach can be used to convert light into electric current. With the significantly reduced activation threshold of the neurons within the micro-well, neural stimulation can be obtained by light irradiance at ambient light levels, or by using a customized projector system with light levels closer to ambient light levels, compared with currently available photovoltaic retinal prostheses.
According to some embodiments, there is thus provided a hybrid retinal prosthesis device which includes a high-density multi-electrode array configured as micro-wells with diameters of between about 5 pm and 50 pm, or any subranges thereof (for example about 5 pm, about 10 pm, about 15 pm, about 5 pm, about 5-15 pm, about 5 pm -20 pm). Each possibility is a separate embodiment. In some embodiments, the wells may be similar, identical or different with respect of size, (e.g., diameter, height), composition, geometrical shape, and/or type of electrode. In some embodiments, the array may include wells of different sizes. In some embodiments, the array may be patterned. In some embodiments, various regions of the array, may have a different distribution of wells. In some embodiments, different regions of the array, may each have a different type of wells (i.e., different with respect of size, composition, shape, etc.). For example, for a rectangular shaped array, the array may include two extreme regions having a same distribution of wells, while the central region, disposed between the two extreme regions, may have a different distribution of wells.
In some embodiments, the micro-well array is spatially patterned such that different regions exhibit distinct well characteristics. These characteristics may include variations in well size, shape, composition, or spatial distribution. For example, a rectangular array may comprise two peripheral regions (at opposite ends) that share the same well configuration, while a central region, positioned between them, contains a different distribution or type of wells. This design allows the array to be tailored for region-specific retinal architecture or stimulation needs. In some embodiments, the wells may be made of suitable photoresist polymers. In some embodiments, SU-8 may be used as a negative photoresist for the microfabrication of the wells, while allowing high aspect ratio, optical transparency, and biocompatibility. In some embodiments, SU-8 is used to define micro-well walls or scaffolding layers. In other embodiments, functionally equivalent photoresists or epoxy-based polymers may be used, including but not limited to KMPR, JSR THB, Ormocomp, EpoCore, or mr-DWL resists, provided they enable comparable resolution, thermal stability, and biocompatibility for retinal implant fabrication.
According to some embodiments, the wells of the device may further include electrodes. The electrodes may be made of any suitable material conductive material, including, for example, but not limited to: gold, platinum, iridium oxide, indium tin oxide (ITO), activated iridium oxide fdm (AIROF), Sputtered iridium oxide fdms (SIROF), Titanium, Titanium nitride (TiN), conductive polymers, (PEDOT:PSS) coated electrodes, and the like, or any combinations thereof. Each possibility is a separate embodiment.
According to some embodiments, as further detailed below, the electrodes may be coated with biomimetic compounds, facilitating the attachment of cells to the electrodes.
According to some embodiments, as further detailed below, the cells may be treated with compositions designed to enhance differentiation and/or growth, thereby enhancing, for example neurite outgrowth, and/or extend cells viability.
According to some embodiments, the wells may be associated with cells. In some embodiments, the cells may be seeded. In some embodiments, the cells may include any retinal or retinal-compatible neuronal cell type that is capable of forming functional synaptic connections with surviving host retinal circuitry. In some embodiments, the cells may be engineered cells (retinal compatible cells or other types of cells). According to some embodiments, such cells may include, for example, but not limited to: Photoreceptor lineage cells (for example, Human embryonic stem cell-derived photoreceptor precursors (hESC- PRPs), Induced pluripotent stem cell-derived photoreceptor precursors (iPSC-PRPs), Fetal or neonatal photoreceptor progenitor cells, Rod or cone photoreceptor precursor cells differentiated from retinal organoids); Retinal progenitor cells (RPCs) (for example, Multipotent progenitor cells isolated from fetal or neonatal retina capable of differentiating into photoreceptors, bipolar cells, or Muller glia, Muller glia-derived progenitors reprogrammed to a photoreceptor lineage); Inner retinal interneuron precursors (for example, Bipolar cell precursors, which can directly relay signals to ganglion cells, Amacrine or horizontal cell precursors for alternative network integration); Other excitatory or inhibitory neurons (for example, Cortical glutamatergic neurons engineered for retinal integration, Neuronal cells (e.g. PRP) genetically modified to express synaptic adhesion molecules favoring connectivity with bipolar cells, Neuronal cells (e.g. PRP) engineered to over-express or downregulate expression of various ionic channels, thus altering the activation threshold); Genetically engineered or optogenetically modified cells (for example, Neurons expressing light-sensitive opsins (e.g., channel rhodopsins) to allow hybrid electrical-optical stimulation, neurons engineered to release glutamate or other excitatory neurotransmitters in response to electrical stimulation), and the like, or any combinations thereof. Each possibility is a separate embodiment. In some exemplary embodiments, the cells may include glutamatergic neurons, photoreceptor precursors, human embryonic stem cell-derived photoreceptor precursors (hESC-PRPs).
In some embodiments, the micro-well system may be configured to accommodate multiple types of cells, including but not limited to neuronal cells, glial cells, photoreceptor precursors, stem cell-derived neurons, or other electrically excitable or support cells.
According to some embodiments, each well is configured to accept a single cell. In some embodiments, each well is capable of holding a single cell. In some embodiments, the microwell geometry provides tight sealing between the neuron membrane and micro-well wall, resulting in amplification of the electric field, elimination of electrode crosstalk, and reduction of neuron activation thresholds to picocoulomb levels, thereby mimicking graded retinal potentials. In some embodiments, the well may be essentially cylindrical. In some embodiments, the well may be essentially conical (e.g., having a conus-like shape, with the narrow portion being closer to the electrode region).
According to some embodiments, the wells may be pre-treated with plasma (e.g., N2- or 02- plasma) prior to seeding the cells.
According to some embodiments, each micro-well has a conical or cylindrical geometry configured to confine a single photoreceptor precursor cell (PRP), and wherein said confinement promotes the formation of an actin cytoskeletal ring at the cell-substrate interface, thereby achieving tight sealing of the cell within the micro-well, amplifying local electric fields, and reducing cross-talk between adjacent wells.
According to some embodiments, the pixel pitch of the micro-well array may be in the range of about 5-50 pm, or any subranges thereof. In some embodiments, the pixel pitch of the micro-well array may be less than about 40 pm, less than about 30 pm, less than about 20 pm, less than about 15 pm, less than about 10 pm. In some embodiments, the array may have a pattemed/spatial distribution of pixel pitches.
According to some embodiments, the hybrid retinal prosthesis is configured to achieve graded levels of visual acuity restoration depending on the pixel pitch of the micro-well electrode array, the degree of neuronal integration, and the disease stage of the host retina. According to some embodiments, the hybrid prosthesis may enable a visual acuity of at least 20/20, 20/40, 20/50, 20/100. Each possibility is a separate embodiment. In one embodiment, the prosthesis may have a pixel pitch of about 10 pm or more, enabling visual acuity of at least 20/40, sufficient for reading standard text and performing daily tasks. In another embodiment, an intermediate -density array with 10-20 pm pitch may provide moderate acuity of 20/50 to 20/100, enabling mobility and large-print reading. In some embodiments, lower-density arrays with over 20 pm pitch may achieve functional vision over 20/400, improving orientation and light perception for advanced retinal degeneration. In some embodiments, ultra-high-density micro-wells with less than about 10 pm (e.g., 5 pm) pitch may achieve near-normal visual acuity (20/20 or better). According to some embodiments, such a range allows the prosthesis to be adapted for varying disease severities and clinical needs, from basic orientation vision to high-resolution central vision restoration.
According to some embodiments, the neuron-micro-well interface may form an actin cytoskeletal ring that enhances seal resistance and neuron-electrode coupling.
According to some embodiments, the glutamatergic neurons may be differentiated photoreceptor precursors expressing CRX marker and capable of synaptogenesis with bipolar cells.
According to some embodiments, the electrical stimulation of the sealed PRPs may trigger a controlled calcium influx and release of glutamate, resulting in selective ON/OFF, color vision, and other bipolar pathway activation.
According to some embodiments, the sealing may provide an activation contrast ratio of over about 1:400 between a target micro-well and adjacent micro-wells, thereby reducing or eliminating crosstalk. In some embodiments, the hybrid retinal prosthesis is configured to provide a stimulation contrast ratio between a target micro-well and adjacent micro-wells of at least 1:50, at least 1: 100, at least 1:400, and in some embodiments at least 1: 1,000. This high contrast ratio may be achieved by sealing resistance created by the close neuron-micro-well contact reducing current leakage, optimized micro-well geometry confining the electric field, selective cell adhesion (using, for example coatings minimizing non-target cell adhesion, as detailed below). According to some embodiments, such contrast ratios effectively reduce or eliminate crosstalk, allowing selective activation of single neurons for high-resolution stimulation.
According to some embodiments, there is provided a micro-well electrode configuration for neural stimulation which includes an insulating micro-well with a neuron-contacting wall having a cell-membrane spacing of less than about 50nm (e.g., less than about 40nm, less than about 40 nm, less than about 30nm, less than about 20nm, less than about 15nm, less than about lOnm); an electrode disposed at the bottom of the micro-well, wherein said sealed configuration yields a charge activation threshold of less than about 500pC (e.g., less than about 400pC, less than about 300pC, less than about 200pC, less than about lOOpC, less that about 75pC, less than about 50pC, less than about 30pC, less than about 20pC, less than about 15 pC, less than about lOpC). In some embodiments, the charge activation threshold may be at least two, three, or more orders of magnitude lower than flat electrode configurations at less than about 40 pm neuron-electrode distance.
According to some embodiments, the array may include multi-layer electrodes with distinct conductive materials to enable multiplexed stimulation.
According to some embodiments, the prosthesis may further include an integrated wireless telemetry system for power delivery, bidirectional data transfer, and real-time monitoring. Each possibility is a separate embodiment.
According to some embodiments, the prosthesis may be fabricated using lithographic, nanoimprint, or 3D printing technologies, enabling customizable well geometries. Each possibility is a separate embodiment.
According to some embodiments, the wells (in particular, the electrodes) may be coated prior to seeding the cells, in order to enhance the attachment of the cells to the electrodes.
According to some embodiments, the coating disclosed herein can promote retinal cell adhesion by the self-assembly monolayer (SAM) of bio-peptidomimetics, which imitates the ECM adhesion motifs and promotes FA (Focal adhesion) formation, to thereby improve neuron-electrode interfaces in the electronic retinal prosthesis or for devising a scaffold integrated with retinal cells. As exemplified herein, cell adhesion occurs through focal adhesion formation, which is regulated via the integrin mechanism, specifically IntasPi and IntavPs, involving the overexpression of adhesion integrins and focal adhesion protein genes. The cell adhesion over the biomolecules was determined by measuring the cell density, the cell area (spreading), the number of focal adhesion sites, and adhesion-related gene expression compared to untreated gold surfaces. According to some embodiments, in order to determine optimal biomolecule type and concentration, the cell density was evaluated after 24h postseeding. As exemplified herein, retinal cells were specifically attracted to surfaces coated with the YIGSR molecules (e.g., molecules having an amino acid sequence as denoted by SEQ ID NOs 4-6), whereas the HEK293 cells were mainly attracted to the RGD-type molecules (GG- RGD and poly-pro-cRGD, e.g., molecules having an amino acid sequence as denoted by SEQ ID NOs 1-3). These examples highlight the surprising cell-specific cell adhesion by the integrin mechanism.
According to some embodiments, surprisingly, as opposed to the superiority of cyclic - RGDfK or linear RGDfK in promoting cell adhesion of HEK293 cells, this effect was not found for the retinal cells, which were preferentially adhering to the YIGSR molecules coating.
According to some embodiments, advantageously, consistent with the density results the YIGSR-type molecules significantly increased the retinal cell surface area, whereas the RGD- type molecules did not.
According to some embodiments, the adhesion biomolecule-coated electrode surfaces can stimulate the recruitment of cytoplasmic proteins (e.g., Vinculin) to form focal adhesion complexes.
According to some embodiments YISGR containing molecules may increase the expression of adhesion integrins IntaV, Inta5, Intpi, and Intp3 as well as the focal adhesion proteins (Vinculin and PTK-2); According to some embodiments, YIGSR-type molecules elicit the cellular adhesion mechanism, leading to an increase in cellular density, cell spreading, and FA expression of neurite cells.
According to some embodiments the adhesion peptide has an amino acid sequence of SEQ ID NO: 6 (YIGSR) or a functionally equivalent variant thereof, wherein one or more conservative substitutions may be made without abolishing retinal cell adhesion activity.
According to some embodiments, the adhesion peptide may have an amino acid sequence of any one of SEQ ID NOs: 4-6, or a functionally equivalent variant thereof. According to some embodiments, the biomimetic adhesion peptide has an amino acid sequence having at least 70%, 80%, 90%, or 95% sequence identity to SEQ ID NO: 6 (YIGSR), SEQ ID NO: 5 (CGG-YIGSR), or SEQ ID NO: 4 (C-(Pro)lO-YIGSR), and retains integrin- mediated retinal neuron adhesion activity.
According to some embodiments, the biomimetic adhesion peptide has an amino acid sequence having at least 70%, 80%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 1-3.
In some embodiments, the amount of biomimetic peptide coating applied to the electrode surface may be selected to achieve optimal retinal neuron adhesion while minimizing nonspecific cell attachment.
In some embodiments, the peptide coating may be applied at a concentration of about 0.05-0.3 mg/mL, for example, about 0.08-0.15 mg/mL, to achieve a surface density of about 0.5-5 pmol/cm2, for example, about l-2 pmol/cm2. The electrode substrate may be incubated for at least 10 minutes, 30 minutes, 1 hour in the peptide solution to form a self-assembled monolayer, after which excess peptide may be removed.
According to some embodiments, the electrode substrate may optionally be cleaned by oxygen or nitrogen plasma, then immersed in a solution of thiol-fiinctionalized YIGSR peptide (SEQ ID NO: 4-6) at a concentration of about 0.05-0.3 mg/mL for about 1-3 hours at room temperature. The thiol anchor may bind covalently to the metal of the electrodes, to form a selfassembled monolayer, optionally combined with PEG-thiols to control peptide spacing. After rinsing and drying, the resulting coated electrode may exhibit a reduced water contact angle of less than about 40°, e.g., 22°, and enhanced retinal neuron adhesion.
According to some embodiments, the biomimetic coating may include controlled-release nanoparticles delivering neuroprotective or anti-inflammatory drugs.
According to some embodiments, the modification may include any thiol-anchor, silane coupling agent, or click-chemistry linker enabling stable peptide immobilization.
According to some embodiments, the spacer length may be in the range of about 1-50 amino acids, or a polyethylene glycol (PEG) linker.
According to some embodiments, there is provided a surface modification for retinal cellspecific adhesion, including: an electrode-like substrate (e.g., conductive electrode); a biomimetic peptide selected from YIGSR-based molecules; wherein the peptide comprises a thiol anchor, a short or long spacer, and a ligand head group, such that the coating promotes preferential adhesion of retinal cells over non-retinal cells by enhancing cell density, spreading, and/or focal adhesion formation.
According to some embodiments, there is provided a method of enhancing retinal cell adhesion to an electrode surface, including: coating the electrode surface with a YIGSR peptide sequence anchored via a cysteine thiol group forming a self-assembled monolayer (SAM), seeding retinal cells on the coated surface, wherein the coating increases retinal cell adhesion density and focal adhesion (Vinculin) formation compared to uncoated or RGD-coated controls.
According to some embodiments, there is provided a biomimetic coating composition for retinal neuroprosthesis, including a peptide sequence CGG-YIGSR (SEQ ID NO: 4) or C- PolyProline( 10) -YIGSR (SEQ ID NO: 5), wherein the spacer length (short GG or long PolyProline) may have lower impact on retinal cell spreading, and the peptide selectively promotes retinal cell adhesion without significantly affecting non-retinal HEK293 cells.
According to some embodiments, there is provided a neuroprosthetic electrode coated with a YIGSR peptide layer that mimics laminin ECM motifs, wherein the coating enhances integrin-mediated adhesion of retinal neurons via a5pi, aVp3, or pi/p3 integrin subunits.
According to some embodiments, the coating may be applied by immersion in a peptide solution (e.g., 1/12 to 1/3 mg/ml) for at least 1-2 hours to achieve optimal surface coverage and hydrophilicity.
According to some embodiments, the coated surface may reduce water contact angle to at least 22°, indicating increased hydrophilicity compared to uncoated electrode.
According to some embodiments, the peptide may further increase cell spreading area at least 2-fold, compared to control surfaces.
According to some embodiments, the coating is configured to provide a biomimetic ECM-mimicking interface for retinal neuroprostheses.
According to some embodiments, axonal elongation of the transplanted photoreceptors has a key role in their integration and creation of functional synapse with the host retina. Neurite elongation is affected by various intrinsic or extrinsic factors, including signals from neighboring cells and the cellular microenvironment, which trigger pathways within the cell. According to some embodiments, enhancing PRP axon elongation may be facilitated by the ROCK pathway. Accordingly, as demonstrated herein, ROCK inhibitors, such as Y-27632 can significantly increase PRP neurite length (for example, up to 27.22 pm ± 3.19). Surprisingly, in addition to axonal elongation, such inhibitor can also increase the percentage of photoreceptor cells extending axons with more than half of the cells extended neurons following treatment.
According to some embodiments, in addition to ROCKi, RCM treatment of PRP cells may also enhance neurite elongation. Surprisingly it was demonstrated herein that the medium containing Muller cell-derived factors, rather than the cells themselves, is sufficient to induce photoreceptor cell extension, without the direct contact between retinal neurons and glial cells.
According to some embodiments, taurine can extend axons in retinal cells.
According to some embodiments, in order to gain a deeper understanding of the specific cellular processes associated with neurite elongation in PRP, RNA-seq was carried out on Y- 27632 treated cultures. Signal pathway analysis revealed augmentation in several pathways associated with neurite elongation, encompassing pathways involving BMP4, CREB, and the phototransduction process. Interestingly, the gene expression analysis did not reveal alterations in the levels of genes directly associated with the Rho pathway, such as ROCK, LIMK, or Cofilin (CFL1).
According to some embodiments, BMP4 may be upregulated in cultures treated with Y- 27632, taurine, and RCM.
According to some embodiments, adding BMP4 protein to PRP cells can results in elongation of the cell’s extensions.
According to some embodiments, a 3D organotypic retinal explant model (harvested from three-month-old RCS animals, devoid of photoreceptors), may be used to seed PRP cells and monitor their outgrowth formation following different treatments.
According to some embodiments, ROCKi and taurine significantly enhance neurite extension compared with the control group.
According to some embodiments, seeding the PRP cells onto a 3D retinal explant in a collagen matrix to mimic the ECM microenvironment can cause a significantly longer neurite when compared to an explant condition (no collagen) after treatment with Y-27632. According to some embodiments, three-dimensional environment is important in influencing the elongation of the PRP extensions.
According to some embodiments, there is provided a method of promoting neurite outgrowth in photoreceptor precursor cells (PRPs), the method includes contacting the PRPs with a ROCK inhibitor, wherein the ROCK inhibitor may be, for example, Y-27632 at a concentration between about 50 pM and 100 pM, thereby increasing neurite length by at least two-fold compared to untreated PRPs and optionally enhancing expression of BMP4 and phototransduction genes.
According to some embodiments, the photoreceptor precursor cells may be treated with a ROCK inhibitor, (such as, Y-27632), at a concentration of about 20-500 pM, e.g., about 50- 100 pM for a time period of about 2-96 hours (e.g., 12-72 hours), prior to and/or after seeding on the biomimetic micro-well prosthesis. In some embodiments,
In some embodiments, the ROCK inhibitor may reduce actin cytoskeletal tension, inducing neurite elongation independently of BMP4 signaling.
In another embodiment, the ROCK inhibitor may be combined with taurine or retinal conditioned medium (RCM) to provide additive neurite outgrowth effects, while BMP4 pathway inhibitors do not block Y-27632-induced elongation.
In some embodiments, additionally or alternatively, the ROCK inhibitor may be delivered post-implantation to enhance PRP survival, enhance neurite extension and/or reduce glial scarring at the implant site. In some embodiments, the ROCK-inhibitor may be provided to the eye, after implantation for a period of time of, for example, 1-28 days, 2-4 weeks, 1-3 months, etc.
In some embodiments, photoreceptor precursor cells may be treated with at least one neurite-promoting compound selected from: ROCK inhibitors (e.g., Y-27632), taurine, retinal conditioned medium, neurotrophic growth factors (e.g., BDNF, CNTF, NGF, GDNF), ECM- derived peptides (e.g., IKVAV), cAMP elevators (e.g., forskolin), mTOR pathway activators (e.g., IGF-1), and small molecule neurogenic compounds (e.g., HDAC inhibitors). These compounds may be used alone or in combination to synergistically enhance neurite elongation, improve PRP survival, and promote synaptic integration with host bipolar cells.
According to some embodiments, there is provided a method for enhancing neurite extension in photoreceptor precursors including: culturing PRPs in a medium containing taurine and/or retinal conditioned medium (RCM) enriched with retinal and Muller glial cell secretome, wherein neurite extension is mediated via BMP4 signalling pathway activation, and wherein inhibition of BMP4 signalling reduces said neurite extension. Each possibility is a separate embodiment.
According to some embodiments, there is provided a three-dimensional (3D) collagen hydrogel or other bio-compatible matrix, wherein the 3D microenvironment provides synergistic mechanical and neurotrophic cues resulting in enhanced neurite outgrowth compared to without the 3D matrix.
According to some embodiments, there is provided a method of stimulating neurite outgrowth in PRPs via BMP4 signaling, including: contacting the PRPs with a BMP4 protein or inducing BMP4 gene expression, wherein the neurite outgrowth effect is blocked by a BMP4 inhibitor such as Noggin, thereby confirming BMP4 pathway mediation in taurine- and RCM- induced axonogenesis.
According to some embodiments, there is provided a 3D bio-compatible matrix for promoting photoreceptor precursor axonal elongation comprising PRPs cells embedded in a collagen hydrogel matrix. According to some embodiments, the 3D matrix may further include a small molecule substance including a ROCK inhibitor, Y-2763, taurine, retinal-conditioned medium (RCM), or any combinations thereof. Each possibility is a separate embodiment.
According to some embodiments, there is provided a method for promoting neurite outgrowth, including contacting PRPs with a neurite-promoting agent under conditions sufficient to increase neurite length by at least 2-, 5-, or 10-fold relative to untreated PRPs.
According to some embodiments, there are provided an ex vivo organoid models, wherein the prosthesis can be applied in retinal organoid cultures for disease modelling or drug screening.
Definitions and embodiments mentioned above and which may be relevant to the treatment method and uses, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.
The term “treating” as used herein refers to means of obtaining a desired physiological effect (e.g., by implanting the hybrid retinal implant as disclosed herein). The effect may be therapeutic in terms of partially or completely restoring retinal function, e.g., at least partially restoring vision. According to some embodiments, there is provided a method for at least partially restoring retinal function or vision in a subject in need thereof, the method includes: implanting the hybrid retinal prosthesis as disclosed herein into a target region; electrically stimulating the glutamatergic neurons within the sealed micro-wells; thereby inducing glutamatergic synaptic signalling to host bipolar cells and restoring graded retinal neurotransmission with high spatial resolution. In some embodiments, electrical stimulation can induce formation of synapses.
According to some embodiments, there is provided a method for at least partially restoring visual function including: seeding photoreceptor precursor cells into a structured electrode array configured for implantation; promoting axonal outgrowth using a combination of ROCK inhibitor and trophic factors; and implanting the array into a subject’s subretinal space to achieve retinal circuit activation.
According to some embodiments, there is provided a method of selectively activating retinal cells or inducing selective retinal stimulation, the method includes applying electrical stimuli through the micro-well electrode array at an amplitude of less than about InC (e.g., less than about lOOpC) to sealed glutamatergic neurons, thereby inducing glutamate or other neurotransmitters release. In some embodiments, the hybrid retinal prosthesis or co-cultured neuronal cells can release neurotransmitters instead of or in addition to glutamate. These may include one or more of GABA, acetylcholine, dopamine, serotonin, nitric oxide, or neuropeptides, either to replicate native retinal processing or to modulate host-graft integration. In further embodiments, neurotransmitter release is patterned spatially or temporally to reproduce natural retinal computations such as directional selectivity or lateral inhibition.
According to some embodiments, the hybrid retinal prosthesis may be used for treating retinal degenerative diseases characterized, for example, by photoreceptor cell loss with at least partial preservation of inner retinal neurons. In some embodiments, the hybrid retinal prosthesis may be used for treating retinal degenerative diseases that include, but not limited to: age- related macular degeneration, retinitis pigmentosa (rod-cone dysterophies), Leber congenital amaurosis, cone-rod dystrophies, Stargardt macular dystrophy, myopic macular degeneration, traumatic or toxic retinopathies, phototoxic injury, and the like, or any combinations thereof. Each possibility is a separate embodiment. In some embodiments, in more advanced stages of retinal diseases where bipolar cells are also compromised, the prosthesis may be seeded with alternative interneurons or optogenetically modified neurons to relay signals directly to retinal ganglion cells. In some embodiments, the term retinal degenerative condition is interchangeable with the term retinal dysfunction condition.
According to some embodiments, the hybrid retinal prosthesis may be implanted in various anatomical locations within or adjacent to the retina depending on the stage of degeneration and surgical accessibility. In some embodiments, the hybrid retinal prosthesis may be implanted into a site selected from the subretinal space, epiretinal space, suprachoroidal space, intraretinal layers, or any combinations thereof, depending on the underlying retinal disease and surgical requirements. Each possibility is a separate embodiment. In some embodiments, subretinal implantation is preferred for diseases with preserved bipolar cells, while epiretinal implantation may be used in advanced degeneration where direct ganglion cell activation is necessary; however in a separate embodiments, epiretinal implantation is used for enabling synapse between the device cells and the bipolar cells. In some embodiments, suprachoroidal placement may be advantageous for minimally invasive procedures.
In some embodiments, the implantation is facilitated such that the electrode surface of the array is facing the retinal inner nuclear layer, optionally anchored via hydrogel adhesion or suture-less geometric fitting.
In some embodiments, there is provided a use of the hybrid retinal prosthesis as disclosed herein, for at least partially restoring retinal function or vision in a subject in need thereof.
In some embodiments, the implant may be used in retinal organoids or ex vivo retinal explants for disease modeling, drug screening, or testing neuroprotective compounds.
In some embodiments, the implant may be used for simultaneously restoring photoreceptor function and monitoring electrophysiological responses of the host retina.
According to some embodiments, the methods may include combining prosthesis implantation with gene therapy or optogenetics. According to some embodiments, the prosthesis may be used in conjunction with viral delivery of opsins, CRISPR-based gene editing, or pharmacological modulation to enhance vision restoration.
According to some embodiments, there is provided a method for modifying a substrate for retinal cell-specific adhesion, the method includes providing a substrate including an electrode; and coating said substrate with a biomimetic peptide having a cell-adhesion motif selected from YIGSR, IKVAV, RGD, or equivalents, a thiol or silane anchor, and a flexible spacer, thereby promoting selective retinal cell adhesion over non-retinal cells. According to some embodiments, there is provided a method for promoting neurite outgrowth in photoreceptor precursor cells (PRPs), the method includes contacting the PRPs with a neurite-promoting agent selected from ROCK inhibitors (e.g., Y-27632, fasudil), BMP modulators, or combinations thereof at 10-100 pM, under conditions sufficient to induce at least a 2- to 10-fold increase in neurite length compared to untreated PRPs.
In some embodiments, there is provided a kit for at least partially restoring retinal function in a subject in need thereof, the kit includes the multi -array electrode as disclosed herein, and instructions for using the same.
In some embodiments, the kit may further include a container including cells (e.g., PRP cells), for seeding on the array.
In some embodiments, the array in the kit is pre-seeded with the cells (e.g., PRP cells).
In some embodiments, the wells and or electrodes of the array are pre-coated with a biomimetic coating, as disclosed herein.
In some embodiments, the wells of the array and/or the cells are treated or incubated in the presence of a neurite-growth enhancing composition as disclosed herein.
In some embodiments, the kit may further include a surgical tool for enhancing implantation.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, governs.
As used herein, the indefinite articles “a” and “an” mean “at least one” or “one or more” unless the context clearly dictates otherwise.
The term "about" when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.
While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims which follow.
In the description and claims of the application, the words “include” and “have”, and forms thereof, are not limited to members in a list with which the words may be associated.
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.
EXAMPLES
Materials and Methods
Generation of photoreceptor precursors (PRPs)
As a choice for the glutamatergic cell, photoreceptor precursors (PRPs) differentiated from hESCs were selected, due to their putative ability to naturally connect with the host retina upon transplantation. PRPs were generated as previously described (Markus, A. et al. An optimized protocol for generating labeled and transplantable photoreceptor precursors from human embryonic stem cells. Exp Eye Res 180, 29-38 (2019)). Briefly, GFP-labelled hESCs (U.S. National Stem Cell Bank [WA09]) were grown on mitomycin C-inactivated STO cells (a murine line derived from embryonic fibroblasts) in NutriStemR hPSC XF Culture Medium. hESCs were trypsinized to single cells and seeded in differentiation medium in uniform-sized agarose micro-wells (9000 cells per well) prepared using silicone micro-molds. One day post seeding on the micro-wells, the medium was replaced with a differentiation medium - GMEM (Gibco, 11710035, Life Technologies, Warrington, UK) supplemented with 20% knockout serum replacement (Gibco, 10828028, Life Technologies, Warrington, UK), O.lmM nonessential amino acids (Biological Industries, 01-340-1B, Israel), 1 mM pyruvate (Biological Industries, 03-042-1B, Israel), 0.1 mM 2-mercaptoethanol (Sigma, M7522, Israel), lOOU/ml penicillin, lOOpg/ml streptomycin and 0.25pg/ml amphotericin (Biological Industries, 03-033- 1B, Israel). Next, 20 mM Y-27632 (TOCRIS,1254, UK) and 3 mM IWRle (TOCRIS, 3532, UK) were added to the medium up to day 12 and 0.1% Matrigel (GFR, BD Biosciences, FAL354230, CA, USA) was added from day 2. On day 12, 10% Fetal Calf Serum (FCS) was added. From day 15 to 24, the medium was supplemented with 3pM CHIR99021 (TOCRIS, 4423/10, UK) and lOOnM Smoothened receptor agonist (SAG) (TOCRIS, 4366, UK). On day 18, the embryoid bodies were seeded on polylysine/laminin-coated plates. The embryoid bodies were trypsinized on day 24 and filtered through a 40-pm strainer (Coming, Cx-431750, NC, USA). Using this protocol, 70-80% of cells expressing CRX, a PRP marker, can be achieved following 30 days of differentiation.
Studying in-vitro PRP synapses using calcium imaging
To test the synapses formed by the differentiated PRPs and verify their glutamatergic nature, calcium imaging was used, which enables the testing of cell population activity at single-cell resolution. To this end, PRPs seeded on 13mm round glass coverslips were incubated at 37°C for 30 min with Oregon Green™ 488 BAPTA-1 (OGB-1 Invitrogen, 06807) or Rhod2 (Invitrogen, R1245). To each 50pg vial of the indicator, 8 pl DMSO (Sigma-Aldrich), 2pl 20% Pluronic acid in DMSO (F-127, Biotium Inc.), and 90pl cell medium were added. Dye solution was then added to the cell culture to a final concentration of 7.92pM (20pl/ml). The coverslip was then placed in the recording chamber of an upright microscope (Slicescope 6000, Scientifica) equipped with a CCD camera (EXI-Blue QIMAGING) and filters to visualize the cells for calcium imaging. Images were captured at 10 frames per second. The chamber contained an extracellular solution consisting of (mM): NaCl (119); KC1 (2.5); MgC12 (2); HEPES (25); CaC12, (2); and D-glucose (30). The OGB/Rhod2 was washed with 1ml of the extracellular solution, after which 3ml of the extracellular solution were added.
The induced calcium currents through the simultaneous intracellular stimulation of a cell through patch clamp (either voltage or current clamp) were studied. Under the voltage clamp configuration, the cell was held at -60mV and voltage steps of 30m V were applied every lOsec. To test the formation of glutamatergic synapses in the cell, the glutamate receptor blockers APV (80pM), CNQX (lOpM), and L-AP4 (30pM) were applied to block the synaptic transmission between the cells and then intracellular stimulations were performed. As control, the simulations without the blockers were repeated under the same conditions.
Implant fabrication and characterization
The micro-well array (1mm diameter) was fabricated using a sequence of conventional photolithography steps in a clean room (Shpun, G. et al. Optimizing the fabrication of a 3D high-resolution implant for neural stimulation. J Biol Eng 17, (2023)). Briefly, the device includes a 3pm-thick flat base layer and a 3D micro-well layer fabricated onto it (10pm, 20pm or 10pm in diameter). Towards this end, an SU-8 negative photoresist polymerized under UV light was spin coated on a pre-cleaned soda lime glass coated with a sacrificial layer (LORI OB, MCC, USA), soft baked and patterned by UV radiation (395nm) using Maskless aligner (Heidelberg MLA150 maskless aligner, Germany) or by mask aligner with a photomask (MA6 Karl Suss, Germany). Next, the exposed photoresist was developed and cured (hard bake) to complete the polymerization process. To enable the fluorescence visualization of the implant, Rhodamine-B (Sigma-Aldrich) was added to the SU-8 before the polymerization process (at a concentration of 1: 10 of the total SU-8). Finally, the polymeric array was stripped from the glass by dissolving the sacrificial layer in organic solution (DMSO) overnight and then rinsed in DDW. Implant characterization was then performed using both confocal and SEM (E-SEM 326, Quanta FEG 250 by FEI) imaging.
Cell-well interface and cell viability
To enhance cell adhesion and cell viability following seeding on the SU8 implants, the devices were N2-plasma-treated several hours before seeding. Then, PRPs (day 24) were trypsinized and separated to single cells using a 10pm strainer (Pluriselect #43-10010-40), and seeded onto SU8 micro-wells in 50pL PRP medium. After two hours of incubation at 37°C, additional PRP medium was added, and the implant was centrifuged (100rcf/4min) and incubated for at least another 24 hours. Following this incubation period, cell viability at day 1 and day 7 post seeding was evaluated using 7AAD assay (GTX85584, GeneTex, CA, USA), which stains dead cells (following the manufacturer’s instructions), combined with Hoechst (to visualize nuclei). Cells were then imaged using confocal microscopy (TCS SP8, Leica, IL, USA), and their viability quantified by counting the number of dead cells.
In addition, to evaluate the interface between the GFP-PRP cells and the micro-wells and to quantify their entrance into the micro-wells, the inherent fluorescence of the GFP-PRP cells was used, in conjunction with actin staining, using Phalloidin (Cat. #PHDN1-A) to visualize the cell cytoskeleton. Three-dimensional images of the cells with implants were acquired using a Leica Stelaris confocal microscope and processed with IMARIS software. To determine the total cell volume, the GFP fluorescence channel was merged with the actin staining fluorescence channel, combining both to define the cell volume and edge and create a "surface" . Similarly, the implant “surface” was obtained using the red fluorescence of the implant. Through the "surface-to-surface contact area" plugin in the IMARIS software, an additional surface representing the contact area between the cell and the implant was created. Additionally, a separate surface was generated based on nuclear staining (Hoechst), with the software automatically segmenting each nucleus into individual objects. The software provided values for the contact area between the cells and the implants, as well as the volume of the cells inside the implants. Finally, the number of nuclei within each implant was manually calculated and assessed whether each implant was sealed.
Sample preparation for Transmission Electron Microscopy (TEM) imaging
To test the cell-well interface at an even higher spatial resolution, TEM imaging was used (Henn, I. et al. SEM/FIB Imaging for Studying Neural Interfaces. Dev Neurobiol (2019) doi: 10.1002/dneu.22707). Briefly, implants were seeded with PRP cells as detailed above. After 5 days of incubation, cells were fixed with a fixation buffer (2.5% (wt/vol) paraformaldehyde, 2.5% glutaraldehyde, 0.1M cacodylate buffer, PH 7) for Ih at 24°C and then left overnight at 4°C. After washing out the fixative three times with 0.1M cacodylate buffer, TEM sample fixation was performed: 1 hour of 1% osmium in a buffer containing CaCO 0.1M, 5mM CaC12, 0.5% potassium dichromate K2Cr2O7, 0.5% potassium hexacyonaferrate K4[Fe(CN)6], followed by three washes with 0.1M cacodylate buffer, and two washes with DDW. Next, the samples were stained with 2% aqueous uranyl acetate for Ih, followed by dehydration in ethanol solutions of 50% 70% and 95% and three times 100%, for ten minutes. Next, samples were embedded using the Epon technique carried out in 5 steps: 30% for 3 hours, 50% overnight, 75% for 3 hours, 100% resin overnight, followed by 100% repeated twice. Next, samples underwent a series of washes with 10 splashes of 5 ml 100% ethanol. Then, samples were placed in an oven for 24 hours at 60°C. Finally, the samples were cut with an ultra-microtome (Leica UC7) and sections of 50-70nm were viewed under an electron microscope (Tecnai G2 Spirit).
Electrophysiological validation of activation threshold reduction in-vitro
To assess the activation threshold of cells seeded on the high-density hybrid retina device, a device prototype, fabricated using the same optimized process, including high-density electrodes (an electrode pitch of 50pm, well diameter of 10 pm and height of 5pm) was used. To validate the activation threshold in-vitro, an excitable HEK293 cell line were rendered excitable through the expression of the voltage-gated sodium channel NaV1.2 (as detailed below). Following transfection, 1,500,000 NaV 1.2-positive cells (as inferred by the GFP fluorescence) were seeded onto polyethylenimine (PEI, Sigma Aldrich, 408727)-coated devices, followed by centrifugation (150rcf/4mins) to ensure cell entrance into wells. Electrical stimulation (24 hours post seeding) was obtained by interfacing with a multielectrode array current injector (MEA 2001, Multi-Channel System, Germany). Cathodic pulses, 0.04ms-lms, IpA-lOOpA, were delivered at a rate of 0.2Hz. The induced responses were recorded using the patch clamp technique, similarly to the description above. The threshold was defined as the first current step inducing an inward membrane current.
Generating NaV1.2 HEK293 -positive cells
HEK239 cell line expressing the voltage-gated sodium channel NaV1.2 was made, by transfecting cells with the plasmids’ CD-splice variant_pcDNA3.1(+) IRES GFP corrected and SCN I B-SCN2B_pcDNA3. 1 (+) IRES (GenScript, NJ, USA), using polyethyleneimine (PEI, Sigma Aldrich, 408727) reagent dissolved in DDW (lOmg/lml). The transfection solution contained 0.5pg DNA, lOpl PEI, 70pl DMEM per well, with the transfection.
The HEK293-GFP cells were then incubated in a medium containing: MEM-eagle (Biological Industries), 1% PSA (Biological Industries), 1% glutamine (Sigma-Aldrich), and 10% fetal bovine serum (Danyel biotech, Rehovot, Israel). Cells were incubated at 37°C with 5% CO2.
Device implantation and in-vivo imaging
All animal experiments were approved by the University Ethics Committee for Animal Research. Animals were immunosuppressed by adding cyclosporine to the animals’ drinking water (Sandimmune Oral Solution lOOug/ml) at a concentration of 0.5mg/ml per 100 kg starting 4 days before and up to 30 days after implantation.
Five (5) Royal College of Surgeons (RCS) rats with retinal degeneration (4-8 weeks) were anesthetized for all procedures with a mix of ketamine (Iml/kg) and xylazine (0.5ml/kg) injected intramuscularly (IM), with anesthesia maintained by ketamine as necessary. Topical lidocaine (2%) was used for local anesthesia of the eye. Body temperature was maintained using a heating pad. Viscous lubricants were used to protect the corneas throughout the surgeries. A Imm-diameter device with cells was implanted in the subretinal space using a technique as previously described (Mandel, Y. etal. Cortical responses elicited by photovoltaic subretinal prostheses exhibit similarities to visually evoked potentials. Nat Commun 4, 1980 (2013). After implantation, imaging was performed using a rodent fluorescence fundus camera combined with an OCT imaging system (Phoenix Research Laboratory, Micron IV). Imaging was repeated on day 30 in order to evaluate the survival of the GFP-labeled cells, as previously described (Markus, A. et al. An Optimized Protocol for Generating Labeled and Transplantable Photoreceptor Precursors from Human Embryonic Stem Cells. Experimental Eye Research vol. 180). Briefly, the fluorescence signal of the cells located on the implant was quantified using Image J software. Toward this end, a binary mask was applied over the image (thresholding) to highlight regions that are GFP positive. The average fluorescence signal at each time point for a region of interest was then normalized to the average fluorescence of the same region evaluated on the first day post transplantation.
Assessing device integration with the host retina
Following imaging on day 30, the rats were sacrificed and the eyes incubated with paraformaldehyde 4% for 24 hours. The eyes were then rinsed using PBS, and the corneas removed to create eyecups. A 5mm sample containing the device was cut out of the eyecups. The nuclei were stained overnight using Hoechst (Hoechst 14533). The samples were then rinsed using PBS and flat-mounted on slides, and imaging performed using an 1X81 Olympus microscope and confocal microscope (Leica TCS SP8).
Following flat mount imaging, the samples were cryo-sectioned. To this end, the samples were incubated in increasing concentrations of sucrose (5%, 15%, 30%) (Millipore, 573113- IKg) at room temperature for 5-30 min, according to the sucrose concentration. They were then incubated with PBS containing 30% sucrose for 24 hours at 4°C. At the end of the process, the samples were frozen in OCT medium (Tissue-Plus, OCT compound embedding matrix Tissue- Tek Scigen) and sectioned into 10pm slices with a cryostat (CM1800 LEICA). Retinal cryosections were rinsed with PBS and then twice with PBST. Sections were then incubated in a blocker solution containing 1% bovine serum albumin (MP Biomedicals, 160069) for 60 min. Following overnight incubation in primary antibodies PKCa (Sigma- Aldrich, P4334), Gluthamine Synthetase (Abeam ab49873), ribeye (BD biosciences 612044), and Mitochondria Monoclonal Antibody (Thermo Fisher MTC02), sections were rinsed with PBS and incubated with secondary antibodies Alexa 594 anti-mouse (ENCO, 711-545-152) and Alexa 647 antiRabbit (ENCO, 711-545-152 ) for 1 hour at room temperature. Samples were imaged using confocal microscopy (Leica TCS SP8).
Computer modelling-simulation: Study physics
The hybrid retina concept was modelled using COMSOL Multiphysics 5.2 with MATLAB R2019a. The electrical potential and electric field for each spatial point in the model were calculated using COMSOL’s electrical current physics model by solving the electric field equation:
V(o V0) = 0 where a is the electrical conductivity and 0 is the potential at a specific location. The outer boundaries of the model were set to an insulating condition in which
— n xj = 0 where n represents the unit’s outward normal vector and J represents the current density; a continuity condition was applied to all other boundaries.
To model the sealed cell, a time-dependent electrical physics was chosen where the cell and the remaining components of the model were assigned two different physics separately. The two parts were then “connected” by a current density assigned to the cell membrane, which is the boundary shared by the two physics. The current density was determined by the well-known Hodgkin-Huxley equations, which govern the active behavior of an excitable cell, and was modelled using Coefficient Form Boundary PDE available in COMSOL. Briefly, Hodgkin- Huxley equations state that where Cm is the membrane capacitance, Ena is the sodium reversal potential, Gna is the maximal sodium channel conductance, m and h are the rate functions governing the activation and inactivation of the sodium channel, respectively, and Gk is the maximal potassium channel conductance.
The m, n and h rate functions were obtained by solving the following exponential rate functions with a time constant: where the equation for each a and is empirically adapted.
The simulations were performed under a 2D axis symmetry geometry to reduce both the computational complexity and the calculation time. Under axis symmetry, the cell was modelled as a 5pm wide, 15pm high rectangle, a shape chosen following the observation that a sealed cell assumed the shape of the well. The microwell was then modelled as a 15 pm high, 1 pm wide rectangle. These parameters were chosen to resemble those of the actual fabricated device. The electrode was then modelled as a 2.5pm wide, 1pm high rectangle on top of the microwell base. An additional rectangle (55pm wide, 1pm high) served as a remote ground, placed far from the cell). Finally, these four domains were surrounded by a large 200pmx200pm rectangle.
The equations were solved governing the electrical field distribution for various pulse durations (0.1, 1, 5, 10 msec) for both a cell expressing the standard Hodgkin-Huxley properties and a cell for which the properties were adjusted to fit the properties of the measured data from a PRP cell (see next section). By varying the distance of the cell from the electrode (i.e., 30nm, lOOOnm) and the spacing between the cell and the well wall (i.e., 50nm, lOOOnm), four different combinations were created. The effect of each combination on the electrical field was then tested.
The electrode was given the properties TiN (electrical conductivity: 5.998e7[S/m]). For comparison, a similar modelling, excluding the SU8 domains, was performed to simulate the non-sealed configuration, which represents the standard cell-flat electrode interface.
Computer modelling using the biophysical parameters of PRPs
To model the hybrid retina with biophysical cell parameters that match those of PRPs, the rate functions a and P to were fit to PRPs by iteratively adjusting the rate function based on previous patch-clamp data acquired from PRPs. The patched cell was held at -60mV and voltage steps ranging from 120mV to 70mV, with a step size of lOmV and width of 200msec, were applied to test ionic currents. The observed electrophysiological signals were amplified (xl) and low pass filtered (Bessel 10kHz) (Multiclamp 700b AXON), sampled at 10kHz and saved (Digidata, Axon). To model the voltage clamp configuration, which was used to characterize the PRP cells, the cell membrane potential was set to steps identical to those used in the electrophysiological experiments, and various parameters employed in the Hodgkin- Huxley equations were iteratively adjusted until the computer modelling data fit the measured parameters.
Cell culture device fabrication for testing of coating
For model electrodes, a gold layer deposited on glass was used. To this end, microscope glass slides (#7105, BOJACK, China) were first cleaned by rinsing in piranha solution (3: 1, Ammonia solution: Hydrogen peroxide), followed by double-distilled water (DDW), and heating to remove moisture in an oven at 120°C for 20min. Next, Cr/Au (lOnm/lOOnm) layers were spatter deposited (Bestec Berlin, Germany) after Ch+Ar (3min, 100W) plasma attaching (Dainer electronics, Pico, Germany) and Ar ion milling (lOsec). For the cell density, cell spreading, and focal adhesion experiments, p-slides (12 wells (#81201, GmbH, Grafelfing, Germany)) were mounted on the gold-coated slides and sealed with SYLGARD®-184 (#761028-5EA, Merck, New Jersey, USA), followed by curing at 80°C overnight. Finally, the samples were rinsed twice in DDW, and uprooted by boiling in DDW for 20 min, followed by a 70% Ethanol wash and exposure to UV radiation for 30min in a biological hood. For the gene expression studies, the slides were uprooted without mounting the p-slides.
Bio-Molecule design
The design of the biomolecules used in this study was based on the following: For the anchor, the thiol group (SH) was used, which is found in Cysteine amino acid, and spontaneously forms a self-assembly monolayer (SAM) of a semi-covalent bond with gold. Aiming to assess the optimal spacer length, short amino acid spacer (GG) and long spacer polyproline (PPPPPPPPPP (SEQ ID NO: 8), which was shown to be more efficient than the PEG spacer at the same length, regarding rigidity.
The amino acid type and sequence (e.g., RGDxx, where xx represents any amino acid or YIGSR (SEQ ID NO: 6), as well as their conformational structure (e.g., linear, cyclic, or branched), greatly influence the molecule’s affinity to the integrin subtype and especially to the adhesion integrins. The RGD motif is an adhesion motif for adhesive basal cells and neurons, YIGSR and IKVAV (SEQ ID NO: 7) are known to specifically promote the adhesion of neuronal cells.
Taking the above-mentioned considerations into account, the efficiency of linear RGD to cyclic RGD (c(RGD)) and YIGSR was compared with both short and long spacers (as per the below). As a control, DDW-coated surfaces were used. The molecules were manufactured by the Hanhong Group (Shanghai Hanhong Chemical Co., Ltd., China):
The following biomolecules were used: a) Short linear RGD; Cys-Gly-Gly-Arg-Gly-Asp-D-Phe-Lys (CGGRGDfK). (SEQ ID NO: 1) b) Short cyclic RGD; Cys-Gly-Gly-cyclo(Arg-Gly-Asp-D-Phe-Ly), (CGG-c(RGDfK)). (SEQ ID NO: 2) c) Long -cyclic RGD; Cys-PolyProline(10)-cyclo(Arg-Gly-Asp-D-Phe-Lys), (C-PolyPro9- c(RGDfK)). (SEQ ID NO: 3) d) Short YIGSR; Cys-Gly-Gly-Tyr-Ile-Gly-Ser-Arg (CGGYIGSR). (SEQ ID NO: 4) e) Long YIGSR; Cys-PolyProline(10)-Tyr-Ile-Gly-Ser-Arg (C-PolyPro9-YIGSR) (SEQ ID NO: 5) Surface functionalization
The peptides were dissolved in aqueous solution 1: 1 (DDW: Acetonitrile (AN), #75058) at a concentration of 1/3 mg/ml and stored until use at -20°C after being divided into several test tubes. Before use, the solutions were thawed and diluted to various concentrations (1/3, 1/6, 1/12, and 1/24 mg/ml). The biomolecule-gold electrode coating was obtained through a self-assembly monolayer (SAM) facilitated by semi-covalent bonds spontaneously forming between the gold and the thiol group (SH) present in the biomolecule, which can be found at cysteine (C) amino acid. Briefly, gold surfaces were coated by immersion for 2 hours in the solutions, followed by rinsing with PBS. For control purposes, untreated gold surfaces were soaked only in DDW. All samples were soaked for 2h at RT and rinsed three times in PBS before cell seeding.
Surface modification analysis:
X-ray photoelectron spectroscopy (XPS)
To evaluate the assembly of the biomolecules to the gold surface, an XPS analysis was performed. Survey and high-resolution spectra were acquired at a pass energy of 80eV and 40eV, respectively. The source power was set to either 75W or 150W. The binding energies of all elements were recalibrated by setting the CC/CH component of the Cl peak to 285eV. Quantitative surface chemical analysis was performed using high-resolution core-level spectra after removing the nonlinear Shirley background. The measurements were carried out under UHV conditions, at a base pressure of 5x 10 torr (and no higher than 3x 10-9torr) . Examinations were performed on gold-coated mica glass disks (Electron Microscopy Sciences, Hatfield, PA, USA).
Contact angle measurement
To evaluate the gold surface modification by the biomolecules, the contact angle between a water droplet and the surface was measured using a Contact Angle Goniometer (System OCA, model OCA20, Data Physics Instruments GmbH, Filderstadt, Germany). Briefly, gold-coated (lOOnm) cover glasses were coated by 2 hours of immersion in Acetonitrile-DDW (1: 1) solutions for each biomolecule at various concentrations. A drop of 5pL of DDW was placed in the center of each sample. The measurements were performed at 25°C and 55% moisture; Laplace-Young curve fitting was used to determine the static water contact angle values.
Fluorescence Microscopy (STEP) Aiming to assess the specific binding of the biomolecules to the gold surface, a chesslike pattern with glass-gold squares (300nm width, lOOnm height) was immersed for 2 hours in a 0. ImM solution of a fluorescent Cys-RGD-NBD (Nitrobenzofurazan, Ex/Em 467/539 nm), which was a generous gift from Rahimipour’s Lab. Following a triple rinse in DDW, the sample was examined using a Leica TCS SP8 STED microscope (Leica-microsystems, Germany) and compared to a non-coated sample.
Cell Culture
Dissociation of rat retinal cells
All animal experiments were approved by the Ethics Committee for Animal Research. The retinas of Sprague Dawley P 1 rats were isolated and dissociated using a papain dissociation kit (# LK003150, Worthington) according to the kit manual. Retinal cells were then incubated in a medium containing DMEM fl2 (Gibco, USA) and neurobasal (Gibco) 1: 1, glutamine 1% (Sigma- Aldrich), pen-strep 1% (Biological Industries, Beit-Haemek, Israel), non-essential amino acid 1%, and horse serum 2% (Biological Industries). B27, N2, and EGF (lOng / pl, Peprotech, Israel), NGF (lOng / pl, Peprotech), NT3(10ng / pl, Peprotech), BDNF (lOng / pl, Peprotech), and FGF (lOng / pl, Peprotech) were added to the medium. The medium was replaced after 24h with fresh medium containing ROCK inhibitor (5pl/lml, #1254/10, Biotest, Dreieich, Germany) and antimetabolite cytosine -D-arabinofuranoside hydrochloride (Ara-c, 0.5pl/lml, #C6645, Merck, USA), aiming to decrease the number of glia cells. Cells were incubated at 37°C with 5% CO2.
HEK293-GFP
HEK293 cells were transfected with polyethyleneimine (PEI, Sigma Aldrich, 408727) reagent dissolved in DDW (lOmg/lml) cells that were seeded on a 24-well plate. The transfection solution contained 0.5pg DNA, lOpl PEI, 70 pl DMEM per well, with the transfection plasmids’ CD-splice variant_pcDNA3.1(+) IRES GFP_corrected and SCN1B- SCN2B_pcDNA3.1(+) IRES (GenScript, NJ, USA). Human HEK293-GFP cell medium contains MEM-eagle (Biological Industries), 1% PSA (Biological Industries), 1% glutamine (Sigma- Aldrich), and 10% fatal bovine serum (Danyel biotech, Rehovot, Israel). Cells were incubated at 37°C with 5% CO2.
Cell adhesion assay
Biomolecule’s effect on cell density Aiming to assess the optimal biomolecule and concentration, the five types of biomolecules were diluted into four concentrations (1/3, 1/6, 1/12, and 1/24 mg/ml) in aqueous solutions DDW: Acetonitrile (1: 1). The gold surfaces were coated as mentioned above in two micro-wells for each concentration (in duplicate). HEK-293-GFP and rat-dissociated retinal cells were seeded at an initial concentration of 0.8M cells/mm2, and incubated for 24h. Then, the cells were gently rinsed with PBS to remove the unattached cells and fixed with 4% paraformaldehyde (#BN15711, Bar Naor, Israel) for 15 min at room temperature. Nuclear staining was performed with Hoechst (#14533, Sigma- Aldrich). Stained samples were rinsed (PBS) and mounted on slides in 90% glycerol (#G9012, Sigma- Aldrich)/ 10% PBS/1% n- propyl-gallate (#P3130, Sigma-Aldrich) and sealed with nail polish. The samples were imaged using a Leica LMD7 Microscope (Leica-microsystems, Germany) at a magnification of x20. The navigation and stitching applications were used to extend the field of view by a factor of 9; three regions of interest (ROI) were imaged from the center of each micro-well, with two duplicates for each concentration, overall, with four repetitions. Cell density was calculated by Image J using a Gaussian filter (3 -pixel mask) and binarization by the “Li dark” method (19 to 255). White (255) pixels were counted, after they referred to the fluorescence of the stained nucleus (no features were removed or added digitally). The average cell density was calculated from n=18 fields for each concentration. For each repetition, the ratio of cells was normalized to the control experiments where electrodes were coated by DDW. An average of all repetitions and the standard error deviations (STDs) were calculated. Statistical significance was determined using either the paired two-tailed t-test or multi-variate two-way ANOVA analysis provided by the MATLAB statistics toolbox.
Biomolecule’s effect on cell spreading
The cell area was measured as a function of the coated biomolecule. To this end, gold surfaces were coated by aqueous solutions (1/6 mg/ml, DDW: Acetonitrile, 1: 1) of the biomolecule and the control as mentioned above with two micro-wells for each molecule (duplicates) and three repetitions. HEK-293-GFP and rat-dissociated retinal cells were seeded at an initial concentration of 0.15M cells/mm2to allow single-cell spreading and incubated for 72h. For the rat-dissociated retinal cells, lOpM Ara-c and 50pM ROCK inhibitors were added after 24h, aiming to restrict the glial cell growth. Then they were gently rinsed with PBS to remove unattached cells; the rat-dissociated retinal cells were stained for cytoplasmic staining using ViaFluor® 488 (#BTM-30086, 1:4000, Biotium, Fremont, CA, USA) as the supplier protocol, fixed with 4% paraformaldehyde (#BN 15711 , Bar Naor, Israel) for 15 min at RT, and nuclear staining was performed by Hoechst (#14533, 1: 1000, Sigma- Aldrich). Stained samples were rinsed (PBS) and mounted on slides in 90% glycerol (#G9012, Sigma- Aldrich)/ 10% PBS/1% n-propyl-gallate (#P3130, Sigma-Aldrich) and sealed with nail polish. Untouched single cells were imaged for each sample using a Leica LMD7 Microscope (Leica- microsystems, Germany) at a magnification of x63. The cell surface area of n=8 cells was calculated based on the cytoplasmic images (green channel, 532nm) by ImageJ. Briefly, the images were blurred by a Gaussian filter (3-pixel mask) and binarized by a “Li dark” filter (no features were removed or added digitally).
The average cell surface area was measured by counting the fluorescence pixels and dividing them by the number of nuclei in the frame; this was further normalized to the average surface area of the “DDW coated” images. Statistical analysis was determined using both multivariate one-way ANOVA analysis and a two-tailed t-test, provided by the MATLAB statistics toolbox.
Biomolecule effect on the focal adhesion spots
The effect of the various biomolecules on the focal adhesion complex formation was estimated by immunocytochemistry. To this end, gold surfaces were coated by aqueous solutions (1/6 mg/ml, DDW:AN, 1: 1) ofthe various biomolecules and the control, as mentioned above. HEK-293-GFP and rat-dissociated-retinal cells were seeded at an initial concentration of 0.8K cells/mm2 to allow single cell spreading and incubated for 72h (for rat-dissociated retinal cells lOpM Ara-c and 50pM ROCK inhibitor were added after 24h). Following incubation, cells were immuno-stained for cytoskeletal markers as follows: Surfaces were rinsed gently with PBS to remove unattached cells; then the cells were fixed with 4% paraformaldehyde (Bar Naor, BN15711) for 15 min at room temperature. Next, the cells were rinsed in PBS with 0.5% Triton-XlOO (Amersham, 22686) and 1% Tween (Amersham, 20605) (PBST). Blocking was performed for 30 min in a blocker solution containing 1% bovine serum albumin (MP Biomedicals, 160069). Cells were incubated overnight at 4 °C with primary antibodies for anti-Vinculin antibody (mouse, #V9131, Sigma-Aldrich, 1:50); the retinal cells were further incubated with anti-CRX antibody (rabbit, #NBP2- 15964, Biotest, 1: 100). The next day the cells were rinsed with PBST, and secondary antibodies coupled to Alexa Fluor 594 anti-mouse (Jackson ImmunoResearch, #711-585-152) and Alexa Fluor 488 anti-rabbit (Jackson ImmunoResearch, #711-545-152) were applied for 30 min at RT and washed with PBS. F-actin staining was performed with #P5282, Sigma-Aldrich, 1:500 and nuclear staining was performed with Hoechst (Sigma-Aldrich, 100MG-14533). Stained samples were rinsed in PBS, mounted on slides in 90% glycerol (Sigma- Aldrich, G9012)/10% PBS/1% n-propyl-gallate (Sigma- Aldrich, P3130), and sealed with nail polish. Cells were imaged using a Leica-Stellaris-5 microscope (Leica-microsystems, Germany) at xlOO oil immersion objective. Aiming to estimate the focal adhesion (FA) spots, the Vinculin channel was binarized using ImageJ and the bright spots for each cell were counted. A DDW- normalized average amount of FA for each molecule was calculated, and the statistical significance was determined using both multi-variate one-way ANOVA analysis and a two- tailed t-test, provided by the MATLAB statistics toolbox.
Evaluation of gene expression
Quantitative PCR analysis was used to evaluate the regulation pathways of focal adhesion. Briefly, HEK293-GFP and rat-dissociated retinal cells were seeded on different biomolecule-coated gold surfaces (1/6 mg/ml), (60M cells in total for each repetition). RNA was extracted after 72h of incubation using the Gene Elute Mammalian Total RNA Miniprep Kit (#RTN70, Sigma-Aldrich) according to the manufacturer’s instructions. RT-PCR was performed on RNA extracted from the cells, which was synthesized to cDNA using M-MLV reverse transcriptase (#M1701, Promega, Madison, WI, USA). Quantitative PCR analysis was then performed using PerfeCTa SYBR Green FastMix (#95074-250, Quantabio, Beverly, MA, USA).
The primers were designed according to the gene sequences database of the National Library of Medicine website, and the specificity of each primer was assessed using the BLAST software and UCSC In-Silico PCR. Primers were manufactured by Sigma Aldrich, checked via gel electrophoresis, and melting curves at 60°C; the efficiency and specificity were assessed using linear calibration curves at various concentrations. The corresponding primers of the genes of interest are listed in Table Supp-Tl in the Supplementary Material.
The gene expression of several adhesion integrin subunits, namely, integrin aiib (ITGA2B), integrin av (ITGAV), integrin as (ITGA5), integrin Pi (ITGB1), integrin 3 (ITGB3), and the focal adhesion-associated proteins Vinculin and protein tyrosine kinase 2 (PTK-2) were tested. The expression level of the gene of interest was compared to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), normalized to the expression level of cells seeded on the control group (DDW coating), and then was calculated by the Pfaffl method, where the primer reaction efficiencies were extracted from the primer’s linear calibration curve. Multivariable one-way ANOVA test, N-way ANOVA, and a t-test were performed. Data analysis
Data are expressed as means±Standard Deviation (SD) and were calculated from the results of at least three independent experiments. Two-way ANOVA and one-way ANOVA were performed for the density experiment, one-way ANOVA for the cell spreading and the FA spot number, N-way ANOVA for the gene expression experiment, and Student’s t-test for all the experiments compared to DDW and to the Optimal biomolecule as described in each section. A value of p<0.05 was considered as statistically significant.
Photoreceptors precursors (PRP) dissociation
The retinas of Pl Sprague Dawley rats were isolated and dissociated using a papain dissociation kit (Worthington #LK003150) following the manufacturer’s protocol with minor adaptations. 1 x 105 cells were seeded on coverslips coated with poly-lysine (Sigma, #P4707) and laminin (Sigma, #L2020). PRP medium contains DMEM/F12 (Gibco) and Neurobasal (Gibco) in a 1: 1 ratio, 1% glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids, and 2% horse serum. The medium was supplemented with B27, N2, EGF (10 ng/pL, PeproTech), NGF (10 ng/pL, PeproTech), NT3 (10 ng/pL, PeproTech), BDNF (10 ng/pL, PeproTech), and FGF (10 ng/pL, PeproTech). The next day, half of the media was replaced with 10 pM ara-C PRP media and treated as mentioned for 3 days.
Flat Mount Organotypic Retinal Culture Explant
The model was based on that previously described in Orlans, H.O., et.al., (2018) (Human Retinal Explant Culture for Ex Vivo Validation of AAV Gene Therapy. Methods Mol Biol 1715, 289-303). Briefly, a 6-well plate was fdled with 1-1.5 mb of non-serum medium containing the PRP media plus treatment. Royal College of Surgeons (RCS) rats with retinal degeneration (P60-90) retinas were gently excised from the eyeballs and mounted on porous inserts (Sigma-Aldrich, PICM0RG50, 0.4 pm PTFE, Millicell cell culture inserts) with the PR facing up. Dissociated retinal cells were labeled with AAV1-GFP virus (Charles River Laboratories, USA) and treated with ara-C (10 pM) for 5 days before being seeded on the retinal explant. For the collagen condition experiments, 1 x 105 retinal cells were seeded in collagen I (pH 7-8), incubated for 15 minutes and then added PRP media for 3 days as described above. Immunocytochemistry
Dissociated rat retinal culture were fixed with 4% PFA (Bar Naor, #BN15711) for 15 minutes, then rinsed twice in PBS (Biological Industries, 02-020-1A) for 5 minutes each, followed by rinsing in PBS with 0.5% Triton X-100 (Amersham, 22686) (PBST) for 5 minutes, and then rinsed again twice in PBS for 5 minutes each. The cells were incubated in a blocker solution containing 1% bovine serum albumin (BSA, MP Biomedicals, 160069) for 45-60 minutes, then incubated overnight with a primary antibody for Rabbit anti-CRX (Biotest, NBP2-15964). The next day, the cells were rinsed with PBST 3 times and incubated with secondary antibodies coupled to Alexa 488 anti-rabbit (ENCO, 711-545-152). Cells were rinsed twice with PBST, and on the third wash, Hoechst (1.8 pM, Sigma- Aldrich, 100MG- 14533) and 140nM Phalloidin (ENCO, PHDNA-1) were added. Stained cells were rinsed with PBS and mounted on slides in 90% glycerol (Sigma- Aldrich, G9012)/10% PBS/1% n-propyl gallate (Sigma-Aldrich, P3130) and sealed with nail polish.
Immunohistochemistry
After incubating the organotypic retinal culture at 37°C and 5% CO2 for 24 hours, the retinas were fixed with 4% PFA (Bar Naor, #BN15711) for 15 minutes, followed by two rinses with PBS (Biological Industries, 02-020-1 A) for 20 minutes each. For the third wash, Hoechst (1.8pM, Sigma-Aldrich, 100MG-14533) was used, followed by one additional rinse with PBS. Subsequently, the retinas were mounted on slides in a solution consisting of 90% glycerol (Sigma-Aldrich, G9012), 10% PBS, and 1% n-propyl -gallate (Sigma-Aldrich, P3130), and then sealed with nail polish.
Quantitative PCR (qPCR)
RNA was extracted from PRP cells on day 4 using Gene Elute Mammalian Total RNA Miniprep Kit (Sigma- Aldrich, Israel, RTN70) according to the manufacturer’s instructions. RT-PCR was performed using MLV reverse transcriptase (Promega, M1701). qPCR analysis was performed using PerfeCTa SYBR® Green FastMix (Quanta, 95074-250).
RNA-seq
RNA was extracted on day 4 from Y-27632 50pM treated PRP cells and untreated cells served as control using Gene Elute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, Israel, RTN70) according to the manufacturer’s instructions. For library preparation Illumina Stranded mRNA library preparation kit (#20040534) was used from 1000 ng of total RNA starting material. All eight RNA samples underwent PolyA selection following the manufacturers protocols, with a 10 PCR cycles in the amplification step. Quantification and quality control of the libraries were done using Denovix fluorimeter and Agilent 4200 TapeStation.
Single-read sequencing of the libraries with a read length of 76 was performed with NextSeq 500 Sequencing System using NextSeq 500/550 High Output v2 kit (75 cycles) (20024906 Illumina), yielding about 22-31 million reads per samples.
RNAseq Bioinformatics analysis
Sequenced reads were mapped to the Rat reference genome sequences (mRatBN7.2) using STAR. The aligned reads were quantitated by Htseq. The normalization and differentially expressed genes test were implemented by DESeq2. An arbitrary cutoff of at least 1-fold and p-value adjusted for multiple testing < 0.1 was chosen to define a differentially expressed gene. Ingenuity IPA tool was used for enrichment analysis on the differentially expressed genes between each group. IPA tool was used for enrichment analysis on the differentially expressed genes between each group [IPA; Ingenuity Systems, http://www.ingenuity.com]. The RNAseq data have been deposited in NCBI's GEO repository. GEO: GSE276397.
Quantification and statistical analysis
Data are presented as the mean ± standard error of the mean (SEM) from at least 3 independent experiments. N indicates the number of cells or fields of view tested. For neurite extension imaging and quantification Cells and retinal imaging were performed using a confocal microscope (STELLARIS, LEICA), equipped with a 63X oil-immersion objective. The length of neurites was measured manually, and quantification was carried out in a blinded manner. Cells with neurites whose total length equaled or exceeded 5 pm were taken into account. For comparisons between the different groups, a two-tailed Student’s t test was utilized, assuming Gaussian distribution. RT-qPCR data were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and analyzed using the 2-AACt method to compare relative gene. Significant levels are denoted as P<0.05, '**' P<0.01, '***' P<0.001. Example 1: micro-well electrode geometry amplifies the electric field around the integrated neurons and eliminates electrode crosstalk
Using finite element computer simulation (as detailed above), the effect of the microwell electrode geometry on the electrode-neuron coupling was tested. More specifically, the electric field around the neurons and the lateral spread of the electric field toward adjacent electrodes (as a measure of potential crosstalk, which can reduce the obtained resolution, visual acuity, and contrast sensitivity) was tested.
To this aim, a computer simulation of a neural cell positioned in a 10pm diameter microwell composed of isolating material (SU-8), at the bottom of which is an electrode (5pm diameter) was conducted. Both the simulated spacing between the cell membrane and the micro-well walls (50nm to 1pm) and the cell-electrode distance (30nm to 1pm) were varied. The configuration in which the space between the cell membrane and the micro-well wall is 50nm and the cell-electrode distance is 30nm is termed as “sealed”; the configuration in which the space between the cell membrane and the micro-well wall is 1 pm and the cell-electrode distance is 30nm is termed as “partially sealed”; and the configuration with no micro-well present is termed as “flat”. The results are presented in Figs. 2A-C. It should be noted that that the actual spacing may be significantly smaller than 50mm, as further exemplified below (see below, Figs. 9A-C).
The selected simulated geometrical parameters were similar to those of the actual fabricated device (as detailed below). The results presented in Fig. 2A show that the induced electrical field around the cell (in response to a 2nA, 5ms pulse) in the sealed configuration is highly amplified compared to the partially sealed and flat configurations. A cross-section at 10pm above the electrode (Fig. 2B) revealed that for a sealed configuration, the electrical field (and the current density) at the cell membrane-well interface (2001.73 V/m) is more than three- orders-of-magnitude higher than that of the flat configuration (1.54V/m) and almost two- orders-of-magnitude higher than that of the partially sealed (56V/m) configuration.
Interestingly, a thorough test of the effect of well-cell spacing on the average electrical field on the cell membrane (Fig. 2C) revealed a field decay with an inverse power function. This finding is associated with the observation that the R seal (the resistance of the extracellular fluid from the stimulating electrode to the ground) is inversely proportional to the cross-section of extracellular fluid between the cell membrane and the insulating micro-well walls. Example 2- Micro-well sealing geometry significantly reduces the neuron activation charge threshold
Following the studies of the induced electrical field distribution, the sealing effect on the neuron activation threshold was studied. To this aim, the Hodgkin-Huxley ion channels’ rate functions and dynamics were incorporated as partial differential equations applicable to the boundary representing the cell membrane. To accurately model the glutamatergic cells, the glutamatergic cells (human embryonic-stem-cell-derived photoreceptor precursors, hESC- PRPs) were patch-clamped, and both ionic currents (under the voltage clamp configuration) and membrane potential (under the current clamp configuration) were measured. These empirical measurements of the biophysical properties of the cells were then used in the simulated cells.
Thereafter, the efficiency with which simulated current injection elicits a change in the cell membrane potential in the sealed configuration were tested compared to the partially sealed and flat (non-sealed) configurations. This entailed applying 1msec current pulses at various current amplitudes, corresponding to charge values of (IpC-lOuC), and simulating the voltage membrane change. These studies (Fig. 3A) revealed that for the sealed configuration (50nm spacing, 30nm height), a depolarization of 18.3mV was elicited by a mere 5pC charge. It should be noted that light-induced voltage membrane change of photoreceptors is in the order of 2mV; similarly, a lOmV electrically induced depolarization of bipolar cells is considered as a target for eliciting visual percepts. For the partially sealed configuration (1pm spacing, 30nm height), the same stimulus (5pC) induced a 1.18mV deflection in membrane potential, more than an order-of-magnitude lower than the sealed configuration. Furthermore, for the non-sealed (flat) configuration (height of 30 nm), a significantly higher charge of lOpC was needed to elicit a deflection of 16.8mV.
To further assess the impact of micro-well geometry on neuron-electrode coupling, the charge activation threshold required to trigger an action potential in neurons was tested. Although photoreceptors operate through graded potentials rather than action potentials, it was decided to simulate the charge activation threshold for inducing an action potential since this metric, rather than the graded potential, is commonly used to evaluate electrode-neuron coupling. This approach allows to more effectively compare this novel geometry with previous data. To this aim, the same micro-well cell geometry modeled with various neuron-wall spacings and neuron-electrode distances was used. The injected cathodic current with a pulse width of 1msec from the electrode situated at the bottom of the well was increased until an action potential was induced.
These simulations further validated the notion that the activation thresholds of a neuron in the sealed configuration (50nm spacing, 30nm distance) were only l lpC, four-orders-of- magnitude lower compared to the case of flat electrodes with a 40pm cell-electrode distance (87nC), and 1.3-orders-of-magnitude lower than with the partially sealed (0.22nC) configuration. More importantly, the simulation results of the sealed configuration are comparable with intracellular activation thresholds of PRPs obtained (5.2pC, Fig. 3B) using the patch-clamp technique. Thus, the simulations suggest that the micro-well electrode configuration yields a tight neural electrode sealing, providing extracellular stimulation of neurons with charge levels comparable to those of intracellular stimulation.
Furthermore, the simulation results for the flat configuration are in agreement with previously published data (Boinagrov, D., Pangratz-Fuehrer, S., Goetz, G. & Palanker, D. Selectivity of direct and network-mediated stimulation of the retinal ganglion cells with epi-, sub- and intraretinal electrodes. J Neural Eng 11, 026008 (2014)).), further validating these simulations. Interestingly, it was observed that for the case of a sealed cell, the cell-electrode distance had no effect on the activation threshold, whereas, for the non-sealed (flat) configuration, the threshold increased with the cell-electrode distance. In contrast to the cellelectrode distance, the percentage of cell membrane covered by the micro-well (which is affected by the cell’s position in the vertical axis), had a significant effect on the activation threshold. These simulations revealed that 25% and 50% coverage ratios of the cell by the well suffice to reduce the activation threshold by almost 1.5 and 0.3 orders of magnitude, respectively, compared to the no membrane coverage scenario (Fig. 3B).
Example 3 - Crosstalk and spatial resolution simulations
To verify the enhanced spatial resolution facilitated by the electrical field confinement, computer simulations were performed to estimate the obtained spatial resolution with the hybrid implant compared with conventional retinal prostheses.
In the first set of simulations, the potential crosstalk between pixels, which is a main determinant of the resolution of patterned retinal activation, was modelled. To this end, two neighbouring pixels (10pm pixel diameter and 5pm diameter electrode) were modelled using the same electrical and biophysical properties used for the single-pixel activation threshold study. The effect of activating a “target” cell on the “adjacent” cell was studied. More specifically, the ratio between the activation threshold of the “target” cell and the activation threshold of the “adjacent” cell when the stimulation is given in the “target” cell electrode, were studied. Further, the effect of various coverage percentages of the cells by the micro-well were studied. In this context, activation contrast ratio is defined as the ratio of the activation threshold of the target pixel to the activation thresholds of the adjacent pixels during electrical stimulation of the target pixel.
The results presented in Figs. 4A-B, show that in the sealed configuration (complete coverage case) there is a difference of more than two orders of magnitude (1 : 430) between the activation charge threshold of the target cell and the adjacent cell (Fig. 4A), suggesting an excellent contrast of patterned neural activation. This ratio decreased as the cell coverage declined, reaching a difference of 5 -fold for the case of almost no coverage (0.1% coverage). It was further found that when the target cell was stimulated by 9.8pC to elicit a lOmV change in the Vm, the change in the adjacent cell was only 0.09mV, corresponding to a ratio that is higher than 100.
Example 4- Electrical stimulation of the PRP elicits an increase in intracellular calcium
Since the glutamatergic neurons in the hybrid retinal implant are activated by electrical stimulation rather than light and should functionally integrate with the host retina, partially differentiated photoreceptors (PRPs) were selected for use as the glutamatergic cells of the device (multi-well electrode array). These cells have the potential to form synapses with the host bipolar cells. PRPs were generated by differentiating hESCs into PRPs using an optimal short 30-day optimized protocol. To evaluate the potential of these cells to be activated by the hybrid implant, current was injected and the resulting calcium changes were measured, as neuronal depolarization accompanied by changes in intracellular and synaptic calcium concentration. This was achieved by applying the whole cell configuration of the patch clamp with the cells held at -60mV and then applying 30mV voltage steps of membrane voltages from -90mV to +30mV while measuring the calcium dynamics using an OGB fluorescence calcium indicator, as shown in Fig. 5A. A robust change in fluorescence (dF/F; magenta trace (Fig. 5B), in response to a voltage step clamp in the cell of interest (magenta circle in Fig. 5A).
Example 5- The PRP cells exhibit glutamatergic synapses
In addition to the increase in intracellular calcium in the electrically stimulated (patched) cell, a synchronous calcium dynamic was observed in some of the cells adjacent (e.g., the cell marked as 2 in Fig 5A) to the stimulated cell (Fig. 5B, cell marked as 1), an indication of synapse-mediated activity in these adjacent cells. To verify the glutamatergic nature of the cells and synapses, a cocktail of glutamate receptor blockers was used (see Methods) to block the synaptic transmission between the stimulated cells and the post-synaptic cell. The outcome was a statistically significant (p< 0.001, as shown in Fig. 5D) reduction in calcium dynamics (indicated by the arrow in Fig. 5C). Overall, these results suggest that electrical stimulation of the PRP cells used in the hybrid retinal implant modulate their intracellular calcium in a controlled manner. They further indicate the capability of the cells to create glutamatergic synapses with other retinal cells.
Example 6- Fabrication and characterization of an implant
Briefly, to produce an implant, multi-step photolithography fabrication process utilizing the negative photoresist SU-8 to obtain the 3D microstructures was used. The fabricated in- vivo implant, with a diameter of 1mm, features a central section containing a multi-electrode array of 3,172 micro-well electrode complexes with a pixel size of 10pm. Each micro-well is 10pm in height (Figs 6A-C) and has an electrode with a diameter of 6pm at the center of its bottom (Figures 6D-E, b', b"). A handle is attached to facilitate its placement beneath the retina and for electrical connectors (Fig. 6A). For ease of fabrication, the experimental implant enables the stimulation of the cells at a single row resolution.
As demonstrated below, implant features enhanced cell -micro-well coupling. As was shown in the simulation studies (Figs. 2A-C, Figs. 3A-B), reducing the activation threshold in the hybrid retina is dependent on the distance between the glutamatergic neurons (PRPs) and the micro-well walls. To this aim, three implant prototypes (no electrodes) were fabricated using the same optimized lithography process with different micro-well diameters (10, 15, and 20pm) (Fig. 7, panes a-c). The photolithography substance SU8 was mixed with rhodamine- fluorophore to enhance the visibility of the micro-well walls. GFP -labelled PRP cells were then seeded onto these devices; cells were fixed 24 hours after the culture and stained for nuclei (Hoechst) and actin (phalloidin).
First the survival of the cells in the micro-wells was studied. Towards this end, cells were stained on days 1 and 7 post-seeding on the implant for 7AAD dead cell staining. It was found that the percentage of viable cells was high on day 1 (92 ± 4), with a similar viability obtained on day 7 post staining (88 ± 3) (n=3, p>0.05, Student’s unpaired).
Next, imaging with a confocal microscope was performed to characterize the cell-well interface and the number of cells per micro-well. Confocal imaging of the cells showed that upon entrance to the micro-well, the cell assumed the micro-well’s shape (Fig. 7, panels a’, b’, c’). The acquired images were then analysed using IMARIS software, as is detailed above. To capture the cell volume accurately the actin and GFP fluorescence channels were combined (Fig. 7, panels a, a’, b, b’, c, c’). The, the percentage of the micro-well wall area that was in contact with the cell membrane was calculated by colocalization analysis of the cell fluorescence (GFP and actin staining) and the SU8 implant fluorescence (rhodamine B, red) (Fig. 7, panels d-f). Using the contact area, a micro-well was defined as sealed by the neuron for wells in which the contact area formed a complete closed circle without any gaps or breaks (e.g., Fig. 7, panels e’, f ) and non-sealed if there was a gap (e.g., Fig. 7 panel d’)
The analysis revealed that the percentage of the micro-well wall area in contact with the cell membrane significantly increased as the micro-well size decreased, reaching 80.6%±31.7 for the 10pm micro-well, significantly larger compared to the 15 pm (57.1%±10.2, P=0.03) and 20pm wells (35.7%±3.4, P=0.0006) (Fig. 8A). Similarly, the percentage of sealed micro-wells is significantly higher in the 10pm (69.7%±12.6) and 15pm (75.6%±9.7) wells compared to the 20pm wells (38.7%±7.6, P=0.005 and P=0.0003, respectively) (Fig. 8B).
The average number of cell nuclei per well decreased significantly as the micro-well diameter decreased (Fig. 7, panels g-h). Specifically, the 10pm wells contained an average of almost a single cell per well (1.2±0.08), which is significantly lower than in the 15pm (1.64±0.09, P=0.003) and 20pm (2.6±0.2, P0.001) wells (Fig. 8C).
Staining for actin (Fig. 7, panels j-1) revealed that actin filaments formed a ring on the well wall, which is probably the cellular mechanism involved in the creation of the tight sealing with the micro-well. Overall, the results showed excellent cell sealing within 10pm microwells.
To further study the cell-micro-well interface distance at nanometer-scale resolution, micro-wells seeded with PRP were fixed and embedded using Epon, cut using an ultramicrotome into 50-70nm sections and then imaged with transmission electron microscopy (TEM; JEM-1400 Flash Electron Microscope). TEM of PRP cells seeded in a 20pm diameter SU8 well revealed several cells inside the well, as inferred by the nuclei in the image (Fig. 9A). A zoom-in on the cell-well interface revealed a close contact between the cells and the well walls on both sides (Figs. 9B-C), with a visible gap present in only a few locations. These results, which are in agreement with the confocal imaging, suggest that the actual distance between the cells and the well surface may well be less than the 50nm, which was in the computational model (Figs. 3A-B).
Example 7 - The micro-well electrode configuration significantly reduces the activation charge
One of the main consequences of tightly sealing the cells within the micro-wells, is the amplification of the electrical field around the cell and reduction of the activation threshold, as predicted by the computer simulations (Figs. 2A-C, Figs. 3A-B). To experimentally validate this prediction, an in-vitro of implant composed of an array of 10pm diameter electrodes, situated at the bottom of 5 pm -high micro-wells, that interface with a commercially available current injection system (MEA2100, Multi-Channel Systems Germany) was fabricated. To study the activation threshold, the cells were seeded into the micro-wells and patch-clamped them (Figs. 10A-B). A stimulating current with pulse durations ranging from 40ps to 1msec and with increasing amplitude was injected (through the electrode at the bottom of the well), while the cell membrane potential was recorded via the intracellular patch-clamp electrode. The activation threshold was defined as the lowest current required to induce an action potential or an inward Na+ current. To overcome the challenge of patching the PRP in 10pm microwells, in this experiment, relatively larger cells, namely, HeK239 cells engineered to express the voltage -sensitive Na+ channel Na2.1 were used. To further simplify the patching, 5pm-high micro-wells were used, rather than ones with the full height, 15pm.
The experiment was conducted under three conditions, namely: micro-well sealed, flat electrode, and distant. In the micro-well sealed condition, the micro-well electrode configuration was used and only cells in which the fluorescence staining pattern suggested complete sealing with the ring of the micro-well were studied (Fig. 10B, yellow arrow). In the flat electrode condition, a similar setup was used, but here the electrodes were not encircled by micro-well structures. In the distant condition, the effect of the cell-electrode distance on activation thresholds was further tested by coating the implant prototype with a uniform layer of collagen (30-50pm, using 10% collagen gel). This layer thickness was selected to reflect the distance between the implanted subretinal electrodes and the bipolar cells (estimated at about 40pm).
The results shown in Figs. IOC and 10D demonstrate the strong effect of the micro-well sealing and the cell-electrode distance on the activation threshold. In the distant condition, which mimics the natural distance between sub-retinal electrodes and the target bipolar cells, the threshold for cell activation was pulses of 5ms and 1.5mA. Most cells were not activated by the maximum current amplitude and duration parameters of the system (10ms, 1.5mA). In contrast, cells in the flat condition, which were cultured directly on flat electrodes (no microwells), exhibited a significantly lower activation current and charges (Fig. 10C-D). The best performance was that of the micro-well sealed configuration, which decreased the threshold even further, to the picocoulomb range (for 40ps pulses, a charge threshold of 420±170pC and 2.6±2.8nC (average±st.dev.) for the sealed and flat configuration, respectively, p<0.01). More importantly, since in this experiment micro-wells with a height of 5pm (rather than 15-20pm) were used, and it is estimated that the cell-micro-well wall coverage is only 25%, based on the simulation results, the expected threshold of a completely sealed configuration is 1.5-orders- of-magnitude lower and is, therefore, expected to be around 13.28pC. Thus, the measured reduction in the activation threshold by the tight-sealing micro-well configuration is within the pC range and fits well with the computer simulation.
Example 8- Implantation of the retinal prosthesis into the sub-retinal space and integration of the glutamatergic cells with the host retina
The in-vivo survival of the PRPs in an implanted hybrid device and their axonal sprouting and synaptic connection with the host retina was tested. The device was seeded with GFP- labeled PRPs before implantation into the sub-retinal space of immunosuppressed RCS rats (a known retinal degeneration model). Fundus imaging and optical coherence tomography (OCT, Spectralis, Heidelberg) revealed the anatomical position of the implant in the subretinal space, in close proximity with the inner nuclear layer (INL), where the target bipolar cells are located (Figs. 11A-B).
In addition, fluorescence fundus images was used (Fig.HC acquired by Micron IV, Phoenix) to estimate the survival of the GFP -labeled PRP cells on the subretinal implant on day 30 by normalizing the fluorescence signal to that on day 1. The cell survival rate on day 30 was ~80%±8.4 st.dev. (n=4, Fig. l id). Interestingly, in a different set of experiments, the survival rate of the same cells following a bolus injection to the subretinal space (without an implant) resulted in lower cell survival (63%±25.91, n=6), although the difference was not statistically significant. These results suggest that the implant, serving as a scaffold, enhances cell survival.
Next, it was sought to ensure the transplanted cells remained inside the device microwells and do not migrate into the inner retina. Synaptic integration of the implanted cells with the host retina is critical for the activation of the host retina and obtaining restored vision with high visual acuity.
Histological analyses of the enucleated eyes revealed that the PRPs survived and remained within the micro-wells throughout the 30-day follow-up period. This was clearly observed in confocal imaging of flat-mounted (Fig. 12A) and 10pm sectioned (Fig. 12B) retinas. Importantly, some of the cells extended axonal -like structures toward the inner retina. Synapse formation between the transplanted cells and the host bipolar cells was tested. To this end, the histology slices were stained with the pre-synaptic marker ribeye and the post-synaptic bipolar cell marker PKC alpha. Co-localization of ribeye expression and GFP cell protrusions was observed in proximity to many bipolar cells (Fig. 12D), suggesting synaptic formation.
Example 9- Surface modification using biomimetic peptides
To assess the specific assembly of the biomolecules to the gold surface, a fluorophore paired RGD molecule (NBD-RGD as a representative molecule) with a Cys anchor was used to coat a checkerboard-patterned gold-glass surface. Figs. 13A-B show a clear checkerboard green-fluorescent pattern with higher fluorescence in the gold compared with bare glasses, suggesting the specific binding of the biomolecule through the thiol group to the gold; X-ray photoelectron spectroscopy (XPS) of the Cys-RGD-coated gold surface revealed energy peaks at 162.5eV and 397eV, which are known to fit S2 (sulfur-gold) and Nis (metal nitrides) energy, respectively, indicating the coating of the gold surface by the peptide biomolecule. Contact angle measurements between the gold surface and a water droplet (Fig. 14A-C) were used to assess the surface energy and wettability relating to the hydrophilicity of the surface. Since the biomolecules are hydrophilic peptides containing amid and carboxylic groups on their chain side, they elicited a decrease in the contact angle compared with the untreated (bare) gold surface, as is shown in Figs 14A-C. All the biomolecules used showed a similar decrease in contact angle compared with the bare gold surface (p<0.05); the lowest contact angle was measured for linear short RGD at 22.2° (Fig. 14D). The contact angle decreased with increasing biomolecule concentration, reaching a plateau at l/12mg/ml (Fig 4E).
Example 10- Surface modification using biomimetic peptides - Retinal Cells are Attracted by YIGSR biomolecules
To evaluate the optimal biomolecule and its concentration for promoting cell adhesion in general, and retinal cells in particular, gold surfaces were coated with the studied biomolecules at concentrations of (l/24mg/ml, l/12mg/ml, l/6mg/ml, and l/3mg/ml); gold surfaces soaked in DDW served as controls. Both cell types were seeded (0.8K cells/cm2) and incubated for 24h onto the pre-coated surfaces. The normalized cell density was evaluated by a self-coded ImageJ image-processing tool (as described in the Methods section) by dividing the number of nuclei counted in each figure by its surface area and by normalizing the average cell density of each biomolecule by the cell density of DDW.
HEK293 and retinal cells were cultured on gold surfaces coated with the tested biomolecules at a concentration of 1/3 [mg/ml]. Quantification of the results, which are presented in Figs. 15A-B suggest that HEK293 cells were mainly attracted to the RGD-type biomolecules, whereas the retinal cells were mainly affected by the YIGSR-type molecules.
Quantification of the normalized average (n=8, normalized to DDW) cell density of HEK293 and retinal cells cultured on the various surfaces is shown in Figs. 15A-B, respectively. For the HEK293 cells (Fig. 15A), all biomolecules elicited a higher cell density compared with DDW (one way ANOVA p<0.001 for all biomolecules). The highest cell density was observed for the long-spacer cRGD of 6.2±1.3 (p<0.005 for a comparison with all other molecules), followed by the short spacer non-cyclic RGD (GG-RGD, (5.02±2.3). For these two biomolecules, cell density significantly increased with increasing biomolecule concentration (both with P for trend=0.001).
In contrast with the HEK293 cells, the retinal cells (Fig. 15B) were mainly attracted to the YIGSR ligand. The highest cell density was found for the long spacer YIGSR (Poly-Pro- YIGSR) at the lowest concentration (3.6±1.6), p<0.001 compared with DDW). Slightly lower cell density was observed for the short spacer YIGSR (2.6± 1.2, p=0.001 compared with DDW).
Example 11- YIGSR biomolecules elicit retinal cell spreading
As an additional measure of the effect of the biomolecules on cell-surface interaction, the effect of the biomolecule on the cell surface area was tested. To this end, both types of cells were seeded on gold surfaces coated with various biomolecules and incubated for 72h, after which the rat retinal cells were stained for Viafluor cytoplasmic indicator; the HEK293 cells constitutively expressed GFP. The average cell surface was calculated by ImageJ and normalized based on the cell surface measurements obtained for DDW (Figs. 16A-B). Quantitative analysis showed that for the HEK293 cells (Fig. 16A), both the RGD and YIGSR molecules led to a significant increase in the cell surface area. The largest cell surface area was observed with the cyclic molecules (2.7±0.7 and 2.6±0.8 for GG-c(RGD) and poly-pro- c(RGD), respectively, p<0.001 compared with DDW). Similarly, the cells seeded on gold coated with YIGSR molecules exhibited an increased surface area compared with those treated with DDW (2.5±0.1 and 1.65±0.5 for GG-YIGSR and poly-pro-YIGSR, respectively, p<0.05 compared with DDW).
In contrast with the HEK293 cells, for the retinal cells, only the YIGSR biomolecules (Fig. 16B) elicited a significant increase in the normalized surface area; an increase of 3.5±0.6, and 3.4±0.7-fold was found for the short and long spacer molecules, respectively (p «0.001 compared with DDW). These results are in alignment with the differential effect of YIGSR and RGD on the cell density observed for HEK293 and the retinal cells, further stressing the importance of a cell-specific coating design for implantable devices and electrodes.
Example 12- Focal adhesion spots
Next, the effect of the biomolecule coating on the focal adhesion (FA) mechanism was tested. To this end, both cell types were seeded on pre-coated gold surfaces with various biomolecules, fixed and stained for Vinculin (the main protein in the FA complex), the cytoskeleton F-actin filament (also integrated into the FA complex), and the nuclei. FA spots (Vinculin clusters) were counted manually.
Characteristic immunocytochemistry confocal images for both cell types indicated representative FA spots. The RGD molecules affected the HEK293 surface attachment, showing developed filopodia and cell spreading. In contrast, YIGSR affected the rat- dissociated-retinal cells more than the RGD-type molecules. Quantitative analysis of the average cellular number of FA spots is presented in Figs 17A-B, for HEK293 (Fig 17A) and retinal cells (Fig. 17B). In line with the results obtained forHEK293 regarding the cell density and cellular area, the cyclic RGD molecules elicited significantly more FA spots compared with the YIGSR molecules (p«0.01 for both cyclic RGD vs both YIGSR and DDW). The largest effect was with the cyclic molecules (33.3±6.9, p<0.01, and 33.5±3.7 p<0.01, compared with DDW for short (GG-cRGD) and long (Poly-Pro-cRGD) spacers, respectively); the short linear (GG-RGD) elicited 29±1.4 focal adhesion spots (p<0.01 compared with DDW). The average cellular number of FAs elicited by the YIGSR molecules was 18.3±7 and 14.4±4.0 spots for the short (GG-YIGSR) and long spacer (Poly-Pro-YIGSR) molecules, respectively, not significantly higher compared with DDW (p>0.4).
In contrast with the HEK293 cells, for the retinal cells (Fig. 17B), all biomolecules elicited a significant increase in the average cellular FA spots, compared with DDW; the largest effect was observed for short-spacer YIGSR (GG-YIGSR, 82.0±15.7, p=0.02 compared with DDW, p=0.04 compared with GG-RGD), further supporting YIGSR as the biomolecule of choice for attracting retinal cells.
Example 13- Gene expression following coating
To better understand the molecular mechanism by which the coating biomolecules regulate cell adhesion via the adhesion integrins and focal adhesions, the expression of genes involved in cell adhesion were quantified. Real-time qPCR of genes associated with various subunits of adhesion integrins (Intaiib, Intav, Intas, IntPi, and Intph ) and focal adhesion proteins (Vinculin and PTK-2) for cells seeded on gold surfaces that were coated with the biomolecules. In these experiments, the molecules that were found to be optimal in the previously described experiments, namely, a long spacer with cyclic-RGD (Poly-Proline-c(RGD) and short spacer YIGSR (GG-YIGSR) were used. Results were normalized to cells seeded on the untreated gold surface after being normalized to GAPHD expression. The relative normalized gene expression levels for both cell types are presented in Figs. 18A-B.
Both biomolecules amplified most of the examined adhesion genes for both cells. For the HEK293 cells (Fig. 18A), most of the adhesion integrins and the focal adhesion genes were affected by the two biomolecules. In general, the effect of RGD was higher than with YIGSR (N-Way ANOVA, P<0.005)T The largest effect of RGD coating on the HEK293 cells’ gene expression was observed for Intav, Intas, and Vinculin (10.7±1.4, 5.2±2.3 and 5.0±1.2-fold, t- test p= 0.001, 0.03, and 0.004, respectively). For the retinal cells, the effect of the various biomolecules on the expression levels of the adhesion integrins and the focal adhesion proteins (Fig. 18B) was a mild increase in the various integrins except for Intaiib.
Example 14- ROCK inhibitor (Y-27632) increases neurite extension in PRP
The effect of a Y-27632 on neurite extension in primary retinal rat dissociated cells was tested. Following dissociation, cells were treated with araC to eliminate all non-neural cells, including Muller glial cells, that can introduce confounding effects and hinder the interpretation of results. To this aim, a dose-dependent effect assay was conducted, comparing axonal length following 72h treatment with Y-27632 at concentrations of 50 pM, 100 pM, and 200 pM (Figs. 19A-D), with untreated samples serving as controls. Staining for CRX, a PRP-specific marker, was carried out to ensure the quantification of PRP neurite length exclusively. Actin staining was used to visualize the cell neurites (Figs. 19A-D). Neurite length and the percent of PRP with neurites were quantified (Figs. 19E, 19F, respectively). A significant increase in PRP extensions length across all concentrations (Fig. 19E) was observed. The larger length was observed at 100 pM (Control: 1.95 pm ± 0.37, 50 pM: 15.86 pm ± 1.19, 100 pM: 27.22 pm ± 3.19, 200 pM: 16.68 pm ± 3.18; P<0.001 for all concentrations compared with control); there was statistic significant difference between the different concentration groups (compared with the lOOpM: 50 pM: P<0.001, 200 pM: P=0.03). The reduced effectiveness of Y-27632 at 200 pM could be due to the molecule's toxicity, as it was reported that ROCK inhibitors at elevated concentrations affect other kinases, such as protein kinases A and C and myosin light chain kinase, potentially leading to unintended side effects.
In addition to the effect of ROCKi on neurite extension, there was a significantly elevated percentage of cells exhibiting neurites in all three concentrations (50 pM: 44.2% ± 5.71, 100 pM: 52.37%± 9.41, 200 pM: 50.41% ± 2.76) compared to the control (0.38%± 1.59; P0.001), with no significant difference observed between doses (Fig. 19F).
Example 15- ROCK inhibitor enhances axonal guidance and phototransduction gene expression in retinal cells.
To elucidate the mechanism by which Y-27632 affects neurite extension, bulk RNA sequencing was conducted on control and 50pM Y -27632 treated samples (n=4). Data analysis revealed significant (>= 2-fold-change) alterations in 645 genes. Notably, among impacted pathways were BMP signaling (including BMP4 and MAPK12 genes), phototransduction genes such as rhodopsin (RHO) and transducin (GNAT1), and CREB signaling (such as DRD4, OXTR and SSTR3).
To verify the RNA-seq findings, PCR analysis was conducted. In line with the RNA- seq, following Y-27632 treatment an elevation in BMP4 was observed (5.87FC±1.18, p=0.018), along with genes related to phototransduction such as RHO (18.79FC±3.82, p=0.01), OPN1SW (3.76FC±0.72, p=0.02), and GNAT1 (113.4FC±15.38, p=0.002).
Example 16-Taurine and RCM media increase neurite extension in PRP
Next, alternative endogenous molecules, which are capable of neurite extension were tested. Taurine, that is found in high concentrations in the retina, and RCM composed of molecules secreted from retinal cells, were selected. To this end, dissociated PRP cells were treated for three days either with taurine (ImM) , RCM, or a combination of both (Figs. 20A- E); their neurite length and percentage of cells with neurites were compared to the untreated control sample (Fig. 201, 20J). A significant effect on neurite extension was observed following both taurine and RCM (Fig. 201, Ctrl: 0.94pm±0.28, Taurine: 10.35pm±2.08, RCM: 11.36pm±1.68, P<0.001 for both treatment); the effect of these the two treatments was similar (P=0.71). In addition, the percentage of CRX-positive cells with neurites was elevated following both treatments; the difference was statistically significant only for RCM (P<0.01) compared to the control (Ctrl: 11.82%±0.74, Taurine: 15.83%±6.64, RCM: 23.41%±1.35).
Notably, the effect of both taurine and RCM was significantly lower than those observed following Y-27632 50pM treatment (RCM: P<0.05, Taurine: P<0.01, compared to Y-27632). To test potential synergistic effects between Y-27632 and taurine or RCM, PRP were treated with RCM or taurine combined with Y -27632 at 50pM. No synergistic effect of Y-27632 with RCM (Fig. 201, 20J) was observed; the combination of taurine and Y-27632 resulted in neurite lengths similar to those observed with taurine alone, but lower than with Y-27632 alone (Taurine+Y-27632: 11.82pm±1.47, RCM+Y-27632: 16.85pm±1.89). The percent of PRP with neurites (Fig. 20J) was not different compared to Y-27632 (Taurine+Y-27632: 37.6%±15.57, RCM+Y-27632: 49.6%±9.87, P=0.62).
Example 17 - BMP4 inhibition reduces neurite extension in taurine and RCM but not Y- 27632-treated cells.
Given that the gene expression analysis suggested the involvement of the BMP4 pathway in Y-27632-induced axonal elongation, qPCR analysis on PRP cultures treated RCM demonstrated that BMP4 was significantly increased; (2.97 FC+0.56, P<0.05); the effect of taurine (ImM) treatment was slightly lower (2.74 FC+0.8) and was not statistically significant.
To validate BMP4 involvement in neurite outgrowth, PRP cell cultures were treated with BMP4 at a 40 ng/mL (Figs. 21A-B). A low but statistically significant increase in axonal length was detected (BMP4 - 3.86pm±0.9, Ctrl - 1.95+0.37, P=0.02). In contrast, no significant effect in the percentage of PRP cells with neurites was observed (BMP4 - 11.27%±3.15, Ctrl - 10.38+1.08, P=0.73) (Figs. 21C-D).
To further verify BMP4 involvement in neurite outgrowth, it was tested whether Noggin, a BMP4 inhibitor, can inhibit neurite extension following treatment with taurine and RCM. Interestingly, lOng/mL Noggin eliminated almost completely PRP neurites outgrowth in both taurine and RCM treated cultures (Figs. 201, J) resulting in axonal lengths comparable to those of the control sample (1.95 pm±0.37, 0.86pm±0.47, 2.47pm±0.61, Avg ± SEM for control, Taurine + Noggin and RCM + Noggin treatment, respectively, P>0.15 for all comparison). These results suggest that the BMP4 pathway is involved with the taurine and RCM derived neurite elongation process. In contrast, Noggin caused only a small (Fig. 20F), but not statistically significant decrease, in the effect of Y -l' l 632 on neurite length (Y-27632: 15.86±1.19, Y-27632 50pM±Noggin: 13.9 pm±2.4, P=0.47) (Fig. 201).
Similar to the effect on neurite length, Noggin elicited a blocking effect on the percentage of PRP that are extending neurites following treatment with RCM or taurine, but had no effect on Y-27632 treatment (Fig. 20J).
Example 18- Y-27632 enhanced neurite elongation in an ex-vivo model of Degenerated Retina
Neuronal cells cultivated in a two-dimensional culture are exposed to different microenvironments and biological signals compared to those grown in a three-dimensional culture or within intact tissue. Therefore, when studying neurite extension, it is important to replicate the natural neural and retinal tissue environment. To achieve this, retinal explants from three- month-old RCS rats, a model of outer retinal degeneration disease, where the photoreceptor cells degenerate, leaving the bipolar and ganglion cell layers relatively preserved, were used (Figs. 22A-D). PRP cells labeled with AAV1-GFP virus were seeded onto the retinal explant, with the bipolar cell layer facing upward. The samples were treated with Y-27632 50pM or Taurine ImM. A significant increase in neurite length in all treated groups compared to the untreated control group (Fig. 22E) was observed (Ctrl: 11.88pm ± 0.72, Y-27632: 17.88pm ± 0.79, Taurine: 16.23pm ±0.8, PO.OOl for all treatment compared to control). Interestingly, in these experiments, the control sample (no treatment) showed axonal lengths significantly longer compared with control group of 2D treated cells discussed above (1.96±0.37 and 11.88±0.72) for retinal explant and 2D cultures controls, respectively (P<0.001). This effect may be mediated via the presence of Muller glia cells in retinal explants secreting growth factors which affect the cells or via three dimensional mechanical cues provided by the tissue. To differentiate between the two potential mechanisms, an artificial photoreceptor outer nuclear layer was generated by applying photoreceptor cells incorporated within a collagen hydrogel mixture and seeded them above the inner nuclear layer (INL). It was found that the length of cell extensions within the collagen was longer compared to cells treated with Y -27632 (22.01±0.71 vs 17.87i0.79, p<0.001, Fig. 22E).
These results demonstrate the combined effect of 3D mechanical cues and neurotrophic retinal factors on promoting axonal extensions.

Claims

CLAIMS What is claimed is:
1. A hybrid retinal prosthesis comprising: a micro-well electrode array having wells with diameters between about 5 pm and about 50 pm for confining neurons; and a biomimetic peptidomimetic coating comprising adhesion motifs associated with the electrode surfaces.
2. The prosthesis according to claim 1, wherein the wells have diameters in the range of about 5 pm- 15 pm.
3. The prosthesis according to claim 1 or 2, wherein the peptidomimetic coating comprising thiol-functionalized adhesion motifs.
4. The prosthesis according to any one of claims 1-3, wherein the peptidomimetic coating comprises thiol-functionalized adhesion motifs selected from YIGSR (SEQ ID NO:6), IKVAV (SEQ ID NO: 7), and cyclic RGD (SEQ ID NO: 1).
5. The prosthesis according to any one of claims 1-4, wherein the peptidomimetic coating comprises a peptide having an amino acid sequence CGG- YIGSR (SEQ ID NO: 4) or C- PolyProline(10)-YIGSR (SEQ ID NO: 5).
6. The prosthesis according to any one of claims 1-5, wherein the peptide density on the electrode surface is at a density sufficient to promote integrin-mediated adhesion of photoreceptors while minimizing non-specific binding of non-retinal cells, as measured by differential adhesion assay.
7. The prosthesis according to any one of claims 1-6, wherein the micro-well electrode array comprises a conductive material electrode at the well base, said electrode comprises or coated with gold, platinum, indium tin oxide (ITO), activated iridium oxide film (AIROF), Sputtered iridium oxide films (SIROF), Titanium, Titanium nitride (TiN), Poly(3,4- ethylenedioxythiophene) polystyrene sulfonate(PEDOT:PSS), or any combinations thereof.
8. The prosthesis according to any one of claims 1-7, wherein the micro-well height is in the range of about 10 pm to 20 pm.
9. The prosthesis according to any one of claims 1-8, further comprising glutamatergic neuronal cells, bipolar cells and/or retinal ganglion cells within said micro-wells.
10. The prosthesis according to claim 9, wherein the cells comprise photoreceptor precursor cells (PRPs).
11. The prosthesis according to claim 10, wherein the PRPs are human embryonic stem cell- derived photoreceptor precursors (hESC-PRPs), induced pluripotent stem cells (iPSCs), fetal retinal progenitors, or primary photoreceptor cells.
12. The prosthesis according to any one of claims 1-11, further comprising a composition comprising small molecule substances and/or growth factors, configured to enhance neurite elongation of neuronal cells.
13. The prosthesis according to claim 12, wherein the small molecule substances comprise a ROCK-inhibitor and/or a BMP4-activator.
14. The prosthesis according to any one of claims 12-13, wherein the small molecule substances comprise Y-2763, fasudil, or hydroxyfasudil, taurine, retinal-conditioned medium (RCM), or any combinations thereof.
15. The prosthesis according to any one of claims 1-14, further comprising collagen, Neuronal growth factor (NGF) and/or extracellular matrix (ECM) hydrogel scaffold to enhance neuronal cells survival and axonal guidance.
16. The prosthesis according to any one of claims 1-15, wherein the micro wells are pretreated with plasma, prior to adding cells to the wells.
17. The prosthesis according to any one of claims 1-16, configured for implantation into a target area of a subject having retinal dysfunction, to thereby at least partially restore vision in the subject.
18. The prosthesis according to claim 17, wherein the target area comprises subretinal, epiretinal, or suprachoroidal space.
19. The prosthesis according to claim 17 or 18, for use in at least partially restoring scotopic, photopic, mesopic, color vision, improving contrast sensitivity, enhancing visual acuity, or any combinations thereof.
20. The prosthesis according to any one of claims 17-19, wherein the subject is having retinitis pigmentosa, age-related macular degeneration, or photoreceptor degeneration.
21. A method for at least partially restoring vision to a subject having retinal disfunction, the method comprising implanting into a target area of the subject the prosthesis according to any one of claims 1-16.
22. The method according to claim 21, for at least partially restoring scotopic, photopic, mesopic, color vision, improving contrast sensitivity, enhancing visual acuity, or any combinations thereof.
23. The method according to claim 21 or 22, wherein the subject is afflicted with retinitis pigmentosa, age-related macular degeneration, or photoreceptor degeneration.
24. The method according to any one of claims 21-23, wherein the target area comprises subretinal, epiretinal, or suprachoroidal space.
25. A hybrid retinal implantable prosthesis comprising: a high-density micro-well electrode array having wells of about 5-30pm diameter; and glutamatergic neurons seeded within said wells; wherein geometry of the microwells provides tight sealing to the cells, to thereby amplify local electric fields, and reduce activation thresholds to about 950pC or less.
26. The prosthesis according to claim 25, wherein the activation thresholds to less than about 500pC.
27. The prosthesis according to any one of claims 25-26, wherein a pixel pitch of the microwell array is about 10 pm or less, thereby enabling a visual acuity of at least 20/40.
28. The prosthesis according to any one of claims 25-27, wherein electrical stimulation of the sealed neurons modulate intracellular calcium, potassium and/or sodium dynamics, leading to neurotransmitter release, resulting in selective ON/OFF bipolar pathway activation and/or activation of other retinal circuitry
29. The prosthesis according to any one of claims 25-28, wherein electrical stimulation of the sealed neurons facilitates controlled calcium influx and release of glutamate, resulting in selective ON/OFF bipolar pathway activation and/or activation of other retinal circuitry.
30. The prosthesis according to any one of claims 25-29, wherein the sealing provides an activation contrast ratio of about 1 :400 or more, between a target micro-well and adjacent micro-wells, thereby reducing crosstalk between adjacent cells, thereby enhancing resolution and contrast of the restored vision.
31. The prosthesis according to any one of claims 25-30, wherein the wells are pretreated with plasma prior to seeding the cells.
32. The prosthesis according to any one of claims 25-31, wherein the wells are made of or coated with photoresists or epoxy-based polymers.
33. A method for surface modification for retinal cell-specific adhesion to a substrate, the method comprising: providing a substrate comprising an electrode; and coating said electrode with a biomimetic peptide comprising an amino acid sequence YIGSR (SEQ ID NO: 6), an anchor and a spacer, wherein the coating promotes preferential adhesion of retinal cells over non-retinal cells by enhancing cell density, spreading, and/or focal adhesion formation.
34. The method according to claim 33, further comprising seeding cells on a surface of the coated substrate.
35. A biomimetic coating composition for promoting retinal cell adhesion to a substrate, the composition comprising a peptide having an amino acid sequence CGG-YIGSR (SEQ ID NO: 4) or C-PolyProline(10)-YIGSR (SEQ ID NO: 5), wherein said peptide selectively promotes retinal cell adhesion to a substrate.
36. The biomimetic coating composition according to claim 35, wherein the substrate is or comprises electrodes.
37. The biomimetic coating composition according to claim 36, wherein the electrode comprises gold, platinum, indium tin oxide (ITO), activated iridium oxide film (AIROF), Sputtered iridium oxide films (SIROF), Titanium, Titanium nitride (TiN), (PEDOT:PSS) coated electrodes, or any combinations thereof.
38. A method of promoting neurite outgrowth in photoreceptor precursor cells (PRPs), the method comprising contacting said PRPs with a ROCK inhibitor for a period of time, under suitable conditions, to thereby induce increase in neurite length by at least 5 -fold compared to untreated PRPs.
39. The method according to claim 38, wherein the ROCK inhibitor is Y-27632, provided at a concentration in the range of about 50 pM and 100 pM.
PCT/IL2025/050652 2024-07-30 2025-07-29 Hybrid retinal neuroprosthesis and methods of using the same Pending WO2026028201A1 (en)

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