Implantable living electrodes

Abstract

To provide implantable living electrodes solving the disadvantage of an inflammatory foreign body response that ultimately diminishes the quality of recording and stimulation.SOLUTION: In one aspect, the invention comprises an implantable living electrode comprising: a substantially cylindrical extracellular matrix core; and one or more neurons implanted along or within the substantially cylindrical extracellular matrix core. The one or more neurons includes one or more optogenetic or magnetogenetic neurons proximal to a first end of the implantable living electrode.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 322,434, filed April 14, 2016, the contents of which are incorporated by reference in their entirety. [Background technology]

[0002] 2. Background of the Invention Brain-machine interfaces (BMIs) allow the nervous system to communicate directly with external devices to mitigate deficits associated with neurodegeneration or to drive peripheral prostheses. Significant progress has been made using penetrating microelectrode arrays and optogenetic strategies. However, these approaches are limited in that they generally rely on placing non-organic electrodes / optrodes in the brain, inevitably leading to an inflammatory foreign body response that ultimately reduces the amount of recording and stimulation. Current BMI strategies suffer from non-permanence, non-specificity, and / or significant foreign body responses when implanted. Summary of the Invention

[0003] BRIEF SUMMARY OF THE INVETION In one aspect, the invention includes an implantable living electrode comprising a substantially cylindrical extracellular matrix core; and one or more neurons implanted along or within the substantially cylindrical extracellular matrix core, the one or more neurons comprising one or more optogenetic or magnetogenetic neurons proximal to a first end of the implantable living electrode.

[0004] In various embodiments, the implantable living electrode is capable of bidirectional stimulation and bidirectional recording.

[0005] In various aspects, the implantable living electrodes are capable of unidirectional stimulation and unidirectional recording.

[0006] In various embodiments, the implantable living electrodes are capable of unidirectional stimulation and bidirectional recording.

[0007] In various embodiments, neurons are stimulated and recorded using different wavelengths of light.

[0008] In various embodiments, the substantially cylindrical extracellular matrix core has a maximum cross-sectional dimension selected from the group consisting of about 10 μm to about 20 μm, about 25 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 150 μm, about 150 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 400 μm, about 400 μm to about 500 μm, about 500 μm to about 700 μm, and about 700 μm to about 1000 μm.

[0009] In various aspects, the implantable living electrode further comprises a hydrogel sheath coaxially surrounding the substantially cylindrical extracellular matrix core.

[0010] In various embodiments, the hydrogel sheath has a maximum cross-sectional dimension selected from the group consisting of about 20 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm to about 800 μm, and about 800 μm to about 1200 μm.

[0011] In various embodiments, the implantable living electrode has a length of about 100 μm to 10 cm or greater.

[0012] In various embodiments, one or more neurons comprise multiple distinct phenotypes targeting multiple targets, and multiple distinct optogenetic or magnetogenetic phenotypes for responding to or emitting distinct wavelengths of light.

[0013] In various embodiments, the one or more neurons comprise one or more selected from the group consisting of primary cerebral cortical neurons, dorsal root ganglion neurons, glutamatergic neurons, GABAergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, peptidergic neurons, thalamic-derived neurons, striatal-derived neurons, hippocampal-derived neurons, substantia nigra-derived neurons, peripheral nervous system-derived neurons, and spinal motor neurons.

[0014] In various embodiments, the primary cerebral cortical neurons comprise one or more selected from the group consisting of neurons from layer I of the cortex, neurons from layer II of the cortex, neurons from layer III of the cortex, neurons from layer IV of the cortex, neurons from layer V of the cortex, neurons from layer VI of the cortex, neurons from the visual cortex, neurons from the motor cortex, neurons from the sensory cortex, and neurons from the entorhinal cortex.

[0015] In various embodiments, the implantable living electrode further comprises one or more non-neuronal cells selected from the group consisting of endothelial cells, myocytes, myoblasts, astrocytes, olfactory ensheathing cells, oligodendrocytes, or Schwann cells.

[0016] In various aspects, the neurons are derived from stem cells.

[0017] In various aspects, the neurons are derived from neuronal progenitor cells.

[0018] In various embodiments, the hydrogel sheath comprises agarose.

[0019] In various embodiments, one or more neurons implanted along or within a substantially cylindrical extracellular matrix core are formed via forced cell aggregation.

[0020] In various embodiments, the present invention includes a method comprising: implanting one or more implantable live electrodes in a subject's brain; and positioning a compatible stimulator proximal to at least one of the one or more implantable live electrodes.

[0021] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to activate or excite brain activity.

[0022] In various embodiments, the method further includes controlling a compatible stimulator to actuate at least one of the implantable living electrodes to activate or excite synaptic activity of the host.

[0023] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to activate or excite neuronal activity.

[0024] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to activate or excite neural network activity.

[0025] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable live electrodes to suppress brain activity.

[0026] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to suppress synaptic activity of the host.

[0027] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to inhibit neuronal activity.

[0028] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to inhibit neural network activity.

[0029] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to modulate synaptic activity of the host.

[0030] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to modulate neuronal activity.

[0031] In various embodiments, the method further includes controlling a compatible stimulator to activate at least one of the implantable living electrodes to modulate neural network activity.

[0032] In various embodiments, at least one of the one or more implantable living electrodes is implanted in the subject's central nervous system, peripheral nervous system, cerebral cortex, striatum, hippocampus, spinal cord, and / or peripheral nerves.

[0033] In various embodiments, the method further comprises selectively exciting or inhibiting cerebral cortical neurons.

[0034] In various embodiments, the method further comprises selectively exciting or inhibiting dopaminergic neurons.

[0035] In various embodiments, the method further comprises selectively exciting or inhibiting dorsal root ganglion neurons.

[0036] In various embodiments, the present invention includes a method comprising: implanting one or more implantable live electrodes in a subject's brain; and positioning a compatible sensor proximal to at least one of the one or more implantable live electrodes.

[0037] In various aspects, the method further comprises reporting the activity of the excitatory neurons.

[0038] In various aspects, the method further comprises reporting inhibitory neuron activity.

[0039] In various embodiments, the method further comprises simultaneously reporting the activity of excitatory and inhibitory neurons.

[0040] In various embodiments, the present invention includes methods comprising implanting one or more implantable living electrodes into the brain of a subject; and applying stimulation to the implantable living electrodes to selectively excite or inhibit one or more selected from the group consisting of glutamatergic neurons, GABAergic neurons, cholinergic neurons, serotonergic neurons, peptidergic neurons, thalamic derived neurons, striatal derived neurons, hippocampal derived neurons, substantia nigra derived neurons, peripheral nervous system derived neurons, spinal motor neurons, cerebral cortical neurons, dopaminergic neurons, and dorsal root ganglion neurons.

[0041] In various aspects, the method further comprises controlling the amount of synaptic input.

[0042] In another aspect, the invention includes an implantable living electrode comprising: a substantially cylindrical extracellular matrix core; a hydrogel sheath coaxially surrounding the substantially cylindrical extracellular matrix core; one or more selected from the group consisting of an electrode, an optrode, a magnetic actuator, a heating probe, a cooling probe, or a chemical applicator disposed between the substantially cylindrical extracellular matrix core and the hydrogel sheath; and one or more neurons implanted along or within the substantially cylindrical extracellular matrix core.

[0043] In another aspect, the invention includes an implantable living electrode comprising: a substantially cylindrical extracellular matrix core; and a plurality of aggregated neurons implanted along or within the substantially cylindrical extracellular matrix core.

[0044] In various aspects, the plurality of aggregated neurons comprises dopaminergic neurons.

[0045] In various embodiments, the extracellular matrix core comprises collagen-laminin.

[0046] In various embodiments, aggregated neurons are formed by centrifugation in pyramidal wells.

[0047] In various embodiments, the implantable living electrode includes a separate neuronal body portion and a separate axon portion.

[0048] In various aspects, the implantable living electrode is a unidirectional or bidirectional implantable living electrode.

[0049] In various embodiments, the extracellular matrix core comprises collagen-laminin.

[0050] In various aspects, the invention includes a method of treating Parkinson's disease in a patient, comprising implanting a living electrode according to any one of the claims into the substantia nigra of the patient.

[0051] In another aspect, the invention includes a method of making an implantable living electrode, comprising the steps of providing an extracellular matrix core; and contacting at least one end of the extracellular matrix core with a plurality of aggregated neurons.

[0052] In various embodiments, the method further comprises maintaining the implantable living electrode under conditions that promote axonal growth within or along the extracellular matrix core.

[0053] In various embodiments, the method further comprises preforming a plurality of aggregated neurons prior to contacting the at least one extracellular matrix core. [The present invention 1001] a substantially cylindrical extracellular matrix core; and one or more neurons implanted along or within said substantially cylindrical extracellular matrix core. 1. An implantable living electrode comprising: An implantable living electrode, wherein the one or more neurons include one or more optogenetic or magnetogenetic neurons proximal to a first end of the implantable living electrode. [The present invention 1002] An implantable living electrode of the present invention 1001 capable of bidirectional stimulation and bidirectional recording. [The present invention 1003] An implantable living electrode of the present invention 1001 capable of unidirectional stimulation and unidirectional recording. [The present invention 1004] An implantable living electrode of the present invention 1001 capable of unidirectional stimulation and bidirectional recording. [The present invention 1005] The implantable living electrode of the present invention 1001, wherein said neurons are stimulated and recorded using different wavelengths of light. [The present invention 1006] 1001. An implantable living electrode according to the present invention, wherein the substantially cylindrical extracellular matrix core has a maximum cross-sectional dimension selected from the group consisting of about 10 μm to about 20 μm, about 25 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 150 μm, about 150 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 400 μm, about 400 μm to about 500 μm, about 500 μm to about 700 μm, and about 700 μm to about 1000 μm. [The present invention 1007] A hydrogel sheath coaxially surrounding a substantially cylindrical extracellular matrix core. The implantable living electrode of the present invention 1001 further comprises: [The present invention 1008] 1007. The implantable living electrode of the present invention, wherein the hydrogel sheath has a maximum cross-sectional dimension selected from the group consisting of about 20 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm to about 800 μm, and about 800 μm to about 1200 μm. [The present invention 1009] An implantable living electrode of the present invention 1001 having a length of about 100 μm to 10 cm or greater. [The present invention 1010] The implantable living electrode of the present invention 1001, wherein the one or more neurons comprise a plurality of different phenotypes targeting a plurality of targets and a plurality of different optogenetic or magnetogenetic phenotypes for responding to or emitting distinct wavelengths of light. [The present invention 1011] The implantable living electrode of the present invention 1001, wherein the one or more neurons include one or more selected from the group consisting of primary cerebral cortical neurons, dorsal root ganglion neurons, glutamatergic neurons, GABAergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, peptidergic neurons, thalamic derived neurons, striatal derived neurons, hippocampal derived neurons, substantia nigra derived neurons, peripheral nervous system derived neurons, and spinal motor neurons. [The present invention 1012] The implantable living electrode of the present invention 1011, wherein the primary cerebral cortical neurons include one or more selected from the group consisting of neurons from layer I of the cortex, neurons from layer II of the cortex, neurons from layer III of the cortex, neurons from layer IV of the cortex, neurons from layer V of the cortex, neurons from layer VI of the cortex, neurons from the visual cortex, neurons from the motor cortex, neurons from the sensory cortex, and neurons from the entorhinal cortex. [The present invention 1013] The implantable living electrode of the present invention 1001 further comprising one or more non-neuronal cells selected from the group consisting of endothelial cells, myocytes, myoblasts, astrocytes, olfactory ensheathing cells, oligodendrocytes, or Schwann cells. [The present invention 1014] The implantable living electrode of the present invention 1001, wherein said neurons are derived from stem cells. [The present invention 1015] The implantable living electrode of the present invention 1001, wherein said neurons are derived from neuronal progenitor cells. [The present invention 1016] 1001. The implantable living electrode of claim 1001, wherein said hydrogel sheath comprises agarose. [The present invention 1017] An implantable living electrode of the present invention 1001, wherein one or more neurons implanted along or within a substantially cylindrical extracellular matrix core are formed via forced cell aggregation. [The present invention 1018] Implanting one or more implantable living electrodes of any of the present inventions 1001-1015 into the brain of the subject; and placing a compatible stimulator proximate to at least one of the one or more implantable living electrodes; A method comprising: [The present invention 1019] controlling a compatible stimulator to activate at least one of said implantable living electrodes to activate or excite brain activity. The method of the present invention 1018 further comprises: [The present invention 1020] controlling a compatible stimulator to activate at least one of said implantable living electrodes to activate or excite synaptic activity in the host. The method of the present invention 1018 further comprises: [The present invention 1021] controlling a compatible stimulator to activate at least one of said implantable living electrodes to activate or excite neuronal activity. The method of the present invention 1018 further comprises: [The present invention 1022] controlling a compatible stimulator to activate at least one of said implantable living electrodes to activate or excite neural network activity. The method of the present invention 1018 further comprises: [The present invention 1023] controlling a compatible stimulator to activate at least one of said implantable living electrodes to inhibit brain activity. The method of the present invention 1018 further comprises: [The present invention 1024] controlling a compatible stimulator to activate at least one of said implantable living electrodes to inhibit synaptic activity of the host. The method of the present invention 1018 further comprises: [The present invention 1025] controlling a compatible stimulator to activate at least one of said implantable living electrodes to inhibit neuronal activity. The method of the present invention 1018 further comprises: [The present invention 1026] controlling a compatible stimulator to activate at least one of said implantable living electrodes to inhibit neural network activity. The method of the present invention 1018 further comprises: [The present invention 1027] controlling a compatible stimulator to activate at least one of said implantable living electrodes to modulate synaptic activity of the host. The method of the present invention 1018 further comprises: [The present invention 1028] controlling a compatible stimulator to activate at least one of said implantable living electrodes to modulate neuronal activity. The method of the present invention 1018 further comprises: [The present invention 1029] controlling a compatible stimulator to activate at least one of said implantable living electrodes to modulate neural network activity. The method of the present invention 1018 further comprises: [The present invention 1030] The method of claim 1018, wherein at least one of the one or more implantable living electrodes is implanted in the subject's central nervous system, peripheral nervous system, cerebral cortex, striatum, hippocampus, spinal cord, and / or peripheral nerves. [The present invention 1031] The method of claim 1018, further comprising the step of selectively exciting or inhibiting cerebral cortical neurons. [The present invention 1032] The method of claim 1018, further comprising the step of selectively exciting or inhibiting dopaminergic neurons. [The present invention 1033] The method of claim 1018, further comprising the step of selectively exciting or inhibiting dorsal root ganglion neurons. [The present invention 1034] Implanting one or more implantable living electrodes of any of the present inventions 1001-1015 into the brain of the subject; and placing a compatible sensor proximate to at least one of the one or more implantable vital electrodes. A method comprising: [The present invention 1035] The method of claim 1034 further comprising the step of reporting activity of the excitatory neurons. [The present invention 1036] The method of claim 1034, further comprising the step of reporting activity of inhibitory neurons. [The present invention 1037] The method of claim 1034, further comprising the step of simultaneously reporting the activity of excitatory and inhibitory neurons. [The present invention 1038] Implanting one or more implantable living electrodes of any of the present inventions 1001-1015 into the brain of the subject; and applying stimulation to said implantable living electrode to selectively excite or inhibit one or more selected from the group consisting of glutamatergic neurons, GABAergic neurons, cholinergic neurons, serotonergic neurons, peptidergic neurons, thalamic derived neurons, striatal derived neurons, hippocampal derived neurons, substantia nigra derived neurons, peripheral nervous system derived neurons, spinal motor neurons, cerebral cortical neurons, dopaminergic neurons, and dorsal root ganglion neurons. A method comprising: [The present invention 1039] The stage that controls the amount of synaptic input The method of the present invention 1038 further comprises: [The present invention 1040] a substantially cylindrical extracellular matrix core; a hydrogel sheath coaxially surrounding said substantially cylindrical extracellular matrix core; one or more selected from the group consisting of an electrode, an optrode, a magnetic actuator, a heating probe, a cooling probe, or a chemical applicator disposed between the substantially cylindrical extracellular matrix core and the hydrogel sheath; and one or more neurons implanted along or within said substantially cylindrical extracellular matrix core. An implantable living electrode, including [The present invention 1041] a substantially cylindrical extracellular matrix core; and a plurality of aggregated neurons implanted along or within said substantially cylindrical extracellular matrix core; An implantable living electrode, including [The present invention 1042] The implantable living electrode of the present invention 1041, wherein said plurality of aggregated neurons comprises dopaminergic neurons. [The present invention 1043] 1041. An implantable living electrode according to the present invention, wherein said extracellular matrix core comprises collagen-laminin. [The present invention 1044] The implantable living electrode of the present invention 1041, wherein said aggregated neurons are formed by centrifugation in pyramidal wells. [The present invention 1045] An implantable living electrode of the present invention 1041 comprising a separate neuronal body portion and a separate axon portion. [The present invention 1046] The implantable living electrode of the present invention 1041, which is a unidirectional or bidirectional implantable living electrode. [The present invention 1047] A method of treating Parkinson's disease in a patient comprising implanting a living electrode of the present invention 1041 into the substantia nigra of the patient. [The present invention 1048] Providing an extracellular matrix core; and contacting at least one end of said extracellular matrix core with a plurality of aggregated neurons. A method for producing an implantable living electrode, comprising: [The present invention 1049] The method of claim 1048 further comprising the step of maintaining the implantable living electrode under conditions that promote axonal growth within or along the extracellular matrix core. [The present invention 1050] The method of claim 1049, further comprising the step of preforming a plurality of aggregated neurons prior to the step of contacting at least one extracellular matrix core. [Brief description of the drawings]

[0054] For a fuller understanding of the nature and desired objects of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings, in which like reference characters refer to corresponding parts throughout the several views, and in which:

[0055] [Figure 1] FIG. 1 illustrates an implantable living electrode (indicated by arrow number 100) comprising a cylindrical extracellular matrix core (102) coaxially surrounded by a hydrogel sheath (104) and implanted with neurons (106a, 106b), according to an embodiment of the invention. In one embodiment, a particular neuronal population (112a) can be excited by one wavelength of light (114), while application of another wavelength of light (116) inhibits another neuronal population (112b). In another embodiment, two different neuronal populations can be excited by two different wavelengths of light. In yet another embodiment, two different neuronal populations can be inhibited by different wavelengths of light. [Diagram 2]2A-2C show cross-sections of electrodes comprising an extracellular matrix core (102) surrounded by a hydrogel sheath, according to an embodiment of the invention. [Diagram 3] 1 illustrates a method of using an implantable living electrode according to an embodiment of the present invention. [Figure 4] Figures 4A-4C show structural and functional analysis of micro-TENNs. Micro-TENNs consist of dense clusters of neuronal cell bodies with dense axonal tracts extending end-to-end within a central column. Figure 4A shows a unidirectional micro-TENN, a single neuron (MAP-2+) population spanned by a tau+ axonal tract. Figure 4B shows a bidirectional micro-TENN displaying two neuronal populations spanned by beta-tubulin-III+ (Tuj 1+) axonal tracts. Figure 4C shows the theoretical framework for in vitro optical stimulation and recording (with genetically encoded opsins and / or fluorescent Ca2+ reporters) and traditional electrophysiology. Activity of micro-TENNs was assessed by stimulating one neuronal population and recording the resulting action potentials in the other population electrophysiologically and optically based on Ca2+-sensitive reporters. [Diagram 5] Figures 5A-5E show the structure, phenotype, and maturation of micro-TENNs for multiple configurations that vary based on the distribution of neuronal cell bodies and axonal penetration. Figure 5A shows a confocal reconstruction of a unidirectional dopaminergic micro-TENN at 2 weeks in vitro (green: all axons (beta-tubulin-III+); red: dopaminergic neurons (tyrosine hydroxylase+); blue: all nuclei). Figure 5B shows a phase-contrast microscopy image of a bidirectional cortical neuronal micro-TENN. Figures 5C and 5D show a unidirectional dorsal root ganglion neuronal micro-TENN. Figure 5E shows a dense unidirectional cortical neuronal micro-TENN with cell body regions externalized to the hydrogel micro-columns. Control of the structure, phenotype, maturation / plasticity, and function of living micro-TENN electrodes is demonstrated in vitro. [Figure 6]Showing extremely long micro-TENNs. In vitro immunohistochemistry of bidirectional micro-TENNs shows robust axonal extension from one end of the neuronal population to the other. Staining of neuronal cell bodies and axons shows axonal extensions of up to 2 cm. [Figure 7] We show micro-TENN survival, ingrowth, and integration in vivo. In Figure 7, panel A, we show that 3 days after delivery to the rat brain, the micro-TENN neurons survived and maintained their axonal organization within the hydrogel tubes. In Figure 7, panels B-D, we show that 28 days after implantation, the micro-TENN neurons survived and integrated with the brain. In Figure 7, panel B, we show that the transplanted neurons extended neurites into the host tissue. In Figure 7, panels C and D, we show a magnified view of a region from Figure 7, panel B, showing putative dendritic spines along the ingrowing neurites, which (Figure 7, panel D) have synapsin-positive puncta in close proximity to the neurites (circles), suggesting synaptic integration. Scale bars: Panel A = 50 μm, Panel B = 40 μm, Panels C and D = 20 μm. [Figure 8]We illustrate the theoretical advantages of axon-based "living electrodes" for neuromodulation, namely the mechanism and specificity of neuronal stimulation for "living electrodes" (left) compared to conventional electrodes (center) and optrodes (right). Living electrodes provide engineered axonal tracts with controlled cellular organization and fully differentiated neurons that, when pre-transfected in vitro, constrain the spatial extent of transfected cells while suppressing the 3D reduction problem derived from the delivery of cell suspensions, both of which are advantageous for clinical deployment. In contrast to conventional electrodes, which essentially stimulate or record from a relatively large 3D volume around the electrode (indicated by the large cloud of stimuli impinging on multiple layers and neurons), living electrodes can also provide high specificity, since the structures can be engineered to synapse with specific neuronal subtypes in a given anatomical region (as indicated by the axons of living electrodes synapsing with neurons of a certain color). Microfiber optrodes can achieve a high level of specificity, whereas in vivo delivery of opsins typically relies on viral injections, which may diffuse and affect non-targeted areas (the spread of optogenetic transduction is illustrated by light grey neurons straying from layer V into layer VI). Optical methods may also have limited advantages due to tissue absorption of light. Finally, living electrodes provide a softer path to route signals to and from deep brain structures compared to the rigid materials used for electrodes / optrodes, minimizing signal issues due to mechanical mismatch / micromotion and glial scarring. [Figure 9] The concepts of target specificity and synaptic integration of living electrodes are illustrated. Living electrodes offer high specificity as structures can be engineered to synapse with specific neuronal subtypes, as demonstrated by micro-TENN axons synapsing only with round neurons and not stellate neurons in a conceptual depiction. [Figure 10] A theoretical framework for recording and stimulation studies using live electrodes in the cerebral cortex is presented. [Figure 11] The concept of a living electrode is shown. Micro-TENNs are used for corticofugal recording (right panel) or cortical afferent stimulation (left panel) interfacing with neural circuits. By penetrating a predefined location in the brain and positioning the other end at the brain surface, the micro-TENN acts as a living electrode. Deep micro-TENN neurons / axons in the brain can then synaptically integrate with local host neurons, whereas axonal projections across the structure act as functional relays to and from the cortical surface, where information is exchanged using optical and / or electrical interfaces. The output (corticofugal) theoretical framework allows for the projection of a replica of deep activity to the cortical surface by local synaptic integration. The input (cortical afferent) theoretical framework allows for the control of excitation or inhibition of specific neural circuits. [Figure 12] Examples of living electrode structures and applications in the brain are shown (purple: tractography of a typical axonal tract in the brain). Initially developed to physically reconstruct lost long-range axonal connections in the brain, micro-tissue engineered neural networks (TENNs) are in vitro grown preformed microstructures consisting of distinct neuronal populations spanned by long axonal tracts. Micro-TENNs can consist of unidirectional or bidirectional axonal tracts. Cortico-thalamic micro-TENNs can be applied as living electrodes to record or modulate sensory-motor information in the cortex or thalamus, which are vulnerable to brain trauma and stroke. Dopaminergic (DA) micro-TENNs can be used to provide / restore dopaminergic input to the striatum, which is important for alleviating motor symptoms in Parkinson's disease. Cortico-hippocampal micro-TENNs can be used to modulate or encode information exchange between the cortex and hippocampus, which is important for learning and memory formation. [Figure 13] 1 provides a perspective view of a macro-TENN electrode according to an embodiment of the present invention. [Figure 14]FIG. 14 shows nerve growth within a macro-TENN structure according to an embodiment of the invention. Nuclei are stained blue with DAPI. Axons are stained green with beta-tubulin-III (Tuj1). Panels A-C of FIG. 14 provide confocal slices along the plane illustrated in Panel D of FIG. 14. Panel A of FIG. 14 is the bottom confocal slice showing nuclei resting on the bottom surface of the macro-TENN structure. Panel B of FIG. 14 is a confocal slice immediately above the slice shown in Panel A of FIG. 14, showing axons (green) growing along the inner edge of the macro-TENN structure. Panel C of FIG. 14 is a confocal slice immediately above the slice shown in Panel B of FIG. 14, showing axons (green) growing only at the interface edges on only the north or south side of the tube. Panel D of FIG. 14 is a schematic looking down the length of the macro-TENN to illustrate the confocal slices shown in Panels A-C of FIG. 14. Panel E of FIG. 14 shows a reconstruction of confocal images of panels AC of FIG. 14, shown in a cross section similar to the perspective of panel D of FIG. [Figure 15] Figures 15A-15C show the deployment of electrodes in a defascicularization device according to an embodiment of the invention. Figure 15A provides a view looking down on the nerve. Figure 15B shows a view along the length of the nerve. Figure 15C shows a tapered device with ends that match the size of the nerve, but with a larger interior structure to increase surface area and separation of the nerve bundle. (Electrodes are not shown in Figure 15C for clarity.) [Figure 16] Phase contrast microscopy images of micro-TENNs constructed using neuronal suspension delivery (panels A and B) versus forced neuronal aggregate delivery (panels C-E) are provided. In panels A and B, an example of a micro-TENN with infiltration of neuronal cell bodies throughout the interior of the micro-column is shown, a result of incomplete extracellular matrix (ECM) continuity in the core. When this occurs, it leads to deviations from ideal micro-TENN cellular organization. In contrast, in panels C-E, when precisely formed neuronal aggregates are used to seed the micro-columns, ideal distribution of cell body (panel D) and axon (panel E) areas is consistently maintained. [Figure 17] Phase contrast and confocal microscopy images of the distribution of neuronal somas and axons in forced neuronal aggregate micro-TENNs. As shown in panels A and B, neuronal aggregates can be precisely seeded at one end of the micro-columns. In panel C, dense axonal extensions projecting from neurons in the aggregates can be observed over several days in vitro. In panel D, confocal microscopy images are provided following immunohistochemistry to label these aggregate micro-TENNs using antibodies recognizing all axons (beta-tubulin III; red) and all cell nuclei (Hoechst; blue), as well as synapses (synapsin; green). The hydrogel that makes up the micro-columns is non-specifically labeled purple. This demonstrates defined and distinct cell body (Hoechst+) and axon (beta-tubulin III+) areas, whereas synapsin+ puncta demonstrate functional maturation and electrochemical activity in the micro-TENNs. The neuronal aggregate seeding methodology consistently results in the formation of unidirectional or bidirectional micro-TENNs with ideal cellular organization, consisting of defined areas with neuronal cell bodies as aggregates at one or both ends of the micro-column and defined areas with axonal projections extending longitudinally across the center of the micro-column. [Figure 18] FIG. 2 shows a longitudinal section of a living electrode according to an embodiment of the present invention. [Figure 19]Long-projection unidirectional axon-based live electrodes for neuromodulation. Panel A shows confocal reconstructions at 28 DIV of live cortical neuronal electrodes immunolabeled for axons (β-tubulin-III; red) and neuronal somas / dendrites (MAP-2; green) with nuclear counterstain (Hoechst; blue). Insets of the aggregate (a') and axon (a") regions are shown on the right with appropriate boxes. Scale bar: 100 μm. Panel B shows confocal reconstructions at 28 DIV of live ventral midbrain (dopaminergic) electrodes immunolabeled for axons (β-tubulin-III; green) and tyrosine hydroxylase (dopaminergic neurons / axons; red) with nuclear counterstain (Hoechst; blue). Insets of the aggregate (b') and axon (b") regions are shown on the left with appropriate boxes. Scale bar: 250 μm. [Figure 20] Figure 1. Potential applications of axon-based living electrodes. Tailored living electrodes consisting of phenotypically controlled neuronal populations extending long axonal tracts through biocompatible microcolumns may be stereotactically implanted across various regions to treat specific disease processes. In panel A, axons projecting from a living dopaminergic electrode may synapse in a localized configuration in the striatum and release dopamine upon in vitro functionalization with channelrhodopsin and perikaryal light stimulation at the brain surface. This mimics the substantia nigra pars compacta input to the striatum in an externally controllable manner. In panel B, axons from a living glutamatergic electrode may preferentially synapse with layer IV neurons in the primary sensory cortex to deliver illusion-based tactile feedback via surface light stimulation to achieve closed-loop control of neuromotor prostheses in patients with paralysis. In panel C, axons from a living GABAergic electrode can also be implanted and juxtaposed to the seizure focus such that light stimulation causes a net suppression of seizure activity in a patient with lesional epilepsy. [Figure 21]Panels A-C illustrate the possibility of exploiting "biological multiplexing" in living electrodes. More sophisticated living electrodes can be developed to further exploit so-called biological multiplexing. By in vitro fabricating structures using microprinting and micropatterning techniques, specific synaptic configurations can be achieved to provide certain fine-grained signal manipulations linking the structures with the brain. In panel a, we show that in its simplest form, a "channel-selective" bundle of axons can transmit a signal to select which other bundles transmit signals into the brain and which are silenced. In panel b, we show that "channel selection" can similarly switch multiple channels converging to one final common output in a biological instantiation most similar to the kind of multiplexing used in electrical communication. Similarly, in panel c, a single input channel can be selected or diverted to one or more parallel outputs to "demultiplex" its signals. In panel d, we show the potential for time-shared "biological multiplexing" in living electrodes. Living electrodes may exploit delay lines emanating from a single "clock" circuit formed by clusters of neurons linked by gap junctions (coupled damped oscillators) and micropatterned inhibitory and excitatory connections. Thus, multiple parallel input channels can be serially multiplexed to a single target output neuron that in turn interfaces with the brain at each clock cycle. The clock rate (and therefore the multiplexed sampling period) can be altered by directly driving the clock circuit. [Figure 22]This is a diffusion tensor imaging representation of long-distance axonal tracts connecting distinct populations of neurons in the human brain. This conceptual depiction shows how unidirectional micro-TENNs (consisting of a population of dopaminergic neurons extending long aligned processes) can be used to recreate the nigrostriatal pathways that degenerate in Parkinson's disease. Axons in the substantia nigra are expected to functionally integrate with dopaminergic neurons grafted into the micro-TENN, whereas grafted dopaminergic axons are expected to functionally integrate with neurons in the striatum. After receiving appropriate input from the substantia nigra, the grafted neurons release dopamine into the striatum, thereby recreating the circuitry lost in Parkinson's disease. [Figure 23]Improved micro-TENN cellular organization using forced aggregation method applied to compositions and methods employing these embodiments. Phase contrast and confocal reconstructions of micro-TENNs plated with primary dopaminergic neurons at 14 DIV. Panel A shows a representative micro-TENN plated with dissociated neurons labeled by immunohistochemistry (with β-tubulin III and Hoechst, respectively) to show neurons / axons and cell nuclei. Dissociated micro-TENNs showed cellular infiltration throughout the entire length of the inner core and therefore did not represent the desired cellular organization. (B,C) Phase contrast images depicting micro-TENNs plated with processed dopaminergic neuron aggregates. Based on the plating technique, aggregates either (B) attached directly to the outside of the agarose micro-column or (C) to the inside of the inner core. High magnification images from the illustrated areas in (B,C) show that (D1,D3) cell body regions differed between the two aggregate plating techniques, but their (D2,D4) axonal regions were similar. (E) Representative aggregated micro-TENNs labeled by immunohistochemistry to display all neurons / axons (β-tubulin III) and dopaminergic neurons / axons (TH) and nuclear counterstain (Hoechst). Aggregated micro-TENNs displayed idealized cellular organization with (E1) distinct cell body regions and (E2, E3) axonal regions. (F) Higher magnification reconstruction from the illustrated area in (E) shows aggregated cell bodies. (G) Micro-TENNs made using aggregates showed greater axonal outgrowth than micro-TENNs plated with dissociated neurons (n=13 micro-TENNs per group; Mann-Whitney test, p<0.0001). Data are presented as mean ± standard deviation. Scale bar (A)=250 μm. Scale bar (B, C)=500 μm. Scale bar (D1)=200 μm. Scale bar (D2-D4) = 100 μm. Scale bar (E) = 250 μm. Scale bar (F) = 50 μm. [Figure 24]Figure 1. Effect of extracellular matrix on axonal outgrowth in micro-TENNs. Representative confocal reconstructions of dopaminergic micro-TENNs plated with different ECM cores. At 14 DIV, all micro-TENNs were immunohistochemically labeled to show all neurons / axons (β-tubulin III) and dopaminergic neurons / axons (TH) along with a nuclear counterstain (Hoechst). The type of ECM strongly influenced axonal outgrowth, with (A) collagen I (n=12 micro-TENNs) and (C) a cocktail of collagen I and laminin (n=12) supporting the longest axonal outgrowth. (B) Micro-TENNs with empty cores (n=9) or (D) cross-linked collagen cores (n=11) showed significantly shorter outgrowth. (a-d) Higher magnification reconstructions from the illustrated areas in (A-D) show similar TH expression between groups. Panel E is a graph showing that one-way ANOVA (p<0.0001) followed by post hoc Turkey's test showed that collagen I and collagen I-laminin cocktail cores were statistically equivalent (p=0.8590) and that they supported axonal outgrowth that was statistically longer than that in empty cores (p<0.0001), laminin coated cores (p<0.0001), or cross-linked collagen cores (p<0.0001), respectively (*denotes significance). Panel F is a graph showing that TH+ axonal length as a percentage of total axonal length was statistically equivalent between collagen I inner cores (n=12) and collagen I and laminin inner cores (n=12) as determined by Mann-Whitney test (p=0.9723). Data are presented as mean ± standard deviation. Scale bar (A, C, D)=500 μm. Scale bar (B) = 250 μm. Scale bar (E–H) = 50 μm. [Diagram 25]Figure 1. Long-projection dopaminergic micro-TENNs. Panels A-F are confocal reconstructions at 28 DIV of representative micro-TENNs plated with dopaminergic aggregates and a collagen I inner core. Micro-TENNs immunohistochemically labeled to display all neurons / axons (β-tubulin III) and dopaminergic neurons / axons (TH) with nuclear counterstain (Hoechst). In panels A-D, long-projection dopaminergic micro-TENNs showed robust viability and axonal outgrowth over 28 DIV. Panels E-F are higher magnification reconstructions from the illustrated area in panel C, showing healthy TH+ neurons and axons with apparent axonal varicosity suggesting sites of dopamine release. In panel G, length measurements of micro-TENNs performed at 28 DIV (n=7 micro-TENNs) show that TH+ axons were 6046±670 μm and total TH+ length was 7264±672 μm, including dopaminergic aggregates. Importantly, these lengths are more than sufficient to cross the nigrostriatal pathway in rats. Data are presented as mean±standard deviation. Scale bars (A-D)=250 μm. Scale bars (E-F)=50 μm. [Figure 26]Figure 1. In vitro synapse formation between micro-TENN dopaminergic axons and striatal neurons. Panel A shows a representative confocal reconstruction at 14 DIV of dopaminergic micro-TENNs plated with aggregated striatal terminal targets. Micro-TENNs were immunohistochemically labeled to display dopaminergic neurons / axons (TH), striatal (medium spiny) neurons (DARPP-32), and synapses (synapsin), with nuclear counterstain (Hoechst). Panels B-E show that higher magnification reconstructions of the illustrated area in (A) display (B) dopaminergic neuronal aggregates, (C) aligned robust TH+ axons, and (D) neurite outgrowth from the striatal neuronal population. (E) Higher degree of synapsin labeling along the projection pathway of TH+ axons suggests that dopaminergic axons have formed synapses with striatal neurons. Panel F is a graph showing that micro-TENNs containing striatal terminal targets did not produce statistically longer axonal outgrowths when compared to unidirectional dopaminergic micro-TENNs without terminal targets (n=9 micro-TENNs per group; Mann-Whitney test, p=0.9182). Data are presented as mean ± standard deviation. In panels G-H, synapsin+ puncta can be seen decorating putative dendrites projecting from striatal neurons shown to have dopaminergic axon varicosity, further suggesting synaptic integration. Scale bar (A)=250 μm. Scale bar (B-E)=50 μm. Scale bar (G-H)=20 μm. [Figure 27]Maintenance of neuronal viability and axonal cellular organization of micro-TENNs in vivo. Panel A shows the projection pathways and dimensions of the micro-TENN implant drawn to scale (Adapted from Gardoni F, Bellone C. 2015, Modulation of the glutamatergic transmission by Dopamine: a focus on Parkinson, Huntington and Addiction diseases, Frontiers in cellular neuroscience, 9: 25). Panel B shows the orientation of the micro-TENN (not to scale). Panel C shows a representative sagittal section 1 week after implantation, showing a longitudinal section of a dopaminergic micro-TENN in which all neurons express GFP on the synapsin promoter and have been labeled by immunohistochemistry to display dopaminergic neurons / axons (TH). This demonstrates that micro-TENN neurons survived and maintained a longitudinally aligned cellular organization. Panel D shows a representative oblique section providing a cross-sectional view of a GFP+ dopaminergic micro-TENN labeled by immunohistochemistry to display dopaminergic neurons / axons (TH) and all neurons / axons (β-tubulin III) one month after implantation. This demonstrates healthy transplanted neurons / axons with robust dopaminergic axonal projections at one month in vivo. Scale bar (A) = 20 μm. Scale bar (B) = 50 μm. [Figure 28]We show micro-TENNs as living electrodes for neuroprosthetic interfaces. Panel a is a conceptual diagram of a micro-TENN. Panel A shows the three-dimensional structure of a micro-TENN, up to several millimeters long, consisting of a hydrogel cylinder that encases an extracellular matrix core of collagen and laminin. The micro-TENN has an outer diameter of 300-400 microns and an inner diameter of 1 micron, but can be made to any size. Neuronal populations are placed at one or both ends of the cylinder, with axonal tracts penetrating the ECM and spanning the length of the cylinder. Panel B shows that neurons from a unidirectional micro-TENN can synapse with host neurons, allowing propagation of signal input to a targeted cortical region. Panel C shows that host neurons can synapse and integrate with a bidirectional micro-TENN, allowing propagation of signal output from a targeted cortical region to a dorsal neuronal population. Panel D shows the in vivo delivery and integration of a micro-TENN as a "living electrode." Micro-TENNs are preformed in vitro, and once implanted in the brain, these living microtubules can serve as input / output channels for sensory-motor information. For input, an LED array (1) photostimulates a unidirectional micro-TENN (2) with channelrhodopsin-positive neurons, which form synapses with host layer IV neurons (3). For output, a host neuron from layer V (4) forms synapses with a neuron in the bidirectional micro-TENN (5); neuronal activity is recorded by a microelectrode array (6). [Figure 29]Fabrication of aggregates of micro-TENNs is shown, comparing conventional cortical micro-TENNs with aggregated cortical micro-TENNs. The living electrodes are fabricated in two steps: formation of agarose micro-columns and aggregation of cortical neurons. Panel (a) shows the formation of agarose micro-columns. 1: A specially designed reusable acrylic mold is used to fabricate agarose micro-columns with specified inner and outer diameters. 2: Top view of the assembled mold. Dashed lines indicate outer diameter (middle) and inner diameter (top; bottom). 3: A needle of specified inner diameter is inserted into the mold. 4: Molten agarose is introduced into the mold and allowed to cool. 5: The needle is removed, the mold is disassembled, and the micro-columns are retrieved. Panel (b) shows the aggregation of cortical neurons. 1: A square pyramidal well is cast in PDMS from the 3D printed positive mold. 2: Image of a PDMS pyramidal well. 3: Single-cell suspensions of rodent embryonic neurons are introduced into pyramidal wells and centrifuged into neuronal aggregates. 4: Phase image of aggregates 24 h after plating. 5: Confocal reconstruction of aggregates at 72 h stained for live and dead neurons. In panel (c) an agarose micro-column is shown that is filled with extracellular matrix (1 mg / ml laminin and collagen; pH 7.2-7.4). Neuronal aggregates are then placed at one or both ends of the micro-column and allowed to grow in vitro. All scale bars: 100 μm. (d, e) Micro-TENNs in previous studies were fabricated using dissociated neurons. Dissociated micro-TENNs showed axonal growth and network formation in vitro over several days, but the control and reproducibility of the micro-TENN configuration was inherently limited. (f, g, h) Using the aggregate method, micro-columns are seeded with one or two neuronal aggregates (for unidirectional or bidirectional micro-TENNs, respectively). Representative bidirectional micro-TENNs after 3 days in vitro. Aggregated micro-TENNs exhibit robust axonal growth and more controllable organization. Specifically, aggregation results in distinct regions housing either cell bodies (g) or neurite projections (h). [Diagram 30]Figure 1 shows axonal growth over time for aggregate-like micro-TENNs. Both unidirectional micro-TENNs (a) and bidirectional micro-TENNs (b) showed robust axonal extension along the ECM core over the first few DIVs. Unidirectional micro-TENNs lacking distal targets showed axonal retraction after approximately 7–8 DIVs. In contrast, axons in bidirectional micro-TENNs traverse the length of the micro-column (2–2.5 mm) and form synapses with the contralateral aggregate by 5 DIVs. Representative micro-TENNs at 1, 3, 5, and 8 DIVs are shown. (c) Longer bidirectional micro-TENNs (5 mm) took longer to develop but still showed robust growth. Representative micro-TENNs at 1, 3, and 5 DIVs are shown. (d) Growth rates quantified at 1, 3, 5, 8, and 10 DIVs for 2 mm unidirectional, 2 mm bidirectional, 5 mm bidirectional, and 2 mm dissociated / conventional micro-TENNs. Growth rates were quantified by identifying the longest neurite from the aggregates in phase microscopy images (10x magnification) at the indicated time points. Sample sizes: n=6 (unidirectional - 2mm), 9 (bidirectional - 2mm), 7 (bidirectional - 5mm), and 7 (dissociated - 2mm). Error bars mean sem. Scale bar: 100 μm. [Diagram 31] Figure 1. Aggregate-specific growth using fluorescent labeling. Confocal reconstructions of bidirectional micro-TENNs labeled with GFP and mCherry to observe axonal growth from each aggregate in vitro. (a, b, c) Micro-TENNs at 1 (a), 3 (b), and 7 (c) DIV. By 3 DIV there are putative axon-axon contacts from each aggregate, followed by more robust outgrowth by 5 DIV. (d) Another micro-TENN at 6 DIV. Insets show axons from each aggregate growing along each other (e) and axons from one aggregate in contact with the opposing population (f). Scale bars: 500 μm (a, d); 100 μm (e, f). [Diagram 32]Figure 1: Viability of micro-TENNs. Viability for unidirectional and bidirectional micro-TENNs and time-matched bidimensional controls from creation was quantified by live-dead (calcein-AM / ethidium homodimer) staining at 10 and 28 DIV. (a, b, c) Representative confocal live-dead images showing an overlay of live cells, dead cells, and unidirectional micro-TENNs at 10 DIV. Schematic insets are shown below. (d, e, f) Representative confocal live-dead images of bidirectional micro-TENNs at 28 DIV. Schematic insets are shown below. All scale bars: 100 μm. (g) Graphs show the mean ratio of live cell body area to total (live+dead) cell area for each experimental group and time point. Two-way ANOVA and post-hoc analysis revealed several statistically relevant pairwise differences (*=p<0.05; **=p<0.01; ***=p<0.001). Error bars mean sem. Sample size: n=4 and 4 (unidirectional); 7 and 4 (bidirectional); 9 and 5 (control) for 10 and 28 DIV, respectively. (h) Live-dead confocal images of stained micro-TENNs at 40 DIV. Scale bar: 100 μm. [Diagram 33] Figure 2: Micro-TENN organization and synapse formation. Confocal reconstructions of representative bidirectional micro-TENNs at 4 DIV (a), 10 DIV (b), and 28 DIV (d) immunolabeled for nuclei (Hoechst), axons (Tuj-1), and synapses (synapsin). Insets in (b) and (d) represent the synapses, axonal networks, and callouts (c) and (e) show zoom-ins of the overlay of the two. (f) Confocal reconstruction of a representative unidirectional micro-TENN at 28 DIV. Scale bar: 200 μm. [Diagram 34]Implantation of corticothalamic micro-TENNs. Brain cross-section 1 month after implantation of a GFP-positive micro-TENN. The implantation here mimics the application of a "living electrode" (Figure 28) where a large dorsal neuronal population extends axons ventrally into the brain. The brain was sectioned and stained to identify the micro-TENN neurons (GFP), dendrites and cell bodies (MAP-2), and axons (Tuj-1). (a) Dorsal view of a micro-TENN. The inset represents the call column showing the lumen of the micro-TENN containing the aggregates (b) and axons (c). Similarly, a cross-section of another micro-TENN implantation reveals the aggregates (d) and axons within the lumen of the micro-column (e). Scale bars: 200 μm (a); 100 μm (b); 50 μm (d); 25 μm (c, e). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] definition The invention is most clearly understood with reference to the following definitions.

[0057] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0058] Unless otherwise stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations from the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified with the term about.

[0059] The terms "comprise," "including," "containing," "having," and the like, as used in the specification and claims, may have the meanings ascribed to them in U.S. patent law and may mean "including," "including," and the like.

[0060] The term "cylinder" or "cylindrical" as used herein includes a surface consisting of lines parallel to a given line, each of which passes through a given curve. In some embodiments, the cylinder has a circular profile. In other embodiments, the cylinder has a cross section selected from the group consisting of square, rectangular, triangular, elliptical, polygonal, parallelogram, rhombus, ring, crescent, semicircular, oval, super-oval, delta-shaped, etc. In other embodiments, the cylinder is the starting point for a more complex three-dimensional structure that can include any number of shapes that can be implemented in computer-aided design, 3D printing, and / or directed evolution approaches of secretory organisms (e.g., corals), including, for example, complex convolutions, spirals, branching patterns, multiple tubular conduits, and various fractal hierarchies.

[0061] The term "living scaffold" as used herein refers to a biological scaffold composed of living neural cells in a preformed, often anisotropic, three-dimensional (3-D) configuration. Living scaffolds can be physically integrated with existing host tissue. Living scaffolds can facilitate targeted neural cell migration and axonal pathfinding by mimicking important developmental mechanisms. Living scaffolds can act based on the simultaneous presentation of structural and soluble cues and / or electrophysiological, ionic, or neurotransmitter-based signaling.

[0062] As used herein, the term "living electrode" refers to a living structure containing nerve cells, generally but not exclusively neurons, having a defined configuration generally consisting of distinct cell body regions with protruding neurite tracts (axons or dendrites) designed to probe or modulate the nervous system.

[0063] Unless expressly stated otherwise or apparent from the context, the term "or" as used herein is understood to be inclusive.

[0064] As used herein, "synapse" refers to the junction between a neuron and another cell across which chemical communication flows.

[0065] As used herein, "synapsed" refers to a neuron that has formed one or more synapses with one or more cells, such as another neuron or a muscle cell.

[0066] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (and fractions thereof unless the context clearly dictates otherwise).

[0067] Detailed Description of the Invention Aspects of the present invention utilize advanced micro-tissue engineering techniques to create the first living biological electrodes for long-term BMI and / or neuromodulation. Novel micro-tissue engineered neural networks (micro-TENNs) act as living electrodes, typically composed of distinct neuronal populations connected by long axonal tracts contained within a microtubular hydrogel. These living micron-scale structures are capable of penetrating the brain to a predefined depth for integration with local neurons / axons, with the rear part remaining externalized on the brain surface, where next-generation optical and electrical interfaces are used to input / control and / or output / collect functional information.

[0068] Micro-TENN neurons survive, integrate with local host neurons, and maintain their axonal organization. These features are exploited to advance living electrodes as functional relays to and from deep brain regions. In this extreme paradigm, only the biological components of these structures would penetrate the brain, attenuating chronic foreign body responses. Moreover, tailored cell and tissue engineering techniques could affect the specific host neuronal subtypes that synapse with micro-TENN neurons, thereby adding a level of specificity to local stimulation and recording that is currently unattainable with conventional microelectrodes.

[0069] Electrophysiological, optogenetic, and advanced microscopy techniques show evidence of micro-TENN synaptic integration with brain neural networks at the cortical surface in rats and cross-communication with micro-TENN neurons. This multifunctional platform technology reads out local brain activity and provides inputs to affect neural activity and function, thereby providing the first demonstration of living tissue-engineered electrodes to functionally integrate with native neural networks and serve as conduits for bidirectional stimulation and recording. This potentially transformative technology at the interface of neuroscience and engineering lays the foundation for preformed implantable neural networks as a viable alternative to conventional electrodes.

[0070] Referring now to Figure 1, one aspect of the present invention provides an implantable living electrode (100). The electrode can include a substantially cylindrical extracellular matrix core (102) and a hydrogel sheath (104) coaxially surrounding the substantially cylindrical extracellular matrix core (102). One or more neurons (106a, 106b) can be implanted along or within the substantially cylindrical extracellular matrix core (102). The one or more neurons (106a, 106b) can include one or more optogenetic or magnetogenetic neurons proximal to a first end (108) of the implantable living electrode.

[0071] The extracellular matrix core (102) can include proteins, nucleic acids, small molecules, hormones, growth factors, etc. that enhance axonal growth, promote survival, reduce host inflammation, or promote integration of the composition into host tissue. Exemplary proteins include collagen, laminin, fibrin, and fibronectin. The extracellular matrix core (102) can additionally or alternatively include hyaluronic acid. The extracellular matrix core can be monolayered (a single material hardened around living axons), bilayered (as described herein), or trilayered.

[0072] The hydrogel sheath (104) can provide sufficient mechanical support for the extracellular matrix core (102) to protect the extracellular matrix core (102) from bending, buckling, collapsing, etc., before, during, and / or after implantation. For example, the hydrogel sheath (104) can have sufficient mechanical stiffness to allow loading within the needle and advancement of the needle into the tissue of the subject. The hydrogel sheath (104), in some embodiments, can be impermeable or substantially impermeable to axonal projections to guide and confine axonal growth within the hydrogel sheath (104) and / or substantially parallel to its central axis. In some embodiments, the hydrogel sheath (104) dissolves, degrades, and / or is absorbed after a period of time (e.g., one month).

[0073] In some embodiments, both the extracellular matrix core 102 and the hydrogel sheath (104) are fabricated from hydrogels, although these hydrogels can have different mechanical and / or chemical properties.

[0074] Additionally, the sheath (104) can be omitted and the core (102) can be fabricated from a hydrogel and / or extracellular matrix in which the cells and axons are embedded and which has sufficient strength to resist bending, buckling, crushing, etc. before, during, and / or after implantation.

[0075] One or more neurons (106a, 106b) can be implanted at various locations within the implantable living electrode (100). In one embodiment, the neurons (106) are implanted at one end (e.g., the first end 108 or the second end 110) and grow to the other end of the electrode after implantation. In another embodiment, the neurons are implanted at the center of the implantable living electrode (100) and grow in both axial directions. In yet other embodiments, the neurons are disposed at the distal surface of the electrode (100) and grow into and through the extracellular matrix core (102). In still other embodiments, the neurons (106) are intermixed with or disposed throughout the extracellular matrix core (102) prior to implantation and form axonal projections that connect with adjacent neurons to facilitate communication across the electrode (100). In one embodiment, the neurons reside at the interface between the extracellular matrix core (102) and the hydrogel sheath (104). In other embodiments, glial cells, or other neural or non-neuronal phenotypes, are implanted to promote neuronal growth and phenotypic differentiation.

[0076] Neurons useful for the compositions and methods provided herein include all neuronal subtypes, including but not limited to motor or sensory PNS, CNS, and stem cells (e.g., induced pluripotent stem cells, embryonic stem cells, etc.) differentiated into neuronal phenotypes.In one aspect of the present invention, neurons are derived from any cell that is a neuronal cell (e.g., cortical neuron, dorsal root ganglion neuron, or sympathetic ganglion neuron) or any cell that can be differentiated into a neuronal cell (e.g., stem cell).Neurons can be autologous, allogeneic, or xenogeneic to the subject.

[0077] In certain embodiments, the neuron is a peripheral or spinal neuron, including dorsal root ganglion neuron or motor neuron. In certain embodiments, the neuron is from the brain, including but not limited to neurons from the cerebral cortex, thalamus, hippocampus, striatum, substantia nigra and cerebellum. In certain embodiments, the primary cerebral cortical neuron includes but is not limited to neurons from cortical layers I, II, III, IV, V and / or VI (separately or any combination thereof), neurons from visual cortex, neurons from motor cortex, neurons from sensory cortex, and neurons from entorhinal cortex. The neuron can be an excitatory or inhibitory neuron. The neuron can be glutamatergic, dopaminergic, GABAergic, serotonergic, cholinergic, or any other type of neuron as classified based on its main neurotransmitter.

[0078] Neurons useful in the present invention may be derived from cell lines or other mammalian sources such as donors or volunteers. In one embodiment, the neurons are human neurons. In one embodiment, the neurons are non-human mammalian neurons, including neurons obtained from mice, rats, dogs, cats, pigs, sheep, horses, or non-human primates. In one embodiment, the neurons are cortical neurons, hippocampal neurons, dorsal root ganglion neurons, or sympathetic ganglion neurons. In another embodiment, the neurons are derived from immortalized cell lines (e.g., NT2, PC12) that are induced to become neuronal-like. In one embodiment, the neurons are derived from cadavers. In another embodiment, the neurons are derived from patients who have undergone ganglionectomy, olfactory epithelium biopsy, temporal lobe resection, tumor border resection, peripheral nerve biopsy, brain biopsy, ventricular shunt implantation with biopsy, or other clinical procedures. Furthermore, the neurons may be single integrated neurons or multiple integrated neurons (i.e., integrated nerve bundles).

[0079] In certain embodiments, glial cells (e.g., astrocytes that can extend processes to modulate host synapses, axons, dendrites, cell bodies, and / or host network activity) are additionally or alternatively incorporated into neurons 106. These can be astrocytes derived from the brain or spinal cord. In certain embodiments, the cells are olfactory ensheathing cells, oligodendrocytes, Schwann cells, endothelial cells, or muscle cells / myoblasts.

[0080] The number or density of cells positioned at either end of the structure will depend on the type of neuron being used and the ultimate use of the structure, for example, in certain embodiments, 1, 100, 1,000, 10,000, 1,000,000, 100,000,000, or more cells are positioned at one end of the structure.

[0081] In certain embodiments, neurons are cultured in vitro or ex vivo. Neurons can be cultured under appropriate conditions to promote axonal growth through the core of the structure. These conditions include, but are not limited to, appropriate temperature and / or pressure, electrical and / or mechanical activity, force, appropriate amounts of O2 and / or CO2, appropriate amounts of humidity, and sterile or near-sterile conditions. For example, cells may require nutritional supplements (e.g., nutrients and / or carbon sources such as glucose), exogenous hormones or growth factors, differentiation factors, and / or a specific pH. Exemplary cell culture media that can support neuronal growth and survival include, but are not limited to, NEUROBASAL® medium, NEUROBASAL® A medium, Dulbecco's Modified Eagle's Medium (DMEM), and Minimum Essential Medium (MEM). In certain embodiments, the culture medium is supplemented with B-27® supplement. In certain embodiments, the culture medium may contain fetal bovine serum or serum from another species at a concentration of at least 1% to about 30%, or about 5% to about 15%, or about 10%. In one embodiment, the culture medium comprises NEUROBASAL® supplemented with about 2% B27 and about 500 μM L-glutamine.

[0082] As shown in FIG. 1, a single electrode 100 can support multiplexing of multiple different types of neurons (106a, 106b). For example, the neurons (106a, 106b) can have different phenotypes designed to target distinct structures (112a, 112b) and / or promote communication along different channels. In one embodiment, the neurons respond to or emit different wavelengths of energy for neural optogenetic control and / or monitoring. For example, the electrode (100) can support bidirectional stimulation and recording, for example, by applying a first wavelength of light from a light source (114) and detecting a second, different wavelength using a detector (116).

[0083] size 2A-2C, the electrode (100) can include multiple layers (102, 104, 206) that can have various diameters and / or thicknesses.

[0084] The extracellular matrix core (102) can have a maximum cross-sectional dimension of about 10 μm to about 1,000 μm. For example, the maximum cross-sectional dimension can be selected from the group consisting of about 10 μm to about 20 μm, about 25 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 150 μm, about 150 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 500 μm, about 500 μm to about 700 μm, and about 700 μm to about 1,000 μm.

[0085] The hydrogel sheath (104) can have a maximum cross-sectional dimension of about 20 μm to about 1,200 μm. For example, the maximum cross-sectional dimension can be selected from the group consisting of about 20 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm to about 800 μm, and about about 800 μm to about 1,200 μm. In other words, the thickness of the hydrogel sheath (104) can be from about 5 μm to about 400 μm.

[0086] The hydrogel sheath (104) can be further surrounded by a layer of carboxymethylcellulose (CMC). CMC is a cellulose derivative in which carboxymethyl groups are bonded with hydroxyl groups. The functional properties depend on the degree of substitution and polymerization of the cellulose structure. CMC has unique properties in that it is rigid in the dehydrated state and gel-like in the hydrated state, with a short transition period between states at small dimensions. CMC is also non-toxic to humans and animals, inexpensive, and widely available. The CMC layer 206, having a thickness of about 15 μm, provides sufficient initial stiffness to allow needle-free insertion into the brain of a subject.

[0087] In some embodiments, the hydrogel sheath (104) is completely replaced by a CMC sheath as shown and described in WO 2015 / 066627.

[0088] The living electrode (100) can have various depths to reflect clinical and anatomical needs. For example, the living electrode (100) can be sized for insertion to a depth sufficient for the first end (108) to be adjacent (e.g., flush, slightly below, or slightly protruding) to the outer surface of the subject's brain (e.g., for manipulation with light and / or magnetic fields) while the second end (110) is adjacent to a neuronal population / nucleus / layer. For example, the electrode (100) can have a length of about 100 μm to about 2 cm or more. In some embodiments, the living electrode can be implanted to a desired depth (e.g., based on imaging and / or feedback) before being trimmed in situ to the desired length relative to the outer surface of the subject's brain.

[0089] Neuron Embodiments of the invention can include optogenetic neurons that allow the use of light to control cells genetically modified to express light-sensitive ion channels in living tissues. For example, the neurons can express one or more optogenetic actuators, such as channelrhodopsin, halorhodopsin, and archaerhodopsin, and / or one or more optogenetic sensors for calcium (e.g., Aequorin, Cameleon, GCaMP), chloride (e.g., Clomeleon), or membrane voltage (e.g., Mermaid).

[0090] Embodiments of the present invention can include magnetogenetic neurons that can be controlled in living tissue through the application of an alternating magnetic field. Magnetogenetic techniques are described in Xiaoyyang Long et al., "Magnetogenetics: remote non-invasive magnetic activation of neuronal activity with a magnetoreceptor," 60(24) Sci. Bull. 2107-19 (2015).

[0091] In certain embodiments, neurons are genetically modified to secrete factors to modulate disease pathophysiology, to render transplanted cells resistant to the pathophysiology of the underlying disease, or to express novel ion channels and receptors to allow for fine biological control. For example, genes can be added / modified to allow neurons to better process / degrade accumulations of proteins such as pathological alpha-synuclein, tau, or amyloid-beta.

[0092] Embodiments of the invention may additionally or alternatively include primary cerebral cortical neurons and / or dorsal root ganglion neurons.

[0093] Biocompatibility The structures described herein can be biocompatible. For example, the structures should not generate chronic immunogenic or inflammatory responses that are harmful to the subject when implanted. In certain embodiments, one or more elements of the structures degrade over time, thereby leaving encapsulated axonal tracts in the subject. In one embodiment, the living electrodes are generated using allogeneic neurons. The allogeneic neurons should not induce any overt immunogenic or inflammatory responses. In one embodiment, the living electrodes are generated using autologous neurons derived from the patient's own stem cells (e.g., induced pluripotent stem cells, or endogenous stem cell populations such as those found in the olfactory epithelium, tongue, superior ventricular, or dentate gyrus).

[0094] Hydrogel Hydrogels are generally capable of absorbing large amounts of liquid, typically consisting of greater than about 60% liquid and less than about 40% polymer at equilibrium. In preferred embodiments, the water content of the hydrogel is about 80-99.9%. Hydrogels are particularly useful due to the inherent biocompatibility of the crosslinked polymer network (Hill-West, et al., 1994, Proc. Natl. Acad. Sci. USA 91:5967-5971). The biocompatibility of hydrogels can be attributed to their hydrophilicity and their ability to absorb large amounts of biological fluids (Preparation and Characterization of Cross-linked Hydrophilic Networks in Absorbent Polymer Technology, Brannon-Peppas and Harland, Eds. 1990, Elsevier: Amsterdam, pp 45-66; Preparation Methods and Structure of Hydrogels in Hydrogels in Medicine and Pharmacy, Peppas, Ed. 1986, CRC Press: Boca Raton, Fla., pp 1-27).

[0095] Hydrogels can be prepared by crosslinking hydrophilic biopolymers or synthetic polymers. Examples of hydrogels formed from physical or chemical crosslinking of hydrophilic biopolymers include, but are not limited to, hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, or agarose (Hennink and van Nostrum, 2002, Adv. Drug Del. Rev. 54, 13-36 and Hoffman, 2002, Adv. Drug Del. Rev. 43, 3-12). These materials consist of high molecular weight backbone chains made of linear or branched polysaccharides or polypeptides. Examples of chemically or physically crosslinked synthetic polymer-based hydrogels include, but are not limited to, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO), PEO-PPO-PEO copolymers (Pluronics), poly(phosphazenes), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethyleneimine), and the like. (Hoffman, 2002, Adv. Drug Del. Rev, 43, 3-12). In some embodiments, the hydrogel comprises poly(ethylene glycol) diacrylate (PEGDA).

[0096] In one embodiment, the hydrogel comprises at least one biopolymer. In another embodiment, the hydrogel scaffold further comprises at least two biopolymers. In yet another embodiment, the hydrogel scaffold further comprises at least one biopolymer and at least one synthetic polymer.

[0097] In one embodiment, the hydrogel comprises agarose. The concentration of agarose may depend, in some cases, on the type of neurons ultimately to be cultured, the desired mechanical properties, etc. For example, increasing the concentration of agarose enhances neuronal survival and neurite outgrowth. In one embodiment, the concentration of agarose is about 0.1% to about 10%. In one embodiment, the concentration of agarose is about 0.5% to about 5%. In one embodiment, the concentration of agarose is about 4%.

[0098] Hydrogels are similar to natural living extracellular matrices (Ratner and Hoffman. Synthetic Hydrogels for Biomedical Applications in Hydrogels for Medical and Related Applications, Andrade, Ed. 1976, American Chemical Society: Washington, DC, pp 1-36). Hydrogels can also be made in vivo degradable by incorporating PLA, PLGA or PGA polymers. Moreover, hydrogels can be modified with fibronectin, laminin, vitronectin, or RGD for example for surface modification, which can promote cell attachment and proliferation (Heungsoo Shin, 2003, Biomaterials 24:4353-4364; Hwang et al., 2006 Tissue Eng. 12:2695-706). Indeed, altering the molecular weight, block structure, degradable linkages, and crosslinking mode can affect the strength, elasticity, and degradation properties of instant hydrogels (Nguyen and West, 2002, Biomaterials 23(22):4307-14; Ifkovits and Burkick, 2007, Tissue Eng. 13(10):2369 85).

[0099] Molecules that can be incorporated into the hydrogel matrix, for example via covalent attachment, encapsulation, etc., include, but are not limited to, vitamins and other nutritional supplements; glycoproteins (e.g., collagen); fibronectin; peptides and proteins; neurotransmitters; growth or neurotrophic factors; differentiation factors; carbohydrates (both simple and / or complex); proteoglycans; antigens; oligonucleotides (sense and / or antisense DNA and / or RNA); antibodies (e.g., against infectious agents, tumors, drugs or hormones); and gene therapy reagents. Hydrogels may be modified with functional groups for covalent attachment of compounds such as a variety of proteins (e.g., collagen) or therapeutic agents. Therapeutic agents that can be incorporated into the matrix include, but are not limited to, analgesics, anesthetics, antifungals, antibiotics, anti-inflammatories, anthelmintics, antidotes, antihistamines, antihypertensives, antimalarials, antimicrobials, antipsychotics, antipyretics, antiseptics, antiarthritics, antituberculous agents, antivirals, chemotherapeutic agents, color or fluorescent imaging agents, corticoids (such as steroids), antidepressants, depressants, diagnostic aids, enzymes, hormones, hypnotics, minerals, nutritional supplements, parasympathomimetics, potassium supplements, radiosensitizers, radioisotopes, imaging contrast agents, sedatives, sulfonamides, stimulants, sympathomimetics, tranquilizers, vasoconstrictors, vasodilators, vitamins, xanthine derivatives, and the like. The therapeutic agent may also be other small organic molecules, naturally occurring isolated entities or their analogs, organometallic agents, chelated metals or metal salts, peptide-based drugs, or agents that target or bind to peptide or non-peptide receptors. In certain embodiments, it is anticipated that the linkage between the therapeutic agent and the matrix may be via a protease-sensitive linker or other biodegradable linkage.

[0100] Other suitable hydrogel components are described in WO 2015 / 066627.

[0101] How to implant Referring now to FIG. 3, another aspect of the present invention provides a method (300).

[0102] In step S302, one or more implantable living electrodes are implanted in the subject. Suitable implantation regions include the subject's brain, spinal cord, peripheral nervous system (e.g., peripheral neurons, peripheral axons, axonal pathways, ganglia, dorsal root ganglia, autonomic ganglia, etc.), and muscle. The electrodes can be implanted with or without the aid of imaging, with or without the aid of stereotactic manual or stereotactic automated delivery systems. In some embodiments, multiple electrodes are implanted in the region of interest.

[0103] In step S304, the implantable living electrode is grown and coupled to one or more deep adjacent host structures (e.g., host neurons or neural networks) over a period of hours, days, weeks, months, years, or decades. In contrast to conventional electrodes, which initially integrate over a period of hours to days but lose effectiveness over time due to chronic foreign body responses, mechanical separation issues, loss of neuronal density near the electrode, and glial scar formation, living electrodes can form a stable interface for weeks, months, years, decades, and / or the lifetime of the implanted individual. Additionally, because living electrodes contain living neurons and axons, synapses formed between neurons of the structure and host neurons can be permanent and / or undergo natural turnover / plasticity consistent with similar synapses in the subject's body. Other cellular communication points can form between the living electrode and the host, including, but not limited to, gap junctions, electrical synapses, and ephaptic junctions.

[0104] In step S306, a compatible stimulator is placed proximal to at least one of the one or more implantable living electrodes. For example, a light source generating a compatible light wavelength can be placed on the subject's brain surface adjacent to the implanted electrode or on the outside of the subject's skull, or through the skull of a specially adapted transparent skull replacement piece. Similarly, a compatible magnetic source can be placed on the subject's brain surface or on the outside of the subject's skull, or through the skull of a specially adapted transparent skull replacement piece. Similarly, an acoustic source such as ultrasound can be placed on the subject's brain surface or on the outside of the subject's skull.

[0105] In steps S308 and S310, a compatible stimulator can be controlled to activate at least one of the implanted electrodes to excite (S308), inhibit (S310), and / or otherwise modulate (S312) brain activity.

[0106] In step S314, a compatible sensor is placed proximate to at least one of the one or more implantable living electrodes. For example, an optical sensor can be placed on the subject's brain surface adjacent to the implanted electrode. Similarly, a compatible magnetic sensor can be placed on the subject's brain surface or on the outside of the subject's skull.

[0107] In step S316, one or more signals emitted by the implanted electrodes are detected by a sensor.

[0108] In certain embodiments, implantable living electrodes are used to interface with the brain and record or stimulate neurons. Neuronal stimulation can be used to treat or diagnose certain functions or dysfunctions of the nervous system. Examples of nervous system dysfunctions can include, but are not limited to, Parkinson's disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, Huntington's disease, prion disease, motor neuron disease, spinocerebellar ataxia, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), encephalitis, epilepsy, head and brain malformations, hydrocephalus, seizures, chronic pain, traumatic brain injury, spinal cord injury, stroke, anoxic brain injury, cerebral palsy, obesity, depression, multiple sclerosis, inflammation, migraine, diabetic neuropathy, locked-in syndrome, glioblastoma, oligodendroglioma, metastasis, etc.

[0109] Macro TENN Another aspect of the present invention provides peripheral nerve interface (PNI) electrodes and techniques that provide greater access to different bundles / axons. Macro-tissue engineered neural networks (macro-TENNs) are larger and specifically engineered for PNS applications. An aspect of the present invention directs axonal regeneration to cause nerve bundles to spread around the inner surface of the macro-TENN. Isolation has the functional utility of being able to isolate bundles and / or axons to obtain a level of neuroselectivity not available with previous techniques.

[0110] In addition to their use in the peripheral nervous system, macro-TENNs can be used to interface with cranial nerves.

[0111] Macro-TENNs employ a technique similar to the micro-TENN technique described herein, but the larger hydrogel column results in a tubular structure in which the axons grow only along the periphery of the tube. In this manner, this embodiment encourages defasciculization and / or spreading of the axons, so that the axons can also more closely interface with multiple electrodes, either inside or outside the tubular column (located at the periphery). Leveraging this method of growth into a tubular structure to spread the host axons and bring them into close contact with surrounding surface electrodes allows for highly selective interfacing (recording or stimulating) with peripheral host axons.

[0112] Similar to the micro-TENNs described herein, macro-TENNs can have a two-part construction with an extracellular matrix (ECM) core and a tubular hydrogel shell (above) as shown in Figures 15A-15C. The ECM core can be composed of several different components, including but not limited to laminin and / or collagen (above). Axons can grow within the core, while the hydrogel allows for the propagation of soluble factors in and out of the core channels.

[0113] Exemplary dimensions include an extracellular matrix core diameter A of about 1 mm to about 5 mm (e.g., about 1 mm to about 2 mm, about 2 mm to about 3 mm, about 3 mm to about 4 mm, about 4 mm to about 5 mm, etc.). Other exemplary dimensions include a hydrogel sheath diameter C of about 2 mm to about 7 mm (e.g., about 2 mm to about 3 mm, about 3 mm to about 4 mm, about 4 mm to about 5 mm, about 5 mm to about 6 mm, about 6 mm to about 7 mm, etc.). In some embodiments, the thickness of the hydrogel sheath B is about 1 mm to about 2 mm.

[0114] Aggregate physical propertiesTENN Another aspect of the invention provides an aggregated TENN comprising a substantially cylindrical extracellular matrix core and a plurality of aggregated neurons implanted along or within the substantially cylindrical extracellular matrix core.

[0115] Another aspect of the invention includes a method of making an implantable living electrode comprising the steps of providing an extracellular matrix core; and contacting at least one end of the extracellular matrix core with a plurality of aggregated neurons.

[0116] In various embodiments, aggregate-type TENNs exhibit a higher axon growth rate than their dissociated counterparts. The term aggregate-type TENNs may include TENNs in which neurons are concentrated to form aggregates prior to TENN construction. The term may include any method of forming aggregates. Centrifugation of neurons in pyramidal wells is one method of forming such aggregates. TENNs produced using this technique may be referred to as centrifuged TENNs, and these terms are used interchangeably herein. However, those skilled in the art will recognize that these terms are not necessarily coextensive. The observed growth rates for dissociated neurons were similar to those reported in the literature. However, the axon growth rates from aggregates greatly exceeded previous reports. Cortical axons typically reach lengths of 100 to 1000 μm over three days in flat culture. The exact cause of this increased growth from aggregates is still under investigation. However, without wishing to be bound by theory, the absence of additional growth factors in the medium and the relatively slow growth of dissociated micro-TENNs implicate features of the aggregation method, such as a more controlled in vivo-like neuronal microenvironment and self-reinforcing, more directed axonal outgrowth that occurs by growing axonal "bundles."

[0117] In various embodiments, the extracellular matrix core is a collagen-laminin extracellular matrix core. Since laminin is an adhesion molecule, it may support rather than accelerate axonal growth. Several studies have examined the relationship between growth cone behavior and axonal growth rate in the mammalian CNS. Notably, axonal branching tends to occur in areas where growth cones temporarily stop forward migration and transiently slow down elongation. Again, without wishing to be limited by theory, growth cones in bidirectional micro-TENNs are presented with two classes of targets as they emerge from the aggregate: either neurons in their own aggregate or neurons in the opposite aggregate. The combined support / growth promotion of the ECM and limited target selection may reduce growth cone arrest / axonal branching and accelerate growth in the interaggregate area until the appropriate target is reached. In various embodiments, the LE BI,5mm The micro-TENNs in the group were LE at 5 DIV. BI,2mm The distance between the axons and their targets may also affect how fast the axons grow, as dissociated micro-TENNs showed faster growth compared to groups. The continued growth of dissociated micro-TENNs up to 10 DIV (even at a slower rate) may be due to a random distribution of neurons throughout the structure, rather than growing collectively towards aggregates (large targets for axon growth and synaptogenesis), where neurons continue to grow individually towards their nearest neighbors.

[0118] In addition to rapid axonal growth, structural evidence of synapses was visible as early as 4 DIV. Because synapses are the primary contact and communication points between neurons, measurements of synapse formation are often used to determine the functional maturity of neuronal cultures. Synapsin-positive puncta were observed from 4 DIV to 28 DIV, suggesting that neurons within micro-TENNs begin to form functional connections that greatly increased with time soon after plating (Figure 33). Indeed, consistent with the literature for planar cortical cultures, where synapse formation tends to increase as a function of time, micro-TENNs at later time points qualitatively expressed higher levels of synapsin. The presence of synapsin as early as 4 DIV may indicate that micro-TENN neurons are forming functional connections earlier, as might be expected from the high growth rate. Alternatively, many of these synapses may be forming within the aggregates themselves, as we did not distinguish between intra-aggregate and inter-aggregate synapses when the experiments that provided the data for Figure 33 were performed. Intra-aggregate synapses being made before inter-aggregate synapses may explain the high synapsin expression at earlier DIV. Overall, micro-TENN neurons are capable of rapidly and consistently forming the desired living electrode configuration in vitro. This allows for rapid and reproducible production and characterization of aggregate-based micro-TENNs. In various aspects, including the method of formation of aggregate-based TENNs, controlling the size of the aggregates (and, at the same time, the number of neurons per structure) allows for more repeatable studies and standardized production methods than when working with single cell suspensions. Experimental details regarding aggregate-based TENNs and their fabrication methods are presented in Figures 16, 17, 19, 23, 25, 26, 29-31 and 34 as well as examples.

[0119] Integrated Sensor An embodiment of the present invention can integrate sensory receptors into the living electrodes described herein to provide an integrated sensor that can both detect stimuli and transmit corresponding signals to the subject's nervous system or external system. For example, the living electrodes can include sensory dorsal root ganglion cells and / or cells with specialized sensors such as baroreceptors, chemoreceptors (e.g., type I / glomus cells and type II glial-like cells), electromagnetic receptors (e.g., infrared receptors, photoreceptors, ultraviolet receptors), electroreceptors, hydroreceptors, magnetoreceptors, mechanoreceptors, nociceptors, odor receptors, osmoreceptors, proprioceptors, taste receptors, thermoreceptors, and the like. The living electrodes can also include cochlear and / or vestibular hair cells.

[0120] Integration with external sensors Embodiments of the present invention can also be utilized to transmit signals from external sensors to a subject's nervous system or external systems. For example, man-made sensors can generate electrical, optical, and / or magnetic signals indicative of conditions such as pressure, temperature, position, force, sound, odor, light, etc. Embodiments of the living electrode structures described herein can receive these signals and transmit / transduce the signals to the subject's nerves.

[0121] Integration of electromechanical devices into living electrodes Although some aspects of the invention are described in relation to external electromechanical devices, such as surface optrodes, that may be adjacent to but separate from the living electrodes described herein, other aspects of the invention may incorporate such devices within the living electrodes. For example, electrical contacts may be grown or inserted within the living electrodes. Similarly, optrodes, electromagnetic devices, thermoelectric (Joule-Thomson resistance) heaters, thermoelectric (Peltier) coolers, and / or chemical applicators may be grown or inserted within the living electrodes. For example, chemical applicators may be adapted and configured to release neurotransmitters, ligands to which neurons respond, and / or ions in response to electrical signals.

[0122] biological multiplexing Biological multiplexing can be broadly defined to encompass biological versions of the kind of channel selection, multiplexing, and demultiplexing used in electrical communication. In some embodiments, biological multiplexing includes both convergent and divergent signaling, where signal processing within the many neurons of a structure can converge to a single host parenchymal target, and one structure neuron can branch its axon divergently to target many host parenchymal neurons (Figure 21, panels A-C). Similarly, since neurons within the host brain are themselves embedded in endogenous neuronal networks, the ability of a living structure to send axonal output to one of these host neurons allows for specific and stable activation of the endogenous neuronal network. Since one axon can essentially synapse on thousands of target neurons, a relatively small population of neurons within a structure can also achieve far-reaching effects. By deploying micropatterning techniques, living electrodes can be fabricated in vitro to enable fine-grained time-division multiplexing when implanted in vivo (Figure 21, panel D).

[0123] Living electrodes formed from stem cells In its various aspects and embodiments, the composition of the present invention can be produced from stem cells. In various embodiments, adverse immune responses can be mitigated by the use of autologous cells from the patient. Neurons, oligodendrocytes, astrocytes, and Schwann cells can be differentiated from human embryonic stem cells, induced pluripotent stem cells, and adipose-derived stem cells. Although direct in vivo delivery of stem cells can replace lost cells and aid in neural regeneration by releasing trophic factors, the mechanism by which they stimulate the nervous system remains unclear, and they have the potential to differentiate into undesirable phenotypes and / or lead to tumor formation. In comparison, there is a distinct advantage to using differentiated neurons in living electrodes. Existing protocols that differentiate stem cells into specific neuronal subtypes, such as cortical projection neurons, interneurons, dopaminergic A9 neurons, and spinal motor neurons, can be used to fabricate living electrodes with specific neuronal composition. Because neurons are terminally differentiated and physically constrained by the 3D configuration of the fabricated structure, this approach may have less risk of tumor formation. In various aspects, differentiated neurons can be genetically modified to enhance regenerative responses. Previous studies have suggested that the low survival rate of transplanted cells may be due to delivery into a degenerative or "hostile" injury environment. In various aspects, the durability and regenerative capacity of differentiated neurons can be enhanced by overexpression of trophic factors using transfection techniques or viral transduction. This approach can make engineered tissues resistant to the pathophysiology underlying neurodegenerative diseases.

[0124] Living electrodes with controlled programmed cell death In various embodiments, the living electrodes described herein can be engineered to include a controlled "kill switch" that drives programmed cell death of the living electrode neurons, and thus of the axons. Indeed, being able to employ different strategies to induce programmed cell death in implanted structures is a potentially important way to enhance the safety of living electrodes. There are multiple suicide gene techniques under development that can be embedded in living electrode structures. These suicide genes are biologically inactive until activated by the introduction of prodrugs, as well as two clinically validated structures, iCasp9 and HSV-TK, and are well suited to different situations based on fast versus slow apoptosis, which may be appropriate in various embodiments.

[0125] Potential applications for axon-based living electrodes In various embodiments, living electrodes can be deployed to enhance or replace conventional neuromodulation, or can be applied for more far-reaching drug delivery applications. For example, living electrodes can be delivered precisely to critical locations that affect the strength of specific connections. In various embodiments, living inhibitory (e.g., GABAergic) electrodes can be designed to form synapses to modulate pathways that are exerting too much influence and causing adverse functional effects, for example, to attenuate hypersynchronous activity in circuits that show epileptiform activity (described in more detail below). Conversely, in some embodiments, excitatory (e.g., glutamatergic) living electrodes can form synapses with axons from structures that release glutamate at targets of degenerative pathways, for example, to strengthen weak pathways. In various embodiments, living electrodes may also act by burst release of neurotransmitters at the axon terminals, either through tonic (self-paced / continuous) activity, by responding to input from the host to the living electrode neuron soma / dendrites, or controlled from externally placed hardware or computer.

[0126] This type of biological neuromodulation can provide direct (i.e., synaptically) excitatory or inhibitory input (or both simultaneously), or can provide controlled release of widespread modulation neurotransmitters (e.g., dopamine) to enhance the function of the circuit. Axon-based living electrodes can uniquely fulfill this role, as compared to, for example, more conventional neuronal grafts, by acting on the basis of network feedback that is relayed and processed between the structure and the surrounding brain, with the possibility of computer-controlled regulation / feedback. Sample applications of the compositions and methods of the present invention are detailed below.

[0127] Friedrich's ataxia In most cases of Friedrich's ataxia, the expansion of trinucleotide (GAA) repeats in intron 1 of both alleles of the frataxin gene on chromosome 9q13 leads to reduced transcription (i.e., silencing) of the gene, reduced expression of the gene product frataxin, and eventual disruption of the dorsal column pathway. As a result, patients develop severe motor impairments in the absence of proprioceptive and discriminatory signals from the periphery. In various embodiments, living electrodes can also provide an artificial sensory arc. By tapping into signals from the periphery (such as from strain gauges, accelerometers and gyroscopes attached to the joints of all four limbs, or implantable cuff recordings of peripheral nerves), living electrodes implanted in the primary sensory cortex can also provide sensory feedback, allowing for improved voluntary movement and functional independence. After being grown with glutamatergic neurons, these live electrodes can also be implanted to terminate in layer IV of the postcentral gyrus, and because the live electrodes themselves are quite small, in various embodiments, multiple structures can be implanted corresponding to different joints (e.g., a gyro from the left knee drives a live electrode implanted in the right medial sensory cortex, the left elbow and shoulder go to the right lateral sensory cortex, and vice versa for the right upper leg and left hemisphere).

[0128] Severe motor and sensory feedback dysfunction In brainstem strokes, spinal cord injuries, muscular dystrophies and amyotrophic lateral sclerosis, people become paralyzed because the substrate of voluntary motor control (the primary motor cortex) is functionally disconnected from skeletal muscles (and in certain cases also from bulbo-pharyngeal muscles). Neuromotor prostheses include a class of brain-computer or brain-machine interfaces that attempt to overcome this paralysis by recording directly from the brain and sending this recorded activity to control devices in the environment for decoding and triggering robotic actuators or driving implanted neuromuscular stimulators. Although several human trials have shown the safety and efficacy of this approach, patients achieve control purely through visual feedback. Although the sensory arc can be preserved in certain patients with motor neuron or muscle diseases, it is lost in complete spinal cord transection and is unavailable to all patients when using external robots. Several groups have attempted to provide tactile feedback by coupling tactile signals to electrical stimulation provided by macro- and microelectrodes implanted in the primary sensory cortex. This type of artificial tactile feedback appears to be effective in non-human primates, but has not yet been tested in humans. As with children and adults with Friedrich's ataxia, live electrodes show promise for repeating and widening sensory arcs by being implanted directly in the sensory cortex (see FIG. 20, panel B). In addition to being driven by externally worn sensors, in other embodiments, live electrode activity can also be triggered by sensors mounted on robotic arms, powered robotic exoskeletons, wheelchair components, and other assistive devices. In this way, paralyzed patients can also virtually "feel" their own limbs and the "limbs" of these devices, facilitating enhanced voluntary control. Additionally, by providing a bidirectional interface to both the motor and sensory cortices routed through an internally implanted microprocessor, in various embodiments, live electrodes can also modulate intercortical communication in a real-time closed loop to restore motor function and sensory / proprioceptive feedback.

[0129] chronic pain In various embodiments, custom living electrodes can be useful for modulating inputs to pain attenuation circuits. In various embodiments, peptidergic neurons secreting endorphins or enkephalins can be used to create living electrodes, which can then be implanted in the spinal cord, aqueductal gray matter, ventroposterior thalamus or substantia gelatinosa of the anterior cingulate cortex. In various embodiments, this replaces the non-specific approach of spinal cord and brain electrical stimulators. In various embodiments, the control of this neuromodulation can also be user-dependent (e.g., similar to a systemic drug pump), so unlike microfluidics that directly inject opiates or other peptides, and unlike electrodes that non-specifically modulate target volumes, living electrodes made of neurons are themselves subject to up- and down-regulation, thus providing additional protection against the development of tolerance, abuse or withdrawal.

[0130] Alzheimer's disease and Lewy body dementia A hallmark of both Alzheimer's disease (amyloid-tauopathy) and Lewy body dementia (alpha-synucleinopathy) is the loss of cholinergic neurons in the basal forebrain. These neurons are interconnected with medial temporal lobe structures, including the hippocampus formation, and are necessary for forming episodic memories. In various embodiments, live electrodes built with cholinergic neurons can also be implanted in the septal nucleus or other adjacent basal forebrain nuclei, such as the basal nucleus of Meynert or the diagonal band of Broca. In various embodiments, live electrodes stereotactically implanted in the basal forebrain and semi-externalized (following the fornix column tract) to brain services can also allow closed-loop control with an external computer. In response to detection of memory interference local field potential signatures decoded from the activity of separate live electrodes implanted in the temporal lobe to enhance contingency encoding, different subpopulations of neurons (cholinergic, GABAergic, glutamatergic) within the live electrodes can also be differentially triggered via optogenetic and intraosseous anchoring waveguides. Similarly, in other embodiments, external cues (e.g., smartphone reminders and user-triggered push-button flagging) can also be used to modulate basal forebrain activity to enhance retention and replay. In various embodiments, a second live electrode can be implanted in the entorhinal cortex and hippocampus, then linked to the live electrode implanted in the basal forebrain via an external computer to functionally re-instantiate the bidirectional fornix septohippocampal pathway.

[0131] Frontotemporal Dementia and Autism Spectrum Disorders In another aspect, for agrammatic primary progressive aphasia, a frontotemporal dementia tauopathy that affects the inferior frontal gyrus of the dominant hemisphere, live electrodes can be implanted to both connect Broca's area with premotor cortex and primary motor cortex (to compensate for motor aphasia and allow motor replacement gestures) and to connect Broca's area with Wernicke's area as an artificial arcuate fasciculus.In an embodiment for behavioral disorder frontotemporal dementia (TDP-43opathy, sometimes tauopathy), structures that connect the degenerated orbitofrontal cortex with intact dorsolateral prefrontal, frontal pole, and anterior cingulate cortex can also reinstantiate behavioral inhibition and self-regulation.In an embodiment for semantic dementia variant FTD (TDP-43 or tau), degeneration of the frontal ventral aspect of the temporal lobe occurs, which can lead to loss of semantic knowledge store and various reading comprehension and perception disorders. In various embodiments, live excitatory glutamatergic electrodes implanted in the visual word-forming area of ​​the fusiform gyrus may boost residual function in this area, and live electrodes can be made into auxiliary axon bundles connecting primary and secondary visual cortical areas to the ventral temporal lobe to recreate the lost "ventral-something" pathway and restore semantic processing.Both autism spectrum disorder and behavioral frontotemporal dementia are impaired in social perception and interaction.In various embodiments, live electrodes built with glutamatergic neurons in the superficial and left dorsolateral prefrontal cortex and oxytocinergic neurons juxtaposed to the supraoptic and paraventricular nuclei of the hypothalamus can also eliminate behavioral disinhibition and restore social behavior, and superficial cortical populations can be triggered by external computers that track social cues decoded from microphones and micro-cameras unobtrusively mounted on eyeglass frames, hearing aids, bracelets, or other attire.

[0132] Stroke and cerebral palsy Both ischemic and hemorrhagic strokes result in focal brain tissue destruction and various degrees of inflammation. In ischemic strokes, the surrounding tissue penumbra remains functional while it may be metabolically vulnerable to further insults (such as from hypotension or hypoxia). When occurring in utero or perinatally, strokes (e.g., germinal matrix hemorrhage) can lead to static insults while the rest of the brain attempts to develop normally around it, and in certain cases, to cerebral palsy with various degrees of motor and cognitive dysfunction. When brain areas are damaged, two aspects of function are lost: the "computation" of the local gray matter and also the "connectivity" of the axons (focal intrinsic fibers and also the crossing fibers that pass through). In various embodiments, micro-TENNs directly restore both computation and connectivity, and serve as "replacement parts" for irreversibly damaged pieces of the brain, and for metabolically, electrically and functionally resurrected and supporting the surrounding penumbra. In an animal model of stroke using middle cerebral artery occlusion, optogenetic grafts have been shown to restore functional mobility. While the grafts were "driven" by an external laser, functionalized living electrodes can also "drive" both intact areas of the brain and external modulation triggered by body sensors or computer-activated rehabilitation to restore activity within the border zone as well as functional mobility and behavior after stroke.

[0133] Treatment-refractory depression Severe clinical depression refractory to pharmacotherapy, psychotherapy and electroconvulsive therapy is characterized by neurometabolic disturbances, including disrupted glucose uptake in limbic structures, including the cingulate gyrus. In various embodiments, micro-TENNs can be implanted to enhance connectivity between the frontopolar cortex and the anterior cingulate, or to connect the suprasegular anterior cingulate cortex to the infragenual anterior cingulate cortex, so that the former can modulate the latter, restoring normal metabolic activity and alleviating symptoms. Similarly, in various embodiments, live electrodes implanted in the nucleus accumbens can be deployed to provide dynamic phase changes in catecholamine tone, thus altering mood salience signatures of thoughts and perceptions, alleviating depressive symptoms without causing rebound dysphoria or post-synaptic upregulation of tolerance.

[0134] Epilepsy The application for epilepsy shows two ways in which the advanced functionality of living electrodes can also achieve treatment goals in ways that are not possible with existing approaches. First, living electrodes can be mimicked, whereby the neuronal population closest to the target field secretes the inhibitory neurotransmitter GABA broadly into the target region. Secretion was either constitutive or triggered from the brain surface based on measurements of early epileptiform activity (see below). In this approach, the living electrode effectively serves as a GABA reservoir and delivery system (see FIG. 20, panel C). In various embodiments, living electrodes can also be seeded with excitatory glutamatergic neurons in an extracellular matrix decorated with neuroligin to synapse coaxially with local endogenous GABAergic neurons. Both approaches can achieve disruption of hypersynchronous activity and thus halt the generation or propagation of pathological seizures from the target field in the brain. As neurons in an epileptiform network in the brain may synapse onto dendrites that radiate from neurons in the structure, various embodiments may achieve a focal closed-loop self-damping circuit, such that focal epileptiform activity will disappear by itself via this self-inhibitory loop mediated by the living inhibitory electrodes. In embodiments directed to multifocal epilepsy, living electrodes may also be implanted in two or more epileptogenic foci (e.g., those identified by intracranial surface and deep recordings). In various embodiments, sensors (e.g., intraosseous or subgaleal leads capturing ongoing local field potentials) may also be used to pick up signatures of pre-ictal or seizure activity and trigger light stimulation of the surface-externalized optogenetically modified micro-TENNs to preemptively stop seizure propagation in ways not possible with conventional electrodes.

[0135] Parkinson's disease In a further aspect, the invention provides micro-TENN compositions and methods for the treatment of Parkinson's disease. In one aspect, the invention includes a substantially cylindrical extracellular matrix core; and one or more neurons implanted along or within the substantially cylindrical extracellular matrix core, where the neurons are dopaminergic neurons. In another aspect, the invention includes a method of treating Parkinson's disease, comprising implanting a dopaminergic micro-TENN into the SNpc of a patient.

[0136] Current treatments for Parkinson's disease, including dopamine replacement strategies and the use of deep brain stimulation, aim to alleviate akinesia rather than correct the underlying causes of motor symptoms. Additionally, dopaminergic neurons and / or fetal graft implantation into the striatum may provide a local dopamine source, and these approaches do not recreate the nigrostriatal circuitry. To address these gaps, tissue engineering solutions have been explored that can also be precisely delivered to physically restore lost dopaminergic neurons in the SNpc and their axonal projections to the striatum. To this end, in one aspect, the present invention includes micro-TENNs that utilize primary dopaminergic neurons. It was found that when fabricated with an optimal inner core ECM, micro-TENNs plated with neuronal aggregates grew greater than 6 mm in length. Additionally, dopaminergic micro-TENNs exhibit evoked dopamine release, are capable of synapsing with striatal cell populations in vitro, and exhibit survival and maintenance of cellular organization when implanted in vivo.

[0137] Dopaminergic micro-TENNs may be fabricated using ventral midbrain neuronal populations that may be enriched in dopaminergic neurons, with some embodiments not being a pure dopaminergic population. Other embodiments include a pure dopaminergic population. In various embodiments, higher purity dopaminergic neurons may be used for functional potency, and in other embodiments, later developmental time points may be used for midbrain isolated cell sorting and / or differentiation from stem cell sources, all of which have been shown to further enrich for dopaminergic populations.

[0138] In another aspect, the invention includes a method for plating dopaminergic "aggregates" into micro-TENNs that can alleviate the lack of separation between neuronal cell bodies and neurites observed in some cases when dissociated cells are plated into micro-TENNs. It was particularly important to ensure that the micro-TENNs exhibited the desired cellular organization, with distinct cell body regions projecting axons throughout the entire length of the inner core, because separate cell body and axon regions are a key feature of the nigrostriatal pathway.

[0139] Without wishing to be limited by theory, closer approximation of the cellular organization of the nigrostriatal pathway leads to improved functional outcomes following implantation of micro-TENNs to reconstitute degenerated dopaminergic neurons in the SNpc and their projections to the striatum. In particular, dopaminergic neurons in the SNpc synapse directly onto striatal neurons and do not exert their effects through intermediate synapses and / or neurons. Thus, for the connectivity and timing of micro-TENNs to be precise when integrated with the host, micro-TENNs may need to achieve modulation of striatal neurons by propagating signals through monosynaptic pathways from the SNpc. Dopaminergic micro-TENNs that approximate the organization of the nigrostriatal pathway are disclosed herein, which increase the likelihood that in vivo functional integration will mimic native monosynaptic pathways.

[0140] In some embodiments, dopaminergic cell aggregates produced neurite extensions that were approximately 10 times longer than projections from individual cells. Again, without wishing to be limited by theory, this may be influenced by the fact that the cell density in neuronal aggregates is more representative of that in the brain. This higher cell density may allow aggregated neurons to better control their 3D microenvironment than dissociated cells, which in turn promoted better cell survival and health, and therefore enhanced axonal extension. Moreover, the gene expression of cells in aggregates may also better represent cells in the brain, allowing cells to enter a developmental program to initiate axonal extension. Furthermore, many neuronal subtypes are programmed for either short- or long-range communication. In dissociated micro-TENNs, "long-range" axons may meet their synaptic partners at intermediate distances along the length of the micro-column. In contrast, for aggregated micro-TENNs, the absence of any intermediate neighbors could have encouraged "long-range" neurons to project the length of the micro-column by upregulating proteins for long-range extension. Finally, aggregates give rise to assembled and fasciculated axonal projections, which may generate a sustained driving force for axonal extension due to focused and persistent growth-promoting signaling and / or physical / structural advantages. The rate and length of dopaminergic axonal extension from neuronal aggregates was unprecedented. Nonetheless, further studies are clearly needed to elucidate the mechanisms of extremely long axonal projections from aggregates.

[0141] In some embodiments, the aggregated micro-TENNs contain collagen I and a cocktail of collagen I and laminin in the inner core. Without wishing to be bound by theory, it is believed that this configuration benefits axonal outgrowth because, unlike laminin coatings and empty cores, both collagen I and the cocktail of collagen I and laminin provide a continuous 3D scaffold that supports axonal outgrowth. Although cross-linked collagen also provides a continuous scaffold, it is much stiffer and more resistant to enzymatic degradation during growth cone outgrowth. However, embodiments that include cross-linked collagen may have ideal structural properties that may be suitable for a variety of applications.

[0142] In various embodiments, dopaminergic micro-TENNs release dopamine both within the neuronal aggregates and at the axon terminals.

[0143] One week and one month after introduction of GFP+ micro-TENNs to cross the nigrostriatal pathway, evidence of micro-TENN survival and maintenance of cellular organization was found. Specifically, surviving GFP+ and TH+ neurons were located within the lumen along the introduced projection pathway. By one month after implantation, tissue sections perpendicular to the projection pathway of the implant revealed dense GFP+ neurons / axons and TH+ axons in cross section. Although the circuitry of the nigrostriatal pathway is complex and the projection pathway of the implant may be refined in future studies, these results are sufficient to demonstrate proof of concept of in vivo implantation and survival.

[0144] In various embodiments, the dopaminergic output of the micro-TENN can be continuously modulated by striatal feedback and SNpc input to mitigate potential runaway dopamine excess and dystonia, which are potential side effects from midbrain dopaminergic cell transplants into the striatum. The micro-TENN is an auxiliary pathway. This engineered pathway is unique in that it mimics the function of dopaminergic axonal projections from the SNpc to the striatum, and seeks to provide dopaminergic input that can be tuned and controlled. In addition to directing circuit remodeling, in various embodiments, optogenetically active micro-TENNs can also be deployed as living dopaminergic electrodes to provide controlled neuromodulatory input via engineered axonal tracts (see FIG. 20, panel A). In FIG. 20, neuronal cell body populations are left semi-externalized at the brain surface to allow for controlled interfacing with subcranial micro-LED arrays. Interfaces other than the nigrostriatal pathway provide a mechanism by which information from other brain areas (e.g., beta oscillations recorded from the primary motor cortex), external sensors (e.g., gyroscopes and accelerometers both located within a battery case in the chest wall, or streamed from sensors implanted or worn externally on the limbs), and external computers (e.g., processing 3D motion capture and force sensors embedded in shoes, treadmills, and gait analysis device surfaces) can also modulate the underlying ganglionic circuits into more healthy activity patterns. EXAMPLES

[0145] The following examples may be useful to those skilled in the art in understanding the present disclosure, but should not be construed as limiting the present invention in any way.

[0146] Various materials and methods employed in some of the following examples are presented here. All procedures were approved by the University of Pennsylvania and the Michael J. Crescenz Veterans Affairs Medical Center IACUC and were performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015).

[0147] Fabrication of three-dimensional micro-TENNs All supplies were from Invitrogen, BD Biosciences, or Sigma-Aldrich unless otherwise noted. Micro-TENNs contained an agarose ECM hydrogel molded into a cylinder into which axons could grow. The outer hydrogel structure consisted of 1% agarose in Dulbecco's phosphate-buffered saline (DPBS). Agarose cylinders with an outer diameter of 398 μm were generated by drawing the agarose solution into a capillary tube (Drummond Scientific) via capillary action. To produce the inner column, an acupuncture needle (diameter: 160 μm) (Seirin) was inserted into the center of the agarose-filled capillary tube. The hardened micro-columns were extruded from the capillary tube and placed into DPBS, where they were cut into 6-12 mm lengths and sterilized under UV light (1 h). 5 μL of the appropriate ECM cocktail was added to each micro-column. The ECM cocktail contained the following: 1.0 mg / mL rat tail type I collagen; 1.0 mg / mL rat tail type I collagen mixed with 1.0 mg / mL mouse laminin; 1.75 mg / mL mouse laminin; and 1.0 mg / mL rat tail type I collagen in 11.70 mM N-(3-dimethylaminopropyl)-N'-ethylcarboimide hydrochloride, 4.3 mM N-hydroxysuccinimide, and 35.6 mM sodium phosphate monobasic. These microcolumns were then incubated at 37°C for 15-30 minutes, after which DPBS was added to the Petri dish.

[0148] Neuronal cell culture Female Sprague-Dawley rats (Charles River) served as a source of primary ventral midbrain neurons, a midbrain region previously shown to be rich in dopaminergic neurons (Weinert et al. 2015, Isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains, Journal of visualized experiments : JoVE, (96)). Timed pregnant rats (embryonic day 14) were euthanized using carbon dioxide, after which the uterus was removed. The brains were removed in Hank's balanced salt solution (HBSS), and the ventral midbrain was isolated. The ventral midbrain was dissociated in accutase at 37°C for 10 min. Cells were centrifuged at 200 relative centrifugal force (RCF) for 5 min and resuspended at 1-2 million cells / mL in standard medium consisting of NEUROBASAL® medium + 2% B27 + 1% fetal bovine serum (Atlanta Biologicals) + 2.0 mM L-glutamine + 100 μM ascorbic acid + 4 ng / mL mouse basic fibroblast growth factor (bFGF) + 0.1% penicillin-streptomycin. The enriched growth medium consisted of NEUROBASAL® medium + 2% B27 + 1% fetal bovine serum (Atlanta Biologicals) + 2.0 mM L-glutamine + 100 μM ascorbic acid + 0.1% penicillin-streptomycin + 12 ng / mL mouse bFGF + 10 ng / mL brain-derived neurotrophic factor (BDNF) + 10 ng / mL glial cell line-derived neurotrophic factor (GDNF) + 10 ng / mL ciliary neurotrophic factor (CNTF) + 10 ng / mL cardiotrophin.Dopaminergic neuronal aggregates were generated based on a protocol adapted from Ungrin MD, Joshi C, Nica A, et al. 2008, Reproducible, ultra high-throughput formation of multicellular tissue from single cell suspension-derived human embryonic stem cell aggregates, PloS one, 3 (2): e1565. Custom arrays of inverted pyramidal wells were fabricated using polydimethylsiloxane (PDMS) (SYLGARD® 184, Dow Corning) cast from a 3D-printed mold and placed in a 12-well plate. 12 μL of cell solution was transferred into each pyramidal well, and the 12-well plate was centrifuged at 1500 rpm for 5 minutes, after which 2 mL of standard medium was placed on top of each array. Centrifugation resulted in forced aggregation of neurons (approximately 3,200 cells per aggregate). The wells were then incubated overnight. At the time of plating, DPBS was removed from the dishes containing the micro-columns and the medium was replaced. Using forceps, aggregates were inserted into one (unidirectional) or both (bidirectional) ends of the micro-columns and the cultures were placed in the incubator (total micro-TENNs made with dopaminergic neurons: n=300).

[0149] For micro-TENNs containing dissociated cells without an ECM core, dopaminergic cells were suspended in standard medium at 10 million cells / mL and 5 μL of this cell suspension was added to each micro-column. The micro-TENNs were incubated for 60 minutes, after which medium was added. For micro-TENNs containing dissociated cells with an ECM core, dopaminergic cells were suspended in 1.0 mg / mL rat tail type 1 collagen at the time of plating (10,000,000 cells / mL) and 5 μL of this mixture was added to each micro-column. The micro-TENNs were incubated for 15 minutes, after which medium was added.

[0150] Culture medium was changed every 3–4 days in vitro (DIV) using pre-warmed medium. In some cases, micro-TENNs were transduced with an adeno-associated virus (AAV) vector (AAV2 / 1.hSynapsin.EGFP.WPRE.bGH, UPenn Vector Core) to express green fluorescent protein (GFP) in neurons. Here, at 3 DIV, micro-TENNs were incubated overnight in medium containing the vector (3.2 × 1010 genome copies / mL), and the cultures were rinsed with medium the next day.

[0151] Female Sprague-Dawley rats (Charles River, Wilmington, MA) were the source of primary striatal neurons. Timed pregnant rats (embryonic day 18) were euthanized using carbon dioxide, and the uterus was then removed. To isolate striatal neurons, the brain was removed in HBSS and the striatum was isolated. The striatum was dissociated in trypsin (0.25%) + ethylenediaminetetraacetic acid (EDTA) (1 mM) at 37°C for 12 min. Trypsin-EDTA was then removed and the tissue was triturated in HBSS containing DNase I (0.15 mg / mL). Cells were centrifuged at 1000 rpm for 3 min and resuspended at 1-2 million cells / mL in NEUROBASAL® medium + 2% B27 + 0.4 mM L-glutamine. Striatal aggregates were prepared and inserted into micro-TENNs as previously described. When testing whether dopaminergic aggregates formed synapses with striatal aggregates, striatal aggregates were inserted into the empty ends of dopaminergic micro-TENNs at 10 DIV, and when testing whether striatal aggregates increased the growth rate of dopaminergic micro-TENNs, they were inserted at 3 DIV.

[0152] immunocytochemistry Micro-TENNs were fixed in 4% formaldehyde for 35 min and permeabilized with 0.3% Triton X100 plus 4% horse serum for 60 min. Primary antibodies (in phosphate-buffered saline (PBS) + 4% serum) were added for 12 h at 4°C. Primary antibodies were markers for the following: (1) β-tubulin III (1:500, Sigma-Aldrich, cat #T8578), a microtubule element expressed primarily in neurons; (2) tyrosine hydroxylase (TH; 1:500, Abcam, cat #AB113), an enzyme involved in the production of dopamine; (3) microtubule-associated protein 2 (MAP-2) (1:500, Millipore, cat #AB5622), a microtubule-associated protein found in dendrites; (4) dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) (1:250, Abcam, cat #AB40801), a protein found in striatal medium spiny neurons; and (5) synapsin 1 (1:1000, Synaptic Systems, cat #106001), a protein expressed in synaptic vesicles in the central nervous system. Add the appropriate fluorescent secondary antibody (Alexa-488, -594 and / or -649, 1:500 in PBS + 30 nM Hoechst + 4% serum) for 2 h at 18–24 °C.

[0153] Implantation of micro-TENNs Male Sprague-Dawley rats (325-350 g) were anesthetized with isoflurane and mounted in a stereotaxic frame. The scalp was cleaned with betadine, bupivacaine was injected along the incision line, and a midline incision was made to expose the Bregma landmark. A 5 mm craniectomy was performed centered on the following coordinates relative to the bregma: +4.8 mm (AP), 2.3 mm (ML). Micro-TENNs were loaded into a needle (OD: 534 μm, ID: 420 μm; Vita Needle, Needham, MA) attached to a Hamilton syringe mounted on the stereotaxic arm. The stereotaxic arm was positioned at 34° to the horizontal, the dura was opened, and the needle was lowered into the brain to a depth of 11.2 mm. The needle was held in place for 10 seconds, at which point the fixation arm was positioned to make contact with the plunger of the Hamilton syringe. The needle containing the micro-TENN was then withdrawn from the brain. The scalp was sutured closed and buprenorphine was given for post-operative analgesia. Animals receiving micro-TENNs were allowed to survive for either 1 week (n=5) or 1 month (n=5). At the time of sacrifice, animals were anesthetized and perfused transcardially with heparinized saline followed by 10% formalin.

[0154] immunohistochemistry After 24 hours of fixation in 4% paraformaldehyde, brains were prepared for either paraffin processing or cryosectioning. Brains were sectioned sagittally and either processed through paraffin or frozen in 30% sucrose until saturation. Sections were cut at 8 μm (paraffin) or 35 μm (cryosection), mounted on slides, and processed for immunohistochemistry.

[0155] Paraffin sections were deparaffinized, then rehydrated and endogenous peroxidase was quenched using 3% hydrogen peroxide in water (Fisher, cat #S25359), followed by heat-induced epitope retrieval in TRIS-EDTA. Sections were blocked with horse serum (ABC Universal Kit, Vector Labs, cat #PK-6200) for 30 min. Rabbit anti-TH (1:750; Abcam, cat #ab112) was applied overnight at 4°C in Optimax buffer. Antigens of interest were visualized using DAB (Vector Labs, cat #SK-4100). Frozen sections were blocked with 5% normal horse serum in 0.1% Triton-x / PBS for 30–45 min. Primary antibodies (rabbit anti-TH, 1:750, Abcam AB112; mouse anti-Tuj1, 1:1000, Sigma T8578) were applied to sections in 2% horse serum / Optimax® buffer for 2 hours at room temperature. Secondary antibodies (1:1000) were applied in 2% horse serum / PBS for 1 hour at room temperature. Sections were counterstained with Hoechst.

[0156] Microscopy and data collection For in vitro analysis, micro-TENNs were imaged using phase contrast fluorescence on a NIKON® ECLIPSE® Ti-S microscope, and image collection used a QICLICK® camera interfaced with NIKON® ELEMENTS™ software. To determine the length of neurite intrusion, the longest observable neurite in each micro-TENN was measured from the proximal end of the neuronal aggregate after fixation. For in vitro immunocytochemistry analysis, cultures and micro-TENNs were fluorescently imaged using a NIKON® A1RSI laser scanning confocal microscope. All micro-TENN confocal reconstructions were from full thickness z-stacks. For analysis of micro-TENNs after implantation into the brain, micro-TENNs were fluorescently imaged using a NIKON® A1RSI laser scanning confocal microscope. Each section was analyzed to assess the presence, organization, and extension / integration of micro-TENN neurons / neurites.

[0157] statistical analysis No method was used to predetermine group sample sizes. In most cases, researchers were not blinded to treatment groups during the experiment or data evaluation, as there were obvious visual differences between experimental groups. For the in vivo implantation study, rats were randomly assigned for use in the experiment. All data were checked for normality, and adjustments were made for non-normal data. Unpaired parametric two-tailed t-tests were performed to determine if there were statistically significant differences in axonal outgrowth between unidirectional vs. bidirectional micro-TENNs containing dopaminergic terminal targets. Unpaired non-parametric two-tailed Mann-Whitney tests were performed to determine if there were statistically significant differences in axonal outgrowth between the following treatment pairs: dissociated vs. aggregated cells, high vs. normal growth factor concentrations, and unidirectional vs. bidirectional micro-TENNs containing striatal terminal targets. Unpaired non-parametric two-tailed Mann-Whitney tests were performed to determine if there were statistically significant differences in TH+ axonal length as a percentage of total axonal length in collagen I inner core vs. collagen I-laminin cocktail inner core. ANOVA was performed for extracellular matrix studies. Tukey's post-hoc pairwise comparisons were performed when differences existed between groups. For all statistical tests, significance was determined at p<0.05. Data are presented as mean ± standard deviation.

[0158] Isolation and culture of cortical neurons Neuronal isolation and culture protocols were similar to those of published studies. Briefly, timed pregnant rats were euthanized and the uterus was removed. Embryonic day 18 fetuses were transferred from the uterus to cold HBSS where the brains were removed and the cerebral cortical hemispheres were isolated under a stereoscope via microdissection. Cortical tissue was dissociated in 0.25% trypsin + 1 mM EDTA at 37°C, after which the trypsin / EDTA was removed and replaced with 0.15 mg / ml DNase in HBSS. The dissociated tissue + DNase was centrifuged at 3000 RPM for 3 minutes, after which the DNase was removed and the cells were resuspended in neuronal culture medium composed of NEUROBASAL® + B27® + Glutamax™ (ThermoFisher) and 1% penicillin-streptomycin.

[0159] Fabrication of micro-TENNs / living electrodes Micro-TENNs were constructed in a three-phase process (Figure 29). First, agarose micro-columns of specified geometry (outer diameter (OD), inner diameter (ID), and length) were formed in a custom-designed acrylic mold (Figure 29, panel A). The mold is an array of cylindrical channels that allows for the insertion of acupuncture needles (Seirin, Weymouth, MA) such that they are concentrically aligned within the channels. Molten agarose in Dulbecco's phosphate-buffered saline (DPBS) was poured into the mold-needle assembly and allowed to cool (agarose: 3% weight / volume). Once the agarose solidified, the needles were removed and the mold disassembled, yielding hollow agarose micro-columns with a specific outer diameter equal to the size of the channel and an inner diameter equal to the outer diameter of the needle. The micro-columns were sterilized via UV light for 30 min and stored until needed in DPBS to prevent dehydration. For these studies, the mold channel was 398 μm in diameter and the acupuncture needle was 180 μm, resulting in a microcolumn with 398 μm OD and 180 μm ID. The microcolumns were cut to lengths of either 2 or 5 mm. Primary cortical neurons were then forced into cell aggregates (FIG. 29, Panel B). These aggregates provide the necessary structure for the growth of long axon bundles that span the length of the microcolumn. Cells were transferred to an array of inverted pyramidal wells made of PDMS (SYLGARD® 184, Dow Corning) cast from a custom-designed 3D-printed mold (FIG. 29, Panel B). Dissociated cortical neurons were suspended at a density of 1-2 million cells / ml and centrifuged in the wells at 200 g for 5 min. This centrifugation resulted in forced aggregation of neurons (or any other cell type) with precisely controlled numbers of neurons per aggregate / sphere (12 μL of cell suspension per well). The pyramidal well and forced aggregation protocols were adapted from Ungrin et al. Finally, the microcolumns were removed from the DPBS, and excess DPBS was removed from inside the microcolumns via a micropipette.The micro-columns were then filled with extracellular matrix (ECM) composed of 1.0 mg / ml rat tail collagen + 1.0 mg / ml mouse laminin (Reagent Proteins, San Diego, CA) (Figure 29, Panel C). Unidirectional or bidirectional micro-TENNs were seeded by carefully placing aggregates at one or both ends of the micro-column, respectively, using fine forceps under a stereoscope and allowed to attach for 45 min at 37°C, 5% CO2. To generate dissociated micro-TENNs, dissociated cortical neurons were transferred via micropipette into the ECM-filled micro-columns as detailed in a previous study. Micro-TENNs were then grown in neuronal culture medium with fresh medium changes every 2 days in vitro (DIV).

[0160] Growth characterization Phase contrast microscopy images of micro-TENNs in culture were acquired at 1, 3, 5, 8, and 10 DIV at 10× magnification using a NIKON® Eclipse Ti-S microscope coupled with a QIClick® camera and NIS Elements BR 4.13.00. Micro-TENNs were fabricated and sorted into one of four groups: dissociated / 2 mm long (LE) or long-term (LOT) groups. DISS,2mm )(n=7), unidirectional aggregate / 2mm length (LE UNI,2mm )(n=6), two-way aggregate / 2mm length (LE BI,2mm ) (n=9), or bidirectional aggregates / 5mm length (LE BI,5mm) (n=7). Growth rate of each group at a given time point was quantified as the change in the longest identifiable neurite length divided by the number of days between the most recent and previous time points. The longest neurite was manually identified in each phase image using functions from the Image Processing Toolbox of MATLAB (MathWorks, MA) and the length was measured from the origin aggregate to the neurite tip. The same starting micro-TENN and starting point were used across all time points for more precise analysis. Mean growth rates were found for each group at the indicated time points and compared with two-way analysis of variance (ANOVA), with post-hoc analysis performed using the Bonferroni procedure as appropriate (significance required p<0.05). All data are presented as mean ± sem. To identify aggregate-specific growth throughout the micro-column, cortical neuronal aggregates were labeled with either green fluorescent protein (GFP) or red fluorescent protein mCherry via adeno-associated virus 1 (AAV1) transduction (Penn Vector Core, Philadelphia, PA). Briefly, after centrifugation of aggregates in pyramidal wells, 1 μL of AAV1 packaged with the human synapsin-1 promoter was added to the aggregate wells (final concentration: approximately 3 × 109 viral copies per aggregate). The aggregates were incubated overnight at 37 °C, 5% CO2, after which the medium was changed twice and the transformed aggregates were then plated as above in microcolumns, one GFP+ and one mCherry+ aggregate each (n = 4 in total). Images of micro-TENNs were acquired over multiple DIV with a NIKON® A1RSI laser scanning confocal microscope in combination with NIS-Elements® AR 4.50.00 software. Consecutive slices of 10-20 μm in the z-plane were collected for each fluorescence channel. All confocal images presented are maximum intensity projections of confocal z-slices.

[0161] Survival assessment To assess neuronal viability, 5 mm-long unidirectional (LE) plates were plated on polystyrene. UNI ) construct and two-way (LEBI ) Constructs and time-matched plates from generation were stained with the calcein-AM / ethidium homodimer-1 (EthD-1) assay (ThermoFisher) at 10 and 28 DIV. Metabolically active cells convert membrane-permeable calcein AM to calcein, which is green fluorescent (λ exc Approximately 495nm;λ em 515 nm), whereas EthD-1 enters membrane-compromised cells and emits red fluorescence (λ exc Approximately 495nm;λ em 635 nm). Briefly, cultures were gently rinsed in DPBS. A solution of calcein-AM (1:2000 dilution; final concentration approx. 2 μM) and ethidium homodimer-1 (1:500; approx. 4 μM) in DPBS was added to each culture, followed by incubation at 37° C., 5% CO2 for 30 minutes. Following incubation, cultures were rinsed twice in fresh DPBS and imaged at 10× magnification on a Nikon® A1RSI laser scanning confocal microscope in combination with NIS-Elements® AR 4.50.00 software. Viability was quantified using ImageJ (National Institutes of Health, MD) as the ratio of the total area of ​​calcein-AM positive cells to the total area of ​​both calcein-AM positive and ethidium homodimer positive cells. The sample sizes for each group were as follows: LE UNI,5mm (n=4, 4);LE BI,5mm (n=7, 4); plated cultures (n=9, 5), 10 and 28 DIV, respectively. All data presented as mean±sem.

[0162] Live calcium imaging As a proof of concept to investigate micro-TENN aggregate connectivity, aggregates were transduced with genetically encoded calcium reporters GCaMP6f or RCaMP1b (Penn Vector Core, Philadelphia, PA). After centrifugation of aggregates in pyramidal wells, 1 μL of AAV1 packaged with the human synapsin-1 promoter was added to the aggregate wells (final concentration: approximately 3 × 109 viral copies per aggregate). Aggregates were incubated overnight at 37 °C, 5% CO2, after which the medium was changed twice and transduced aggregates were plated as above in microcolumns. Micro-TENNs were imaged after 7–10 DIV using a NIKON® Eclipse Ti® microscope combined with an ANDOR Neo / Zyla camera® and Nikon® Elements AR 4.50.00, and calcium translocations from recordings were then identified with NIKON® Instruments® Elements AR 4.50.00. The intensity of selected regions of interest (ROIs) of micro-TENNs was plotted against the background (defined as ROIs without cell bodies or axons) over time.

[0163] Functional analysis Fluorescent calcium recordings were collected as described above to generate tiff stacks of micro-TENN activity. Each tiff stack consisted of 120 seconds of activity recorded at 20 frames per second (2400 frames in total). Functional analysis was performed using three MATLAB® software toolboxes - FluoroSNNAP, MATLAB® software statistical toolbox, and SIFT for EEGLAB. FluoroSNNAP is an interactive software package designed by Meaney et al. for performing calcium imaging-based neuronal network analysis in vitro. Briefly, time-averaged images were generated from the tiff stacks, and then ROIs of approximately 20 μm diameter were manually selected. FluoroSNNAP was used to extract and normalize the intensity of the ROIs to assess functional connectivity patterns within the bidirectional micro-TENNs; specifically, normalized Pearson cross-correlation and normalized phase synchrony matrices were generated from the calcium translocation patterns of the ROIs. Additionally, to assess information flow across micro-TENNs, we fitted a multivariate autoregressive (MVAR) model using the SIFT toolbox, which was used to create a normalized Direct Transfer Function (nDTF) connectivity matrix. nDTF has been used in the literature to determine the direction and frequency content of EEG activity. Here, we applied it to detect information flow from one aggregate to another in a bidirectional micro-TENN. A numerically stable 10th-order MVAR model was used to obtain nDTF estimates with a sliding window of 80 s and a time step of 40 s. The Nyquist limit yielded nDTF coefficients between 1 and 9 Hz.

[0164] immunocytochemistry Micro-TENNs were fixed in 4% formaldehyde for 35 min at 4, 10, and 28 DIV. Micro-TENNs were then rinsed in 1x PBS and permeabilized with 0.3% Triton X100 + 4% horse serum in PBS for 60 min before incubation with primary antibodies overnight at 4 °C. Primary antibodies were Tuj-1 / beta-III tubulin (T8578, 1:500, Sigma-Aldrich) to label axons and synapsin-1 (A6448, 1:500, Invitrogen) to label presynaptic specializations. Following incubation with primary antibodies, micro-TENNs were rinsed in PBS and incubated with fluorescently labeled secondary antibodies (1:500; supplied by Life Technologies, Invitrogen, and Jackson ImmunoResearch) for 2 h at 18–24 °C. Finally, Hoechst (33342, 1:10,000, ThermoFisher) was added for 10 min at 18–24 °C, followed by rinsing in PBS. Micro-TENNs were imaged with a NIKON® A1RSI laser scanning confocal microscope combined with NIS Elements AR 4.50.00. Consecutive slices of 10–20 μm in the z-plane were collected for each fluorescent channel. All confocal images presented are maximum intensity projections of confocal z-slices.

[0165] Cortical and cortical thalamic implantations As a proof of concept of the in vivo behavior of micro-TENNs, preformed micro-TENNs with GFP+ neurons / axons were delivered to the brain via stereotaxic microinjection similar to that described in previous studies. Male Sprague-Dawley rats weighing 325–350 grams were anesthetized with 1.0–2.0 liters per minute of isoflurane (inhalation: 5.0%, maintenance: 1–5–2.0%) and mounted in a stereotaxic frame. Meloxicam (2.0 mg / kg) and bupivacaine (2.0 mg / kg) were given subcutaneously at the base of the neck and along the incision line, respectively. The area was shaved and cleaned with betadine solution, after which a small craniotomy was made over the sensory cortex (coordinates: +4.8 mm AP, ±2.3 mm ML). Bidirectional micro-TENNs (5 mm long) were loaded into a needle connected to a Hamilton syringe and mounted on a stereotaxic arm for precise placement. To deliver the constructs to the brain without forcible expulsion, the needle was slowly inserted into the cortex to a depth of 6 mm. The plunger of the Hamilton syringe was then fixed while the needle containing the micro-TENN was manually raised 5 mm at approximately 1.5 mm / min. This process allowed for low-force micro-TENN delivery to connect the deep thalamic structures, the ventral posteromedial nucleus (VPM) and the whisker barrel cortex.

[0166] Tissue harvesting and histology On days 7 and 28 after implantation, rats were euthanized and perfused with cold heparinized saline and 10% formalin. After overnight fixation of the heads, brains were harvested to assess micro-TENN survival and host / micro-TENN synaptic integration. Briefly, brains were sectioned sagittally and cut into 40 μm slices for cryosectioning. For cryosectioning, slices were air-dried for 30 min, treated twice with ethanol for 3 min, and rehydrated twice in PBS for 3 min. Sections were blocked for 30–45 min with 0.1% Triton-x / 5% normal horse serum in PBS (ABC Universal Kit, Vector Labs, cat #PK-6200). Primary antibodies were applied to sections in 2% normal horse serum / Optimax buffer for 2 h at room temperature. Primary antibodies were goat anti-GFAP (1:1000), rabbit anti-IBA1 (1:1000), chicken anti-MAP2 (1:1000), and mouse anti-Tuj1 (1:1000). Sections were rinsed three times with PBS for 5 min, and then secondary antibodies (1:1000) were applied in 2% normal horse serum / PBS for 1 h at room temperature. Sections were counterstained with DNA-specific fluorescent Hoechst 33342 for 10 min and then rinsed with PBS. After immunostaining, slides were mounted on coverslips with Fluoromount-G mounting medium.

[0167] In vitro development of micro-TENNs Hydrogel micro-columns were optimized in vitro to support neuronal survival and directed growth of axons. The micro-columns were 5-30 mm long and consisted of hollow agarose tubes (outer diameter 350-500 µm) that orient axonal outgrowth through a central extracellular matrix (ECM; inner diameter 150-400 µm). Dissociated neurons were delivered to the proteinaceous matrix at one or both ends of the micro-column and cultured for 7-42 days in vitro (DIV) based on the desired length of axonal outgrowth (Figure 4). Electrical stimulation and Ca 2+Using a photosensitive dye in a bidirectional micro-TENN, we demonstrated that one neuronal population could be stimulated and the resulting action potential could travel across the axonal field to the other population. Micro-TENNs were fabricated using multiple neuronal subtypes, including primary cortical neurons, dopaminergic neurons, and dorsal root ganglion neurons (Figure 5).

[0168] Long micro-TENNs with axonal bundles (approximately 1 cm at 14 DIV and >2 cm at 28 DIV) were also fabricated as depicted in Figure 6. Additionally, smaller micro-TENNs spanning hundreds of microns to millimeters were fabricated as depicted in Figure 5. These were more appropriately scaled for one of the current applications of live electrodes, to pass through layers IV or V of the rat cortex.

[0169] Figure 17 provides a representation of an immunolabeled long-projection unidirectional axon-based living electrode. These axon-based living electrode constructs are on the order of hundreds of microns in diameter (similar to the diameter of a human hair), but may extend at least to the order of centimeters to reach deep brain layers / nuclei with a relatively small microinjection area.

[0170] In vivo delivery and survival of micro-TENNs Micro-TENNs were delivered to the brain via stereotaxic microinjection, providing viable axonal bridges reconnecting distinct anatomical regions. For in vivo delivery, the hydrogel shell provided structural support to protect the microtissue during transportation and implantation. Furthermore, the small size allows for minimally invasive implantation in delicate regions of the nervous system. Micro-TENNs of the desired length were aspirated into a needle, slowly inserted into the cortex, and ejected using a plunger. Micro-TENNs were stereotaxically inserted to connect cortical barrel fields and thalamic structures to assess survival and integration of the constructs.

[0171] At 3, 7, and 28 days after implantation, immunohistochemistry and fluorescence microscopy revealed surviving neurons within the micro-TENNs that maintained dense clusters with axon bundles extending parallel to the cortico-thalamic axis (Figure 6). This demonstrated that the micro-TENN neurons survived and maintained their axonal organization. Additionally, micro-TENN neurons demonstrated cortical integration, as dendrites derived from implanted neurons penetrated the cortex, along with structural evidence of synapse formation between micro-TENN neurons and host neurons (Figure 7).

[0172] Recording / stimulation array Living electrodes are used as biological conduits to convey information to / from neurons from deep within the brain at the brain's surface. At the surface, arrays of devices are used to record or stimulate the superficial ends of the micro-TENNs. These arrays can also include electrical, optical, magnetic, chemical, acoustic, or other modalities that create specific, widespread arrays that can interface with the superficial ends of the micro-TENNs in precise temporal and spatial fashions. These arrays can be placed on the brain's surface (subdural), on top of the dura (epidural), inside skull defects (intramyosseous or periosseous), outside the skull under the galea (subgaleal), or outside the skin (non-invasively on the scalp). These arrays can be coupled to or integrated with microelectronics. These can include power batteries, radio frequency and / or infrared induction coils, light sources, multiplexing / demultiplexing circuitry, cerebrospinal fluid flow or vascular beds utilizing heat sinks, and / or implanted waveguides.

[0173] Method of implementation FIG. 8 describes the advantages of living electrodes over conventional electrodes and optrodes, highlighting the enhanced specificity due to the synaptic integration of living electrodes.

[0174] Preformed 3-D living electrodes offer at least two clinical deployment advantages: (1) avoidance of spatial constraints of transfection agents and cells / axons due to in vitro transduction and (2) avoidance of maturation / decrease issues derived from cell suspensions.

[0175] Living electrodes provide a flexible pathway to transmit signals to / from deep brain structures compared to the rigid materials used in electrodes / optrodes, thus reducing chronic foreign body reactions, mechanical detachment issues, and glial scarring.

[0176] Optrodes can achieve high levels of specificity, but optical methods can be limited in range due to tissue absorption. Electrodes essentially stimulate or record from a fixed volume around the electrode (red area in Figure 8). The spread of optogenetic transduction is exemplified by yellow neurons in multiple layers (Figure 8). In contrast, living electrodes offer high specificity, as their constructs can be designed to synapse with specific neuronal subtypes, which in turn can form many synapses with surrounding neurons (Figures 8 and 9).

[0177] Figures 10 and 11 show examples of living electrodes to be used in a paradigm of recording or stimulation in the rat cortex. The implementation of living electrodes is not limited to the cortex, but can be employed anywhere in the brain where non-surface signals need to be recorded or stimulated. Additionally, input / stimulation versions of the micro-TENNs can be designed to interface with specific types of neurons to selectively activate excitatory or inhibitory neurons. Although rats are depicted here, the technology can ultimately be used in non-human primates and humans. Further iterations of living electrodes can also include the use of multiple neuronal types to achieve interaction with multiple neural subtypes. For example, one may desire a living electrode that excites one neuronal population while inhibiting another (Figure 1). As mentioned earlier, living electrodes can also be developed to be a true two-way interface modality that allows for closed-loop interaction with the brain. This living electrode could record, process, and then stimulate the brain in a controlled, closed-loop fashion providing sophisticated and well-controlled stimulation of the nervous system, delicately treating disease instead of the current brute force methods using constant application of electrical stimulation.

[0178] Tissue engineered constructs for neurosurgical implantation As shown in Figure 12, live electrodes can be implanted in the brain and interfaced to record or stimulate neurons for treatment or as a diagnosis of specific functions or dysfunctions of the nervous system (i.e., Parkinson's disease, obesity, inflammation, migraine, diabetes, epilepsy, etc.). For example, in Parkinson's disease patients, dopaminergic live electrodes restore dopamine input to deep brain structures (e.g., striatum) while controlling activation at the brain surface. For epilepsy patients, GABAergic live electrodes can also be used to activate upon detection of early epileptiform activity to suppress seizure foci (e.g., inhibitory live electrodes can precisely deliver large amounts of GABA directly to the foci at the earliest signs of preictal neural activity).

[0179] Implanted living electrodes can also affect thought-mediated behavior or behavior related to sensory input by recording or stimulating neurons for precise and selective coupling between biological and non-biological entities for restoring or enhancing function in individuals with or without locked-in syndrome. For example, motor control (reading activity of neurons in the motor cortex or motor output sites in the brain or spinal cord) for device (e.g., robotic hand / arm) actuation can be monitored. Sensory feedback (input information to sensory cortex or sensory processing sites in the brain) related to the feel and / or proprioception of an external device (e.g., robotic hand / arm) can also be recorded.

[0180] Also, living electrodes can be implanted and interfaced with the brain, using their activation as a way to enhance regrowth or improvement of function. For example, implantable living electrodes can control local neural activity to promote / induce endogenous regeneration. In another example, living electrodes can control local neural activity to enhance healthy network function and / or attenuate / block deleterious network function (e.g., correct circuit timing problems).

[0181] Confocal reconstruction of macro-TENNs in vitro: Proof-of-concept Applicants grew primary dorsal root ganglion neurons in the macro-TENN constructs for 7 days in vitro (DIV). Following this period, the cells were fixed and immunolabeled with a marker for cell nuclei (DAPI) and their axons (beta-tubulin-III / Tuj-1). The constructs were then imaged with a confocal microscope. The images were reconstructed and stitched together to provide a detailed view of the construct on all sides ( FIG. 14 ). What the images showed is best illustrated pictorially in panel D of FIG. 14 , which represents a reconstruction looking in the y-direction of the construct (i.e., looking down the length of the construct) (depicted in panel E of FIG. 14 ). Panels D and E of FIG. 14 depict that the axons grow only along the inner surface of the tube and not within the extracellular matrix that fills the core of the construct. In smaller constructs, the axons grow through the entire cross-section of the construct, not just at the interface as seen here. Viewing the construct from the top, panels A-C of Figure 14 show successive slices of confocal images starting from the bottom (A) to the top (C). In panel C of Figure 14, the axons grow only along the sidewall, while in panel B of Figure 14, at the bottom of the construct, the axons spread out along the entire surface of the bottom.

[0182] Proposed device in PNS Based on findings showing axonal regrowth only on the inner surface of macro-TENNs, applicants propose to fabricate a representative electrode as depicted in Figures 15A-15C. Deployment of this device would allow axonal growth through the channel unimpeded (as opposed to a sieve electrode). The channel may be the same size as the nerve or larger to provide further spreading and denervation of a particular nerve and its bundles. Electrodes can also be placed along the periphery and along the length of the construct, allowing selective stimulation and recording (Figures 15A and 15B). If the construct is sized larger than the nerve, it will taper at the proximal and distal ends, allowing suturing and attachment of the selected nerve (Figure 15C). The larger cross-sectional area advantageously allows the axons / bundles to spread further, thereby providing greater selectivity.

[0183] Aspects of the present invention provide superior conformation to the interior of the tube sidewall. Without wishing to be bound by theory, Applicants believe that the conformation is superior due to the greater surface area provided by using the interior of the tube compared to midplane techniques. Again, without wishing to be bound by theory, Applicants believe that the greater surface area allows for greater separation of axons / bundles and therefore greater selectivity.

[0184] Further Micro-TENN Fabrication Techniques Further embodiments of the invention provide novel microtissue engineering methodologies to more consistently create micro-TENNs of desired configurations composed of distinct neuronal populations connected by pure axonal tracts. In particular, embodiments of the invention utilize "forced cell aggregation" in custom-made pyramidal microwells to create neuronal "aggregates" or "spheres" with precisely controlled numbers of neurons (and therefore diameter) per aggregate / sphere, as depicted in panels C-E of FIG. 16. To accomplish this, dissociated neurons were transferred to a chamber containing an array of inverted pyramidal microwells made of PDMS (SYLGUARD® 184, Dow Corning) cast from a custom-designed 3D-printed mold. The wells were then centrifuged at 200 g for 5 minutes. This centrifugation resulted in the forced aggregation of neurons (or any other cell type, if desired) with precisely controlled numbers of neurons per aggregate / sphere based on the density of neurons used and the volume added to each well. 12 μL of neuronal suspension per well at a density of 1-2 million cells / mL was suitable to create aggregates / spheres of appropriate diameter to fit within the micro-column. The aggregates / spheres were then carefully placed within one or both ends of the micro-column and allowed to attach for 45 min at 37°C, 5% CO2.The seeded micro-columns were then grown in vitro for at least several days in neuronal growth medium composed of NEUROBASAL® medium, GLUTAMAX™ medium, and B-27® medium (ThermoFisher™) to form micro-TENNs (as described in Laura A. Struzyna et al., “Rebuilding Brain Circuitry with Living Micro-Tissue Engineered Neural Networks”, 21(21-22): 2744-2756 Tissue Engineering Part A (2015) and JP Harris et al., “Advanced biomaterial strategies to transplant preformed micro-tissue engineered neural networks into the brain,” 13(1) J. Neural Eng. 016019 (2016)). This methodology resulted in the formation of unidirectional or bidirectional micro-TENNs with defined neuronal soma areas and axonal extensions of several millimeters over several days in vitro ( FIG. 17 ). Remarkably, this methodology consistently produces ideal micro-TENN cellular configurations consisting of defined regions with neuronal cell bodies as aggregates at one or both ends of the micro-column and defined regions with axonal projections extending longitudinally across the center of the micro-column (Figure 17). This ideal distribution was further verified by immunocytochemistry and confocal microscopy, labeling these aggregate micro-TENNs using an antibody that recognizes all axons (beta-tubulin III) and all cell nuclei (Hoechst), and non-specifically labeling the hydrogel containing the micro-column. The micro-TENNs were also labeled for a synaptic marker (synapsin), indicative of functional maturation and electrochemical activity in the micro-TENNs.

[0185] Applicants have advanced microtissue engineering methodologies to improve the consistency of biomaterial construction of micro-columns. Here, micro-columns were printed from hyaluronic acid or similar hydrogels using a commercially available BIOBOTS™ 3D printer designed for tissue engineering applications. Micro-columns were printed with an outer diameter C of 200 μm and an inner diameter A of 100-150 μm while simultaneously filling with a bioactive extracellular matrix (typically 1 mg / mL collagen and / or 1 mg / mL laminin) to provide a gap with a depth G of 100 μm for cell aggregate delivery at one or both ends of the micro-column, as depicted in FIG. 18. Micro-TENNs were printed with a central radial axis oriented horizontally or vertically. Horizontal printing may be preferred for speed and / or because any ridges, grooves, or other artifacts resulting from printing can be parallel to the central radial axis and act as guides for nerve growth.

[0186] Use as a biological interface Embodiments of the present invention can be utilized as an interface between a host and various electronic devices. Although embodiments of the present invention have been described in the context of optical or magnetic sensors, embodiments of the present invention can also support engagement via electrical impulses applied to neurons in living electrodes.

[0187] As discussed herein, tissue engineered "living electrodes" enable stable long-term interfaces to probe and modulate the nervous system. "Living electrodes" increase target specificity while mitigating the foreign body response inherent in non-organic electrodes.

[0188] Without wishing to be bound by theory, Applicants believe that the invention described herein operates by one or more of the following mechanisms of action: First, aspects of the invention provide targeting specificity by integrating with specific neuronal subtypes while mitigating chronic foreign body reactions. Second, aspects of the invention provide synaptic integration that provides persistence not possible with prior approaches. Third, biological multiplexing is possible through robust effects elicited by relatively few axons.

[0189] The integration of custom-made "living electrodes" with specific cells / regions in the brain is mediated by synapses. Synaptic integration via engineered axonal tracts provides persistence and target specificity not possible with traditional approaches.

[0190] Synapse-based interfaces using engineered neurons and / or axonal tracts can form a biological link between the host and electronics, ultimately enabling prosthetic control, sensory / proprioceptive feedback, and / or neuromodulation.

[0191] Robust effects, i.e., recruitment of large numbers of host neurons, can be elicited by relatively few axons via a novel mechanism termed "biological multiplexing." As an example, a single micro-TENN axon can (theoretically) synapse with hundreds or even thousands of neurons to produce a remarkable amplifying effect. Applicants are currently building micro-TENNs with approximately 50,000 neurons in a column approximating the diameter of a human hair, thus demonstrating the potential to affect millions of host neurons with a single construct.

[0192] In various embodiments, neuronal phenotypes can be selected to release / secrete certain neurotransmitters to restore levels associated with certain disease processes. In various embodiments, constructs can be tailored to specific lengths to achieve connectivity at specific anatomical targets and synapse with specific neuronal subtypes within those regions. In various embodiments, proteinaceous matrices and co-delivered factors can be modified to haptotactically and chemotactically attract specific host neuronal types targeted for modulation. In various embodiments, construct neurons can target specific neurons without any external artificial actuation (provided synaptic specificity is achieved). In various embodiments, constructs can be seeded with one or more neuronal populations, and these living neural networks can perform multiplexing of functions within themselves and by achieving targeted output of advanced information to parenchymal tissues.

[0193] 3. Micro-TENNs Plating Dissociated Neuron Suspensions Dopaminergic neurons were isolated from the ventral midbrain of embryonic rats. In plate culture, these neurons displayed healthy neuronal morphology, the presence of dopaminergic neurons (based on TH expression), prominent neurite outgrowth (based on β-tubulin III expression), and network formation by 28 DIV. To create dopaminergic micro-TENNs, initial seeding micro-columns with dissociated neuron suspensions were used. These dissociated cells infiltrated the length of the lumen but generally did not create the desired cellular organization of distinct cell body regions projecting axons the length of the inner core.

[0194] However, dissociated neurons within the microtissue constructs displayed healthy morphology and sometimes spontaneously self-organized into the desired cellular configuration. In these cases, unidirectional axonal projections reached lengths of several millimeters, and importantly, the integrity of these constructs was also maintained up to 28 DIV. To see if the inclusion of additional ECM in the inner core increased the consistency with which precise configurations were generated, dissociated cells were suspended in collagen and the mixture was injected into the gel inside the microcolumn.

[0195] Unfortunately, the presence of collagen did not support production of the desired cellular organization, and the dissociated cells continued to spread throughout the length of the inner core (Panels A1-A3 of Figure 23). While these results demonstrated that dopaminergic neurons that formed extensive neurite networks could be cultured within hydrogel microcolumns, these techniques were not sufficient to consistently generate the desired cellular organization.

[0196] Forced neuronal aggregation method Previous micro-TENN fabrication methods did not reliably produce the desired cellular organization, so a method was adapted to mechanically aggregate neurons into aggregates (Ungrin MD, Joshi C, Nica A, et al. 2008, Reproducible, ultra high-throughput formation of multicellular tissue from single cell suspension-derived human embryonic stem cell aggregates, PloS one, 3 (2): e1565). After dissociating the embryonic tissue into a single cell suspension, the solution was centrifuged in an inverted pyramidal well to pellet the cells at the bottom of the well. The well was left in an incubator overnight, during which time the pelleted cells became neuronal aggregate spheres. Once formed, the aggregates were inserted into the end of an agarose micro-column. This method consistently yielded micro-TENNs with distinct cell body and axon regions. Furthermore, it was found that it was possible to create micro-TENNs that exhibited externalized or internalized cell body regions based on the depth and placement of the aggregates within the micro-column (Figure 23, panels B-D). Furthermore, this technique resulted in long-projecting unidirectional axon tracts, as demonstrated by TH and β-tubulin III immunoreactivity (Figure 23, panels E-F). Indeed, axons projecting from the aggregates grew approximately 10-fold longer than similar axonal extensions in micro-columns seeded with dissociated neurons, as judged by the longest neurite length in each micro-TENN (Figure 23, panel G).

[0197] Optimization of micro-TENN length The nigrostriatal tract is approximately 6 mm in rats, therefore, micro-TENNs of at least 6 mm in length are desirable. To optimize growth conditions for length, the effects of ECM components in the lumen, the presence of growth factors, and the orientation of the micro-TENNs on outgrowth were tested. Collagen I and collagen I and laminin were found to have yielded the longest axonal outgrowth as judged by the longest neurite length in each micro-TENN (Figure 24). The average axonal outgrowth for collagen I and collagen I and laminin cores was 4892 ± 703 μm and 4686 ± 921 μm, respectively. In contrast, cross-linked collagen cores (1227 ± 481 μm), laminin-coated cores (205 ± 615 μm), and empty cores (approximately 0 μm) were found to have yielded significantly reduced neurite outgrowth. For the two high-growth groups (lumens made of collagen or collagen and laminin), TH+ dopaminergic axon projections were confirmed to have reached at least 60% of maximum axon length (Figure 24, panel F). We also tested the effect of medium growth factor concentration on axon outgrowth within the micro-columns. Medium containing a relatively low concentration of bFGF (4 ng / mL) was compared to medium containing high concentrations of growth factors previously shown to increase the outgrowth and survival of dopaminergic neurons. At 14 DIV, it was found that the high growth factor concentration medium did not result in denser or longer axon outgrowth compared to the low concentration medium (n=14 micro-TENNs in each group). Finally, we investigated whether the use of a targeted population of dopaminergic cells would increase axon outgrowth. Bidirectional dopaminergic micro-TENNs were plated by inserting dopaminergic aggregates at both ends of the micro-column. On the other hand, to determine whether chemotactic signaling between the populations increases outgrowth, the two dopaminergic neuron populations were separated by 1.2 cm. At 14 DIV, axon outgrowth in bidirectional micro-TENNs was equal to or less than that in unidirectional micro-TENNs (n = 14 micro-TENNs in each group).Thus, the use of engineered neuronal aggregates and specific ECM components were important factors in axonal outgrowth, while the presence of high growth factor medium and target neuronal populations did not affect axonal outgrowth. Of note, the average neuronal aggregate length at 14 DIV was 1165 ± 212 µm; therefore, the total micro-TENN length (neuronal aggregate + axon length) achieved using dopaminergic aggregates in collagen was >6 mm at 14 DIV, which is suitable for connecting the nigrostriatal tract in rats. Following optimization studies, dopaminergic micro-TENNs with an inner core of collagen I were fabricated and grown for 28 DIV to determine whether axonal outgrowth further proceeded within the micro-columns. Continued axonal outgrowth by 28 DIV was found with lengths of 6046±670 μm for dopaminergic axons, 7697±1085 μm for all axons, and 8914±1187 μm for total aggregate+axon length. In some cases, the maximum total micro-TENN length at this time point exceeded 10 mm, well beyond the length required to encompass the nigrostriatal pathway in rats (FIG. 25).

[0198] Formation of synapses with striatal populations Since dopaminergic axons comprising the nigrostriatal tract synapse with striatal neurons in the brain, we explored the ability of tissue engineered nigrostriatal tracts to synapse with populations of striatal neurons in vitro. Dopaminergic micro-TENNs were fabricated and after 10 DIV, aggregates of embryonic rat striatum were inserted into the empty end of the micro-column. After 4 DIV, immunocytochemistry was performed to assess potential synaptic integration between the two populations. This analysis confirmed the presence of appropriate neuronal subtypes in the two aggregate populations, specifically TH+ dopaminergic neurons and DARPP-32+ medium spiny striatal neurons (Figure 26). Furthermore, confocal microscopy revealed extensive axodendritic integration and putative synapse formation involving dopaminergic axons and striatal neurons (Panels D, E, G, H of Figure 26). Immunocytochemistry also confirmed that most striatal (DARPP-32+) neurites were also MAP-2+, suggesting that they were dendritic (data not shown). To determine whether chemotactic cues generated by the striatal population influenced axonal outgrowth from dopaminergic neuronal aggregates, the length of axonal outgrowth was quantified with and without striatal terminal targets. Axonal outgrowth in dopaminergic micro-TENNs containing a targeted population of striatal neurons was found to be statistically not greater than axonal outgrowth in unidirectional dopaminergic micro-TENNs (n=9 micro-TENNs in each group; FIG. 26, panel F).

[0199] In vivo implantation and survival of preformed dopaminergic micro-TENNs To demonstrate the ability to precisely deliver preformed dopaminergic micro-TENNs to the brain, as well as their survival and organization at various time points after implantation, dopaminergic aggregate micro-TENNs with a collagen I-containing lumen were transduced to express GFP and grown for 14 DIV, after which they were aspirated into custom-made needles and stereotactically macroinjected near the nigrostriatal tract of adult male Sprague-Dawley rats. Sacrificing animals at 1 week and 1 month (n=5 each) revealed viable GFP+ neurons and axons within the micro-TENN lumen that were easily identified throughout the nigrostriatal tract as the agarose micro-columns were only partially degraded at these time points (FIG. 27). Tissue sections were co-labeled for the axonal marker β-tubulin III and the dopaminergic marker TH, revealing the preservation of robust neuronal and dopaminergic axon populations. Notably, there were longitudinally projecting TH+ axons, confirming that micro-TENNs were able to maintain their cellular organization after implantation into the brain (Figure 27).

[0200] Quantifying living electrode growth in vitro In previous studies, micro-TENNs were seeded with single cell suspensions of primary cortical neurons, which often formed clusters at random sites throughout the micro-TENN interior (Figure 29, panels C-D). This generation of micro-TENNs was formed with pre-formed cortical aggregates prior to plating into the micro-columns, allowing for greater control and reproducibility of the desired cellular organization of distinct soma and axonal regions (Figure 29, panels F-H). This reproducibility in itself leads to robust characterization of micro-TENNs in vitro, a necessary step in their application as living electrodes. Aggregate micro-TENNs were plated with approximately 8,000-10,000 neurons per aggregate, with micro-column lengths of 2 mm and 5 mm. Both unidirectional (one aggregate) and bidirectional (two aggregates) 2 mm long micro-TENNs (LEs) were fabricated using a 3D-based ... UNI,2mm and L.E. BI,2mm) were plated, while all 5 mm long LEs (LE BI,5mm ) were plated as a bidirectional construct. 2 mm long dissociated micro-TENNs (LE DISS,2mm The growth characteristics of previous dissociated micro-TENNs were compared to current aggregate-based constructs by plating them on the ECM core of the LE (Table 1, FIG. 30). Through analysis of phase contrast microscopy images, robust axonal outgrowth was found along the ECM core throughout all aggregate LEs within the first few days in vitro (FIG. 30). BI,5mm Aggregate LE neurons in the group exhibited rapid axon growth rates that peaked at 1087.7±84.3 microns / day over all DIVs measured. UNI,2mm Neurons in the group showed an initial peak axon growth rate of 358 ± 19.8 microns / day on day 1, which steadily decreased over time. BI,2mm Within groups, neuronal processes traversed the length of the microcolumn and formed synapses with the contralateral population by 5 DIV (Figure 30, Panel A), with a concomitant decrease in growth rate (Figure 30, Panel D). BI,5mm Neurons from the group were cultured in the LE for the first 3 DIV. BI,2mm Although they grew at a similar rate to neurons, they showed a large increase in growth rate until 5 DIV, after which axonal growth slowed as synapses formed between aggregates (Figure 30, panels C-D). The growth rates in all aggregate micro-TENN groups exceeded that of dissociated micro-TENNs, which reached a maximum growth rate of 61.7 ± 5 microns / day at 1 DIV (Table 1).

[0201] Two-way ANOVA revealed significant main effects (all p<0.0001) from DIV (F-statistic=15.97) and LE group (F-statistic=27.4), as well as their interaction (F-statistic=5.92). Therefore, Bonferroni analysis was used for subsequent pairwise comparisons, revealing several statistical differences both within and between LE groups at different DIV. In general, growth rates for bidirectional micro-TENNs were greater at earlier time points than at later time points, whereas growth rates for dissociated and unidirectional micro-TENNs did not change significantly with time. Notably, LE at 5 DIV showed a significant increase in growth rates. BI,5mm The growth rate of LE DISS,2mm (p<0.0001), L.E. UNI,2mm (p<0.0001), and L.E. BI,2mm The overall growth rate for LE was greater than that for all other groups (p<0.001). BI,5mm Within the group itself, the growth rate at 5 DIV was statistically greater than the growth rate at all other DIVs (p<0.01). BI,2mm The growth rate of LE DISS,2mm The growth rates of LE were significantly higher than those of all other growth rates (p<0.01). BI,5mm (p<0.001) and LE BI,2mm The growth rate at 1 DIV was greater than that at 10 DIV for both (p<0.01).

[0202] Table 1 Growth rate of micro-TENNs TIFF0007656642000001.tif32160 Data presented as mean ± sem in microns / day.

[0203] Bidirectional micro-TENNs labeled with GFP and mCherry were imaged over time to observe interactions between axonal projections from each aggregate (Figure 31). Confocal images revealed that when contacted with opposing axons, projections continued to grow in parallel to each other toward opposing aggregates, supporting a physical interaction between the two neuronal populations (Figure 31).

[0204] Acute and chronic viability of living electrodes Via live / dead staining and confocal microscopy, survival was quantified at 10 and 28 DIV for short unidirectional and short bidirectional LEs (Figure 32). Plated cultures matched in time from preparation served as controls. Viability was defined as the ratio of the integrated area of ​​calcein-AM positive cells to the integrated area of ​​all stained cells (i.e., both calcein-AM+ and ethidium homodimer+ cells). We confirmed that neuronal survival in live electrodes persisted until at least 28 DIV, with evidence of survival until 40 DIV (Figure 32). ANOVA showed that DIV was a significant main effect (F statistic = 32.21, p < 0.0001), but not in the LE / culture group (p > 0.84). An interaction effect was also found to be significant (p < 0.01), so Bonferroni analysis was used to compare groups at each time point (Figure 32, panel C). Survival of plated cultures at 28 DIV was significantly greater than that of LEs at 10 DIV. UNI (p<0.05), L.E. BI The survival rates of plate cultures at 10 DIV were found to be statistically lower than that of LE at 28 DIV (p<0.001) and plate cultures (p<0.0001). Furthermore, plate culture survival rates at 10 DIV were statistically lower than that of LE at 28 DIV (p<0.0001). UNI and L.E. BI The survival rates of both groups were significantly higher (p<0.01).

[0205] Assembly of living electrodes and synaptogenesis over time To characterize LE organization over time, bidirectional LEs were fixed and immunolabeled at 4, 10, and 28 DIV to identify cell nuclei, axons, and synapses (Figure 33). Neuronal somata were almost exclusively localized in aggregates connected by long axons, as indicated by Tuj-1 (Figure 33); axons and dendrites were also found within aggregates, likely from intraaggregate connections formed at or shortly after plating. The total area of ​​synapsin 1-positive puncta across specific time points was used to qualitatively assess the presence of synapses. We identified a moderate distribution of synapsin within micro-TENN aggregates as well as increased synapsin expression within the lumen of the micro-columns, suggesting that neurons within bidirectional micro-TENNs may have the ability to communicate between aggregates.

[0206] Corticothalamic implantation Preformed micro-TENNs (fabricated as described above to consist of aligned axonal tracts projecting from neuronal aggregates encased in tubular hydrogel micro-columns) were implanted into the recurrent corticothalamic pathway by connecting the whisker barrel cortex with the VPM. One month after injection into the rodent brain, GFP+ micro-TENNs were found to have survived and maintained the preformed cell body-axon distribution configuration (Figure 34). Large dense clusters (aggregates) of GFP+ cell bodies were found in the dorsal and ventral implantation regions, with axons and dendrites within the lumen spanning the two locations (Figure 34).

[0207] Equivalent While specific terminology has been used to describe preferred embodiments of the invention, it is to be understood that such description is for purposes of illustration only, and that changes and modifications may be made without departing from the spirit or scope of the following claims.

[0208] INCORPORATION BY REFERENCE The entire contents of all patents, published patent applications, and other references cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. Cylinder-shaped extracellular matrix core; a hydrogel sheath coaxially surrounding the cylindrical extracellular matrix core; one or more electrodes and / or optrodes disposed between the cylindrical extracellular matrix core and the hydrogel sheath; and one or more neurons implanted along or within said cylindrical extracellular matrix core; An implantable living electrode, including

2. The implantable living electrode of claim 1 , wherein the one or more neurons include one or more optogenetic neurons proximal to a first end of the implantable living electrode.

3. 3. The implantable living electrode of claim 2, comprising an optrode disposed between the cylindrical extracellular matrix core and the hydrogel sheath.

4. 2. The implantable living electrode of claim 1, wherein the one or more neurons include one or more magnetogenetic neurons proximal to a first end of the implantable living electrode.

5. 2. The implantable living electrode of claim 1, wherein said cylindrical extracellular matrix core has a maximum cross-sectional dimension of between 10 μm and 1,000 μm.

6. 2. The implantable living electrode of claim 1, wherein said cylindrical extracellular matrix core has a maximum cross-sectional dimension of 300 μm to 350 μm or 350 μm to 400 μm.

7. 2. The implantable living electrode of claim 1, wherein said cylindrical extracellular matrix core has a maximum cross-sectional dimension of 400 μm to 500 μm or 500 μm to 700 μm.

8. The implantable living electrode of claim 4, wherein said hydrogel sheath has a maximum cross-sectional dimension of between 20 μm and 1,200 μm.

9. The implantable living electrode of claim 4, wherein said hydrogel sheath has a maximum cross-sectional dimension of between 800 μm and 1,200 μm.

10. controlling a compatible stimulator to activate the implantable living electrode to activate or excite brain activity; controlling a compatible stimulator to actuate the implantable living electrode to activate or excite synaptic activity in the host; controlling a compatible stimulator to activate the implantable living electrode to activate or excite neuronal activity; controlling a compatible stimulator to activate the implantable living electrode to inhibit brain activity; controlling a compatible stimulator to activate the implantable living electrode to inhibit synaptic activity of the host; controlling a compatible stimulator to activate the implantable living electrode to inhibit neuronal activity; controlling a compatible stimulator to activate the implantable living electrode to inhibit neural network activity; controlling a compatible stimulator to activate the implantable living electrode to modulate synaptic activity in the host; controlling a compatible stimulator to activate the implantable living electrode to modulate neuronal activity; and controlling a compatible stimulator to activate the implantable living electrode to modulate neural network activity; 10. The implantable living electrode of claim 1 for use in a method comprising at least one step selected from the group consisting of:

11. The implantable living electrode of claim 10 , wherein the compliant stimulator is a light source.

12. The implantable living electrode of claim 10 , wherein the compliant stimulator is a magnetic source.

13. 2. The implantable living electrode of claim 1, wherein the one or more neurons comprise one or more neurons selected from the group consisting of glutamatergic neurons, GABAergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, and peptidergic neurons.

14. The implantable living electrode of claim 1 , wherein the one or more neurons include a dopaminergic neuron.

15. 2. The implantable living electrode of claim 1, wherein the one or more neurons comprise one or more neurons selected from the group consisting of primary cerebral cortical neurons, dorsal root ganglion neurons, thalamic-derived neurons, striatal-derived neurons, hippocampal-derived neurons, and substantia nigra-derived neurons.

16. 2. The implantable living electrode of claim 1, wherein the one or more neurons comprise primary cerebral cortical neurons, the primary cerebral cortical neurons comprising one or more members selected from the group consisting of neurons from layer I of the cortex, neurons from layer II of the cortex, neurons from layer III of the cortex, neurons from layer IV of the cortex, neurons from layer V of the cortex, neurons from layer VI of the cortex, neurons from the visual cortex, neurons from the motor cortex, neurons from the sensory cortex, and neurons from the entorhinal cortex.

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