Preformed neural tissue to restore or enhance auditory input to the brain

Abstract

Provided herein is a system, e.g., a living electrode, that includes a biocompatible construct that includes a matrix and a plurality of auditory neurons. Also disclosed herein are methods of making the system and methods of using the system for implantation in a subject to modulate auditory neurons in the subject and / or to treat or alleviate symptoms of hearing impairment. Additionally, provided herein is a kit that includes the system described herein. TIFF2024509088000002.tif124128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 153,321, filed February 24, 2021, and No. 63 / 234,048, filed August 17, 2021, which are incorporated by reference in their entireties herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under NS094340 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] background Hearing impairment affects a large percentage of the world's population. One of the treatment options for the hearing impaired is hearing implants. However, currently available implants have several limitations, including the inability to restore the patient's natural hearing, complications during implantation, and difficulty understanding complex sounds. Summary of the Invention

[0004] overview Provided herein, inter alia, is a system, such as a living electrode, that includes a biocompatible construct comprising a matrix and a plurality of auditory neurons. Also disclosed herein are methods of making the system, such as at least one living electrode, that includes a biocompatible construct comprising a matrix and a plurality of auditory neurons, and methods of using the system for implantation in a subject to modulate auditory neurons in the subject and / or treat or alleviate symptoms of hearing impairment. Also provided herein are kits that include the systems described herein.

[0005] The present disclosure provides a system including: (a) a biocompatible construct including a matrix having a first end, a second end, and a body; and (b) at least one living electrode including a plurality of auditory neurons (e.g., vestibular neurons and / or cochlear neurons).

[0006] In some embodiments, the biocompatible construct comprises an interior surface and / or an exterior surface.

[0007] In some embodiments, the inner surface of the biocompatible construct defines a luminal core.

[0008] In some embodiments, the exterior surface of the biocompatible construct comprises at least one hydrogel. In some embodiments, the hydrogel comprises a hydrophilic biopolymer and / or a synthetic polymer. In some embodiments, the hydrogel is at least partially crosslinked, where crosslinking optionally increases stiffness, reduces porosity, and / or increases degradation time. In some embodiments, the hydrophilic biopolymer comprises one or more of agarose, hydrogel, hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, fibrin, and methylcellulose. In some embodiments, the hydrophilic biopolymer comprises agarose. In some embodiments, the hydrophilic biopolymer comprises a hydrogel. In some embodiments, the hydrophilic biopolymer comprises chitosan. In some embodiments, the hydrophilic biopolymer comprises alginate. In some embodiments, the hydrophilic biopolymer comprises collagen. In some embodiments, the hydrophilic biopolymer comprises dextran. In some embodiments, the hydrophilic biopolymer comprises pectin. In some embodiments, the hydrophilic biopolymer comprises carrageenan. In some embodiments, the hydrophilic biopolymer comprises polylysine. In some embodiments, the hydrophilic biopolymer comprises gelatin. In some embodiments, the hydrophilic biopolymer comprises hyaluronic acid. In some embodiments, the hydrophilic biopolymer comprises fibrin. In some embodiments, the hydrophilic biopolymer comprises methylcellulose.

[0009] In some embodiments, the hydrophilic biopolymer comprises agarose. In some embodiments, the agarose is about 0.25-30%, about 0.25%-3%, about 0.5%-3%, about 1-20%, about 1.5-10%, about 2-9%, about 2.5-8%, or about 3-7%. In some embodiments, the agarose is about 0.25-29%, about 0.25-28%, 0.25%, about 0.25-27%, about 0.25-26%, about 0.25-25%, about 0.25-24%, about 0.25-23%, about 0.25-22%, about 0.25-21%, about 0.25-20%, about 0.25-19%, about 0. 25~18%, about 0.25~17%, about 0.25~16%, about 0.25~15%, about 0.25~14%, about 0.25~13%, about 0.25~12%, about 0.25~11%, about 0.25~10%, about 0.25~9%, about 0.25~8%, about 0.25~7%, about 0.25~6%, about 0.25~5%, about 0.25~ 4%, approx. 0.25-3%, approx. 0.25-2%, approx. 0.25-1%, approx. 0.25-0.5%, approx. 0.5-30%, approx. 1-30%, approx. 2-30%, approx. 3-30%, approx. 4-30%, approx. 5-30%, approx. 6-30%, approx. 7-30%, approx. 8-30%, approx. 9-30%, approx. 10-30%, approx. 11-30%, approx. 12-30%, approx. 13-30%, approximately 14-30%, approximately 15-30%, approximately 16-30%, approximately 17-30%, approximately 18-30%, approximately 19-30%, approximately 20-30%, approximately 21-30%, approximately 22-30%, approximately 23-30%, approximately 24-30%, approximately 25-30%, approximately 26-30%, approximately 27-30%, approximately 28-30%, approximately 29-30%.

[0010] In some embodiments, the agarose is about 0.25%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%. In some embodiments, the agarose is about 3%.

[0011] In some embodiments, the hydrophilic biopolymer comprises at least one synthetic hydrogel (e.g., a pluronic hydrogel) or at least one synthetic hydrogel made of an amphiphilic copolymer consisting of ethylene oxide (PEO) and polypropylene oxide (PPO) units. In some embodiments, the hydrogel is about 2-50% w / v, about 3-40% w / v, about 4-30% w / v, about 5-25% w / v, or about 10-20% w / v. In some embodiments, the hydrogel is about 2-45%, about 2-40%, about 2-35%, about 2-30%, about 2-29%, about 2-28%, about 2-27%, about 2-26%, about 2-25%, about 2-24%, about 2-23%, about 2-22%, about 2-21%, about 2-20%, about 2-19%, about 2-18%, about 2-17%, about 2-16%, about 2-15%, about 2-14%, about 2-13%, about 2-12%, about 2-11%, about 2-10%, about 2-9%, about 2-8%, about 2-7%, about 2-6%, about 2-5%, about 2-4%, about 2-3%, about 3-50%. , about 4-50%, about 5-50%, about 6-50%, about 7-50%, about 8-50%, about 9-50%, about 10-50%, about 11-50%, about 12-50%, about 13-50%, about 14-50%, about 15-50%, about 16-50%, about 17-50%, about 18-50%, about 19-50%, about 20-50%, about 21-50%, about 22-50%, about 23-50%, about 24-50%, about 25-50%, about 26-50%, about 27-50%, about 28-50%, about 29-50%, about 30-50%, about 35-50%, about 40-50%, and about 45-50% w / v. In some embodiments, the hydrogel is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 40%, about 45%, or about 50% w / v. In some embodiments, the hydrogel is about 28.1%, about 28.2%, about 28.3%, about 28.4%, about 28.5%, about 28.6%, about 28.7%, about 28.8%, about 28.9%, about 30% w / v.In some embodiments, the hydrogel is about 28.6% w / v.

[0012] In some embodiments, the inner surface of the biocompatible construct comprises one or more extracellular matrix (ECM) components. In some embodiments, the ECM components comprise collagen, laminin, fibronectin, hyaluronic acid, or combinations thereof. In some embodiments, the ECM components comprise collagen. In some embodiments, the ECM components comprise laminin. In some embodiments, the ECM components comprise fibronectin. In some embodiments, the ECM components comprise hyaluronic acid.

[0013] In some embodiments, the ECM component comprises collagen, hi some embodiments, the collagen is at a concentration of about 0.1-10 mg / ml. In some embodiments, collagen is about 0.1-9 mg / ml, about 0.1-8 mg / ml, about 0.1-7 mg / ml, about 0.1-6 mg / ml, about 0.1-5 mg / ml, about 0.1-4 mg / ml, about 0.1-3 mg / ml, about 0.1-2 mg / ml, about 0.1-1 mg / ml, about 0.1-0.9 mg / ml, about 0.1-0.8 mg / ml, about 0.1-0.7 mg / ml, about 0.1-0.5 mg / ml, about 0.5-10 mg / ml, about 0.6-10 mg / ml, about 0.7-10 mg / ml, about 0.8-10 mg / ml, about 0.9-10 mg / ml, about 1-10 mg / ml, about 2-10 mg / ml, about 3-10 mg / ml, about 4-10 mg / ml, about 5-10 mg / ml, about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, about 9-10 mg / ml, about 10-10 mg / ml, about 20-10 mg / ml, about 30-10 mg / ml, about 40-10 mg / ml, about 50-10 mg / ml, about 60-10 mg / ml, about 70-10 mg / ml, about 80-10 mg / ml, about 90-10 mg / ml, about 10 ... The concentrations are about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, and about 9-10 mg / ml. In some embodiments, the collagen is at a concentration of about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml. In some embodiments, the collagen is at a concentration of about 1 mg / ml.

[0014] In some embodiments, the ECM component comprises laminin. In some embodiments, the laminin is about 0.1-9 mg / ml, about 0.1-8 mg / ml, about 0.1-7 mg / ml, about 0.1-6 mg / ml, about 0.1-5 mg / ml, about 0.1-4 mg / ml, about 0.1-3 mg / ml, about 0.1-2 mg / ml, about 0.1-1 mg / ml, about 0.1-0.9 mg / ml, about 0.1-0.8 mg / ml, about 0.1-0.7 mg / ml, about 0.1-0.5 mg / ml, about 0.5-10 mg / ml, about 0.6-10 mg / ml, about 0.7-10 mg / ml, about 0.8-10 mg / ml, about 0.9-10 mg / ml, about 1-10 mg / ml, about 2-10 mg / ml, about 3-10 mg / ml, about 4-10 mg / ml, about 5-10 mg / ml, about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, about 9-10 mg / ml, about 10-10 mg / ml, about 20-10 mg / ml, about 30-10 mg / ml, about 40-10 mg / ml, about 50-10 mg / ml, about 60-10 mg / ml, about 70-10 mg / ml, about 80-10 mg / ml, about 90-10 mg / ml, about 10 ... The concentrations are about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, and about 9-10 mg / ml. In some embodiments, the laminin is at a concentration of about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml. In some embodiments, the laminin is at a concentration of about 1 mg / ml.

[0015] In some embodiments, the plurality of auditory neurons includes about 50-5000 auditory neurons (e.g., vestibular neurons and / or cochlear neurons, e.g., auditory neurons described herein). In some embodiments, the plurality of auditory neurons includes about 50-4500, about 50-4000, about 50-3500, about 50-3000, about 50-2500, about 50-2000, about 50-1500, about 50-1000, about 50-900, about 50-800, about 50-700, about 50-600, about 50-550, about 50-500, about 50-600, about 50-700, about 50-800, about 50-900, about 50-900, about 50-1000, about 50-15 ... about 0-450, about 50-400, about 50-300, about 50-200, about 50-100, about 100-5000, about 200-5000, about 300-5000, about 400-5000, about 500-5000, about 1000-5000, about 2000-5000, about 3000-5000, or about 4000-5000 auditory neurons. In some embodiments, the plurality of auditory neurons comprises at least 50 auditory neurons, at least 100 auditory neurons, at least 200 auditory neurons, at least 300 auditory neurons, at least 400 auditory neurons, at least 450 auditory neurons, at least 500 auditory neurons, at least 550 auditory neurons, at least 600 auditory neurons, at least 700 auditory neurons, at least 800 auditory neurons, at least 900 auditory neurons, at least 1000 auditory neurons, at least 2000 auditory neurons, at least 3000 auditory neurons, at least 4000, or at least 5000 auditory neurons. In some embodiments, the plurality of auditory neurons comprises at least 500 auditory neurons.

[0016] In some embodiments, the auditory neuron is or comprises a neuron having one or more characteristics of a neuron present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of an organism, such as a human. In some embodiments, the auditory neuron is or comprises a neuron derived from a neuron present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of an organism, such as a human, or a combination thereof.

[0017] In some embodiments, at least one of the plurality of auditory neurons is in contact with a biocompatible construct.

[0018] In some embodiments, at least one neuron in the plurality of neurons comprises a cell body and / or an axon. In some embodiments, at least one neuron in the plurality of neurons comprises a cell body. In some embodiments, at least one neuron in the plurality of neurons comprises an axon. In some embodiments, at least one neuron in the plurality of neurons comprises a cell body and an axon. In some embodiments, the axon is about 0.5-50 cm in length.

[0019] In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned (i) at a first end of the biocompatible construct; (ii) at a second end of the biocompatible construct; (iii) at, along or within a portion of the body of the biocompatible construct; and / or (iv) throughout the body of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at a first end of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at a second end of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at, along or within a portion of the body of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned throughout the body of the biocompatible construct.

[0020] In some embodiments, the portion of at least one neuron in the plurality of auditory neurons positioned at the first end or the second end of the biocompatible construct comprises a cell body.

[0021] In some embodiments, a portion of at least one neuron in the plurality of auditory neurons positioned at, along or within a portion of the body of the biocompatible construct comprises an axon. In some embodiments, the body of the biocompatible construct comprises an interior surface of the body. In some embodiments, the interior surface comprises a luminal core.

[0022] In some embodiments, a portion of at least one neuron in the plurality of auditory neurons positioned throughout, along, or within the body of the biocompatible construct comprises an axon. In some embodiments, the axon extends unidirectionally or bidirectionally. In some embodiments, the axon extends unidirectionally. In some embodiments, the axon extends bidirectionally.

[0023] In some embodiments, at least a portion of the plurality of auditory neurons comprises a clump of auditory neurons.

[0024] In some embodiments, the plurality of auditory neurons comprises spiral ganglion neurons, inferior colliculus (IC) auditory neurons, cortical auditory neurons, type I auditory neurons, or type II auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of spiral ganglion neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of IC auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of cortical auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of type I auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of type II auditory neurons.

[0025] In some embodiments, the systems disclosed herein, such as, for example, living electrodes, further comprise one or more additional cells, such as astrocytes, oligodendrocytes, Schwann cells, microglia, or endothelial cells.

[0026] In some embodiments, the plurality of auditory neurons is isolated from a subject.

[0027] In some embodiments, the plurality of auditory neurons is or comprises at least one cell derived from a stem cell. In some embodiments, the stem cell comprises one or more of induced pluripotent stem cells (iPSCs), embryonic stem cells, or tissue stem cells. In some embodiments, the stem cell comprises an iPSC. In some embodiments, the stem cell comprises embryonic stem cells. In some embodiments, the stem cell comprises tissue stem cells.

[0028] In some embodiments, the plurality of auditory neurons is autologous, allogeneic, or xenogeneic to the subject. In some embodiments, the plurality of auditory neurons is autologous to the subject. In some embodiments, the plurality of auditory neurons is allogeneic to the subject. In some embodiments, the plurality of auditory neurons is xenogeneic to the subject.

[0029] In some embodiments, at least one neuron in the plurality of auditory neurons is genetically engineered or reprogrammed using non-genetic techniques. In some embodiments, at least one genetically engineered neuron comprises a sensor, an actuator, a receptor, and / or a reporter.

[0030] In some embodiments, at least one engineered neuron in said plurality of auditory neurons comprises a reporter. In some embodiments, the reporter comprises a fluorescent reporter.

[0031] In some embodiments, at least one engineered neuron in the plurality of auditory neurons comprises a receptor, hi some embodiments, the receptor comprises one or more of a magnetoreceptor, an electroreceptor, an electroreceptor, a hydroreceptor, a mechanoreceptor, an osmoreceptor, a thermoreceptor, and a piezoelectric ion channel.

[0032] In some embodiments, the electromagnetic receptor comprises one or more of an infrared receptor, a photoreceptor, or an ultraviolet receptor.

[0033] In some embodiments, the mechanoreceptors include one or both of mechanosensory receptors or proprioceptors.

[0034] In some embodiments, at least one engineered neuron in the plurality of auditory neurons comprises a sensor, hi some embodiments, the sensor comprises an optical sensor comprising one or more of a light-gated ion channel, a calcium sensor, and a membrane voltage sensor.

[0035] In some embodiments, the sensor comprises a calcium sensor comprising one or more of Aequorin, Cameleon, and GCaMP.

[0036] In some embodiments, the sensor comprises a chloride sensor comprising Clomeleon.

[0037] In some embodiments, the sensor comprises a membrane voltage sensor comprising Mermaid.

[0038] In some embodiments, at least one engineered neuron in the plurality of auditory neurons comprises an actuator, hi some embodiments, the actuator comprises an optical actuator comprising one or more of channelrhodopsin, halorhodopsin, and archerhodopsin.

[0039] In some embodiments, at least one engineered neuron in the plurality of auditory neurons comprises an optical sensor and / or the optical actuator is an optogenetic neuron.

[0040] In some embodiments, the biocompatible construct is a tubular construct. In some embodiments, the biocompatible construct is helical or wye shaped. In some embodiments, the biocompatible construct has a curvature of about 240-900 degrees. In some embodiments, the curvature of the biocompatible construct is about 270 degrees.

[0041] In some embodiments, the system, including, for example, at least one living electrode, has an outer diameter of about 0.1 to 40 mm. In some embodiments, the system is about 0.1-35 mm, about 0.1-30 mm, about 0.1-25 mm, about 0.1-20 mm, about 0.1-15 mm, about 0.1-12 mm, about 0.1-11 mm, about 0.1-10 mm, about 0.1-9 mm, about 0.1-8 mm, about 0.1-7 mm, about 0.1-6.5 mm, about 0.1-6 mm, about 0.1-5 mm, about 0.1-4 mm, about 0.1-3 mm, about 0.1-2 mm, about 0.1-1 mm, about 0.1-0.5 mm, about 0.2-40 mm, about 0.3-40 mm, about 0.4-40 mm, about 0.5-40 mm, about 1-40 mm, about 2-40 mm, about 3-40 mm, about 4-40 mm, about 5-40 mm, about 6-40 mm, about 7-40 mm, about 8-40 mm, about 9-40 mm, about 10-40 mm, about 11-40 mm, about 12-40 mm, about 13-40 mm, about 14-40 mm, about 15-40 mm, about 16-40 mm, about 17-40 mm, about 18-40 mm, about 19-50 mm, about 21-40 mm, about 22-40 mm, about 23-40 mm, about 24-40 mm, about 25-40 mm, about 26-40 mm, about 27-40 mm, about 28-40 mm, about 29-40 mm, about 31-40 mm, about 32-40 mm, about 33-40 mm, about 34-40 mm, about In some embodiments, the system has an outer diameter of about 0.1 mm, about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 6.5 mm, about 7 mm, about 8 mm, about 9 mm, about 40 mm, about 10 mm, about 40 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, or about 40 mm. In some embodiments, the system has an outer diameter of about 1.5 mm.

[0042] In some embodiments, the system, including at least one living electrode, has a diameter of about 0.5-70 mm, about 0.5-60 mm, about 0.5-50 mm, about 0.4-50 mm, about 0.5-30 mm, about 0.5-20 mm, about 0.5-10 mm, about 1-70 mm, about 2-70 mm, about 3-70 mm, about 4-70 mm, about 5-70 mm, about 6-70 mm, about 7-70 mm, about 8-70 mm, about 9-70 mm, about 10-70 mm, about 20-70 mm, about 30-70 mm, about 40-70 mm, about 50-70 mm. In some embodiments, the system has a diameter of about 0.1-10 mm, about 0.2-9 mm, about 0.3-8 mm, about 0.4-7 mm, about 0.5-6.5 mm. In some embodiments, the system has a diameter of about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 6.5 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm. In some embodiments, the system has a diameter of about 7 mm.

[0043] In some embodiments, a system including, e.g., at least one living electrode, has a pitch of about 0.1-10 mm, about 0.1-8 mm, about 0.1-6 mm, about 0.1-4 mm, about 0.1-3 mm, about 0.1-2 mm, about 0.1-1.5 mm, about 0.1-1 mm, about 0.5-10 mm, about 0.6-10 mm, about 0.7-10 mm, about 0.8-10 mm, about 0.9-10 mm, about 1-10 mm, about 2-10 mm, about 4-10 mm, about 6-10 mm, or about 8-10 mm. In some embodiments, the system has a pitch of about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm. In some embodiments, the system has a pitch of about 1 mm.

[0044] In some embodiments, the system, including, for example, at least one living electrode, is manufactured, e.g., assembled, ex vivo or in vitro. In some embodiments, the system is manufactured, e.g., assembled, ex vivo. In some embodiments, the system is manufactured, e.g., assembled, in vitro.

[0045] In some embodiments, the system, including, for example, at least one living electrode, can modulate and / or assess, or can be used to modulate and / or assess, at least one activity of at least one neuron in said plurality of neurons in the system and / or at least one neuron in a subject. In some embodiments, the system can modulate or can be used to modulate, at least one activity of at least one neuron in said plurality of neurons in the system. In some embodiments, the system can modulate or can be used to modulate, at least one activity of at least one neuron in a subject. In some embodiments, the system can modulate or can be used to modulate, at least one activity of at least one neuron in said plurality of neurons in the system and at least one neuron in a subject.

[0046] In some embodiments, the modulating step is or includes unidirectional or bidirectional modulation. In some embodiments, the modulating step is or includes unidirectional modulation. In some embodiments, the modulating step is or includes bidirectional modulation.

[0047] In some embodiments, the modulating step comprises stimulating or inhibiting at least one activity of at least one neuron in said plurality of auditory neurons in the system and / or at least one neuron in the subject. In some embodiments, the modulating step comprises stimulating or inhibiting at least one activity of at least one neuron in said plurality of auditory neurons in the system. In some embodiments, the modulating step comprises stimulating or inhibiting at least one activity of at least one neuron in the subject. In some embodiments, the modulating step comprises stimulating or inhibiting at least one activity of at least one neuron in said plurality of auditory neurons in the system and at least one neuron in the subject.

[0048] In some embodiments, the stimulating step comprises electrical stimulation; light stimulation; sound or vibration stimulation; magnetic stimulation; or a combination thereof. In some embodiments, the stimulating step comprises electrical stimulation. In some embodiments, the stimulating step comprises light stimulation. In some embodiments, the stimulating step comprises sound or vibration stimulation. In some embodiments, the stimulating step comprises magnetic stimulation. In some embodiments, the stimulating step comprises electrical stimulation and light stimulation.

[0049] In some embodiments, the assessing step comprises monitoring and / or recording. In some embodiments, the assessing step comprises monitoring. In some embodiments, the assessing step comprises recording. In some embodiments, the assessing step comprises monitoring and recording.

[0050] In some embodiments, at least one neuron in the subject is or comprises an auditory neuron. In some embodiments, the auditory neuron comprises a vestibular neuron, a cochlear neuron, a spiral ganglion neuron, an IC neuron, or a combination thereof.

[0051] In some embodiments, the system, including, for example, at least one living electrode, can stimulate or inhibit at least one activity of at least one auditory pathway in a subject. In some embodiments, the system can stimulate at least one activity of at least one auditory pathway in a subject. In some embodiments, the system can inhibit at least one activity of at least one auditory pathway in a subject.

[0052] In some embodiments, the system, including, for example, at least one living electrode, can stimulate or inhibit at least one activity of at least one peripheral auditory pathway in a subject. In some embodiments, the system can stimulate at least one activity of at least one peripheral auditory pathway in a subject. In some embodiments, the system can inhibit at least one activity of at least one peripheral auditory pathway in a subject.

[0053] In some embodiments, a system disclosed herein, including, for example, at least one live electrode, is implanted at, near, or in the cochlea of ​​a subject; at, near, or in the inferior colliculus (IC) of a subject; at, near, or in the cochlear nucleus of a subject; and / or at, near, or in the auditory cortex of a subject. In some embodiments, the system is implanted at, near, or in the cochlea of ​​a subject. In some embodiments, the system is implanted in the cochlea of ​​a subject. In some embodiments, the system is implanted near the cochlea of ​​a subject. In some embodiments, the system is implanted in the cochlea of ​​a subject. In some embodiments, the system is implanted at, near, or in the inferior colliculus (IC) of a subject. In some embodiments, the system is implanted in the inferior colliculus (IC) of a subject. In some embodiments, the system is implanted near the inferior colliculus (IC) of a subject. In some embodiments, the system is implanted in the inferior colliculus (IC) of a subject. In some embodiments, the system is implanted at, near, or in the cochlear nucleus in a subject. In some embodiments, the system is implanted in the cochlear nucleus in a subject. In some embodiments, the system is implanted near the cochlear nucleus in a subject. In some embodiments, the system is implanted within the cochlear nucleus in a subject. In some embodiments, the system is implanted at, near, or in the auditory cortex in a subject. In some embodiments, the system is implanted at the auditory cortex in a subject. In some embodiments, the system is implanted near the auditory cortex in a subject. In some embodiments, the system is implanted within the auditory cortex in a subject.

[0054] In some embodiments, a plurality of systems disclosed herein, including, for example, at least one live electrode, are implanted in, near, or in the cochlea in a subject; in, near, or in the inferior colliculus (IC) in a subject; in, near, or in the cochlear nucleus in a subject; and / or in, near, or in the auditory cortex in a subject. In some embodiments, a first system disclosed herein and a second system disclosed herein are implanted in a subject. In some embodiments, the first system disclosed herein and the second system disclosed herein are the same, for example, two similar systems disclosed herein are implanted in a subject. In some embodiments, the first system disclosed herein and the second system disclosed herein are different, for example, two different systems disclosed herein are implanted in a subject. In some embodiments, a third or further (for example, a fourth, fifth, sixth, seventh, eighth, ninth, tenth, etc.) system disclosed herein is implanted in a subject.

[0055] In some embodiments, a first system is implanted at, near, or in the cochlea in a subject. In some embodiments, a first system is implanted in a cochlea in a subject. In some embodiments, a system is implanted near the cochlea in a subject. In some embodiments, a first system is implanted in a cochlea in a subject. In some embodiments, a second system is implanted at, near, or in the inferior colliculus (IC) in a subject. In some embodiments, a second system is implanted in the inferior colliculus (IC) in a subject. In some embodiments, a second system is implanted near the inferior colliculus (IC) in a subject. In some embodiments, a system is implanted in the inferior colliculus (IC) in a subject.

[0056] In some embodiments, the second system is implanted at, near, or in the cochlear nucleus in the subject. In some embodiments, the second system is implanted in the cochlear nucleus in the subject. In some embodiments, the second system is implanted near the cochlear nucleus in the subject. In some embodiments, the system is implanted in the cochlear nucleus in the subject. In some embodiments, the system is implanted in the cochlear nucleus in the subject. In some embodiments, the second system is implanted at, near, or in the auditory cortex in the subject. In some embodiments, the second system is implanted in the auditory cortex in the subject. In some embodiments, the second system is implanted near the auditory cortex in the subject. In some embodiments, the system is implanted in the auditory cortex in the subject.

[0057] In some embodiments, the implanted system or implanted systems disclosed herein contact at least one cell in a subject, or contact at least one cell in, near, or within the area of ​​the auditory cortex in a subject. In some embodiments, the at least one cell in a subject is an endogenous cell. In some embodiments, the at least one cell comprises a neuron. In some embodiments, the neuron comprises an auditory neuron, for example, as described herein. In some embodiments, the auditory neuron comprises a neuron having one or more characteristics of a neuron present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei, or combinations thereof, of an organism (e.g., a human). In some embodiments, the auditory neuron comprises one or more of a spiral ganglion neuron or an IC neuron.

[0058] In some embodiments, the step of contacting the system or systems disclosed herein with at least one cell in a subject is determined to create a synapse between at least one neuron of the plurality of auditory neurons of the system and at least one cell in the subject.In some embodiments, the synapse is or includes a neuronal synapse.

[0059] In some embodiments, the implanted system or the plurality of implanted systems disclosed herein provides an accessible interface for modulating at least one neuron in the plurality of auditory neurons in the system and / or at least one cell in a subject. In some embodiments, the implanted system or the plurality of implanted systems disclosed herein provides an accessible interface for modulating at least one neuron in the plurality of auditory neurons in the system. In some embodiments, the implanted system or the plurality of implanted systems disclosed herein provides an accessible interface for modulating at least one cell in a subject.

[0060] In some embodiments, the biocompatible construct has a non-linear shape.

[0061] In some embodiments, the systems disclosed herein further comprise a stimulator array. In some embodiments, the stimulator array is or comprises an optrode array.

[0062] Provided herein is a system, such as a cochlear implant, comprising: with stimulator array; a plurality of living electrodes, at least one of the plurality of living electrodes being in optical and / or electrical communication with an element of the stimulator array, each of the plurality of living electrodes including: (1) a proximal end including a stimulator-complementary neuron; and (2) a distal end including an auditory neuron in neural communication with an optogenetic neuron in the living electrode; Including, The plurality of living electrodes have staggered distal ends.

[0063] In some embodiments, the stimulator array is or comprises an optrode array.

[0064] In some embodiments, the stimulator complementary neuron is or comprises an optogenetic neuron.

[0065] Also provided herein is a method of manufacturing a system including, for example, at least one living electrode, the method including: (a) providing a biocompatible construct including a matrix having a first end, a second end, and a body; and (b) associating the biocompatible construct with a plurality of auditory neurons (e.g., vestibular neurons and / or cochlear neurons).

[0066] In some embodiments, the methods described herein include maintaining the system under conditions that promote the growth of at least one neuron in the plurality of auditory neurons. In some embodiments, the methods described herein include maintaining the system under conditions that maintain the viability of at least one neuron in the plurality of auditory neurons.

[0067] In some embodiments, the methods described herein further include forming an aggregate of at least a portion of the plurality of auditory neurons. In some embodiments, the aggregate of at least a portion of the plurality of auditory neurons is formed prior to contacting the plurality of auditory neurons with a biocompatible construct.

[0068] The present disclosure provides a method for implanting in a subject a system including, e.g., at least one live electrode, comprising: (a) a biocompatible construct including a matrix having a first end, a second end, and a body; and (b) a plurality of auditory neurons (e.g., vestibular neurons and / or cochlear neurons), the method comprising implanting the system at, near, or within the cochlea, inferior colliculus (IC), cochlear nucleus, or auditory cortex in the subject.

[0069] In some embodiments, the implantation is determined to be less likely to cause inflammation as compared to implantation of conventional microelectrodes.

[0070] In some embodiments of any of the methods disclosed herein, the subject is a human.

[0071] Provided herein is a method of modulating and / or assessing at least one activity of at least one neuron in a subject, the method comprising: (i) providing to the subject a system comprising: (a) a biocompatible construct comprising a matrix having a first end, a second end, and a body; and (b) at least one living electrode, the system comprising a plurality of auditory neurons (e.g., vestibular neurons and / or cochlear neurons); and (ii) modulating the at least one neuron with electrical stimulation, light stimulation, or both.

[0072] In some embodiments, the assessing step comprises monitoring, recording, stimulating, and / or inhibiting at least one neuron in the subject. In some embodiments, the assessing step comprises monitoring at least one neuron in the subject. In some embodiments, the assessing step comprises recording at least one neuron in the subject. In some embodiments, the assessing step comprises stimulating at least one neuron in the subject. In some embodiments, the assessing step comprises inhibiting at least one neuron in the subject.

[0073] In some embodiments, the at least one neuron in the subject comprises an auditory neuron.

[0074] In some embodiments, the auditory neurons include neurons having one or more characteristics of neurons present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, cortical brainstem nuclei, or combinations thereof. In some embodiments, the auditory neurons include one or more of vestibular neurons, cochlear neurons, spiral ganglion neurons, or IC neurons.

[0075] In some embodiments of any of the methods disclosed herein, the subject is a human.

[0076] The present disclosure further provides a method of treating a subject having a hearing impairment, the method comprising: (a) a biocompatible construct comprising a matrix having a first end, a second end, and a body; and (b) providing to the subject a system comprising a plurality of auditory neurons, such as at least one living electrode, thereby treating the subject.

[0077] In some embodiments, the hearing impairment comprises one or more of conductive hearing loss, sensorineural hearing loss, or mixed hearing loss. In some embodiments, the hearing impairment comprises one or more of acquired hearing loss, sudden hearing loss, secondary hearing loss (medication-, noise-, or trauma-induced hearing loss), autoimmune inner ear disease, otosclerosis, Meniere's disease, presbycusis, or acoustic neuroma.

[0078] In some embodiments of any of the methods disclosed herein, the subject is a human.

[0079] Provided herein is a method of ameliorating symptoms of hearing impairment in a subject, the method comprising providing to the subject a system comprising: (a) a biocompatible construct comprising a matrix having a first end, a second end, and a body; and (b) at least one living electrode comprising a plurality of auditory neurons (e.g., vestibular neurons and / or cochlear neurons, e.g., neurons as described herein).

[0080] In some embodiments, the hearing impairment comprises one or more of conductive hearing loss, sensorineural hearing loss, or mixed hearing loss. In some embodiments, the hearing impairment comprises one or more of acquired hearing loss, sudden hearing loss, secondary hearing loss (drug-induced or trauma-induced hearing loss), autoimmune inner ear disease, otosclerosis, Meniere's disease, presbycusis, or acoustic neuroma.

[0081] In some embodiments of any of the methods disclosed herein, the subject is a human.

[0082] Also provided herein is a method of preventing worsening of symptoms of hearing impairment in a subject, the method comprising providing to the subject a system comprising: (a) a biocompatible construct comprising a matrix having a first end, a second end, and a body; and (b) at least one living electrode comprising a plurality of auditory neurons (e.g., vestibular neurons and / or cochlear neurons, e.g., neurons as described herein).

[0083] In some embodiments, the hearing impairment comprises one or more of conductive hearing loss, sensorineural hearing loss, or mixed hearing loss. In some embodiments, the hearing impairment comprises one or more of acquired hearing loss, sudden hearing loss, secondary hearing loss (drug-induced or trauma-induced hearing loss), autoimmune inner ear disease, otosclerosis, Meniere's disease, presbycusis, or acoustic neuroma.

[0084] In some embodiments of any of the methods disclosed herein, the subject is a human.

[0085] The present disclosure provides a kit including a system including (a) a biocompatible construct including a first end, a second end, and a body, and (b) a plurality of auditory neurons and instructions for using the same.

[0086] In some embodiments, the kit further comprises instructions for its use.

[0087] In some embodiments, a kit described herein comprises a system described herein.

[0088] In some embodiments, any of the methods or kits disclosed herein include a system comprising, e.g., at least one living electrode, as disclosed herein. In some embodiments, the system disclosed herein comprises: (a) a biocompatible construct comprising a matrix comprising a first end, a second end, and a body; and (b) a plurality of auditory neurons.

[0089] In some embodiments of any of the methods or kits disclosed herein, the biocompatible construct comprises an interior surface and / or an exterior surface. In some embodiments, the interior surface of the biocompatible construct defines a luminal core.

[0090] In some embodiments of any of the methods or kits disclosed herein, the exterior surface of the biocompatible construct comprises at least one hydrogel. In some embodiments, the hydrogel comprises a hydrophilic biopolymer and / or a synthetic polymer. In some embodiments, the hydrogel is at least partially crosslinked, where crosslinking optionally increases stiffness, reduces porosity, and / or increases degradation time. In some embodiments, the hydrophilic biopolymer comprises one or more of agarose, hydrogel, hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, fibrin, and methylcellulose. In some embodiments, the hydrophilic biopolymer comprises agarose. In some embodiments, the hydrophilic biopolymer comprises a hydrogel. In some embodiments, the hydrophilic biopolymer comprises chitosan. In some embodiments, the hydrophilic biopolymer comprises alginate. In some embodiments, the hydrophilic biopolymer comprises collagen. In some embodiments, the hydrophilic biopolymer comprises dextran. In some embodiments, the hydrophilic biopolymer comprises pectin. In some embodiments, the hydrophilic biopolymer comprises carrageenan. In some embodiments, the hydrophilic biopolymer comprises polylysine. In some embodiments, the hydrophilic biopolymer comprises gelatin. In some embodiments, the hydrophilic biopolymer comprises hyaluronic acid. In some embodiments, the hydrophilic biopolymer comprises fibrin. In some embodiments, the hydrophilic biopolymer comprises methylcellulose.

[0091] In some embodiments of any of the methods or kits disclosed herein, the hydrophilic biopolymer comprises agarose. In some embodiments, the agarose is about 0.25-30%, about 0.25%-3%, about 0.5%-3%, about 1-20%, about 1.5-10%, about 2-9%, about 2.5-8%, or about 3-7%. In some embodiments, the agarose is about 0.25-29%, about 0.25-28%, 0.25%, about 0.25-27%, about 0.25-26%, about 0.25-25%, about 0.25-24%, about 0.25-23%, about 0.25-22%, about 0.25-21%, about 0.25-20%, about 0.25-19%, about 0. 25~18%, about 0.25~17%, about 0.25~16%, about 0.25~15%, about 0.25~14%, about 0.25~13%, about 0.25~12%, about 0.25~11%, about 0.25~10%, about 0.25~9%, about 0.25~8%, about 0.25~7%, about 0.25~6%, about 0.25~5%, about 0.25~ 4%, approx. 0.25-3%, approx. 0.25-2%, approx. 0.25-1%, approx. 0.25-0.5%, approx. 0.5-30%, approx. 1-30%, approx. 2-30%, approx. 3-30%, approx. 4-30%, approx. 5-30%, approx. 6-30%, approx. 7-30%, approx. 8-30%, approx. 9-30%, approx. 10-30%, approx. 11-30%, approx. 12-30%, approx. 13-30%, approximately 14-30%, approximately 15-30%, approximately 16-30%, approximately 17-30%, approximately 18-30%, approximately 19-30%, approximately 20-30%, approximately 21-30%, approximately 22-30%, approximately 23-30%, approximately 24-30%, approximately 25-30%, approximately 26-30%, approximately 27-30%, approximately 28-30%, approximately 29-30%.

[0092] In some embodiments of any of the methods or kits disclosed herein, the agarose in the systems disclosed herein is about 0.25%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%. In some embodiments, the agarose is about 3%.

[0093] In some embodiments of any of the methods or kits disclosed herein, the hydrophilic biopolymer comprises at least one synthetic hydrogel (e.g., a pluronic hydrogel) or at least one synthetic hydrogel made of an amphiphilic copolymer consisting of ethylene oxide (PEO) and polypropylene oxide (PPO) units. In some embodiments, the hydrogel is about 2-50% w / v, about 3-40% w / v, about 4-30% w / v, about 5-25% w / v, or about 10-20% w / v. In some embodiments, the hydrogel is about 2-45%, about 2-40%, about 2-35%, about 2-30%, about 2-29%, about 2-28%, about 2-27%, about 2-26%, about 2-25%, about 2-24%, about 2-23%, about 2-22%, about 2-21%, about 2-20%, about 2-19%, about 2-18%, about 2-17%, about 2-16%, about 2-15%, about 2-14%, about 2-13%, about 2-12%, about 2-11%, about 2-10%, about 2-9%, about 2-8%, about 2-7%, about 2-6%, about 2-5%, about 2-4%, about 2-3%, about 3-50%. , about 4-50%, about 5-50%, about 6-50%, about 7-50%, about 8-50%, about 9-50%, about 10-50%, about 11-50%, about 12-50%, about 13-50%, about 14-50%, about 15-50%, about 16-50%, about 17-50%, about 18-50%, about 19-50%, about 20-50%, about 21-50%, about 22-50%, about 23-50%, about 24-50%, about 25-50%, about 26-50%, about 27-50%, about 28-50%, about 29-50%, about 30-50%, about 35-50%, about 40-50%, and about 45-50% w / v. In some embodiments, the hydrogel is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 40%, about 45%, or about 50% w / v.In some embodiments, the hydrogel is about 28.1%, about 28.2%, about 28.3%, about 28.4%, about 28.5%, about 28.6%, about 28.7%, about 28.8%, about 28.9%, about 30%. In some embodiments, the hydrogel is about 28.6% w / v.

[0094] In some embodiments of any of the methods or kits disclosed herein, the inner surface of the biocompatible construct comprises one or more extracellular matrix (ECM) components. In some embodiments, the ECM components comprise collagen, laminin, fibronectin, hyaluronic acid, or a combination thereof. In some embodiments, the ECM components comprise collagen. In some embodiments, the ECM components comprise laminin. In some embodiments, the ECM components comprise fibronectin. In some embodiments, the ECM components comprise hyaluronic acid.

[0095] In some embodiments, the ECM component comprises collagen, hi some embodiments, the collagen is at a concentration of about 0.1-10 mg / ml. In some embodiments, collagen is about 0.1-9 mg / ml, about 0.1-8 mg / ml, about 0.1-7 mg / ml, about 0.1-6 mg / ml, about 0.1-5 mg / ml, about 0.1-4 mg / ml, about 0.1-3 mg / ml, about 0.1-2 mg / ml, about 0.1-1 mg / ml, about 0.1-0.9 mg / ml, about 0.1-0.8 mg / ml, about 0.1-0.7 mg / ml, about 0.1-0.5 mg / ml, about 0.5-10 mg / ml, about 0.6-10 mg / ml, about 0.7-10 mg / ml, about 0.8-10 mg / ml, about 0.9-10 mg / ml, about 1-10 mg / ml, about 2-10 mg / ml, about 3-10 mg / ml, about 4-10 mg / ml, about 5-10 mg / ml, about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, about 9-10 mg / ml, about 10-10 mg / ml, about 20-10 mg / ml, about 30-10 mg / ml, about 40-10 mg / ml, about 50-10 mg / ml, about 60-10 mg / ml, about 70-10 mg / ml, about 80-10 mg / ml, about 90-10 mg / ml, about 10 ... The concentrations are about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, and about 9-10 mg / ml. In some embodiments, the collagen is at a concentration of about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml. In some embodiments, the collagen is at a concentration of about 1 mg / ml.

[0096] In some embodiments, the ECM component comprises laminin. In some embodiments, the laminin is about 0.1-9 mg / ml, about 0.1-8 mg / ml, about 0.1-7 mg / ml, about 0.1-6 mg / ml, about 0.1-5 mg / ml, about 0.1-4 mg / ml, about 0.1-3 mg / ml, about 0.1-2 mg / ml, about 0.1-1 mg / ml, about 0.1-0.9 mg / ml, about 0.1-0.8 mg / ml, about 0.1-0.7 mg / ml, about 0.1-0.5 mg / ml, about 0.5-10 mg / ml, about 0.6-10 mg / ml, about 0.7-10 mg / ml, about 0.8-10 mg / ml, about 0.9-10 mg / ml, about 1-10 mg / ml, about 2-10 mg / ml, about 3-10 mg / ml, about 4-10 mg / ml, about 5-10 mg / ml, about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, about 9-10 mg / ml, about 10-10 mg / ml, about 20-10 mg / ml, about 30-10 mg / ml, about 40-10 mg / ml, about 50-10 mg / ml, about 60-10 mg / ml, about 70-10 mg / ml, about 80-10 mg / ml, about 90-10 mg / ml, about 10 ... The concentrations are about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, and about 9-10 mg / ml. In some embodiments, the laminin is at a concentration of about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml. In some embodiments, the laminin is at a concentration of about 1 mg / ml.

[0097] In some embodiments of any of the methods or kits disclosed herein, a system including, e.g., at least one living electrode, includes a plurality of auditory neurons, e.g., about 50-5000 auditory neurons, as described herein. In some embodiments, the plurality of auditory neurons is about 50-4500, about 50-4000, about 50-3500, about 50-3000, about 50-2500, about 50-2000, about 50-1500, about 50-1000, about 50-900, about 50-800, about 50-700, about 50-600, about 50-550, about 50-500, about 50-600, about 50-700, about 50-800, about 50-900, about 50-900, about 50-1000, about 50-15 ... about 0-450, about 50-400, about 50-300, about 50-200, about 50-100, about 100-5000, about 200-5000, about 300-5000, about 400-5000, about 500-5000, about 1000-5000, about 2000-5000, about 3000-5000, or about 4000-5000 auditory neurons. In some embodiments, the plurality of auditory neurons comprises at least 50 auditory neurons, at least 100 auditory neurons, at least 200 auditory neurons, at least 300 auditory neurons, at least 400 auditory neurons, at least 450 auditory neurons, at least 500 auditory neurons, at least 550 auditory neurons, at least 600 auditory neurons, at least 700 auditory neurons, at least 800 auditory neurons, at least 900 auditory neurons, at least 1000 auditory neurons, at least 2000 auditory neurons, at least 3000 auditory neurons, at least 4000, or at least 5000 auditory neurons. In some embodiments, the plurality of auditory neurons comprises at least 500 auditory neurons.

[0098] In some embodiments of any of the methods or kits disclosed herein, at least one of the plurality of auditory neurons is in contact with a biocompatible construct.

[0099] In some embodiments of any of the methods or kits disclosed herein, at least one neuron in the plurality of neurons comprises a cell body and / or an axon. In some embodiments, at least one neuron in the plurality of neurons comprises a cell body. In some embodiments, at least one neuron in the plurality of neurons comprises an axon. In some embodiments, at least one neuron in the plurality of neurons comprises a cell body and an axon. In some embodiments, the axon is about 0.5-50 cm in length.

[0100] In some embodiments of any of the methods or kits disclosed herein, a portion of at least one neuron in the plurality of auditory neurons is positioned (i) at a first end of a biocompatible construct; (ii) at a second end of a biocompatible construct; (iii) at, along, or within a portion of the body of the biocompatible construct; and / or (iv) throughout, along, or within the body of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at a first end of a biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at a second end of a biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at, along, or within a portion of the body of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned throughout, along, or within the body of the biocompatible construct.

[0101] In some embodiments of any of the methods or kits disclosed herein, a portion of at least one neuron in the plurality of auditory neurons positioned at the first end or the second end of the biocompatible construct comprises a cell body.

[0102] In some embodiments of any of the methods or kits disclosed herein, a portion of at least one neuron in the plurality of auditory neurons positioned along or within a portion of a body of a biocompatible construct comprises an axon. In some embodiments, the body of a biocompatible construct comprises an interior surface of the body. In some embodiments, the interior surface comprises a luminal core.

[0103] In some embodiments of any of the methods or kits disclosed herein, a portion of at least one neuron in the plurality of auditory neurons positioned throughout, along, or within the body of the biocompatible construct comprises an axon. In some embodiments, the axon extends unidirectionally or bidirectionally. In some embodiments, the axon extends unidirectionally. In some embodiments, the axon extends bidirectionally.

[0104] In some embodiments of any of the methods or kits disclosed herein, at least a portion of the plurality of auditory neurons comprises an aggregate of auditory neurons.

[0105] In some embodiments of any of the methods or kits disclosed herein, the plurality of auditory neurons comprises neurons having one or more characteristics of neurons present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of an organism, such as a human. In some embodiments, the plurality of auditory neurons comprises neurons derived from neurons present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of an organism, such as a human. In some embodiments, the plurality of auditory neurons comprises spiral ganglion neurons, inferior colliculus (IC) auditory neurons, cortical auditory neurons, type I auditory neurons, or type II auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of spiral ganglion neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of IC auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of cortical auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of type I auditory neurons. In some embodiments, the plurality of auditory neurons is or comprises a plurality of type II auditory neurons.

[0106] In some embodiments of any of the methods or kits disclosed herein, the system including, e.g., at least one living electrode, further comprises one or more additional cells, such as astrocytes, oligodendrocytes, Schwann cells, microglia, or endothelial cells.

[0107] In some embodiments of any of the methods or kits disclosed herein, the plurality of auditory neurons is isolated from a subject.

[0108] In some embodiments of any of the methods or kits disclosed herein, the plurality of auditory neurons is or comprises at least one cell derived from a stem cell. In some embodiments, the stem cell comprises one or more of induced pluripotent stem cells (iPSCs), embryonic stem cells, or tissue stem cells. In some embodiments, the stem cell comprises an iPSC. In some embodiments, the stem cell comprises an embryonic stem cell. In some embodiments, the stem cell comprises a tissue stem cell. In some embodiments, the auditory neuron derived from a stem cell has one or more characteristics of a neuron present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of an organism, such as a human.

[0109] In some embodiments of any of the methods or kits disclosed herein, the plurality of auditory neurons is autologous, allogeneic, or xenogeneic to the subject. In some embodiments, the plurality of auditory neurons is autologous to the subject. In some embodiments, the plurality of auditory neurons is allogeneic to the subject. In some embodiments, the plurality of auditory neurons is xenogeneic to the subject.

[0110] In some embodiments of any of the methods or kits disclosed herein, at least one neuron in the plurality of auditory neurons is genetically engineered or reprogrammed using non-genetic techniques. In some embodiments, at least one genetically engineered neuron comprises a sensor, an actuator, a receptor, and / or a reporter.

[0111] In some embodiments of any of the methods or kits disclosed herein, at least one engineered neuron in the plurality of auditory neurons comprises a reporter. In some embodiments, the reporter comprises a fluorescent reporter.

[0112] In some embodiments, at least one engineered neuron in the plurality of auditory neurons comprises a receptor, hi some embodiments, the receptor comprises one or more of a magnetoreceptor, an electromagnetic receptor, an electroreceptor, a hydroreceptor, a mechanoreceptor, an osmoreceptor, a thermoreceptor, and a piezoelectric ion channel.

[0113] In some embodiments, the electromagnetic receptor comprises one or more of an infrared receptor, a photoreceptor, or an ultraviolet receptor.

[0114] In some embodiments, the mechanoreceptors include one or both of mechanosensory receptors or proprioceptors.

[0115] In some embodiments, at least one engineered neuron in the plurality of auditory neurons comprises a sensor, hi some embodiments, the sensor comprises an optical sensor comprising one or more of a light-gated ion channel, a calcium sensor, and a membrane voltage sensor.

[0116] In some embodiments, the sensor comprises a calcium sensor comprising one or more of Aequorin, Cameleon, and GCaMP.

[0117] In some embodiments, the sensor comprises a chloride sensor comprising Clomeleon.

[0118] In some embodiments, the sensor comprises a membrane voltage sensor comprising Mermaid.

[0119] In some embodiments, at least one engineered neuron in the plurality of auditory neurons comprises an actuator, hi some embodiments, the actuator comprises an optical actuator comprising one or more of channelrhodopsin, halorhodopsin, and archerhodopsin.

[0120] In some embodiments, at least one engineered neuron in said plurality of auditory neurons comprises an optical sensor and / or the optical actuator is an optogenetic neuron.

[0121] In some embodiments of any of the methods or kits disclosed herein, the biocompatible construct is a tubular construct.

[0122] In some embodiments of any of the methods or kits disclosed herein, the biocompatible construct is helical or Y-shaped. In some embodiments, the biocompatible construct has a curvature of about 240-900 degrees. In some embodiments, the curvature of the biocompatible construct is about 270 degrees.

[0123] In some embodiments, the system including the biocompatible construct has an outer diameter of about 0.1 to 40 mm. In some embodiments, the system is about 0.1-35 mm, about 0.1-30 mm, about 0.1-25 mm, about 0.1-20 mm, about 0.1-15 mm, about 0.1-12 mm, about 0.1-11 mm, about 0.1-10 mm, about 0.1-9 mm, about 0.1-8 mm, about 0.1-7 mm, about 0.1-6.5 mm, about 0.1-6 mm, about 0.1-5 mm, about 0.1-4 mm, about 0.1-3 mm, about 0.1-2 mm, about 0.1-1 mm, about 0.1-0.5 mm, about 0.2-40 mm, about 0.3-40 mm, about 0.4-40 mm, about 0.5-40 mm, about 1-40 mm, about 2-40 mm, about 3-40 mm, about 4-40 mm, about 5-40 mm, about 6-40 mm, about 7-40 mm, about 8-40 mm, about 9-40 mm, about 10-40 mm, about 11-40 mm, about 12-40 mm, about 13-40 mm, about 14-40 mm, about 15-40 mm, about 16-40 mm, about 17-40 mm, about 18-40 mm, about 19-50 mm, about 21-40 mm, about 22-40 mm, about 23-40 mm, about 24-40 mm, about 25-40 mm, about 26-40 mm, about 27-40 mm, about 28-40 mm, about 29-40 mm, about 31-40 mm, about 32-40 mm, about 33-40 mm, about 34-40 mm, about In some embodiments, the system has an outer diameter of about 0.1 mm, about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 6.5 mm, about 7 mm, about 8 mm, about 9 mm, about 40 mm, about 10 mm, about 40 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, or about 40 mm. In some embodiments, the system has an outer diameter of about 1.5 mm.

[0124] In some embodiments of any of the methods or kits disclosed herein, the system including the biocompatible construct has a diameter of about 0.5-70 mm, about 0.5-60 mm, about 0.5-50 mm, about 0.4-50 mm, about 0.5-30 mm, about 0.5-20 mm, about 0.5-10 mm, about 1-70 mm, about 2-70 mm, about 3-70 mm, about 4-70 mm, about 5-70 mm, about 6-70 mm, about 7-70 mm, about 8-70 mm, about 9-70 mm, about 10-70 mm, about 20-70 mm, about 30-70 mm, about 40-70 mm, about 50-70 mm. In some embodiments, the system has a diameter of about 0.1-10 mm, about 0.2-9 mm, about 0.3-8 mm, about 0.4-7 mm, or about 0.5-6.5 mm. In some embodiments, the system has a diameter of about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 6.5 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm. In some embodiments, the system including the biocompatible construct has a diameter of about 7 mm.

[0125] In some embodiments, the system including the biocompatible construct has a pitch of about 0.1-10 mm, about 0.1-8 mm, about 0.1-6 mm, about 0.1-4 mm, about 0.1-3 mm, about 0.1-2 mm, about 0.1-1.5 mm, about 0.1-1 mm, about 0.5-10 mm, about 0.6-10 mm, about 0.7-10 mm, about 0.8-10 mm, about 0.9-10 mm, about 1-10 mm, about 2-10 mm, about 4-10 mm, about 6-10 mm, or about 8-10 mm. In some embodiments, the system has a pitch of about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm. In some embodiments, the system including the biocompatible construct has a pitch of about 1 mm.

[0126] In some embodiments, the system used in any method or kit disclosed herein is produced, e.g., assembled, ex vivo or in vitro. In some embodiments, the system is produced, e.g., assembled, ex vivo. In some embodiments, the system is produced, e.g., assembled, in vitro.

[0127] In some embodiments, the system used in any method or kit disclosed herein can regulate and / or assess, or can be used to regulate and / or assess, at least one activity of at least one neuron in said plurality of neurons in the system and / or at least one activity of at least one neuron in a subject.In some embodiments, the system can regulate or can be used to regulate, at least one activity of at least one neuron in said plurality of neurons in the system.In some embodiments, the system can regulate or can be used to regulate, at least one activity of at least one neuron in a subject.In some embodiments, the system can regulate or can be used to regulate, at least one activity of at least one neuron in said plurality of neurons in the system and at least one neuron in a subject.

[0128] In some embodiments, the modulating step is or includes unidirectional or bidirectional modulation. In some embodiments, the modulating step is or includes unidirectional modulation. In some embodiments, the modulating step is or includes bidirectional modulation.

[0129] In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in said plurality of auditory neurons in the system and / or at least one neuron in the subject. In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in said plurality of auditory neurons in the system. In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in the subject. In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in said plurality of auditory neurons in the system and at least one neuron in the subject.

[0130] In some embodiments, the stimulating step comprises electrical stimulation; light stimulation; sound or vibration stimulation; magnetic stimulation; or a combination thereof. In some embodiments, the stimulating step comprises electrical stimulation. In some embodiments, the stimulating step comprises light stimulation. In some embodiments, the stimulating step comprises sound or vibration stimulation. In some embodiments, the stimulating step comprises magnetic stimulation. In some embodiments, the stimulating step comprises electrical stimulation and light stimulation.

[0131] In some embodiments, the assessing step comprises monitoring and / or recording. In some embodiments, the assessing step comprises monitoring. In some embodiments, the assessing step comprises recording. In some embodiments, the assessing step comprises monitoring and recording.

[0132] In some embodiments, at least one neuron in the subject is or comprises an auditory neuron, hi some embodiments, the auditory neuron comprises a spiral ganglion neuron, an IC neuron, or a combination thereof.

[0133] In some embodiments, the system used in any of the methods or kits disclosed herein can stimulate or inhibit an auditory pathway in a subject. In some embodiments, the system can stimulate an auditory pathway in a subject. In some embodiments, the system can inhibit an auditory pathway in a subject.

[0134] In some embodiments, the system used in any of the methods or kits disclosed herein can stimulate or inhibit the peripheral auditory pathway in a subject. In some embodiments, the system can stimulate the peripheral auditory pathway in a subject. In some embodiments, the system can inhibit the peripheral auditory pathway in a subject.

[0135] In some embodiments, a system including, for example, at least one live electrode, used in any of the methods or kits disclosed herein is implanted at, near, or in the cochlea in a subject; at, near, or in the inferior colliculus (IC) in a subject; at, near, or in the cochlear nucleus in a subject; and / or at, near, or in the auditory cortex in a subject. In some embodiments, the system is implanted at, near, or in the cochlea in a subject. In some embodiments, the system is implanted in the cochlea in a subject. In some embodiments, the system is implanted near the cochlea in a subject. In some embodiments, the system is implanted in the cochlea in a subject. In some embodiments, the system is implanted at, near, or in the inferior colliculus (IC) in a subject. In some embodiments, the system is implanted in the inferior colliculus (IC) in a subject. In some embodiments, the system is implanted near the inferior colliculus (IC) in a subject. In some embodiments, the system is implanted in the inferior colliculus (IC) in a subject. In some embodiments, the system is implanted at, near, or in the cochlear nucleus in a subject. In some embodiments, the system is implanted in the cochlear nucleus in a subject. In some embodiments, the system is implanted near the cochlear nucleus in a subject. In some embodiments, the system is implanted within the cochlear nucleus in a subject. In some embodiments, the system is implanted at, near, or in the auditory cortex in a subject. In some embodiments, the system is implanted at the auditory cortex in a subject. In some embodiments, the system is implanted near the auditory cortex in a subject. In some embodiments, the system is implanted within the auditory cortex in a subject.

[0136] In some embodiments, a plurality of systems disclosed herein, including, for example, at least one live electrode, used in any method or kit disclosed herein, are implanted in, near, or in the cochlea in a subject; in, near, or in the inferior colliculus (IC) in a subject; in, near, or in the cochlear nucleus in a subject; and / or in, near, or in the auditory cortex in a subject. In some embodiments, a first system disclosed herein and a second system disclosed herein are implanted in a subject. In some embodiments, the first system disclosed herein and the second system disclosed herein are the same, for example, two similar systems disclosed herein are implanted in a subject. In some embodiments, the first system disclosed herein and the second system disclosed herein are different, for example, two different systems disclosed herein are implanted in a subject. In some embodiments, a third or additional system disclosed herein is implanted in a subject.

[0137] In some embodiments, a first system is implanted at, near, or in the cochlea in a subject. In some embodiments, a first system is implanted in a cochlea in a subject. In some embodiments, a system is implanted near the cochlea in a subject. In some embodiments, a first system is implanted in a cochlea in a subject. In some embodiments, a second system is implanted at, near, or in the inferior colliculus (IC) in a subject. In some embodiments, a second system is implanted in the inferior colliculus (IC) in a subject. In some embodiments, a second system is implanted near the inferior colliculus (IC) in a subject. In some embodiments, a system is implanted in the inferior colliculus (IC) in a subject.

[0138] In some embodiments, the first system is implanted at, near, or in the cochlea in the subject. In some embodiments, the first system is implanted in the cochlea in the subject. In some embodiments, the system is implanted near the cochlea in the subject. In some embodiments, the first system is implanted in the cochlea in the subject. In some embodiments, the second system is implanted at, near, or in the cochlear nucleus in the subject. In some embodiments, the second system is implanted in the cochlear nucleus in the subject. In some embodiments, the second system is implanted near the cochlear nucleus in the subject. In some embodiments, the system is implanted in the cochlear nucleus in the subject. In some embodiments, the system is implanted in the cochlear nucleus in the subject. In some embodiments, the second system is implanted at, near, or in the auditory cortex in the subject. In some embodiments, the second system is implanted in the auditory cortex in the subject. In some embodiments, the second system is implanted near the auditory cortex in the subject. In some embodiments, the system is implanted in the auditory cortex in the subject.

[0139] In some embodiments, the implanted system used in any of the methods or kits disclosed herein contacts at least one cell in a subject, or contacts at least one cell in, near, or within the area of ​​the auditory cortex in a subject. In some embodiments, the at least one cell in a subject is an endogenous cell. In some embodiments, the at least one cell comprises a neuron. In some embodiments, the neuron comprises an auditory neuron, for example, as described herein. In some embodiments, the auditory neuron comprises one or more of a spiral ganglion neuron or an IC neuron.

[0140] In some embodiments, the step of contacting the system used in any method or kit disclosed herein with at least one cell in a subject is determined to create a synapse between at least one neuron of the plurality of auditory neurons of the system and at least one cell in the subject.In some embodiments, the synapse is or comprises the synapse of a neuron.

[0141] In some embodiments, the implanted system used in any of the methods or kits disclosed herein provides an accessible interface for regulating at least one neuron in the plurality of auditory neurons in the system and / or at least one cell in a subject.In some embodiments, the implanted system provides an accessible interface for regulating at least one neuron in the plurality of auditory neurons in the system.In some embodiments, the implanted system provides an accessible interface for regulating at least one cell in a subject.

[0142] In some embodiments of any of the methods or kits disclosed herein, the biocompatible construct has a non-linear shape.

[0143] These and other aspects encompassed by the present disclosure are described more fully below and in the appended claims. [Brief description of the drawings]

[0144] The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, in which: For the purpose of illustrating the invention, non-limiting embodiments are shown in the drawings, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0145] [Figure 1]We provide a schematic of a micro-TENN biohybrid neural interface: the hydrogel scaffold guides axonal growth from a population of neurons toward a target tissue of interest (in this case, spiral ganglion cells in the cochlea or native auditory neurons in the inferior colliculus) while the cell bodies remain accessible for electrical or optical stimulation. [Figure 2A] Figure 2 shows simultaneous optical stimulation and recording in micro-TENNs. Cortical neuronal micro-TENNs transduced to express both an optical actuator and a fluorescent calcium reporter can be controlled and monitored with light. Figure 2A: Phase images and confocal reconstructions at 10 DIV. The aggregate on the left is transduced with ChR2, while the aggregate on the right is transduced with the calcium reporter RCaMP. Figure 2B: RCaMP+ aggregates from Figure 2A under fluorescence microscopy during recording. Figure 2C: Confocal reconstruction of Figure 2B after stimulation. ROIs containing single neurons were manually defined (white outline). Figure 2D: Normalized pixel intensity of ROIs within RCaMP+ aggregates from Figures 2A-2C during stimulation. Grey lines indicate representative user-defined ROIs that were averaged to obtain the mean ROI of the aggregates (solid black line). Time stamps of a single series of 1 Hz, 100 ms stimulation pulses are shown as blue bands along the abscissa. Changes in pixel intensity due to stimulation of the input aggregates are seen as sharp spikes within the endogenous high amplitude slow wave activity. Figure 2E: Zoomed in red inset showing micro-TENN activity during (left) and after light stimulation (right). Figure 2F: Average maximum ΔF / Fo across stimulation intensities. Statistical comparison revealed that stimulation at the control wavelength (620 nm) resulted in a significantly lower maximum ΔF / Fo than stimulation at 465 nm (*=p<0.05). Scale bar: 100 μm. These results validate optically evoked responses in constructs similar to those featured in the current proposal. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E]See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Diagram 3] Anatomically-inspired micro-TENNs are shown. Confocal reconstructions of dopaminergic micro-TENNs emulating the nigrostriatal pathway in vitro: distinct populations of neurons with long projection axon tracts. [Figure 4] Combined immunohistochemistry micrograph of SGN explants at 7 DIV. Antibodies against Hoechst (nuclei), GFAP (astrocytes), tuj-1 (axons / neuronal cell bodies), and synapsin (synapses) are shown. Scale bar = 100 μm. [Figure 5-1] Figure 5 shows calcium imaging. Figure 5A: Phase images of spiral ganglion aggregates (1, 2) plated at 18 DIV. Figure 5B: Aggregates were transduced with GCaMP and live calcium imaging recordings revealed neuronal activity (B, B', B''). (Figure 5C, 5D) For both aggregates, ROIs containing single neurons were identified and average pixel intensity was analyzed in MATLAB. [Figure 5-2] See description of Figure 5-1. [Figure 6] SGN LE with aggregated SGNs in implantable micro-columns. Figure 6A: Phase image of spiral ganglion LE projecting thick axonal tracts at 14 DIV. Figure 6B: Immunolabeled LE from Figure 6A staining for nuclei (DAPI) (Figure 6C), axons (beta-tubulin-III) (Figure 6D), and peripherin (Figure 6E). Scale bar: 500 μm. [Figure 7]The cochlear construct design is shown. Figure 7A: Phase contrast micrograph of a live electrode showing a bend with the central microcolumn off-center. The curvature of the microcolumn is visible due to the off-center orientation of the axonal tracts. The material is flexible and can deform to accommodate the curvature of the cochlea. The bottom panels show the 3D printed 270° construct, shown at 50% (Figure 7B) and 75% (Figure 7C) completion levels to better highlight the central channel (arrow). [Figure 8] We present a novel neuronal "aggregate" method to create micro-TENNs. We applied a "forced cell aggregation" method using gentle centrifugation in inverted pyramidal microwells. These neuronal aggregates were then precisely seeded onto the ends of hydrogel micro-columns. Over a period of several days DIV, we observed robust unidirectional axonal extension along the length of the micro-columns. [Figure 9] Surgical approach to the right cochlea of ​​an adult Sprague-Dawley rat. Figure 9A: The otic bulla was opened to expose the stapedial artery (striped arrow) which runs above the cochlear promontory and between the stapedial peduncle. Figure 9B: Cochleostomy at the basal turn of the cochlea (white arrow). Figure 9C: The otic bulla was removed to expose the bony modiolus surrounding the spiral ganglion (black arrow). [Figure 10] Schematics of future construct designs are provided. FIG. 10A: Single optrode and neuronal cell body aggregate. FIG. 10B: Two-dimensional array of optrode and cell body pairs. This portion of the construct resides within the otic bulla or outside the brainstem parenchyma. FIG. 10C: Schematic of the cochlear construct, showing the optrode and neuronal aggregate located within the otic bulla. The black lines represent the axonal tracts terminating at various positions along the cochlea. FIG. 10D: Schematic of the inferior colliculus construct, showing the optrode and neuronal aggregate located outside the brainstem parenchyma. The lines represent the axonal tracts terminating at various positions within the inferior colliculus volume. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0146] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0147] Unless specifically 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 of the mean. "About" may 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 by the term about.

[0148] As used herein, "synapse" refers to the junction between a neuron (eg, an auditory neuron) and another cell across which chemical communication flows.

[0149] Ranges provided herein are understood as shorthand notations of 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 (as well as fractions thereof unless the context clearly indicates otherwise).

[0150] The term "biocompatible construct" as used herein refers to a construct including a matrix that, when placed in contact with living tissue, e.g., in vivo (e.g., over a period of time), does not cause significant harm to such tissue. In certain embodiments, the biocompatible construct is not toxic to cells (e.g., over a period of time). In certain embodiments, the biocompatible construct does not induce significant inflammatory or other immune responses, or other adverse effects, when placed or implanted in a subject (e.g., over a period of time). In some embodiments, the matrix of the biocompatible construct includes a scaffold. In some embodiments, the matrix of the biocompatible construct includes an inner surface, an outer surface, or both an inner surface and an outer surface. In some embodiments, the inner surface of the matrix of the biocompatible construct defines a luminal core. In some embodiments, the inner surface of the matrix of the biocompatible construct includes a component disclosed herein. In some embodiments, the outer surface of the matrix of the biocompatible construct includes a component disclosed herein. In some embodiments, the biocompatible construct includes one or more additional components. In some embodiments, the biocompatible construct is a tubular construct, a curved construct, a Y-shaped construct, or a non-linear construct.

[0151] Generally, the term "engineered" refers to the aspect of being manipulated by the hand of man. For example, a cell or organism is considered to be "engineered" if it has undergone manipulation to alter its genetic, epigenetic, and / or phenotypic identity compared to an appropriate reference cell, such as a non-manipulated but otherwise identical cell. In some embodiments, an engineered cell is a cell that has been engineered to contain and / or express a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in a different amount and / or at a different timing compared to such an appropriate reference cell. As is customary and understood in the art, progeny of an engineered cell are also typically referred to as "engineered", even if the actual manipulation was performed in the progeny.

[0152] As used herein, the term "living electrode" refers to a system comprising a biocompatible construct comprising a matrix; and a plurality of cells (e.g., auditory neurons). In some embodiments, the living electrode comprises one or more neurite extensions, e.g., axons and / or dendrites, extending unidirectionally or bidirectionally. In some embodiments, the living electrode can be implanted in a subject. In some embodiments, the living electrode comprises an accessible interface for modulating the activity of at least one neuron in said plurality of neurons or at least one cell in a subject in which the living electrode is implanted.

[0153] The term "subject" as used herein refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is afflicted with hearing impairment. In some embodiments, the subject is susceptible to hearing impairment. In some embodiments, the subject exhibits one or more symptoms or characteristics of hearing impairment. In some embodiments, the subject does not exhibit symptoms or characteristics of hearing impairment. In some embodiments, the subject is a person with one or more characteristics that are characteristic of susceptibility or risk of hearing impairment. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom diagnosis and / or therapy is and / or has been performed.

[0154] The term "auditory neuron" as used herein refers to a neuron having one or more characteristics of a neuron present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of an organism, such as a human. In some embodiments, the characteristics include physical characteristics and / or cellular characteristics. In some embodiments, the physical characteristics include size, shape, aggregation pattern, and / or motility. In some embodiments, the cellular characteristics include expression patterns of entities (e.g., lipids, polypeptides, and / or polynucleotides); differentiation potential; proliferation potential; signaling pathways typically associated with auditory neurons as described herein; and / or the ability to respond to stimuli, such as physiological or electrophysiological responses to stimuli. In some embodiments, the characteristics include the ability to form synapses (e.g., with cells, such as neurons) and / or the ability to transmit signals. In some embodiments, the auditory neuron is a neuron derived from or obtained from a neuron present in the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of an organism, such as a human. In some embodiments, the auditory neuron is or comprises a neuron present in the cochlea. In some embodiments, the auditory neuron is or comprises a neuron present in the spiral ganglion. In some embodiments, the auditory neuron is or comprises a neuron present in the auditory nerve. In some embodiments, the auditory neuron is or comprises a neuron present in the auditory brainstem nuclei. In some embodiments, the auditory neuron is or comprises a neuron present in the cortical brainstem nuclei. In some embodiments, the auditory neuron is or comprises a spiral ganglion neuron, an inferior colliculus (IC) auditory neuron, a cortical auditory neuron, a type I auditory neuron, a type II auditory neuron. In some embodiments, the plurality of auditory neurons comprises a spiral ganglion neuron, an inferior colliculus (IC) auditory neuron, a cortical auditory neuron, a type I auditory neuron, a type II auditory neuron, or a combination thereof.

[0155] Detailed Description It is estimated that 15% of the world's population has some degree of hearing loss, and over 5% suffer from disabling deafness. When hearing loss progresses to the point where amplification alone is not sufficient, cochlear implantation has become the standard of care. A cochlear implant (CI) bypasses the damaged parts of the cochlea and directly stimulates the auditory nerve. The impact of cochlear implantation cannot be overstated, and the vast majority of recipients benefit greatly from their device. However, despite their success, cochlear implants do not restore natural hearing, and even the most successful patients have difficulty understanding speech in noise, talking on the phone, and appreciating complex sounds such as music.

[0156] The basis of CI electrode design has remained essentially unchanged since the first implants were developed in the 1960s: a linear array with a variable number of leads inserted from the base of the cochlea along the length of the cochlea. Advances in electrode design include multichannel arrays that exploit the frequency-specific mechanisms of the cochlea; pre-curved designs to mimic the shape of the cochlear duct and position the leads closer to the spiral ganglion; and reduced stiffness to reduce insertion trauma and ensure proper placement within the scala cochlea. Even with these advances, current spillover and cross-stimulation limit the number of independent channels available. It has been estimated that 30-50 independent channels are needed to approximate normal hearing, but current technology often achieves fewer than 10.

[0157] Several strategies have been developed to compensate for these limitations. Preoperative imaging is used to more precisely select electrode size for each individual patient; postoperative image-guided programming techniques can inform selective deactivation of interfering electrodes and provide an estimate of initial frequency programming; and intraoperative electrocochleography can monitor insertion trauma in real time. Stimulation and encoding strategies have also been developed to more precisely guide the current and stimulation site within the cochlea and mimic biosignal patterns.

[0158] For patients who cannot benefit from cochlear implantation, only one central implant has been approved in the United States. The Auditory Brainstem Implant (ABI) has a flat array of electrodes intended to be placed against the surface of the cochlear nucleus (CN). In contrast to the success of cochlear implants, only slightly more than 1000 ABIs have been placed worldwide to date, and in most cases performance has not matched that of cochlear implants. Modifications of the ABI, including both surface and penetrating electrodes, have been attempted to take advantage of the frequency-specific placement of the CN, but no improvement in outcomes was achieved. An alternative central implant, the Auditory Midbrain Implant (AMI), with 20 ring electrodes along a single shank, was intended to be implanted within the inferior colliculus (IC). Although preclinical data was promising, outcome goals were not met in small clinical trials, and the device remains investigational. Examples of novel bioengineering efforts intended to overcome the obstacles mentioned above and deliver the next generation of hearing implants are also disclosed herein.

[0159] Modifications to the cochlea Several methods have been developed to modify the cochlear environment and improve the performance of existing electrode technology. Many of these are based on inducing neuronal growth between the electrode and the remaining spiral ganglion cells, thereby reducing the potential for current spillover. In vitro studies have found several factors that can promote neurite outgrowth in spiral ganglion cells, including growth hormone, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, and erythropoietin. Alternatively, coatings on the electrode itself have been evaluated, including oriented collagen and microtexturing to promote neurite outgrowth. Laminin-coated electrodes, BDNF gene transfer, and chronic administration of growth factors into the scala tympani have been shown to induce nerve outgrowth into the scala tympani and may allow for direct contact with the implanted electrode.

[0160] Techniques that attempt to preserve residual hearing after implantation and provide both acoustic and electrical hearing have shown superior outcomes compared to electrical stimulation alone. Therefore, there is great interest in methods to reduce the immediate and long-term inflammatory response in the cochlea to prevent delayed hearing loss. Coating electrodes with polymers or silicone fibers has helped to prevent fibroblast growth, and there are also several techniques to slowly release anti-inflammatory drugs in the cochlea after implantation.

[0161] Regenerative Medicine Techniques Damage to cochlear hair cells and spiral ganglion neurons that leads to sensorineural hearing loss is currently irreversible except in certain rare conditions. The problems mentioned above with cochlear implants could be avoided by being able to restore the natural function of the cochlea. Gene therapy has been proposed for preserving or regenerating hair cells and auditory neurons in the cochlea, as well as for certain genetic forms of hearing loss. These techniques require that therapeutic agents be able to be introduced into the inner ear without causing additional damage, and several nanotechnology techniques have been developed to address this limitation. Stem cell-based therapies, including induced pluripotent stem cells, have also shown promise for the regeneration of both hair cells and auditory neurons. Decellularized cochleae are being investigated to act as scaffolds to support such regenerative medicine strategies.

[0162] light stimulation Optical stimulation has been proposed as an alternative to conventional electrical stimulation, as it potentially avoids the problems associated with current spillover and has the frequency and intensity resolution required to simulate complex sounds. Viral-based optogenetic transformation of spiral ganglion cells and linear multichannel microLED arrays have been shown to stimulate the auditory system in animal models. Several significant technical challenges remain to be overcome before such techniques can be applied in clinical settings.

[0163] Piezoelectric Piezoelectric materials have the ability to convert between electrical and mechanical energy, a quality that naturally leads to hearing applications. Piezoelectric materials are being investigated for hearing applications as thin films, electrospun fibers, and nanoparticles. Piezoelectric materials have also been proposed as the key to developing fully implantable hearing systems. As with optogenetic technologies, the potential of piezoelectric materials to form the basis of next-generation hearing implants is great, but significant technological hurdles remain to be overcome.

[0164] Therefore, there is an unmet need to develop hearing implants that can overcome the obstacles mentioned above.

[0165] In recent years, there has been great progress in the development of implantable micro-tissue engineered neural networks (micro-TENNs) for the reconstruction of brain pathways and the regeneration of the central nervous system. In some embodiments, a micro-TENN is also referred to as a system that includes a biocompatible construct, e.g., including at least one living electrode, and a plurality of auditory neurons. In some embodiments, the constructs disclosed herein are generated in vitro by seeding a population of neurons within a biocompatible construct, e.g., a customizable soft hydrogel scaffold, whose neuronal phenotype can be tailored to the application of interest. In some embodiments, the neurons contained within these scaffolds extend neurites along preformed channels, while the cell bodies remain in a defined location. In some embodiments, the axons of each implanted neuron within the micro-TENN synapse directly, e.g., onto the cell bodies of one or more neurons within the target tissue, thereby eliminating the potential for current spillover. In some embodiments, the constructs may be placed such that the axons are directed to the location of interest, while the cell bodies remain accessible. The non-organic components of the neural interface, including the conventional electrodes or optrodes used to stimulate the implanted neurons, remain outside the brain parenchyma or cochlea. This technique causes much less trauma at implantation and is less likely to cause chronic inflammation from the biomechanical mismatch of conventional electrodes. In some embodiments, this configuration of micro-TENNs, combined with synapse-specific stimulation where axons interact with native neurons, creates an accessible interface for stimulating or recording neuronal tissue - also referred to in some embodiments as a "living electrode" (LE). In some embodiments, the neurons utilized in the constructs may be optogenetically functionalized to take advantage of the benefits of optical stimulation described above.

[0166] The present disclosure provides for the first time a system including a biocompatible construct and a plurality of auditory neurons (e.g., including at least one living electrode) that can be implanted in a subject, such as to modulate auditory neurons and / or treat or prevent hearing disorders. In some embodiments, application to the auditory system has several potential advantages over existing solutions. In some embodiments, a biocompatible construct including a matrix, such as a scaffold, may be designed with the complex 3D anatomical structure of the IC or CN. While both the IC and CN have basic frequency-specific structures, complex sounds such as speech may not follow this simplified model. In some embodiments, reproducing these complex sounds will likely require precise simulations throughout the volume of the nucleus. In some embodiments, the biocompatibility of the systems disclosed herein, such as living electrodes, may allow access to a larger volume of the auditory nucleus while avoiding the issue of chronic inflammation. In some embodiments, for cochlear applications, prior studies have shown that axons can be grown to several centimeters in length to extend along the length of the cochlea and utilize established implantation techniques. In some embodiments, the cell bodies of implanted neurons may remain within the mastoid process, where they may be stimulated, for example, either optically or electrically, while their axons are positioned to terminate at various depths along the length of the cochlea. In some embodiments, the systems disclosed herein, such as living electrode constructs, may physiologically interact with the neuronal populations of the spiral ganglion. In some embodiments, such in vivo interactions benefit from the native SGN outgrowth strategies described above. In some embodiments, the specificity of stimulation has the potential to be superior, for example, to options using standard electrode techniques.

[0167] Hearing implants are the only widely available method to restore sensation in humans. Since the first single-channel cochlear implants were shown to provide reliable auditory sensation, significant progress has been made in the decades since, and they have progressed to more anatomically and physiologically friendly designs, generally inspired by the natural structure and function of the cochlea. To date, however, these designs remain constrained within the limitations of conventional electrode technology. Although results are encouraging for hundreds of thousands of patients suffering from hearing loss, advancing outcomes to something approaching natural hearing will require a fundamental rethinking of the prosthetic interface. While methods continue to advance for optimizing conventional electrode technology, including electrode properties, drug elution, surface coatings, and cochlear adaptation, multiple additional lines of research, including optogenetics, piezoelectric materials, nanotechnology, and living electrodes, may hold promise for forming the basis of entirely novel methods to restore hearing. The present disclosure thus provides, inter alia, a system including a biocompatible construct and a plurality of auditory neurons (e.g., including at least one living electrode) that can be implanted within a subject, e.g., to modulate auditory neurons and / or to treat or prevent hearing impairment.

[0168] System Comprising Biocompatible Constructs and Auditory Neurons Disclosed herein, among other things, is a system (e.g., living electrode) that includes a biocompatible construct that includes a matrix and a plurality of auditory neurons. The system disclosed herein can be manufactured, e.g., assembled, in vitro or ex vivo. The system disclosed herein can be implanted in a subject and can be used to modulate auditory neurons in the subject. In some embodiments, the system disclosed herein is or includes at least one living electrode.

[0169] Biocompatible constructs The biocompatible construct disclosed herein comprises a matrix, for example as disclosed herein. In some embodiments, the biocompatible construct disclosed herein comprises a first end, a second end, and a body. In some embodiments, the matrix of the biocompatible construct comprises an interior surface and / or an exterior surface. In some embodiments, the interior surface of the biocompatible construct defines a luminal core.

[0170] In some embodiments, the matrix of the biocompatible construct comprises a scaffold. In some embodiments, the matrix of the biocompatible construct comprises a component described herein, such as, for example, an internal component as described herein, and / or an external component as described herein. In some embodiments, the matrix of the biocompatible construct comprises one or more additional components, such as, for example, as disclosed herein.

[0171] As an example, the exterior surface of the biocompatible construct disclosed herein comprises at least one hydrogel (e.g., as described herein). In some embodiments, the hydrogel comprises a hydrophilic biopolymer and / or a synthetic polymer. In some embodiments, the hydrogel is at least partially crosslinked, where crosslinking optionally increases stiffness, reduces porosity, and / or increases degradation time. In some embodiments, the hydrophilic biopolymer comprises one or more of agarose, hydrogel, hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, fibrin, and methylcellulose. In some embodiments, the hydrophilic biopolymer comprises agarose. In some embodiments, the hydrophilic biopolymer comprises a hydrogel. In some embodiments, the hydrophilic biopolymer comprises chitosan. In some embodiments, the hydrophilic biopolymer comprises alginate. In some embodiments, the hydrophilic biopolymer comprises collagen. In some embodiments, the hydrophilic biopolymer comprises dextran. In some embodiments, the hydrophilic biopolymer comprises pectin. In some embodiments, the hydrophilic biopolymer comprises carrageenan. In some embodiments, the hydrophilic biopolymer comprises polylysine. In some embodiments, the hydrophilic biopolymer comprises gelatin. In some embodiments, the hydrophilic biopolymer comprises hyaluronic acid. In some embodiments, the hydrophilic biopolymer comprises fibrin. In some embodiments, the hydrophilic biopolymer comprises methylcellulose.

[0172] In some embodiments, the hydrophilic biopolymer comprises agarose. In some embodiments, the agarose is about 0.25-30%, about 0.25%-3%, about 0.5%-3%, about 1-20%, about 1.5-10%, about 2-9%, about 2.5-8%, or about 3-7%. In some embodiments, the agarose is about 0.25-29%, about 0.25-28%, 0.25%, about 0.25-27%, about 0.25-26%, about 0.25-25%, about 0.25-24%, about 0.25-23%, about 0.25-22%, about 0.25-21%, about 0.25-20%, about 0.25-19%, about 0. 25~18%, about 0.25~17%, about 0.25~16%, about 0.25~15%, about 0.25~14%, about 0.25~13%, about 0.25~12%, about 0.25~11%, about 0.25~10%, about 0.25~9%, about 0.25~8%, about 0.25~7%, about 0.25~6%, about 0.25~5%, about 0.25~ 4%, approx. 0.25-3%, approx. 0.25-2%, approx. 0.25-1%, approx. 0.25-0.5%, approx. 0.5-30%, approx. 1-30%, approx. 2-30%, approx. 3-30%, approx. 4-30%, approx. 5-30%, approx. 6-30%, approx. 7-30%, approx. 8-30%, approx. 9-30%, approx. 10-30%, approx. 11-30%, approx. 12-30%, approx. 13-30%, approximately 14-30%, approximately 15-30%, approximately 16-30%, approximately 17-30%, approximately 18-30%, approximately 19-30%, approximately 20-30%, approximately 21-30%, approximately 22-30%, approximately 23-30%, approximately 24-30%, approximately 25-30%, approximately 26-30%, approximately 27-30%, approximately 28-30%, approximately 29-30%.

[0173] In some embodiments, the agarose is about 0.25%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%. In some embodiments, the agarose is about 3%.

[0174] In some embodiments, the hydrophilic biopolymer comprises at least one synthetic hydrogel (e.g., a pluronic hydrogel) or at least one synthetic hydrogel made of an amphiphilic copolymer consisting of ethylene oxide (PEO) and polypropylene oxide (PPO) units. In some embodiments, the hydrogel is about 2-50% w / v, about 3-40% w / v, about 4-30% w / v, about 5-25% w / v, or about 10-20% w / v. In some embodiments, the hydrogel is about 2-45%, about 2-40%, about 2-35%, about 2-30%, about 2-29%, about 2-28%, about 2-27%, about 2-26%, about 2-25%, about 2-24%, about 2-23%, about 2-22%, about 2-21%, about 2-20%, about 2-19%, about 2-18%, about 2-17%, about 2-16%, about 2-15%, about 2-14%, about 2-13%, about 2-12%, about 2-11%, about 2-10%, about 2-9%, about 2-8%, about 2-7%, about 2-6%, about 2-5%, about 2-4%, about 2-3%, about 3-50%. , about 4-50%, about 5-50%, about 6-50%, about 7-50%, about 8-50%, about 9-50%, about 10-50%, about 11-50%, about 12-50%, about 13-50%, about 14-50%, about 15-50%, about 16-50%, about 17-50%, about 18-50%, about 19-50%, about 20-50%, about 21-50%, about 22-50%, about 23-50%, about 24-50%, about 25-50%, about 26-50%, about 27-50%, about 28-50%, about 29-50%, about 30-50%, about 35-50%, about 40-50%, and about 45-50% w / v. In some embodiments, the hydrogel is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 40%, about 45%, or about 50% w / v. In some embodiments, the hydrogel is about 28.1%, about 28.2%, about 28.3%, about 28.4%, about 28.5%, about 28.6%, about 28.7%, about 28.8%, about 28.9%, about 30%.In some embodiments, the hydrogel is about 28.6% w / v.

[0175] In some embodiments, the inner surface of the biocompatible construct disclosed herein comprises one or more extracellular matrix (ECM) components. In some embodiments, the ECM components comprise collagen, laminin, fibronectin, hyaluronic acid, or combinations thereof. In some embodiments, the ECM components comprise collagen. In some embodiments, the ECM components comprise laminin. In some embodiments, the ECM components comprise fibronectin. In some embodiments, the ECM components comprise hyaluronic acid.

[0176] In some embodiments, the ECM component comprises collagen, hi some embodiments, the collagen is at a concentration of about 0.1-10 mg / ml. In some embodiments, collagen is about 0.1-9 mg / ml, about 0.1-8 mg / ml, about 0.1-7 mg / ml, about 0.1-6 mg / ml, about 0.1-5 mg / ml, about 0.1-4 mg / ml, about 0.1-3 mg / ml, about 0.1-2 mg / ml, about 0.1-1 mg / ml, about 0.1-0.9 mg / ml, about 0.1-0.8 mg / ml, about 0.1-0.7 mg / ml, about 0.1-0.5 mg / ml, about 0.5-10 mg / ml, about 0.6-10 mg / ml, about 0.7-10 mg / ml, about 0.8-10 mg / ml, about 0.9-10 mg / ml, about 1-10 mg / ml, about 2-10 mg / ml, about 3-10 mg / ml, about 4-10 mg / ml, about 5-10 mg / ml, about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, about 9-10 mg / ml, about 10-10 mg / ml, about 20-10 mg / ml, about 30-10 mg / ml, about 40-10 mg / ml, about 50-10 mg / ml, about 60-10 mg / ml, about 70-10 mg / ml, about 80-10 mg / ml, about 90-10 mg / ml, about 10 ... The concentrations are about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, and about 9-10 mg / ml. In some embodiments, the collagen is at a concentration of about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml. In some embodiments, the collagen is at a concentration of about 1 mg / ml.

[0177] In some embodiments, the ECM component comprises laminin. In some embodiments, the laminin is about 0.1-9 mg / ml, about 0.1-8 mg / ml, about 0.1-7 mg / ml, about 0.1-6 mg / ml, about 0.1-5 mg / ml, about 0.1-4 mg / ml, about 0.1-3 mg / ml, about 0.1-2 mg / ml, about 0.1-1 mg / ml, about 0.1-0.9 mg / ml, about 0.1-0.8 mg / ml, about 0.1-0.7 mg / ml, about 0.1-0.5 mg / ml, about 0.5-10 mg / ml, about 0.6-10 mg / ml, about 0.7-10 mg / ml, about 0.8-10 mg / ml, about 0.9-10 mg / ml, about 1-10 mg / ml, about 2-10 mg / ml, about 3-10 mg / ml, about 4-10 mg / ml, about 5-10 mg / ml, about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, about 9-10 mg / ml, about 10-10 mg / ml, about 20-10 mg / ml, about 30-10 mg / ml, about 40-10 mg / ml, about 50-10 mg / ml, about 60-10 mg / ml, about 70-10 mg / ml, about 80-10 mg / ml, about 90-10 mg / ml, about 10 ... The concentrations are about 6-10 mg / ml, about 7-10 mg / ml, about 8-10 mg / ml, and about 9-10 mg / ml. In some embodiments, the laminin is at a concentration of about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml. In some embodiments, the laminin is at a concentration of about 1 mg / ml.

[0178] Physical properties of biocompatible constructs The biocompatible construct disclosed herein may have one or more properties described herein and / or may be part of the system disclosed herein (e.g., a living electrode). In some embodiments, the biocompatible construct disclosed herein is a tubular construct. In some embodiments, the biocompatible construct disclosed herein is a spiral or Y-shaped. In some embodiments, the biocompatible construct disclosed herein has a nonlinear shape.

[0179] The helical or Y-shaped biocompatible construct may have a curvature of about 240 to 900 degrees. In some embodiments, the curvature of the biocompatible construct is about 240 to 850 degrees, about 240 to 800 degrees, about 240 to 750 degrees, about 240 to 700 degrees, about 240 to 650 degrees, about 240 to 600 degrees, about 240 to 550 degrees, about 240 to 500 degrees, about 240 to 450 degrees, about 240 to 400 degrees, about 240 to 350 degrees, about 240 to 300 degrees, about 240 to 290 degrees, about 240 to 280 degrees, about 240 to 270 degrees, about 240 to 260 degrees. , approximately 240 to 250 degrees, approximately 250 to 900 degrees, approximately 260 to 900 degrees, approximately 270 to 900 degrees, approximately 280 to 900 degrees, approximately 290 to 900 degrees, approximately 300 to 900 degrees, approximately 350 to 900 degrees, approximately 400 to 900 degrees, approximately 450 to 900 degrees, approximately 500 to 900 degrees, approximately 550 to 900 degrees, approximately 600 to 900 degrees, approximately 650 to 900 degrees, approximately 700 to 900 degrees, approximately 750 to 900 degrees, approximately 800 to 900 degrees, and approximately 850 to 900 degrees. In some embodiments, the curvature of the biocompatible construct is about 240 degrees, about 250 degrees, about 260 degrees, about 270 degrees, about 280 degrees, about 290 degrees, about 300 degrees, about 350 degrees, about 400 degrees, about 450 degrees, about 500 degrees, about 550 degrees, about 600 degrees, about 650 degrees, about 700 degrees, about 750 degrees, about 800 degrees, about 850 degrees, or about 900 degrees.

[0180] In some embodiments, a system comprising a biocompatible construct disclosed herein has an outer diameter of about 0.1 to 40 mm. In some embodiments, the system is about 0.1-35 mm, about 0.1-30 mm, about 0.1-25 mm, about 0.1-20 mm, about 0.1-15 mm, about 0.1-12 mm, about 0.1-11 mm, about 0.1-10 mm, about 0.1-9 mm, about 0.1-8 mm, about 0.1-7 mm, about 0.1-6.5 mm, about 0.1-6 mm, about 0.1-5 mm, about 0.1-4 mm, about 0.1-3 mm, about 0.1-2 mm, about 0.1-1 mm, about 0.1-0.5 mm, about 0.2-40 mm, about 0.3-40 mm, about 0.4-40 mm, about 0.5-40 mm, about 1-40 mm, about 2-40 mm, about 3-40 mm, about 4-40 mm, about 5-40 mm, about 6-40 mm, about 7-40 mm, about 8-40 mm, about 9-40 mm, about 10-40 mm, about 11-40 mm, about 12-40 mm, about 13-40 mm, about 14-40 mm, about 15-40 mm, about 16-40 mm, about 17-40 mm, about 18-40 mm, about 19-50 mm, about 21-40 mm, about 22-40 mm, about 23-40 mm, about 24-40 mm, about 25-40 mm, about 26-40 mm, about 27-40 mm, about 28-40 mm, about 29-40 mm, about 31-40 mm, about 32-40 mm, about 33-40 mm, about 34-40 mm, about In some embodiments, the system has an outer diameter of about 0.1 mm, about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 6.5 mm, about 7 mm, about 8 mm, about 9 mm, about 40 mm, about 10 mm, about 40 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, or about 40 mm. In some embodiments, the system has an outer diameter of about 1.5 mm.

[0181] In some embodiments, the system comprising the biocompatible construct disclosed herein has a diameter of about 0.5-70 mm, about 0.5-60 mm, about 0.5-50 mm, about 0.4-50 mm, about 0.5-30 mm, about 0.5-20 mm, about 0.5-10 mm, about 1-70 mm, about 2-70 mm, about 3-70 mm, about 4-70 mm, about 5-70 mm, about 6-70 mm, about 7-70 mm, about 8-70 mm, about 9-70 mm, about 10-70 mm, about 20-70 mm, about 30-70 mm, about 40-70 mm, about 50-70 mm. In some embodiments, the system has a diameter of about 0.1-10 mm, about 0.2-9 mm, about 0.3-8 mm, about 0.4-7 mm, about 0.5-6.5 mm. In some embodiments, the system has a diameter of about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 6.5 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm. In some embodiments, the system has a diameter of about 7 mm.

[0182] In some embodiments, a system comprising a biocompatible construct disclosed herein has a pitch of about 0.1-10 mm, about 0.1-8 mm, about 0.1-6 mm, about 0.1-4 mm, about 0.1-3 mm, about 0.1-2 mm, about 0.1-1.5 mm, about 0.1-1 mm, about 0.5-10 mm, about 0.6-10 mm, about 0.7-10 mm, about 0.8-10 mm, about 0.9-10 mm, about 1-10 mm, about 2-10 mm, about 4-10 mm, about 6-10 mm, or about 8-10 mm. In some embodiments, the system has a pitch of about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm. In some embodiments, the system has a pitch of about 1 mm.

[0183] Auditory neurons Auditory neurons convey information from the cochlea to the cortex. Exemplary auditory neurons include spiral ganglion neurons, inferior colliculus (IC) auditory neurons, cortical auditory neurons, type I auditory neurons, or type II auditory neurons.

[0184] A system disclosed herein for use in treating hearing impairment and / or restoring hearing in a subject with hearing loss includes a biocompatible construct (e.g., as described herein) and a plurality of auditory neurons. In some embodiments, the plurality of auditory neurons includes spiral ganglion neurons, inferior colliculus (IC) auditory neurons, cortical auditory neurons, type I auditory neurons, or type II auditory neurons. In some embodiments, the plurality of auditory neurons is or includes a plurality of spiral ganglion neurons. In some embodiments, the plurality of auditory neurons is or includes a plurality of IC auditory neurons. In some embodiments, the plurality of auditory neurons is or includes a plurality of cortical auditory neurons. In some embodiments, the plurality of auditory neurons is or includes a plurality of type I auditory neurons. In some embodiments, the plurality of auditory neurons is or includes a plurality of type II auditory neurons.

[0185] In some embodiments, the plurality of auditory neurons comprises about 50-5000 auditory neurons, e.g., as described herein. In some embodiments, the plurality of auditory neurons comprises about 50-4500, about 50-4000, about 50-3500, about 50-3000, about 50-2500, about 50-2000, about 50-1500, about 50-1000, about 50-900, about 50-800, about 50-700, about 50-600, about 50-550, about 50-500, about about 50-450, about 50-400, about 50-300, about 50-200, about 50-100, about 100-5000, about 200-5000, about 300-5000, about 400-5000, about 500-5000, about 1000-5000, about 2000-5000, about 3000-5000, or about 4000-5000 auditory neurons. In some embodiments, the plurality of auditory neurons comprises at least 50 auditory neurons, at least 100 auditory neurons, at least 200 auditory neurons, at least 300 auditory neurons, at least 400 auditory neurons, at least 450 auditory neurons, at least 500 auditory neurons, at least 550 auditory neurons, at least 600 auditory neurons, at least 700 auditory neurons, at least 800 auditory neurons, at least 900 auditory neurons, at least 1000 auditory neurons, at least 2000 auditory neurons, at least 3000 auditory neurons, at least 4000, or at least 5000 auditory neurons. In some embodiments, the plurality of auditory neurons comprises at least 500 auditory neurons.

[0186] In some embodiments, at least one of the plurality of auditory neurons is in contact with a biocompatible construct.

[0187] In some embodiments, at least one neuron in said plurality of neurons comprises a cell body and / or an axon. In some embodiments, at least one neuron in said plurality of neurons comprises a cell body. In some embodiments, at least one neuron in said plurality of neurons comprises an axon. In some embodiments, at least one neuron in said plurality of neurons comprises a cell body and an axon.

[0188] In some embodiments, the axon has a length of about 0.5-50 cm, about 0.5-49 cm, about 0.5-48 cm, about 0.5-47 cm, about 0.5-46 cm, about 0.5-45 cm, about 0.5-44 cm, about 0.5-43 cm, about 0.5-42 cm, about 0.5-41 cm, about 0.5-40 cm, about 0.5-39 cm, about 0.5-38 cm, about 0.5-37 cm, about 0.5-36 cm, about 0.5-35 cm, about 0.5-34 cm, about 0.5-33 cm, about 0.5-32 cm, about 0.5-31 cm, about 0.5-30 cm, about 0.5-29 cm, about 0.5-28 cm, about 0.5-27 cm, about 0.5-26 cm, about 0.5-25 cm, about 0.5-24 cm, about 0.5-23 cm, about 0.5-22 cm, about 0.5-21 cm, about 0.5-20 cm, about 0.5-19 cm, about 0.5-18 cm, about 0.5-17 cm, about 0.5-16 cm, about 0.5-15 cm, about 0.5-14 cm, about 0.5-13 cm, about 0.5-12 cm, about 0.5-11 cm, about 0.5-10 cm, about 0.5-9 cm, about 0.5-8 cm, about 0.5-7 cm, about 0.5-6 cm, about 0.5-5 cm, about 0.5-4 cm, about 0.5-3 cm, about 0.5-2 cm, about 0.5-1, about 0.6-50 cm, about 0.7-50 cm, about 0.8-50 cm, approximately 0.9~50 cm, 1~50 cm, 2~50 cm, 3~50 cm, 4~50 cm, 5~50 cm, 6~50 cm, 7~50 cm, 8~50 cm, 9~50 cm, 10~50 cm, 11~50 cm, 12~50 cm, 13~50 cm, 14~50 cm, 15~50 cm, 16~50 cm, 17~50 cm, 18~50 cm, 19~50 cm, 20~50 cm, 21~50 cm, 22~50 cm, 23~50 cm, 24~50 cm, 25~50 cm, 26~50 cm, 27~50 cm, 28~50 cm, 29~50 cm, about 30~50 cm, about 31~50 cm, about 32-50 cm, about 33-50 cm, about 34-50 cm, about 35-50 cm, about 36-50 cm, about 37-50 cm, about 38-50 cm, about 39-50 cm, about 40-50 cm, about 41-50 cm, about 42-50 cm, about 43-50 cm, about 44-50 cm, about 45-50 cm, about 46-50 cm, about 47-50 cm, about 48-50 cm, or about 49-50 cm.

[0189] In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned (i) at a first end of a biocompatible construct; (ii) at a second end of a biocompatible construct; (iii) at, along, or within a portion of the body of the biocompatible construct; and / or (iv) throughout, along, or within the body of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at a first end of a biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at a second end of a biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned at, along, or within a portion of the body of the biocompatible construct. In some embodiments, a portion of at least one neuron in the plurality of auditory neurons is positioned throughout, along, or within the body of the biocompatible construct.

[0190] In some embodiments, the portion of the at least one neuron in the plurality of auditory neurons positioned at the first end or the second end of the biocompatible construct comprises a cell body.

[0191] In some embodiments, the portion of the at least one neuron in the plurality of auditory neurons positioned at, along or within a portion of the body of the biocompatible construct comprises an axon. In some embodiments, the body of the biocompatible construct comprises an interior surface of the body. In some embodiments, the interior surface comprises a luminal core.

[0192] In some embodiments, a portion of the at least one neuron in the plurality of auditory neurons positioned throughout, along, or within the body of the biocompatible construct comprises an axon. In some embodiments, the axon extends unidirectionally or bidirectionally. In some embodiments, the axon extends unidirectionally. In some embodiments, the axon extends bidirectionally.

[0193] In some embodiments, at least a portion of the plurality of auditory neurons comprises a clump of auditory neurons.

[0194] In some embodiments, the plurality of auditory neurons is isolated from a subject.

[0195] In some embodiments, the plurality of auditory neurons is or comprises at least one cell derived from a stem cell. In some embodiments, the stem cell comprises one or more of induced pluripotent stem cells (iPSCs), embryonic stem cells, or tissue stem cells. In some embodiments, the stem cell comprises an iPSC. In some embodiments, the stem cell comprises embryonic stem cells. In some embodiments, the stem cell comprises tissue stem cells.

[0196] In some embodiments, the plurality of auditory neurons is autologous, allogeneic, or xenogeneic to the subject. In some embodiments, the plurality of auditory neurons is autologous to the subject. In some embodiments, the plurality of auditory neurons is allogeneic to the subject. In some embodiments, the plurality of auditory neurons is xenogeneic to the subject.

[0197] In some embodiments, at least one neuron in the plurality of auditory neurons is genetically engineered or reprogrammed using non-genetic techniques. In some embodiments, the at least one engineered neuron comprises a sensor, an actuator, a receptor, and / or a reporter.

[0198] In some embodiments, the at least one engineered neuron in the plurality of auditory neurons comprises a reporter. In some embodiments, the reporter comprises a fluorescent reporter.

[0199] In some embodiments, the at least one engineered neuron in the plurality of auditory neurons comprises a receptor. In some embodiments, the receptor comprises one or more of a magnetoreceptor, an electromagnetic receptor, an electroreceptor, a hydroreceptor, a mechanoreceptor, an osmoreceptor, a thermoreceptor, and a piezoelectric ion channel.

[0200] In some embodiments, the electromagnetic receptor comprises one or more of an infrared receptor, a photoreceptor, or an ultraviolet receptor.

[0201] In some embodiments, the mechanoreceptors include one or both of mechanosensory receptors or proprioceptors.

[0202] In some embodiments, the at least one engineered neuron in the plurality of auditory neurons comprises a sensor, hi some embodiments, the sensor comprises an optical sensor comprising one or more of a light-gated ion channel, a calcium sensor, and a membrane voltage sensor.

[0203] In some embodiments, the sensor comprises a calcium sensor comprising one or more of Aequorin, Cameleon, and GCaMP.

[0204] In some embodiments, the sensor comprises a chloride sensor comprising Clomeleon.

[0205] In some embodiments, the sensor comprises a membrane voltage sensor comprising Mermaid.

[0206] In some embodiments, the at least one engineered neuron in the plurality of auditory neurons comprises an actuator, hi some embodiments, the actuator comprises an optical actuator comprising one or more of channelrhodopsin, halorhodopsin, and archerhodopsin.

[0207] In some embodiments, the at least one engineered neuron in the plurality of auditory neurons comprises an optical sensor and / or the optical actuator is an optogenetic neuron.

[0208] Methods of using the systems disclosed herein A system comprising a biocompatible construct and a plurality of auditory neurons as disclosed herein can be used to modulate auditory neurons in a subject, treat a subject with hearing impairment, ameliorate symptoms of hearing loss in a subject, or restore hearing in a subject. In some embodiments, the subject has been determined to have a hearing impairment. In some embodiments, the subject has symptoms that are characteristic of a hearing impairment. In some embodiments, the subject is at risk of developing a hearing impairment. In some embodiments, the subject has a hearing impairment.

[0209] In some embodiments, the hearing impairment comprises one or more of conductive hearing loss, sensorineural hearing loss, or mixed hearing loss. In some embodiments, the hearing impairment comprises one or more of acquired hearing loss, sudden hearing loss, secondary hearing loss (drug-induced or trauma-induced hearing loss), autoimmune inner ear disease, otosclerosis, Meniere's disease, presbycusis, or acoustic neuroma.

[0210] In some embodiments, the subject is a human.

[0211] In some embodiments, the methods described herein further include forming an aggregate of at least a portion of the plurality of auditory neurons. In some embodiments, the aggregate of at least a portion of the plurality of auditory neurons is formed prior to contacting the plurality of auditory neurons with a biocompatible construct.

[0212] In some embodiments of any of the methods disclosed herein, the method includes implanting a system disclosed herein in a subject. In some embodiments, the system is implanted at, near, or in the cochlea in the subject; or at, near, or in the inferior colliculus (IC) in the subject. In some embodiments, the system is implanted at, near, or in the cochlea in the subject. In some embodiments, the system is implanted at, near, or in the inferior colliculus (IC) in the subject. In some embodiments, the implanted system contacts at least one cell in the subject, or contacts at least one cell at, near, or in an area of ​​the auditory cortex in the subject. In some embodiments, the at least one cell in the subject is an endogenous cell. In some embodiments, the at least one cell comprises an auditory neuron, e.g., as described herein. In some embodiments, the auditory neuron comprises one or more of a spiral ganglion neuron or an IC neuron.

[0213] In some embodiments, the step of contacting the system with at least one cell in the subject is determined to create a synapse between at least one neuron of the plurality of auditory neurons of the system and at least one cell in the subject. In some embodiments, the synapse is or includes a neuronal synapse.

[0214] In some embodiments, the implanted system provides an accessible interface for modulating at least one activity of at least one neuron in said plurality of auditory neurons in the system and / or at least one activity of at least one cell in the subject. In some embodiments, the implanted system provides an accessible interface for modulating at least one activity of at least one neuron in said plurality of auditory neurons in the system. In some embodiments, the implanted system provides an accessible interface for modulating at least one activity of at least one cell in the subject. In some embodiments, the implanted system provides an accessible interface for modulating at least one activity of at least one neuron in said plurality of auditory neurons in the system and at least one activity of at least one cell in the subject.

[0215] The method of using the system disclosed herein includes modulating and / or assessing at least one activity of at least one neuron. In some embodiments, the system can modulate or be used to modulate at least one activity of at least one neuron in said plurality of neurons in the system and / or at least one activity of at least one neuron in a subject. In some embodiments, the system can modulate or be used to modulate at least one activity of at least one neuron in said plurality of neurons in the system. In some embodiments, the system can modulate or be used to modulate at least one activity of at least one neuron in a subject. In some embodiments, the system can modulate or be used to modulate at least one activity of at least one neuron in said plurality of neurons in the system and at least one activity of at least one neuron in a subject.

[0216] In some embodiments, the modulating step is or includes unidirectional or bidirectional modulation. In some embodiments, the modulating step is or includes unidirectional modulation. In some embodiments, the modulating step is or includes bidirectional modulation.

[0217] In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in said plurality of auditory neurons in the system and / or at least one neuron in the subject. In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in said plurality of auditory neurons in the system. In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in the subject. In some embodiments, the modulating step comprises stimulating or inhibiting at least one neuron in said plurality of auditory neurons in the system and at least one neuron in the subject.

[0218] In some embodiments, the stimulating step comprises electrical stimulation; light stimulation; sound or vibration stimulation; magnetic stimulation; or a combination thereof. In some embodiments, the stimulating step comprises electrical stimulation. In some embodiments, the stimulating step comprises light stimulation. In some embodiments, the stimulating step comprises sound or vibration stimulation. In some embodiments, the stimulating step comprises magnetic stimulation. In some embodiments, the stimulating step comprises electrical stimulation and light stimulation.

[0219] In some embodiments, the assessing step comprises a monitoring and / or a recording step. In some embodiments, the assessing step comprises a monitoring step. In some embodiments, the assessing step comprises a recording step. In some embodiments, the assessing step comprises a monitoring step and a recording step.

[0220] In some embodiments, at least one neuron in the subject is or comprises an auditory neuron, hi some embodiments, the auditory neuron comprises a spiral ganglion neuron, an IC neuron, or a combination thereof.

[0221] In some embodiments of any of the methods disclosed herein, the system is capable of stimulating or inhibiting an auditory pathway in the subject. In some embodiments, the system is capable of stimulating an auditory pathway in the subject. In some embodiments, the system is capable of inhibiting an auditory pathway in the subject.

[0222] In some embodiments of any of the methods disclosed herein, the system is capable of stimulating or inhibiting a peripheral auditory pathway in the subject. In some embodiments, the system is capable of stimulating a peripheral auditory pathway in the subject. In some embodiments, the system is capable of inhibiting a peripheral auditory pathway in the subject. EXAMPLES

[0223] The present invention will be further described by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless so specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing all variations that become evident as a result of the teachings provided herein.

[0224] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, utilize the components of the present invention and practice the claimed methods. The following working examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0225] Example 1: Study Design Background and Significance It is estimated that 15% of the world's population has some degree of hearing loss, and more than 5% suffer from disabling deafness. Furthermore, there is growing evidence that hearing loss leads to social isolation and has been correlated with dementia and cognitive decline. The most common causes of adult hearing loss include aging, noise exposure, ototoxicity, and inner ear disease, with acquired hearing loss often being associated with deterioration and loss of cochlear hair cells. Disability due to hearing loss is particularly prevalent in the veteran population, where significant noise exposure has led to hearing loss, with tinnitus being the most frequent service-related disability. In many cases, mild to moderate hearing loss is sufficient for reintegration into society with conventional hearing aids. However, when hearing loss becomes severe or speech discrimination is impaired, hearing aids are no longer sufficient. Cochlear implantation is the only technique that can restore human sensation and is the standard of care for patients with significant deafness. Over 200,000 patients have been implanted worldwide, with over 60,000 in the United States alone. There is also evidence that cochlear implants can provide tinnitus suppression during operation. Cochlear implants were recently approved by the FDA for patients with unilateral deafness, and one of the primary therapeutic benefits in this population is tinnitus suppression. Although encouraging for many with profound hearing loss, there is a patient population that cannot benefit from cochlear implants due to damage to either the cochlear nerve or the cochlea itself. For these patients, the only option for auditory perception is direct stimulation of the central auditory pathways through an auditory brainstem implant.

[0226] Cochlear implants:Cochlear implants use electrical impulses to directly stimulate the auditory nerve, bypassing damaged areas of the cochlea. Advances in surgical techniques and coding strategies have enabled the majority of implant recipients to derive significant benefit from their devices. These improvements have been so significant that many of the measures used to measure outcomes do not provide a complete picture of patient outcomes, and quality of life outcomes have increasingly been emphasized to better define the impact implants may have. Most cochlear implant users report a marked improvement in quality of life, with clear improvements also being shown in certain subpopulations, including children and the elderly. Interestingly, quality of life measures have been found to only modestly correlate with traditional outcome measures, such as speech understanding scores, indicating an impact beyond traditional measures of success. However, despite their success, cochlear implants do not restore natural hearing. Even the most successful patients have difficulty understanding speech in noise, talking on the telephone, and appreciating complex sounds such as music. Although there have been advances in electrode design, the basic principle has remained essentially unchanged since the first implants were developed in the 1960s: a linear electrode array with a variable number of leads inserted along the length of the cochlea from the base of the cochlea at or near the round window.

[0227] Currently available standard electrode designs in the United States include 12-lead (MED-EL), 16-lead (Advanced Bionics), and 22-lead (Cochlear Ltd). In theory, a higher number of channels leads to increased stimulation specificity and improved performance. In practice, current spillover and cross-stimulation between electrodes limits the benefit of additional channels. All modern designs leave a space between the electrode and the auditory nerve. This allows current to spill over into the surrounding electrolyte-rich fluid, thus reducing the number of electrodes that can be independently activated at a given time. This problem, combined with inconsistencies in electrode placement, has necessitated the development of image-guided programming techniques; these selectively inactivate electrodes that are likely to interfere with each other, thereby reducing crosstalk, but also limit the number of channels available to convey auditory information. It is estimated that approximately 30-50 independent channels are needed to approximate normal hearing, but current technology often achieves fewer than 10. Increasing the number of independent channels available for stimulation is necessary for more challenging listening situations, such as speech understanding in noise and music listening. Attempts have been made to reduce the distance between the lead and the spiral ganglion cells by pre-bending the electrodes, but there has been no significant increase in clinical outcomes, and pre-bent electrodes are more likely to cause damage to the delicate structures of the cochlea during insertion than lateral electrodes. Another method being tried to reduce the potential for current spillover is to induce neuronal growth between the electrode and the remaining spiral ganglion cells. In vitro studies have found several factors that can promote neurite outgrowth in cultured spiral ganglion cells, including growth hormone, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, and erythropoietin. In addition, coatings on the electrode itself have been evaluated, including, among others, polymer or silicone fibers to prevent fibroblast growth, as well as oriented collagen (38) and microtexturing to promote neurite outgrowth.Specifically, laminin-coated electrodes have been found to promote "neurite outgrowth through the bony spiral lamina and into the scala tympani," BDNF gene transfer could induce "regeneration of [SGN] fibers throughout the fiber track in the bony spiral lamina, as well as ectopic branching into both the scala tympani and scala media," and chronic administration of growth factors into the scala cochlea "induced fibers to penetrate through the canaliculi perforantes at the modal wall and even into the scala tympani." Light stimulation has also been proposed as an alternative to avoid current spillover and has been shown to have better temporal and dynamic range characteristics for auditory stimulation, but induction of light sensitivity in native cells in the cochlea and introduction of a light source have proven to be significant technical challenges. Although these methods show promise in the laboratory, none have yet reached clinical applicability. It appears that a limit has been reached in the hearing fidelity achievable through current cochlear implant electrode designs, and fundamental advances in electrode technology are required to provide a hearing experience that can approximate natural hearing.

[0228] Central implant: For patients who cannot benefit from cochlear implantation, there is currently one central implant approved in the United States. This device is FDA-approved for patients with neurofibromatosis type II (NFII) over the age of 12, but it could potentially benefit patients who cannot have a cochlear implant for anatomical reasons, such as those whose cochlear nerve has been severed or damaged due to surgery or trauma, whose cochlea is inaccessible due to scarring or ossification, or whose cochlea has been removed to provide surgical access to a base of the skull tumor.

[0229] Auditory brainstem implants (ABIs) are based on the cochlear implant platform, but instead of a linear array intended for insertion into the cochlea, there is a flat array of electrodes placed against the surface of the brainstem, intended to stimulate second-order neurons in the cochlear nucleus (CN). In contrast to cochlear implants, which have been implanted in hundreds of thousands of patients, only slightly more than 1000 ABIs have ever been placed worldwide. Although a small number of patients achieve open set speech (understanding speech without visual cues), the majority of patients rarely achieve more than sound awareness. Most patients implanted with these devices have large vestibular schwannomas that distort the normal anatomy of the brainstem, which can adversely affect the accuracy of placement. Patients implanted with ABIs for other indications have somewhat better outcomes, but in most cases the results do not match those of cochlear implants.

[0230] To circumvent some of the anatomical problems associated with insertion into the cochlear nucleus and to introduce a novel electrode design, an alternative central implant has been created. The Auditory Midbrain Implant (AMI) has an electrode intended to be implanted within the inferior colliculus (IC), a single shank with 20 ring electrodes along its length. Preclinical data was promising, but in a clinical trial with five NF-2 patients, none achieved open-set speech recognition, although some sound awareness was achieved. This device is still investigational and not clinically available.

[0231] Criticism of both types of electrode arrays centers on their inability to specifically interact with the complex 3D anatomical structure of the CN or IC. Even the penetrating shank electrodes of AMI can only provide separate stimulation at different depths along a single axis, but not along its length or width. Although both IC and CN have a basic frequency-specific structure, complex sounds such as speech may not follow this simplified model. Reproducing these complex sounds will likely require precise simulation throughout the volume of the nucleus. To overcome this limitation, alternative electrode designs with both surface and penetrating electrodes (called penetrating ABI or PABI) have been attempted. Penetrating electrodes were found to require lower charge levels to achieve auditory sensations than surface electrodes, but few were able to provide any auditory sensations, and PABI patients performed rather poorly compared to patients implanted with standard ABI surface electrodes. Thus, no central implant has ever been able to approach the outcomes of cochlear implants, and in many cases only sound awareness is achieved.

[0232] The theoretical advantage of central implants over cochlear implants is that the delicate 3D anatomy of the cochlea is highly susceptible to damage during electrode insertion and to the chronic inflammatory response common with long-term placement of foreign bodies, both of which can result in loss of residual hearing. Bypassing the cochlea to place a central implant avoids these problems and may potentially allow the implant to enhance natural hearing early in the hearing loss process. This advantage is offset by the increased morbidity of brainstem access. Advances in minimally invasive skull base techniques may overcome this limitation and make central implantation feasible for patients who are not adequately benefited from hearing aids, with similar surgical risks as current cochlear implants.

[0233] Regenerative medicine techniques for hearing restoration:Acoustic energy entering the ear is converted to electrical signals in spiral ganglion neurons via inner hair cells (IHCs) located in the organ of Corti. Acquired hearing loss is most often secondary to the deterioration and eventual loss of these cells, which in humans have no ability to regenerate. There are also certain rare forms of hearing loss that may respond to medical intervention. Sudden deafness is a sensorineural hearing loss that develops over hours to days and is thought to result from a viral invasion of the inner ear and a subsequent inflammatory response. If detected early, there is some evidence that steroids may improve the chances of recovery. Similarly, immunosuppression may restore hearing or prevent further hearing loss in patients with autoimmune inner ear disease. This is an extremely rare disorder characterized by bilateral progressive hearing loss that is responsive to steroid treatment. The disease may occur alone or as part of a more systematic hearing loss. Both of these conditions are rare, and sensorineural hearing loss is generally considered irreversible with treatments available in the clinic today.

[0234] Gene therapy for hearing restoration has been proposed for several potential goals, including preserving or regenerating hair cells and auditory neurons in the cochlea, or treating genetic forms of hearing loss. These techniques require the ability to introduce therapeutic agents into the inner ear without causing additional damage, and several novel nanotechnology techniques have been developed to address this constraint. Stem cell-based therapies, including induced pluripotent stem cells, have shown promise for the regeneration of both hair cells and auditory neurons. Currently, there is one ongoing US gene therapy clinical trial utilizing CGF166, a recombinant adenovirus 5 (Ad5) vector containing human Atonal transcription factor (Hath1) cDNA, but no gene or cell-based therapies have been approved for clinical use at this time.

[0235] Live Electrode Technology:The inventors of this disclosure have pioneered techniques to create anatomically inspired micro-tissue engineered neural networks (micro-TENNs) for reconstructing brain pathways (Struzyna, LA et al. Tissue Eng Pt A 21, 2744-2756 (2015), Cullen, DK et al. Tissue Eng Pt A 18, 2280-2289 (2012), Struzyna, LA et al. J Vis Exp Jove e55609 (2017) doi:10.3791 / 55609, Harris, JP et al. J Neural Eng 13, 016019 (2016)). These are implantable, tissue engineered "living scaffolds" that can promote regeneration of the central nervous system. The constructs are generated in vitro by seeding modest-sized populations of neurons into soft hydrogel scaffolds. The neuronal phenotype and composition of the hydrogel scaffolds are customized for the application of interest. Neurons contained within these scaffolds extend neurites along preformed channels within the scaffolds, while the cell bodies remain in a defined location. Conventional electrodes induce currents that depolarize the target tissue for all cell bodies within the volume surrounding the electrode lead, while the axons of each implanted neuron in the micro-TENN synapse directly onto the cell bodies of one or more neurons in the target tissue, thereby eliminating the potential for current spillover. The construct is placed such that the axons are directed to the location of interest within the brain parenchyma, while the cell bodies remain accessible at the surface (Figure 1). The non-organic components of the neural interface, including the conventional electrodes or optrodes used to stimulate the implanted neurons, remain outside the brain parenchyma. In some embodiments, this technique causes much less trauma upon implantation (58) and is less likely to cause chronic inflammation from the biomechanical mismatch of conventional electrodes.

[0236] In some embodiments, this configuration of micro-TENNs, combined with synapse-specific stimulation where axons interact with native neurons, creates an accessible interface for stimulating or recording neuronal tissue—also referred to herein as essentially a “living electrode” (LE), in some embodiments, as a system comprising a biocompatible construct and multiple auditory neurons. In some embodiments, the construct is further functionalized via transduction with channelrhodopsin to allow light-based activation or monitoring of accessible neuronal somata (FIGS. 2A-2F). FIG. 3 shows an example of a micro-TENN that was generated—in this case built—to restore dopaminergic axonal input in a Parkinson's disease patient. Using multiple neuronal subtypes, micro-TENNs have been successfully generated that include primary cortical (glutamatergic) neurons, dorsal root ganglion neurons, GABAergic neurons, and dopaminergic neurons.

[0237] Importantly, the application of this technology to the auditory system has yet to be explored, and has specific advantages over current central and cochlear implants in several key ways. In some embodiments, the most direct transition from existing efforts is to a central implant. In some embodiments, hydrogel scaffolds can be designed with the complex 3D anatomical structure of the IC or CN in mind. For example, groups of constructs described above can be combined into custom three-dimensional configurations to create arrays with multiple living electrodes. In some embodiments, axons can be guided to synapse at precise locations along the length and width of the nucleus, as well as to precise depths to reach the frequency-specific configuration of either nucleus, while the transplanted neuronal soma remains accessible on the brainstem surface, or even on the skull surface. Previous studies have shown that it is possible to grow axons up to several centimeters in length. This length can be utilized to spatially separate individual aggregates of neuronal soma on the brain surface, while precisely targeting axonal projections to match the more compact three-dimensional arrangement of the IC or CN volume. In some embodiments, the specificity of stimulation has the potential to surpass any option using standard electrode technology. Rather, it has been shown that optical and electrical stimulation may have complementary advantages, and an ideal implant may involve elements of both. Similar principles would apply to the application of this technology to the unique anatomical limitations of the cochlea, but additional innovations as described herein would be required.

[0238] Clinical Transition and Importance:The experiments described herein represent the first steps in applying a fundamentally new regenerative medicine interface technology to hearing restoration. Even with recent significant technological advances in cochlear implants, the resulting hearing experience falls short of normal hearing, and central implants lag even further behind. Due to these performance limitations, there are many patients who suffer from hearing loss that cannot be adequately addressed by hearing aids, but are not candidates for implantation. Given that systems including biocompatible constructs and multiple auditory neurons, such as LE, can be implanted using minimally invasive techniques, and that there is generally no neuronal tissue damage associated with implantation, the systems disclosed herein, such as LE, may be implanted in patients with significant residual hearing, or in patients with tinnitus and hearing preservation as a requirement. In some embodiments, an improvement in the fidelity of hearing provided by the implant, or the ability to be implanted without risk to residual hearing, may provide options for patients living in the gap between hearing aids and cochlear implants, or may have a significant impact on the quality of life of such patients. It is believed that advanced implants for impaired deafness will benefit the entire target population.

[0239] In some embodiments, application of a system comprising a biocompatible construct and a plurality of auditory neurons, such as a living electrode, to the auditory system may enhance the quality of the hearing experience made available with an implantable hearing device.

[0240] In some embodiments, micro-TENNs are constructs, e.g., living 3D neural constructs, consisting of a population of discrete neurons with long projecting axonal tracts encapsulated within small tubular hydrogels (Struzyna, LA et al. Tissue Eng Pt A 21, 2744-2756 (2015), Cullen, DK et al. Tissue Eng Pt A 18, 2280-2289 (2012), Struzyna, LA et al. J Vis Exp Jove e55609 (2017) doi:10.3791 / 55609, Harris, JP et al. J Neural Eng 13, 016019 (2016), Struzyna, L. A et al. Curr Opin Solid State Mater Sci 18, 308-318 (2014), Struzyna, LA, et al Neural Regen Res 10, 679-685 (2015). In some embodiments, the interior of the hydrogel column contains a customizable extracellular matrix (ECM) that is optimized to support neuronal survival and neurite outgrowth in vitro, but degrades in vivo, for example over a period of weeks. While previous attempts at artificial stimulation of the auditory system have utilized light or electrical stimulation, the present disclosure provides a fundamentally different approach in that in some embodiments, direct neuronal synapses are utilized to stimulate native neurons and induce auditory perception. In some embodiments, this provides targeted and specific stimulation that does not suffer from current spillover or field effects. Furthermore, the size and form factors of the construct allow for minimally invasive implantation using stereotactic guidance, and also allow all non-organic components of the system to remain outside of the brain parenchyma. The construct has been shown to be well tolerated, survive, and integrate within the native nervous system. In some embodiments, the disclosure provides the use of spiral ganglion cells to create micro-TENNs and apply these as "living electrodes" for stimulation of central and peripheral auditory pathways.In some embodiments, SGN micro-TENNs may be generated using induced pluripotent stem cells for future applications in humans, e.g., to avoid the limitations of relying on harvested tissue. In some embodiments, living electrode technology offers an innovative approach to auditory system stimulation that may enable hearing restoration beyond current techniques.

[0241] summary: Impaired hearing affects 5% of the world's population and is one of the most common health concerns for US veterans. Highly specific stimulation of the auditory system is not possible with today's technology, and the hearing experience with implantable hearing devices falls far short of normal hearing. Although refinements in technology and surgical techniques have improved outcomes for cochlear implant recipients, there appear to be limitations to the performance achievable with current designs. In some embodiments, the present disclosure provides for the application of living electrode technology, such as for restoring hearing via implantation, e.g., in the cochlea or inferior colliculus, of a biocompatible construct, e.g., a hydrogel construct, pre-seeded with, e.g., spiral ganglion cells. In some embodiments, the present approach has the potential to restore hearing for patients suffering from, e.g., hearing loss, and to change how hearing loss is treated.

[0242] Example 2: Use of a system including a biocompatible construct and a plurality of auditory neurons in an auditory setting This example describes the application of a system, such as a living electrode, comprising a biocompatible construct and a plurality of auditory neurons in an auditory setting.

[0243] Spiral ganglion cell collection and culture: Spiral ganglion cells were harvested from the temporal bones of P0 Sprague-Dawley rat pups. To isolate spiral ganglion cells, cochleae were isolated under a stereoscope by microdissection. After removal from the cochlea, the spiral ganglion was either further sectioned into 200–300 μm segments for explant culture (Figure 4) or enzymatically dissociated after gentle micropipette trituration. For the latter, the spiral ganglion 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 before removing the DNase and resuspending the cells in neuronal medium. Neurons were plated onto polystyrene Petri dishes soaked overnight in PLL (0.05 mg / mL, sterile cell culture water solution). Petri dishes were then rinsed in sterile cell culture water and soaked overnight in laminin (20 μg / mL, sterile cell culture water solution) before plating and culturing. Cells were then incubated under standard cell culture conditions (37°C, 5% CO2). Half-medium changes were performed every 2 days in vitro (DIV). Histological examination was performed as described below. The medium consisted of Neurobasal medium, B-27 (1x), glutamax (0.5 mM), penicillin-streptomycin (20 U / mL), and BDNF (5 ng / ml). Representative aggregates are shown in Figure 4.

[0244] To examine the ability of distinct SGN populations to interact with each other, spiral ganglia were isolated and aggregated as described above. Aggregates were then plated onto polystyrene Petri dishes precoated with PLL (50 μg / mL) and laminin (20 μg / mL) and grown in medium (as described above) at 37°C, 5% CO2, with half-medium changes every 48 h. After positive attachment of aggregates to the medium surface and axonal outgrowth was confirmed by phase contrast microscopy up to 5 DIV, aggregate cultures were virally transduced overnight to express the fluorescent calcium reporter GCaMP6 (Addgene / Penn Vector Core) (titer: approx. 5x109 vg / mL) to enable optical imaging of neuronal activity. After transduction, aggregates were grown and GCaMP expression / neuronal activity was monitored using a Nikon Eclipse Ti-S microscope coupled with a QIClick camera and NIS Elements BR 4.13.00. Recordings were acquired at 10–15 frames per second to visualize calcium transients.

[0245] Synchronous and spontaneous signaling activity was observed between adjacent aggregates, indicating functional synapse formation between two distinct populations of SGNs (Figures 5A-5D).Similarly, synchronous and spontaneous recordings were measured between SGNs and aggregates of central (both cortical and brainstem) origin.

[0246] Fabrication of SGN micro-TENNs: Micro-TENNs were constructed in a three-step process as described above. Briefly, 3% agarose micro-columns were molded with glass capillaries and acupuncture needles to the desired size (outer diameter: 398 μm; inner diameter: 160 μm) and cut to the desired length. The formed micro-column channels were sterilized under UV light (30 min) and filled with ECM containing rat tail type I collagen (1.0 mg / mL) and mouse laminin (1.0 mg / mL).

[0247] To seed cultured SGNs onto micro-TENNs, cells were dissociated and single-cell suspensions were made into spherical aggregates by centrifugation (200 x g, 5 min) in inverted pyramidal wells made of PDMS. After centrifugation, SGN aggregates were incubated in neuronal medium for 48 h and then carefully placed on one end of an ECM-filled micro-column using precision tweezers under a stereoscope. Micro-TENNs were grown in neuronal medium with half-medium replacement every 2 DIV.

[0248] Immunocytochemistry: Plate cultures and micro-TENNs were fixed in 4% formaldehyde for 35 min, permeabilized with 0.3% Triton X100 + 4% horse serum in PBS for 60 min, and incubated with primary antibodies overnight at 4°C. After primary antibody incubation, samples were incubated with fluorescently labeled secondary antibodies (1:500; Life Technologies & Invitrogen) 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. Samples were imaged on a Nikon A1RMP+ multiphoton confocal microscope coupled with NIS Elements AR 4.60.00. For each fluorescence channel, consecutive slices of 10-20 μm in the Z plane were acquired. All confocal images presented are maximum intensity projections of confocal z slices. Primary antibodies were Tuj-1 / beta-III tubulin (1:500, T8578, Sigma-Aldrich) for labeling axons, peripherin (1:500, ab4666, Abcam) for labeling type II SGNs, and / or glial fibrillary acidic protein (GFAP) for labeling astrocytes (1:500, ab53554, Abcam). This demonstrated that we were able to generate micro-TENNs using SGN aggregate seeding in micro-columns (Figures 6A-6E).

[0249] Cochlear Constructs: A novel technique was developed to create constructs more amenable to implantation within the curved structure of the cochlea. Single-lumen spiral microcolumns with curvatures up to 270 degrees were designed via computer-aided modeling (Autodesk Inventor, Autodesk Inc.) using the human cochlear geometry as a reference. All variants were modeled with an outer diameter of 1.5 mm, an overall diameter of 7 mm, and a vertical pitch of 1 mm. Single-lumen constructs were molded with one lumen of 850 μm diameter and 3D printed by extrusion bioprinting (BioAssemblyBot, Advanced Solutions) from Pluronic hydrogel (28.6% w / w, Advanced Solutions). The geometry of the support material was automatically generated as needed by the bioprinter front-end (TSIM, Advanced Solutions) and printed from the same Pluronic hydrogel prior to printing the microcolumn itself. Printing parameters were optimized to achieve sustained extrusion and minimize gap formation or overprinting (nozzle pressure 55 psi, nozzle speed 4 mm / s, line width 0.159 mm, layer height 0.05 mm, nozzle diameter 0.159 mm). To characterize build fidelity, constructs were printed directly onto leveled 40x70 mm glass microscope slides (Fisher) for 6 minutes, measured, and imaged. Because Pluronic hydrogels are opaque and therefore difficult to image by optical microscopy, constructs were printed to 50, 75, and 100% completion so that the inner and outer dimensions could be directly verified with digital calipers (Mitutoyo) throughout. Constructs were then imaged (D3400, Nikon); constructs at 50 and 75% completion, including the deposited support material, are presented for reference (Figures 7B and 7C). All printed constructs were determined to be within ±100 microns of the model specifications in all dimensions listed.

[0250] Implantation: In vivo implantation of live electrodes was performed in the IC of live rats. Adult male Sprague-Dawley rats were anesthetized and immobilized in a stereotaxic frame. An incision was made above the skull and a craniotomy was performed above the inferior colliculus. The live electrode was inserted using stereotaxic coordinates such that its terminals were located within the central nucleus of the inferior colliculus and the cell body aggregates remained accessible at the surface of the cerebellum. Animals were healthy at 3 weeks, intravital multiphoton microscopy revealed live cells within the implanted aggregates, and spontaneous firing was monitored by high-speed fluorescence microscopy of spontaneous activity of GCaMP-expressing neurons.

[0251] The data disclosed herein show that cultured SGNs can be isolated, that SGN aggregates can be maintained in culture, and that interactions between distinct SGN cell populations can be induced. Furthermore, axonal growth through hydrogel microcolumn scaffolds was successful, and pre-bent constructs for cochlear implantation could be developed. Furthermore, implantation of living electrodes into ICs was performed, and both constructs and animals were viable at 3 weeks. SGN cells were shown to behave similarly to previously used cortical-derived neurons and to interact in vitro with both centrally derived neuronal aggregates; this indicated a high probability of success for further studies. Importantly, these data show that living electrodes can be reliably fabricated and implanted with SGNs.

[0252] Study design and methods Overview and hypotheses:Current interfaces for hearing rehabilitation suffer from significant shortcomings. Cochlear implants, while clinically successful, have relied on similar electrode designs since inception and have achieved only about 20% of the independent channels required to approximate normal hearing. Central implants are inferior and provide little improvement over sound awareness in the majority of cases. One of the major limitations to the advancement of these technologies is the interface between the electrode and the neural tissue. This disclosure provides a new technology that promises to change the nature of machine-neural interfaces for artificial stimulation of the auditory system through bioengineering and regenerative medicine techniques. One hypothesis is that living electrode technology can be applied to hearing rehabilitation via integration into the cochlea and inferior colliculus. We developed two separate micro-TENN designs that act as living electrodes that, when seeded with spiral ganglion cells, are implanted either in the inferior colliculus or in the cochlea of ​​rats. This work builds on previous work on living neuronal scaffolds and their application for the restoration of central nervous system function after trauma and neurodegenerative diseases. This example utilizes a novel cell type for seeding the scaffold (spiral ganglion neurons instead of centrally derived neurons) and develops a new form factor of the microcolumn scaffold designed to be placed in the cochlea rather than centrally as before. In some embodiments, one of the outcomes proposed in this study is a two-biohybrid neural interface. In some embodiments, SGNs are pre-grown on an implantable scaffold designed to be inserted centrally into the inferior colliculus or peripherally into the cochlea. In some embodiments, both constructs can orient neurites from the transplanted SGNs to interact with the target tissue, while the cell bodies of the transplanted SGNs remain accessible for stimulation - thereby creating a so-called "living electrode" for auditory stimulation. The techniques developed in this process also serve as the basis for multiple avenues of future research in regenerative medicine bioengineering that will be applied to broaden our understanding of the hearing process and ultimately restore hearing.

[0253] Scientific evidence:There are no clinically available treatments to reverse damage within the cochlea to restore hearing. Although hair cell regenerative medicine techniques are improving, there are significant hurdles to overcome. The physical structure of the cochlea must be re-established, and degenerated spiral ganglion neurons must be induced to regenerate and synapse with new hair cells. Currently, the most successful strategy for the rehabilitation of profound hearing loss is to directly stimulate the cochlear nerve, bypassing the complex structure of the cochlea entirely. This approach has in many cases enabled patients with significant hearing loss to regain speech comprehension without lip reading, but still falls far short of normal hearing. One of the main limitations of current technology is positioning the implanted electrode close enough to the spiral ganglion cells to limit current spread along the cochlea and the subsequent loss of fidelity of the transmitted signal. Numerous methods have been attempted to reduce this distance, but none have had significant clinical impact so far. The direct interaction with these cells and increased selectivity of stimulation is believed to create the potential for dramatic improvements in hearing outcomes after implantation. The present disclosure combines tissue engineering and regenerative medicine techniques for hearing restoration. In some embodiments, the technology of the system disclosed herein, e.g., living electrodes, overcomes some of the limitations of current options. First, instead of standard electrodes in the cochlea that receive current spillover, direct synaptic stimulation of native neuronal tissue is achieved in some embodiments. In some embodiments, this increases the number of independent channels available for stimulation, thus increasing the fidelity of the hearing experience. Second, previous studies have shown the safety of implantation in the brain parenchyma, potentially enhancing hearing without the risk of further hearing loss due to damage to natural structures. In some embodiments, designing constructs that keep in mind the complex 3D anatomy of the inferior colliculus or cochlear nucleus in a well-tolerated package may allow for central implant hearing outcomes to approach those of cochlear implantation. Furthermore, successful demonstration of the living electrode concept is believed to open the door to further applications such as restoring vestibular function or vision.

[0254] This example describes three objectives to develop living electrodes for implantation within the peripheral or central auditory system. The first objective focuses on optimizing the recovery and culture of spiral ganglion neurons from fetal or neonatal rats; seeding these cells onto microcolumn scaffolds designed for implantation into either the cochlea or inferior colliculus; and describing the optical stimulation parameters of the cell aggregates. The second objective is to characterize the in vivo behavior of the transplanted cells. The objectives of these studies are to quantify cell survival and characterize the behavior of the transplanted neurons. The final objective is to quantify the effects of stimulation of these scaffolds implanted onto the auditory system via electrophysiological recordings and innate behavioral models in the auditory cortex, and to compare living electrodes to standard electrode stimulation.

[0255] Biofabrication of living electrodes using spiral ganglion neurons and validation of optical control of their function in vitro (a) Spiral ganglion neurons (SGNs) recovery and culture, and assessment of in vitro survival, synaptogenesis, and optogenetic transformation; and (b) in vitro seeding of SGNs onto custom microcolumn scaffolds. SGNs can be recovered from the temporal bones of neonatal rat pups, cultured, transduced for optical stimulation, and induced to synaptically form in vitro with distinct SGN- and centrally derived neuronal populations. These cultured SGNs can then be grown within custom microcolumn scaffolds to form living SGN electrodes designed for cochlear or central implantation.

[0256] Given their natural anatomical and physiological position in the auditory pathway, we utilized spiral ganglion cells as the primary neuronal population for these studies. Several groups have reported successful recovery and culture of spiral ganglion cells from prenatal and postnatal rodents. However, significant differences exist in the techniques used for these cultures, including whether the cells are dissociated prior to plating. Variables for optimization include the timing of SGN recovery, whether the spiral ganglia are cultured intact or dissociated, and the duration of culture prior to plating. All of these conditions are tested, along with immunohistochemistry to assess cell viability, the proportion of neurons of interest (relative to supporting cells) in culture, and the propensity for neurite outgrowth. Viability is assessed with calcein AM (to label live cells) and ethidium homodimer (to label nuclei of dead cells), and neurite outgrowth is assessed by immunohistochemistry (using antibodies that recognize axonal cytoskeletal components).

[0257] SGC recovery and culture: To isolate spiral ganglion cells, cochleae are isolated from Sprague-Dawley rats at specific embryonic and neonatal time points (E18, P0, P5, P10) under a stereoscope by microdissection. After removal from the cochlea, the spiral ganglion is either further divided into 200-300 μm segments for explant culture or enzymatically dissociated after gentle micropipette trituration. For the latter, the spiral ganglion is dissociated in 0.25% trypsin + 1 mM EDTA at 37°C, followed by removal of trypsin / EDTA and replacement with 0.15 mg / ml DNase in HBSS. The dissociated tissue is centrifuged with DNase at 3000 RPM for 3 minutes, before removing the DNase and resuspending the cells in neuronal medium. Neurons are plated on polystyrene Petri dishes soaked overnight in PLL (0.05 mg / mL, sterile cell culture water solution). Petri dishes are then rinsed in sterile cell culture water and soaked overnight in laminin (20 μg / mL, sterile cell culture water solution) prior to plating and culturing. They are then cultured under standard cell culture conditions (37°C, 5% CO 2) and incubate the cells under 5% CO. Half-medium changes are performed every 2 DIV. As described in preliminary data, the medium consists of Neurobasal medium, B-27 (1x), glutamax (0.5 mM), penicillin-streptomycin (20 U / mL), and BDNF (5 ng / mL). Similar techniques to the isolation protocol described here were used for the recovery and culture of centrally derived neurons, except that brain cortex tissue was collected instead of spiral ganglion tissue.

[0258] Different media formulations for SGN culture have been reported in the literature; further optimization will require direct comparison of growth, neuronal survival, and function between SGN cultures and / or constructs grown in different media. BDNF has been shown to promote SGN survival both in vitro and in vivo, suggesting its potential in functional reinnervation (69, 70). However, the benefit of BDNF may depend on both the concentration and the age of the SGNs, as determined by the embryonic / postnatal isolation timepoint, and may affect type II vs. type I SGNs predominantly. In addition to media and conditions, in some embodiments, the aggregation protocol will also require optimization to determine the best centrifugation speed, duration, and post-centrifugation incubation period for SGN constructs. Additionally, the micro-columns may be seeded with either aggregates or similarly sized spiral ganglion explants, and it remains an open question how the choice will affect the growth of the constructs.

[0259] To test whether axon-axon interactions are sufficient for physiological connectivity of separate SGN aggregates, following isolation and aggregation of spiral ganglia as described above, spiral ganglion aggregates are seeded into agarose micro-columns to generate bidirectional (i.e., two aggregates) micro-TENNs. As a control for potential growth constraints inside the micro-TENN architecture, aggregates are also plated in 2D culture. After at least 7 DIV of culture, micro-TENNs and control aggregates are fixed with formaldehyde for fluorescent immunolabeling. The antibodies selected target presynaptic (synaptophysin) and postsynaptic (PSD-95) markers, as well as neuronal cell bodies and axons (beta-tubulin-III). Immunolabeled samples are imaged using confocal or two-photon microscopy, with axon-axon synapses identified through overlap of synaptic markers with axons. Further validation of axonal synapses through immunolabeling of dendrites (e.g., with MAP-2) may be indicated to further distinguish axonal synapses from axon-dendritic and axon-somatic synapses.

[0260] Optogenetic transduction: Two optogenetic transductions are performed; one to report activation monitoring and one to allow light stimulation. For calcium imaging, SGNs are transduced with an AAV vector (Penn Viral Vector Core) carrying the GCaMP6 gene for 16–24 h. A full medium exchange is then performed to remove any remaining viral particles. Calcium activity can be observed using standard fluorescence or confocal microscopy starting 4–5 days after viral transduction. This activity is then recorded as a movie using the microscope software (NIS-Elements, Nikon). Fluorescence activity is then measured using regions of interest drawn manually in NIS-Elements or using automated Matlab scripts. For optogenetic stimulation, 1.34 x 10 SGNs are transduced with an AAV vector (Penn Viral Vector Core) carrying a light-activated opsin gene (e.g. ChR2, CrimsonR). 10SGNs are transduced for 16–24 h with titers of genomic copies / mL. Stimulation experiments are delayed for approximately 2 weeks after viral transduction to allow the opsin protein to mature. Light stimulation is performed using an LED array consisting of LEDs of appropriate wavelengths coupled to a 200 μm optical fiber (Thorlabs). The system is driven by a multichannel LED driver (Thorlabs) and a digital acquisition board (National Instruments). Stimulation parameters are defined below. Ca 2+ Cells transfected with light-dependent activity reporters can be used to monitor neuronal activation in real time, while cells with light-activated opsins can be stimulated using light instead of electrical stimulation (see examples in Figures 2A-2F).

[0261] The techniques disclosed herein rely on the ability of recovered SGNs to interact with and stimulate native SGNs in the host cochlea. SGN-SGN interaction and stimulation has not previously been reported to be possible. It has been shown that peripherally derived neurons can be induced to perform this behavior (e.g., dorsal root ganglion neurons synapsing with other dorsal root ganglion neurons), and synchronous passive neuronal activity between neighboring SGN populations has been observed. To test this further, one SGN population was injected with Ca 2+ In one experiment, a population of SGNs is induced to carry light-dependent activity while another population carries light-activated opsins. The two populations are induced to form spherical aggregates (see below) and cultured in close proximity to each other. Axon outgrowth is monitored. Once an interaction between the two populations is demonstrated, the Ca ion release upon optical stimulation of the opsin population is monitored. 2+ The first group will be monitored for Ca-dependent light activity. 2+If dependent light activity is confirmed, it indicates an interaction between the two populations and that the axons of transplanted SGNs can induce the activity of native SGNs in vivo. As an indication that transplanted SGNs can excite native neurons in the inferior colliculus (and thus better represent the in vivo circuitry), a similar experimental design tests the ability of SGNs to interact with and induce the activity of the centrally derived population.

[0262] Light Stimulation Parameters: Although the response of in vivo transduced auditory system neurons to optogenetic stimulation has been assessed in previous studies, it is possible that cell aggregates formed for LE generation may perform differently than transduced native neurons. To assess this, aggregates are cultured in close proximity to each other as described above. Light stimulation patterns derived from previous auditory system optogenetic studies are then performed to establish activation thresholds, as well as frequency and intensity response curves. Stimulation of opsin-transduced aggregates is via 473 nm pulses of light applied via the optical fiber described above, positioned 2 mm from the aggregate of interest. Light intensity is calibrated by positioning the fiber 2 mm from a high-sensitivity thermophile sensor connected to a power meter. Stimulation is performed with exposures ranging from 1 to 20 uJ / mm 2 , pulse duration 0.1-10 ms, frequency 0-70 Hz, while recording the luminescence from the GCaMP-derived aggregates. Generate response curves to determine threshold (above 10% of background) and maximum for each of the parameters of interest. Repeat this test for all combinations of reporter aggregates with stimulated SGNs and central neurons (cortex and brainstem). Perform control tests in similar culture conditions, omitting opsin transduction in the stimulated aggregates.

[0263] The experiments described herein are expected to optimize harvesting and culture conditions that provide consistent, high-quality populations of SGNs. Furthermore, co-culturing separate populations of these neurons in close proximity induces axonal elongation and synapse formation between them, which is related to Ca 2+ It is expected that the signal transduction from the ChR2-dependent photic activity will be detectable through the ChR2-dependent photic activity. This may suggest that the transplanted SGNs have the capacity to synapse with native SGNs in the cochlea of ​​living rats. Similar behavior is expected when SGNs are co-cultured with centrally derived neurons in anticipation of central implantation. The latter pair will then be reversed to test the ability of the centrally derived neurons to induce depolarization in SGNs. This may allow for increased yield of constructs, as the number of central neurons obtained from a given animal will far exceed that of SGNs. Furthermore, the use of photic stimulation to activate spiral ganglion cells is well established, and responses to variations in input stimuli are predictable. Given the reported success of photic stimulation of the auditory pathway, it is predicted that ChR2-transfected neuronal aggregates may exhibit similar stimulation parameters.

[0264] After the above steps are successfully completed, the SGN population is seeded into the microcolumn scaffold. Two custom hydrogel scaffolds are developed. In some embodiments, the first scaffold is used for implantation into the inferior colliculus and is similar to the one used previously. This scaffold allows the neurites of the implanted SGNs to synapse with the inferior colliculus while allowing their cell bodies to remain on the surface and accessible for recording or stimulation. In some embodiments, the second scaffold is similar but also conforms to the curvature of the cochlea and guides the SGN axons towards the native spiral ganglion cells that run through the modiolus.

[0265] Design of the construct: In some embodiments, the scaffolds contain 3D hydrogel microcolumns designed to guide the extension of neurites from transplanted SGNs to interact with native neuronal tissue while the cell bodies of these SGNs remain accessible. The microcolumns contain 3% agarose with a collagenous ECM inside to allow neurite attachment and extension. To create these scaffolds, the agarose is dissolved in warmed Dulbecco's phosphate buffered saline (DPBS). A mold of the appropriate shape for the external dimensions of the scaffold is then made, and a fine needle is used to create a space that will become the lumen of each microcolumn. The mold is then filled with dissolved agarose and allowed to gel. The mold is then removed, leaving an agarose shell with a hollow bore. The microcolumns are then sterilized by UV light exposure for 15 minutes. The microcolumns are then filled with a collagen ECM solution (rat tail type I collagen in neuronal growth medium, 0.2-2.0 mg / mL). The collagen is then polymerized and seeded with cells that are cultured in a tissue culture incubator (37° C., 5% CO2). Previous studies constructing hydrogels have noted that the physical properties of the scaffold allow deformation without cracking or buckling, allowing some conformity to the shape of the cochlea beyond a preformed shape. In some embodiments, 3D printing technology is utilized for additional customization of the construct design.

[0266] SGN seeding on custom scaffolds: To seed cultured SGNs onto micro-columns, cells are dissociated and single-cell suspensions are made into spherical aggregates by centrifugation (200 x g, 5 min) in inverted pyramidal wells made of PDMS (Figure 8). After centrifugation, SGN aggregates are incubated in neuronal medium for 48 h and then carefully placed at one end of an ECM-filled micro-column using precision tweezers under a stereoscope. The resulting constructs are then grown in neuronal medium as described above (with half-medium replacement every 2 DIV) to form living SGN electrodes.

[0267] Given the unique physical constraints of the cochlea, multiple scaffold geometries are fabricated and tested on harvested live rat cochleae with various implantation techniques. In vivo insertion of microcolumns is performed to simulate insertion into a live animal, and the cochlea is histologically analyzed for placement location and damage induced upon insertion. Areas to optimize for the live electrode include microcolumn diameter and shape; technique for 3D printing custom microcolumn geometries; type and concentration of ECM to fill the microcolumns; seeding density of SGNs; and timing of culture. The portion of the construct housing the SGN cell bodies remains within the otic bulla of the temporal bone for cochlear implantation and on the brainstem surface for IC implantation, allowing access for recording and stimulation.

[0268] Once the final configuration is established for the micro-columns, SGNs are seeded and grown for 1, 2, 4, 7, and 14 DIV prior to histological analysis. At given time points, neurite number, neurite length, and cell viability are assessed. Constructs are then fixed in 4% formaldehyde and permeabilized with Triton X. Tissues are stained with antibodies to specifically assess axonal growth; these include MAP-2, a microtubule-associated protein found in dendrites, β-tubulin III, an abundant microtubule element in axons, and glial fibrillary acidic protein, an intermediate filament protein expressed in astrocytes, as previously described. To characterize growth in SGN constructs, phase images of live SGN electrodes are taken using a Nikon Eclipse Ti-S microscope coupled with a QIClick camera and NIS Elements BR 4.13.00. The longest neurite is manually identified within each phase image using ImageJ (National Institutes of Health, MD). The change in length of this longest process at each time point divided by the number of DIVs since the previous time point is quantified as the growth rate. All length measurements are normalized as the longitudinal distance of the end of the longest process relative to the edge of the source aggregate (identified 1 DIV).

[0269] The fabrication of living SGN electrodes for the inferior colliculus is similar to those previously fabricated for applications in the nervous system. In some embodiments, it is expected that living electrodes for the cochlea can be fabricated in a manner that allows them to conform to the anatomy of the cochlea - through a combination of pre-designed shapes and flexibility of the construct. In some embodiments, the harvested SGNs are expected to be seeded onto the scaffold with high viability and to exhibit neurite outgrowth at a rate similar to previous studies with centrally derived neurons.

[0270] Demonstration of in vivo delivery, survival, and integration of living electrodes In vivo implantation of (a) cochlear and (b) central living SGN electrodes and assessment of survival and integration. In some embodiments, living SGN electrodes can be implanted into the cochlea or inferior colliculus of rats, and the transplanted neurons can survive and synapse with appropriate target neurons.

[0271] Implantation: After successful seeding of the microcolumns and sufficient evidence of SGN viability and neurite outgrowth, live electrodes optimized as described above are implanted in live rats. For cochlear implantation, the scaffold is inserted through the round window of the cochlea of ​​anesthetized rats (or through an alternative approach to the cochlea as described below) similar to the protocol previously described for more standard electrodes. Rats are anesthetized with isoflurane, the hair behind one ear is removed, and the area is cleaned with betadine. The otocyst bulla of the temporal bone is exposed by a postauricular incision and dissection of the underlying musculature. Next, with the aid of an operating microscope, the otocyst bulla is drilled away to expose the round window. It is anticipated that insertion will occur through the round window. If so, the round window fossa is removed and the round window membrane is incised. The construct is then gently advanced through the round window until resistance is encountered or the construct is fully inserted. The aggregate of neuronal cell bodies remains accessible within the otocyst bulla. The post-auricular skin incision is then closed.

[0272] For insertion into the inferior colliculus, rats are anesthetized with isoflurane and mounted in a stereotaxic frame. After shaving the head and cleaning with betadine, an incision and a small craniotomy are made above the inferior colliculus. A live SGN electrode is then drawn into a Hamilton syringe mounted on the stereotaxic frame and delivered via needle injection; afterwards, to deliver the construct with minimal force, the Hamilton syringe is immobilized while the needle is slowly lifted. Again, aggregates of neuronal cell bodies remain on the brain surface to allow for subsequent light stimulation. After removal of the needle from the brain, the craniotomy is sealed with bone wax and the skin is sutured. For both protocols, rats are administered meloxicam (2.0 mg / kg) and monitored postoperatively until recovery. This general procedure was performed routinely.

[0273] After implantation, animals are allowed to recover from surgery and survive for 1, 2, 4, 8, or 12 weeks post-implantation to harvest brains or temporal bones at the time points. To assess the ability to monitor physiological activity with the LE, in vivo fluorescence microscopy is performed prior to euthanasia and tissue harvest to quantify spontaneous GCaMP activity from the IC-implanted electrodes. Rats are anesthetized and securely fixed in a stereotaxic frame with a palate bar. Fluorescence microscopy is then performed as described above. The use of GCaMP-induced live electrodes to monitor intrinsic neuronal activity was performed as previously and was used for the successful IC implants described above. Anesthetized animals are also subjected to broadband auditory stimulation during monitoring of cell bodies of live electrodes. In some embodiments, increased activity following auditory stimulation confirms physiological interactions with native neuronal populations in the auditory pathway, and the timing of activation is compared to known timing of ABR signals from the IC. For each time point, the extent of stimulation recording is compared to histological assessment; this may allow for non-invasive monitoring of synaptogenesis in future studies. For histological analysis, animals are perfused transcardially with heparinized saline and then with 10% neutral buffered formalin. The excised brains or temporal bones are rinsed in PBS and then immersed in 30% sucrose. After flash freezing in 2-methylbutane, brains are stored at -80°C or immediately sectioned (20 μm thick). For histological analysis of the cochlea, the cochlea are demineralized and fixed as described previously. For immunohistochemistry, tissues are fixed in 4% formaldehyde for 35 min, permeabilized with 0.3% Triton X100 + 4% horse serum in PBS for 60 min, and incubated with primary antibodies overnight at 4°C. Samples after primary antibody incubation are incubated with fluorescently labeled secondary antibodies (1:500; Life Technologies & Invitrogen) 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. Samples were imaged on a Nikon A1RMP+ multiphoton confocal microscope coupled with NIS Elements AR 4.60.00.For each fluorescence channel, consecutive slices of 10-20 µm in the Z plane were acquired. All confocal images presented are maximum intensity projections of confocal z slices. Primary antibodies were Tuj-1 / beta-III tubulin (1:500, T8578, Sigma-Aldrich) for labeling axons, peripherin (1:500, ab4666, Abcam) for labeling type II SGNs, and / or GFAP (1:500, ab53554, Abcam) for labeling astrocytes.

[0274] Live electrodes will be implanted either intracochleaeally or intracollicularly in live rats. Histological analysis will be performed at 1, 2, 4, 8, and 12 weeks with 10 replicates of each time point. Two additional rats per time point will be used as controls by implanting acellular hydrogel constructs. An additional 5 rats will be used to test alternative approaches to cochlear implants. Therefore, it is estimated that 110 rats (2 groups x 5 time points x 10 replicates + 5 controls + 5 alternative cochlear implants) will be used over the course of the grant.

[0275] In some embodiments, implantation of live SGN electrodes into the inferior colliculus is expected to lead to successful axonal extension and synapse formation on native cells. This technique is similar to that previously performed. As disclosed above, implantation into the IC has already been achieved with viability of both animals and implanted neurons at 3 weeks. Although technically different from central implantation, in some embodiments, SGNs transplanted through the cochlea are expected to survive as well, extend neurites, and form synapses on native spiral ganglion cells. Furthermore, cell viability and axonal growth rate are expected to be similar to previous studies.

[0276] Even if direct axonal interactions within the scala tympani are not yet available, in some embodiments, direct access to the spiral ganglion cell bodies can be achieved through multiple cochlear fenestration procedures as described above or by completely removing the superficial portion of the cochlear otic capsule (Figure 9). In some embodiments, this method may disrupt the natural structure of the cochlea, but potentially allows direct access to the entire length of the spiral ganglion. It may also potentially allow access to the cochlear nerve itself as it enters the cochlea. While these techniques are more applicable to non-human implantation, it would be a major advancement in the technology if implanted live electrodes were shown to interact with native SGNs, while less invasive implantation techniques could be developed. As disclosed above, axonal interactions within cultured SGN populations have already been established. In some embodiments, SGNs may not remain viable after seeding on the scaffold or may not show proper neurite outgrowth, in which case neurotrophic factors may be added to the ECM used to grow the axonal tracts. In some embodiments, if SGNs cannot be induced to grow effectively, centrally or peripherally derived neurons may be used instead. In some embodiments, if native SGN axons can be induced into the scala tympani, but the insertion of the construct through the round window is hindered by the physical constraints of the cochlea, a round window widening or cochleostomy approach is undertaken. In these cases, either an anterior-inferior extension of the round window is drilled or a completely separate anterior-inferior cochleostomy is performed. In some embodiments, if the construct is properly placed and the SGNs remain viable, but there is no evidence of axonal extension along the cochlea with synapse formation onto the SGNs, centrally derived neurons may be utilized instead of SGNs. In some embodiments, altering the composition of the ECM contained within the lumen of the living electrode or introducing neurotrophic factors may also induce improved synaptic interactions.

[0277] Assessing the effectiveness of living electrodes to transmit auditory input to the brain Neuroelectrophysiological and behavioral evaluation of implanted living SGN electrode stimulation In some embodiments, after implantation and integration of living SGN electrodes within the cochlea or inferior colliculus of rats, either electrical or optical stimulation of the transplanted neurons can lead to detectable signals in the auditory cortex and measurable behavioral changes, indicating effects on the auditory pathway.

[0278] Electrophysiological recordings: An initial confirmation of the ability of implanted live SGN electrodes to generate signals in the auditory cortex is completed in deafened rats. Single channel constructs are implanted in the cochlea or inferior colliculus as described above and allowed to integrate for the optimal time period described above. During electrophysiological recordings, rats are anesthetized with isoflurane and deafened by direct bilateral cochlear perfusion with the selectively cochleotoxic antibiotic neomycin. Deafness is verified in both ears by the absence of auditory brainstem responses (ABRs) to monaural click stimuli at 93 dB peak equivalent sound pressure level (83). The animal's head is then securely fixed in a stereotaxic frame. The area above the implanted live SGN electrodes is exposed (via reopening of the craniotomy incision for the IC and a postaural incision for the cochlea) and a second craniotomy is made in the upper layers of the auditory cortex. The exposed somata of the implanted SGNs are optically stimulated and recordings are made from the auditory cortex. Light stimulation parameters will be tested similar to those described in Aim 1, and a stimulus-response comparison will be made via recordings in the auditory cortex. To compare LE stimulation to standard electrode stimulation, a single-channel Teflon-insulated platinum / iridium wire with the distal 1 mm of insulation removed will be placed in the IC via stereotaxic coordinates as described above, or in the cochlea via a round window or cochleostomy approach. Stimulation parameters will be guided by previously published similar studies. Briefly, the amplitude threshold will be defined as the minimum amplitude that can generate a reproducible signal in the auditory cortex. Initial parameters will be 100 microsecond biphasic pulses with a 100 microsecond interpulse interval. Current will start at 0.1 mA and be increased in 0.1 mA steps until a reliable signal is detected. Once the threshold amplitude level is established, the frequency and pulse width will be adjusted and a stimulus-response curve will be generated. This will allow a direct comparison of live single-channel stimulation to standard single-channel stimulation in the cochlea or IC. For all studies, electrophysiological signals are continuously recorded at 32 kHz using an Intan 128ch stimulation / recording system (Intan) from a planar 32-channel 4-prong probe (Neuronexus) placed within the auditory cortex via stereotaxic coordinates. Wideband signals are used for analysis of local field potentials (LFPs).For initial spike detection, the broadband signal is passed online through a digital high-pass filter (0.6–6 kHz). Spike sorting is performed offline using KlustaKwik and SpikeSort 3D software (Neuralynx), followed by manual refinement of the clusters. Subsequent cluster analysis is performed using custom Matlab scripts.

[0279] Behavioral Responses: Although the electrophysiological recordings described above confirm that auditory pathway activation is possible with the live electrode technique, they do not provide insight into the auditory experience induced by such activation. To further test the fit of live electrodes within the auditory system, we examine the elicitation and modification of the acoustic startle reflex (ASR). The ASR is an innate behavioral response in rodents to sudden loud acoustic stimuli that does not require training and is well established as a model for studying the central auditory pathway. This process can be easily measured without the need for complex tasks or behavioral training. The ASR can be reliably attenuated by preceding the auditory stimulus with a weaker stimulus - either via an auditory stimulus or electrical stimulation of the auditory pathway - this is known as prepulse inhibition (PPI). To assess whether live electrodes can elicit these responses, we induce the ASR with stimulation of the cochlea with live electrodes and evaluate the ability of live electrode simulations of the cochlea or IC implants to modify the PPI.

[0280] At the time of implantation of the live electrodes as described above, a custom head fixation device as described above is attached, modified to avoid covering the areas overlying the IC or cochlear implant sites. The implants are allowed to integrate for an appropriate period of time as disclosed above. For testing, the animals are placed in a custom restraining rig mounted on a previously developed continuous force transducer. This rig allows for a firm fixation of the head of an awake animal while monitoring a continuous recording of the downward force exerted by the animal. The rig is housed in a sound-isolating booth with a calibrated speaker for the introduction of auditory stimuli and access for fiber optic wiring to optically stimulate the implanted live electrodes. Non-implanted animals are tested to validate the experimental setup and establish baseline recordings. Animals implanted with either cochlear or IC live electrodes are then tested for the possibility of modulating prepulse inhibition of the ASR induced by auditory stimuli with live electrode stimulation. Stimuli are presented to the contralateral ear for both groups. It has been shown that the position of PPI stimulation relative to the auditory stimulation does not affect the degree of PPI. To establish a stimulus-response relationship for PPI, the light stimulation parameters are adjusted within the parameters defined above. In addition, a similar test is performed with a single-channel standard electrode as described above, and a comparison is made between the live electrode and the standard electrode. Finally, the ability of the stimulation of the cochlea standard electrode and the live electrode to induce ASR is evaluated. The stimulation parameters are defined as described above, with increasing amplitude until a startle response is elicited. There is no published data on the ability of non-acoustic direct stimulation of the cochlea to elicit or suppress the ASR. In some embodiments, electrical stimulation of the IC has been shown to modulate the PPI of the ASR, although it is not expected that IC stimulation will trigger the ASR, since the neuronal arcs involved in this reflex do not require IC.

[0281] Behavioral testing protocols have been developed and central auditory processing in rodents has been reported previously. Recordings from the central auditory pathway, the design and interpretation of auditory-based animal behavioral models, and processing of neuronal recording data have also been disclosed previously.

[0282] Number of animals: Electrophysiological and behavioral recordings will be performed on rats after integration of live electrodes either in the cochlea or in the inferior colliculus. For electrophysiological recordings, it is expected that 20 implanted rats and 10 controls per group will be required. To validate the setup, 5 additional rats without live electrode implants will be subjected to a similar recording setup. These same animals will then be implanted with standard electrodes to perform the comparative tests outlined above. Similarly, for behavioral analysis, 20 implanted rats, 10 controls and 5 non-implanted rats will be used. Thus, 130 rats will be used for these experiments.

[0283] The experiments disclosed herein are expected to establish the efficacy of live electrodes in either the cochlea or IC to induce reliable and predictable signal changes in the auditory cortex. Furthermore, basic behavioral analysis of ASR activation and its inhibition via prepulse inhibition will be assessed. Statistical analysis: Across all experiments described herein, the minimum number of animals necessary for scientific validity will be used, with animals used in multiple experiments where appropriate. Power analyses were performed to estimate group sizes required to obtain significant differences (if any existed) (power = 0.90, α = 0.05). All data were tested for normality, with adjustments made for non-normal data. ANOVAs were performed as appropriate, with live SGN electrode type, treatment, and time as independent variables, and outcome measures (as above) as dependent variables. Post-hoc pairwise comparisons were performed where differences existed between groups. For all statistical tests, p < 0.05 was used as the requirement for significance.

[0284] Conclusion:Clinically meaningful advances in hearing restoration are predicted to require novel techniques in bioengineering and regenerative medicine. The application of living electrode technology to this important clinical need is described herein.

[0285] Scientific Advances: Although the experiments as described are primarily intended for the development of living electrode implants for hearing rehabilitation, this work also forms the basis for multiple avenues for further investigation. This work also enables the development of research protocols for preclinical and translational evaluations comparing the efficacy of multiple approaches to peripheral and central hearing rehabilitation, including neuroengineering, surgical techniques, functional outcome measures, behavior, neurophysiology, and histology. This experimental framework can be used to innovate and test future technologies in the coming decades.

[0286] More specifically, advanced tissue engineering techniques that can be utilized may, in some embodiments, be used to create a biofidelic test bed for systematically evaluating alternative hearing therapies and potentially reducing the reliance on time- and resource-intensive in vivo models. In some embodiments, one possible manifestation of this is a modular and customizable "in vitro cochlea" that contains a multicellular living electrode. In some embodiments, this contains SGN aggregates in the center with axons extending to centrally derived aggregates on one end and to cultured cochlear hair cells on the other end. In some embodiments, the three early cell types of the auditory pathway are represented, each of which can be tested and manipulated independently, as well as the connections between them. In some embodiments, despite lacking the complex anatomical structures for transmitting sound, multiple important lines of exploration can be explored: electrical stimulation of distal axons / SGN cell bodies as they associate with the cochlear implant, regenerative medicine techniques for hair cells or SGNs, computational models of signal processing within the cochlea, ototoxic and otoprotective drugs, and many more. In some embodiments, as techniques for fabricating constructs improve, the model will continue to evolve to more closely resemble a living cochlea. In some embodiments, one additional technique that is likely to advance during the execution of this project is the use of living electrodes as monitors of physiological neuronal activity. In some embodiments, living electrodes are biological implants that can be functionalized as luminescent monitors as described above. Further refinements promise to open multiple new avenues for studying the central auditory pathway without the limitations currently posed by conventional electrode techniques. Furthermore, these in vitro and small animal techniques can also contribute to ongoing primate models with more advanced behavioral training, and ultimately human clinical trials.

[0287] In some embodiments, further advances are expected to be made in the culture and evaluation of SGNs, which are a key cell type for multiple hearing pathologies and will be crucial in advancing multiple approaches to hearing restoration. In some embodiments, as described above, effective extension of SGN axons into the scala tympani has the potential to dramatically improve the performance of existing cochlear implant technologies. In some embodiments, the techniques described above help advance these studies further by introducing modalities that combine both growth factors and ECM proteins, each of which have independently shown promise. Second, hair cell loss has been shown to lead to spiral ganglion cell loss. In some embodiments, regenerative medicine techniques for cochlear hair cells will rely on the ability of SGNs to regrow axons in order to be utilized by the auditory pathway, and regenerative medicine techniques for SGNs will require both interaction with hair cells and integration into the central auditory pathway. Additionally, hidden hearing loss and synaptopathy are increasingly recognized causes of hearing loss, but are currently poorly understood. The ability to directly study SGNs in vitro, as well as their interactions with both hair cells and brainstem neurons in in vitro cochlear models such as those described above, may pave the way for improved diagnostic and therapeutic modalities. Collectively, the work described herein holds the promise of contributing to advances in hearing science far beyond the development of living electrode implants.

[0288] Future Directions:In some embodiments, the proposed research forms the basis for two novel living electrodes for hearing rehabilitation. In some embodiments, one is intended to be implanted peripherally in the cochlea and one is intended to be implanted centrally in the inferior colliculus. Although the method presented herein results in a single channel implant, in some embodiments, multi-channel implants will be required to improve clinical applicability, and their configuration must be customized to the morphology of the target tissue. In some embodiments, the multi-channel implant will be based on nano 3D printed constructs utilizing NanoScribe Photonic Professional GT (Nanoscribe GmbH, Eggenstein-Leopoldshafen, Germany), a 3D printing system capable of dealing with features smaller than a micrometer.

[0289] In some embodiments, the IC and cochlear designs will share common elements in terms of the distribution of neuronal soma aggregates and the optrode interface, but will differ in the conformation of the axonal channel. In some embodiments, the neuronal soma aggregates are spaced apart so that each aggregate can be stimulated by a triplet of optrodes that deliver light at a distinct wavelength. In some embodiments, each of the neuronal soma aggregates contains a cell population of cells that are optogenetically transformed to respond to each of these wavelengths of light. In some embodiments, a two-dimensional array of triplet optrodes is paired with a similar array of neuronal soma aggregates. In some embodiments, the channels emanating from the underside of the soma aggregates are designed to distribute the axons throughout the three-dimensional volume of the inferior colliculus or along the length of the cochlea. In some embodiments, even when fully implanted, the optrode / aggregate portion of the construct sits on the surface of the brainstem or within the otic bulla outside the cochlea (Figure 10). In some embodiments, the inorganic portion of the implant is not introduced into the parenchyma of the brainstem and does not need to conform to the anatomy of the cochlear lumen.

[0290] List of Aspects Below is provided a list of embodiments, the numbering of which should not be construed as denoting importance. Aspect 1 provides the following: (a) a biocompatible construct including a first end, a second end, and a body; and (b) Multiple auditory neurons A system including: Aspect 2 provides the following: The system of embodiment 1, wherein the biocompatible construct comprises an inner surface and an outer surface. Aspect 3 provides the following: The system of embodiment 1 or 2, wherein the inner surface of the biocompatible construct defines a luminal core. Aspect 4 provides the following: The system of embodiments 1-3, wherein the exterior surface of the biocompatible construct comprises at least one hydrogel. Aspect 5 provides the following: The system of any one of embodiments 1 to 4, wherein the hydrogel comprises a hydrophilic biopolymer and / or a synthetic polymer. Aspect 6 provides the following: The hydrogel is at least partially crosslinked; The crosslinking optionally increases stiffness, reduces porosity, and / or increases degradation time. A system according to embodiments 1 to 5. Aspect 7 provides the following: The system of any one of embodiments 1 to 6, wherein the hydrophilic biopolymer comprises one or more of agarose, hydrogel, hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, fibrin, and methylcellulose. Aspect 8 provides the following: The system of any one of embodiments 1 to 7, wherein the hydrophilic biopolymer comprises agarose. Aspect 9 provides the following: The system according to any one of claims 1 to 8, wherein the agarose is about 0.25 to 30%, about 0.25% to 3%, about 0.5% to 3%, about 1 to 20%, about 1.5 to 10%, about 2 to 9%, about 2.5 to 8%, or about 3 to 7%. Aspect 10 provides the following: The system according to any one of embodiments 1 to 9, wherein the agarose is about 3%. Aspect 11 provides the following: The hydrophilic biopolymer At least one synthetic hydrogel or synthetic hydrogels made of amphiphilic copolymers consisting of ethylene oxide (PEO) and polypropylene oxide (PPO) units. The system according to any one of embodiments 1 to 10, comprising: Aspect 12 provides the following: 12. The system of any one of embodiments 1 to 11, wherein the hydrogel is about 2-50% w / v, about 3-40% w / v, about 4-30% w / v, about 5-25% w / v, or about 10-20% w / v. Aspect 13 provides the following: 13. The system of any one of claims 1 to 12, wherein the hydrogel is about 28.6% w / v. Aspect 14 provides the following: The system of any one of embodiments 1 to 13, wherein the inner surface of the biocompatible construct comprises one or more extracellular matrix (ECM) components. Aspect 15 provides the following: The system of any one of embodiments 1 to 14, wherein the ECM component comprises collagen, laminin, fibronectin, hyaluronic acid, or a combination thereof. Aspect 16 provides the following: 16. The system of any one of embodiments 1 to 15, wherein the ECM comprises collagen. Aspect 17 provides the following: 17. The system of claim 1, wherein the collagen is at a concentration of about 0.1 to 10 mg / ml, or 0.1 to 9 mg / ml. Aspect 18 provides the following: 18. The system of any one of claims 1 to 17, wherein the collagen is at a concentration of about 1 mg / ml. Aspect 19 provides the following: 19. The system of any one of embodiments 1 to 18, wherein the ECM comprises laminin. Aspect 20 provides the following: The system of any one of embodiments 1 to 19, wherein the laminin is at a concentration of about 0.1 to 10 mg / ml, or 0.1 to 9 mg / ml. Aspect 21 provides the following: 21. The system of any one of claims 1 to 20, wherein the laminin is at a concentration of about 1 mg / ml. Aspect 22 provides the following: 22. The system of claim 1, wherein the plurality of auditory neurons comprises at least 500 auditory neurons. Aspect 23 provides the following: 23. The system of any one of aspects 1 to 22, wherein at least one of the plurality of auditory neurons is in contact with a biocompatible construct. Aspect 24 provides the following: 24. The system of any one of embodiments 1 to 23, wherein at least one neuron in the plurality of neurons comprises a cell body and / or an axon. Aspect 25 provides the following: The system of any one of embodiments 1 to 24, wherein the axon is about 0.5 to 50 cm in length. Aspect 26 provides the following: The at least one portion of neurons in the plurality of auditory neurons comprises: (i) at a first end of the biocompatible construct; (ii) at a second end of the biocompatible construct; (iii) on, along or within a portion of the body of a biocompatible construct; and / or (iv) throughout, along, or within the body of a biocompatible construct A system according to any one of embodiments 1 to 25, which is positioned. Aspect 27 provides the following: 27. The system of claim 1, wherein the portion of the at least one neuron in the plurality of auditory neurons positioned at a first end or a second end of a biocompatible construct comprises a cell body. Aspect 28 provides the following: The system of any one of embodiments 1 to 27, wherein the portion of at least one neuron in the plurality of auditory neurons positioned at, along, or within a portion of a body of a biocompatible construct comprises an axon. Aspect 29 provides the following: The system of any one of embodiments 1 to 28, wherein the body of the biocompatible construct comprises an interior surface of the body. Aspect 30 provides the following: The system of any one of embodiments 1 to 29, wherein the inner surface comprises a luminal core. Aspect 31 provides the following: The system of any one of embodiments 1 to 30, wherein the portion of at least one neuron in the plurality of auditory neurons positioned throughout, along, or within the body of a biocompatible construct comprises an axon. Aspect 32 provides the following: The system of any one of embodiments 1 to 31, wherein the axons extend unidirectionally or bidirectionally. Aspect 33 provides the following: The system of any one of embodiments 1 to 32, wherein at least a portion of the plurality of auditory neurons comprises an aggregate of auditory neurons. Aspect 34 provides the following: the plurality of auditory neurons (a) a spiral ganglion neuron, an inferior colliculus (IC) auditory neuron, a cortical auditory neuron, a type I auditory neuron, or a type II auditory neuron, or a combination thereof; or (b) a neuron present within the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, or cortical brainstem nuclei of the organism, or any combination thereof; The system of any one of aspects 1 to 33, comprising: Aspect 35 provides the following: 35. The system of claim 1, wherein the plurality of auditory neurons is or comprises a plurality of spiral ganglion neurons. Aspect 36 provides the following: 36. The system of claim 1, wherein the plurality of auditory neurons is or includes a plurality of IC auditory neurons. Aspect 37 provides the following: The system of any one of embodiments 1 to 36, further comprising one or more additional cells, such as astrocytes, oligodendrocytes, Schwann cells, microglia, or endothelial cells. Aspect 38 provides the following: The system of any one of aspects 1 to 37, wherein the plurality of auditory neurons are isolated from a subject. Aspect 39 provides the following: The system of any one of embodiments 1 to 38, wherein the plurality of auditory neurons is or comprises at least one cell derived from a stem cell. Aspect 40 provides the following: The system according to any one of embodiments 1 to 39, wherein the stem cells comprise one or more of induced pluripotent stem cells (iPSCs), embryonic stem cells, or tissue stem cells. Aspect 41 provides the following: The system of any one of aspects 1 to 40, wherein the plurality of auditory neurons are autologous, allogeneic, or xenogeneic to the subject. Aspect 42 provides the following: 42. The system of any one of embodiments 1 to 41, wherein said at least one neuron in said plurality of auditory neurons is genetically engineered or reprogrammed using non-genetic techniques. Aspect 43 provides the following: 43. The system of any one of embodiments 1 to 42, wherein the at least one engineered neuron comprises a sensor, an actuator, a receptor, and / or a reporter. Aspect 44 provides the following: 44. The system of any one of embodiments 1 to 43, wherein the at least one engineered neuron in the plurality of auditory neurons comprises a reporter. Aspect 45 provides the following: 45. The system of any one of embodiments 1 to 44, wherein the reporter comprises a fluorescent reporter. Aspect 46 provides the following: 46. ​​The system of any one of embodiments 1 to 45, wherein the at least one genetically engineered neuron in the plurality of auditory neurons comprises a receptor. Aspect 47 provides the following: The system of any one of embodiments 1 to 46, wherein the receptor comprises one or more of a magnetic receptor, an electromagnetic receptor, an electroreceptor, a hydroreceptor, a mechanoreceptor, an osmoreceptor, a thermoreceptor, and a piezoelectric ion channel. Aspect 48 provides the following: 48. The system of any one of embodiments 1 to 47, wherein the electromagnetic receptor comprises one or more of an infrared receptor, a photoreceptor, or an ultraviolet receptor. Aspect 49 provides the following: The system of any one of embodiments 1 to 48, wherein the mechanoreceptors include one or both of mechanosensory receptors or proprioceptors. Aspect 50 provides the following: 50. The system of claim 1, wherein the at least one engineered neuron in the plurality of auditory neurons comprises a sensor. Aspect 51 provides the following: The sensor, Optical sensors including one or more of a light-sensitive ion channel, a calcium sensor, and a membrane voltage sensor. The system according to any one of claims 1 to 50, comprising: Aspect 52 provides the following: The sensor, Calcium sensors that include one or more of Aequorin, Cameleon, and GCaMP The system according to any one of claims 1 to 51, comprising: Aspect 53 provides the following: The system of any one of embodiments 1 to 52, wherein the sensor comprises a chloride sensor comprising Clomeleon. Aspect 54 provides the following: The system of any one of embodiments 1 to 53, wherein the sensor comprises a membrane voltage sensor comprising Mermaid. Aspect 55 provides the following: 55. The system of any one of embodiments 1 to 54, wherein the at least one engineered neuron in the plurality of auditory neurons comprises an actuator. Aspect 56 provides the following: The actuator, Optical actuators including one or more of channelrhodopsin, halorhodopsin, and archerhodopsin The system according to any one of claims 1 to 55, comprising: Aspect 57 provides the following: The system of any one of embodiments 1 to 56, wherein the at least one genetically engineered neuron in the plurality of auditory neurons comprises an optical sensor and / or the optical actuator is an optogenetic neuron. Aspect 58 provides the following: The system of any one of embodiments 1 to 57, wherein the biocompatible construct is a tubular construct. Aspect 59 provides the following: The system of embodiments 1 to 58, wherein the biocompatible construct is helical or wye shaped. Aspect 60 provides the following: The system of any one of embodiments 1 to 59, wherein the biocompatible construct has a curvature of about 240 to 900 degrees. Aspect 61 provides the following: The system of any one of embodiments 1 to 60, wherein the curvature of the biocompatible construct is about 270 degrees. Aspect 62 provides the following: The system of any one of embodiments 1 to 61, having an outer diameter of about 0.1 to 40 mm. Aspect 63 provides the following: The system of any one of embodiments 1 to 62, having an outer diameter of about 1.5 mm. Aspect 64 provides the following: The system of any one of embodiments 1 to 63, having a diameter of about 0.5 to 70 mm, or 0.1 to 10 mm. Aspect 65 provides the following: The system of any one of embodiments 1 to 64, having a diameter of about 7 mm. Aspect 66 provides the following: The system of any one of embodiments 1 to 65, having a pitch of about 0.1 to 10 mm. Aspect 67 provides the following: The system of any one of embodiments 1 to 66, having a pitch of about 1 mm. Aspect 68 provides the following: The system of embodiments 1 to 67, which is produced or assembled ex vivo or in vitro. Aspect 69 provides the following: The system of embodiments 1 to 68, wherein at least one neuron in the plurality of neurons in the system and / or at least one neuron in a subject can be modulated or used for modulation. Aspect 70 provides the following: The system of embodiments 1-69, wherein the regulating is or includes unidirectional or bidirectional regulation. Aspect 71 provides the following: The system of any one of embodiments 1 to 70, wherein regulating comprises stimulating or inhibiting at least one neuron in the plurality of auditory neurons in the system or at least one neuron in the subject. Aspect 72 provides the following: 72. The system of any one of embodiments 1 to 71, wherein the stimulating comprises electrical stimulation, light stimulation, sound / vibration stimulation, magnetic stimulation, or a combination thereof. Aspect 73 provides the following: The system of any one of embodiments 1 to 72, wherein the stimulating comprises electrical stimulation and light stimulation. Aspect 74 provides the following: The system of any one of embodiments 1 to 73, wherein regulating includes monitoring and / or recording. Aspect 75 provides the following: The system of any one of embodiments 1 to 74, wherein the at least one neuron in the subject is or comprises an auditory neuron. Aspect 76 provides the following: The auditory neurons (a) spiral ganglion neurons, IC neurons, or a combination thereof; or (b) neurons present within the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of the organism, or combinations thereof; The system according to any one of claims 1 to 75, comprising: Aspect 77 provides the following: A system according to any one of embodiments 1 to 76, capable of stimulating or inhibiting an auditory pathway in a subject. Aspect 78 provides the following: A system according to any one of embodiments 1 to 77, capable of stimulating or inhibiting the peripheral auditory pathway in a subject. Aspect 79 provides the following: The system of embodiments 1-78, wherein the system is implanted at, near, or in the cochlea of ​​a subject; at, near, or in the inferior colliculus (IC) of a subject; at, near, or in the cochlear nucleus of a subject; and / or at, near, or in the auditory cortex of a subject. Aspect 80 provides the following: The system of embodiments 1-79, wherein the implanted system contacts at least one cell within the subject, or contacts at least one cell in, near, or within the area of ​​the auditory cortex within the subject. Aspect 81 provides the following: The system according to any one of aspects 1 to 80, wherein the at least one cell in the subject is an endogenous cell. Aspect 82 provides the following: 82. The system of any one of embodiments 1 to 81, wherein the at least one cell comprises a neuron. Aspect 83 provides the following: 83. The system of embodiment 1-82, wherein the neurons comprise neurons present in the cochlea, auditory nerve, auditory brainstem nuclei, cortical brainstem nuclei, spiral ganglion, IC, or combinations thereof. Aspect 84 provides the following: The system of any one of aspects 1 to 83, wherein contacting the system with the at least one cell in the subject is determined to create a synapse between at least one of the plurality of auditory neurons of the system and the at least one cell in the subject. Aspect 85 provides the following: The system according to embodiments 1 to 84, wherein the synapse is or comprises a neuronal synapse. Aspect 86 provides the following: The system of any one of embodiments 1 to 85, wherein the implanted system provides an accessible interface for modulating the at least one neuron in the plurality of auditory neurons within the system, or the at least one cell within a subject. Aspect 87 provides the following: The system of any one of embodiments 1 to 86, wherein the biocompatible construct has a non-linear shape. Aspect 88 provides the following: 1. A method for manufacturing a system, comprising the steps of: (a) providing a biocompatible construct comprising a first end, a second end, and a body; and (b) associating the biocompatible construct with a plurality of auditory neurons. Aspect 89 provides the following: 89. The method of any one of embodiments 1 to 88, comprising maintaining the system under conditions that promote growth of at least one neuron in said plurality of auditory neurons. Aspect 90 provides the following: The method of any one of embodiments 88 to 89, comprising maintaining the system under conditions that maintain the viability of the at least one neuron in the plurality of auditory neurons. Aspect 91 provides the following: The method of any one of aspects 88 to 90, further comprising forming an aggregate of at least a portion of the plurality of auditory neurons. Aspect 92 provides the following: The method of any one of aspects 88 to 91, wherein the aggregate of at least a portion of the plurality of auditory neurons is formed prior to the step of contacting the plurality of auditory neurons with a biocompatible construct. Aspect 93 provides the following: The method of any one of embodiments 88 to 92, wherein the system comprises a system provided in any one of embodiments 1 to 87. Aspect 94 provides the following: (a) a biocompatible construct including a first end, a second end, and a body; and (b) Multiple auditory neurons 1. A method for implanting in a subject a system comprising: A method comprising implanting the system at, near or within the cochlea, cochlear nucleus, auditory cortex, or inferior colliculus (IC) in a subject. Aspect 95 provides the following: The method of embodiment 94, wherein the implantation is determined to be less likely to cause inflammation as compared to implantation of conventional microelectrodes. Aspect 96 provides the following: The method of embodiment 94 or 95, wherein the subject is a human. Aspect 97 provides the following: The method of any one of embodiments 94 to 96, wherein the system comprises a system provided in any one of embodiments 1 to 87. Aspect 98 provides the following: 1. A method of modulating at least one neuron in a subject, the method comprising: (i) Below: (a) a biocompatible construct including a first end, a second end, and a body; and (b) Multiple auditory neurons providing to the subject a system comprising: (ii) modulating at least one neuron with electrical stimulation, light stimulation, or both. Aspect 99 provides the following: The method of embodiment 98, wherein the modulating step comprises monitoring, recording, stimulating, and / or inhibiting. The embodiment 100 provides: The method of embodiment 98 or 99, wherein the at least one neuron in the subject comprises an auditory neuron. Aspect 101 provides the following: The method of any one of embodiments 98 to 100, wherein the auditory neurons include one or more of spiral ganglion neurons or IC neurons. Aspect 102 provides: The method according to any one of embodiments 98 to 101, wherein the subject is a human. Aspect 103 provides the following: The method of any one of embodiments 98 to 102, wherein the system comprises a system provided in any one of embodiments 1 to 87. Aspect 104 provides the following: 1. A method of treating a subject having a hearing impairment, comprising the steps of: (a) a biocompatible construct including a first end, a second end, and a body; and (b) Multiple auditory neurons providing to the subject a system comprising the steps of: Aspect 105 provides the following: The method of embodiment 104, wherein the hearing impairment comprises one or more of conductive hearing loss, sensorineural hearing loss, or mixed hearing loss. Aspect 106 provides the following: The method of any one of embodiments 104 to 105, wherein the hearing impairment comprises one or more of acquired hearing impairment, sudden hearing impairment, secondary hearing loss (drug-induced or trauma-induced hearing loss), autoimmune inner ear disease, otosclerosis, Meniere's disease, presbycusis, or acoustic neuroma. Aspect 106 provides the following: The method according to any one of embodiments 104 to 106, wherein the subject is a human. Aspect 108 provides the following: The method of any one of embodiments 104 to 107, wherein the system comprises a system provided in any one of embodiments 1 to 87. Aspect 109 provides the following: 1. A method of ameliorating a symptom of hearing impairment in a subject, comprising: (a) a biocompatible construct including a first end, a second end, and a body; and (b) Multiple auditory neurons providing to the subject a system comprising: Aspect 110 provides the following: The method of embodiment 109, wherein the hearing impairment comprises one or more of conductive hearing loss, sensorineural hearing loss, or mixed hearing loss. Aspect 111 provides the following: The method of embodiment 109 or 110, wherein the hearing impairment comprises one or more of acquired hearing impairment, sudden hearing impairment, secondary hearing loss (drug-induced or trauma-induced hearing loss), autoimmune inner ear disease, otosclerosis, Meniere's disease, presbycusis, or acoustic neuroma. Aspect 112 provides the following: The method according to any one of embodiments 109 to 111, wherein the subject is a human. Aspect 113 provides the following: The method of any one of embodiments 109 to 112, wherein the system comprises a system provided in any one of embodiments 1 to 87. Aspect 114 provides the following: 1. A method for preventing worsening of symptoms of hearing impairment in a subject, comprising: (a) a biocompatible construct including a first end, a second end, and a body; and (b) Multiple auditory neurons providing to the subject a system comprising: Aspect 115 provides the following: The method of embodiment 114, wherein the hearing impairment comprises one or more of conductive hearing loss, sensorineural hearing loss, or mixed hearing loss. Aspect 116 provides the following: The method of embodiment 114 or 115, wherein the hearing impairment comprises one or more of acquired hearing impairment, sudden hearing impairment, secondary hearing loss (drug-induced or trauma-induced hearing loss), autoimmune inner ear disease, otosclerosis, Meniere's disease, presbycusis, or acoustic neuroma. Aspect 117 provides the following: The method according to any one of embodiments 114 to 116, wherein the subject is a human. Aspect 118 provides the following: The method of any one of embodiments 114 to 117, wherein the system comprises a system provided in any one of embodiments 1 to 87. Aspect 119 provides the following: (a) a biocompatible construct comprising a first end, a second end, and a body; and (b) a plurality of auditory neurons and instructions for using the same; A kit comprising a system including: Aspect 120 provides the following: 120. The kit of embodiment 119, further comprising instructions for using the kit. Aspect 121 provides the following: The kit according to embodiment 119 or 120, wherein the system comprises a system provided in any one of embodiments 1 to 87. Aspect 122 provides the following: Stimulator array; and a plurality of living electrodes, at least one of the plurality of living electrodes being in optical communication with an element of the stimulator array; a proximal end including a stimulator-complementary neuron; a distal end including an auditory neuron in neural communication with the optogenetic neuron within the living electrode; a plurality of living electrodes, each of the plurality of living electrodes including Including, A cochlear implant, wherein the plurality of living electrodes have staggered distal ends. Aspect 123 provides the following: the stimulator array is an optrode array; and The stimulator complementary neuron is an optogenetic neuron; 123. The cochlear implant of embodiment 122.

[0291] equivalent Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. It should be understood that, unless otherwise stated or unless a contradiction or inconsistency would be apparent to one skilled in the art, the invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims, which are introduced into another claim dependent on the same base claim (or into any other claim, as appropriate). Moreover, it should also be understood that any aspect or aspect of the invention may be expressly excluded from the claims, regardless of whether a specific exclusion is recited herein. The scope of the invention is not intended to be limited to the above description, but is as set forth in the appended claims.

Claims

1. (a) a biocompatible construct including a first end, a second end, and a body; and (b) Multiple auditory neurons Including, the system.

2. The system of claim 1 , wherein the biocompatible construct comprises an inner surface and an outer surface.

3. The system of claim 2 , wherein the inner surface of the biocompatible construct defines a luminal core.

4. 3. The system of claim 2, wherein the exterior surface of the biocompatible construct comprises at least one hydrogel.

5. The system of claim 4, wherein the hydrogel comprises a hydrophilic biopolymer and / or a synthetic polymer.

6. The hydrogel is at least partially crosslinked; The crosslinking optionally increases stiffness, reduces porosity, and / or increases degradation time.

6. A system according to claim 4 or 5.

7. 6. The system of claim 5, wherein the hydrophilic biopolymer comprises one or more of agarose, hydrogel, hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, fibrin, and methylcellulose.

8. 8. The system of claim 7, wherein the hydrophilic biopolymer comprises agarose.

9. 9. The system of claim 7 or 8, wherein the agarose is about 0.25-30%, about 0.25%-3%, about 0.5%-3%, about 1-20%, about 1.5-10%, about 2-9%, about 2.5-8%, or about 3-7%.

10. 10. The system of claim 9, wherein the agarose is about 3%.

11. The hydrophilic biopolymer At least one synthetic hydrogel or synthetic hydrogels made of amphiphilic copolymers consisting of ethylene oxide (PEO) and polypropylene oxide (PPO) units.

8. The system of claim 7, comprising:

12. 12. The system of claim 7 or 11, wherein the hydrogel is about 2-50% w / v, about 3-40% w / v, about 4-30% w / v, about 5-25% w / v, or about 10-20% w / v.

13. 13. The system of claim 12, wherein the hydrogel is about 28.6% w / v.

14. The system of any one of claims 1 to 13, wherein the inner surface of the biocompatible construct comprises one or more extracellular matrix (ECM) components.

15. The system of claim 14, wherein the ECM component comprises collagen, laminin, fibronectin, hyaluronic acid, or a combination thereof.

16. The system of claim 15, wherein the ECM comprises collagen.

17. 17. The system of claim 16, wherein the collagen is at a concentration of about 0.1 to 10 mg / ml, or 0.1 to 9 mg / ml.

18. 18. The system of claim 16 or 17, wherein the collagen is at a concentration of about 1 mg / ml.

19. The system of claim 15, wherein the ECM comprises laminin.

20. 20. The system of claim 19, wherein the laminin is at a concentration of about 0.1 to 10 mg / ml, or 0.1 to 9 mg / ml.

21. 21. The system of claim 19 or 20, wherein the laminin is at a concentration of about 1 mg / ml.

22. 2. The system of claim 1, wherein the plurality of auditory neurons comprises at least 500 auditory neurons.

23. 2. The system of claim 1 , wherein at least one of the plurality of auditory neurons is in contact with a biocompatible construct.

24. 2. The system of claim 1 , wherein at least one neuron in the plurality of neurons comprises a cell body and / or an axon.

25. 25. The system of claim 24, wherein the axon is about 0.5 to 50 cm in length.

26. The at least one portion of neurons in the plurality of auditory neurons comprises: (i) at a first end of the biocompatible construct; (ii) at a second end of the biocompatible construct; (iii) at, along or within a portion of the body of a biocompatible construct; and / or (iv) throughout, along, or within the body of a biocompatible construct.

2. A system according to any one of the preceding claims, which is positioned.

27. 27. The system of claim 26, wherein the portion of the at least one neuron in the plurality of auditory neurons positioned at a first end or a second end of a biocompatible construct comprises a cell body.

28. 27. The system of claim 26, wherein the portion of the at least one neuron in the plurality of auditory neurons positioned at, along or within a portion of a body of a biocompatible construct comprises an axon.

29. 26. The system of claim 26(iii) or (iv), wherein the body of the biocompatible construct comprises an interior surface of the body.

30. 30. The system of claim 29, wherein the inner surface comprises a luminal core.

31. 31. The system of claim 26(iii) or (iv), or claim 30, wherein the portion of the at least one neuron in the plurality of auditory neurons positioned throughout, along or within the body of a biocompatible construct comprises an axon.

32. 32. The system of claim 31, wherein the axons extend unidirectionally or bidirectionally.

33. 2. The system of claim 1, wherein at least a portion of the plurality of auditory neurons comprises a clump of auditory neurons.

34. the plurality of auditory neurons (a) spiral ganglion neurons, inferior colliculus (IC) auditory neurons, cortical auditory neurons, type I auditory neurons, or type II auditory neurons, or any combination thereof; or (b) a neuron present within the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, or cortical brainstem nuclei of an organism, or any combination thereof; 13. The system of any one of the preceding claims, comprising:

35. 35. The system of claim 34, wherein the plurality of auditory neurons is or includes a plurality of spiral ganglion neurons.

36. 35. The system of claim 34, wherein the plurality of auditory neurons is or includes a plurality of IC auditory neurons.

37. 20. The system of any one of the preceding claims, further comprising one or more additional cells, such as astrocytes, oligodendrocytes, Schwann cells, microglia, or endothelial cells.

38. 2. The system of claim 1, wherein the plurality of auditory neurons are isolated from a subject.

39. 2. The system of claim 1, wherein the plurality of auditory neurons is or comprises at least one cell derived from a stem cell.

40. 40. The system of claim 39, wherein the stem cells comprise one or more of induced pluripotent stem cells (iPSCs), embryonic stem cells, or tissue stem cells.

41. 2. The system of claim 1 , wherein the plurality of auditory neurons are autologous, allogeneic, or xenogeneic to the subject.

42. 2. The system of claim 1, wherein the at least one neuron in the plurality of auditory neurons is genetically engineered or reprogrammed using non-genetic techniques.

43. 43. The system of claim 42, wherein the at least one engineered neuron comprises a sensor, an actuator, a receptor, and / or a reporter.

44. 44. The system of claim 43, wherein the at least one engineered neuron in the plurality of auditory neurons comprises a reporter.

45. 45. The system of claim 44, wherein the reporter comprises a fluorescent reporter.

46. 44. The system of claim 43, wherein the at least one engineered neuron in the plurality of auditory neurons comprises a receptor.

47. 47. The system of claim 46, wherein the receptors include one or more of a magnetic receptor, an electromagnetic receptor, an electroreceptor, a hydroreceptor, a mechanoreceptor, an osmoreceptor, a thermoreceptor, and a piezoelectric ion channel.

48. 48. The system of claim 47, wherein the electromagnetic receptor comprises one or more of an infrared receptor, a photoreceptor, or an ultraviolet receptor.

49. 48. The system of claim 47, wherein the mechanoreceptors include one or both of mechanosensory receptors or proprioceptors.

50. 44. The system of claim 43, wherein the at least one engineered neuron in the plurality of auditory neurons comprises a sensor.

51. The sensor, Optical sensors including one or more of a light-sensitive ion channel, a calcium sensor, and a membrane voltage sensor.

51. The system of claim 50, comprising:

52. The sensor, Calcium sensors that include one or more of Aequorin, Cameleon, and GCaMP 51. The system of claim 50, comprising:

53. 51. The system of claim 50, wherein the sensor comprises a chloride sensor comprising Clomeleon.

54. 51. The system of claim 50, wherein the sensor comprises a membrane voltage sensor comprising Mermaid.

55. 44. The system of claim 43, wherein the at least one engineered neuron in the plurality of auditory neurons comprises an actuator.

56. The actuator, Optical actuators including one or more of channelrhodopsin, halorhodopsin, and archerhodopsin 56. The system of claim 55, comprising:

57. 57. The system of claim 51 or 56, wherein the at least one genetically engineered neuron in the plurality of auditory neurons comprises an optical sensor and / or the optical actuator is an optogenetic neuron.

58. The system of any one of the preceding claims, wherein the biocompatible construct is a tubular construct.

59. The system of any one of claims 1 to 57, wherein the biocompatible construct is helical or wye shaped.

60. The system of any one of claims 1-57 or 59, wherein the biocompatible construct has a curvature of about 240-900 degrees.

61. 61. The system of claim 60, wherein the curvature of the biocompatible construct is about 270 degrees.

62. 62. The system of claim 60 or 61, having an outer diameter of about 0.1 to 40 mm.

63. 63. The system of claim 62, having an outer diameter of about 1.5 mm.

64. 62. The system of any one of claims 60-61, having a diameter of about 0.5 to 70 mm, or 0.1 to 10 mm.

65. 65. The system of claim 64, having a diameter of about 7 mm.

66. 66. The system of any one of claims 60 to 65, having a pitch of about 0.1 to 10 mm.

67. 67. The system of claim 66, having a pitch of about 1 mm.

68. 20. The system of any one of the preceding claims, which is manufactured or assembled ex vivo or in vitro.

69. 10. A system according to any preceding claim, wherein at least one neuron in the plurality of neurons in the system and / or at least one neuron in a subject can be modulated or used for modulation.

70. 70. The system of claim 69, wherein the modulating is or includes unidirectional or bidirectional modulation.

71. 71. The system of claim 69 or 70, wherein modulating comprises stimulating or inhibiting at least one neuron in the plurality of auditory neurons in the system or at least one neuron in the subject.

72. 72. The system of claim 71, wherein the stimulating comprises electrical stimulation, light stimulation, sound / vibration stimulation, magnetic stimulation, or a combination thereof.

73. 72. The system of claim 71, wherein the stimulating comprises electrical stimulation and light stimulation.

74. 70. The system of claim 69, wherein regulating comprises monitoring and / or recording.

75. 75. The system of any one of claims 69-74, wherein the at least one neuron in a subject is or includes an auditory neuron.

76. The auditory neurons (a) spiral ganglion neurons, IC neurons, or a combination thereof; or (b) neurons present within the cochlea, spiral ganglion, auditory nerve, auditory brainstem nuclei, and / or cortical brainstem nuclei of the organism, or combinations thereof; 76. The system of claim 75, comprising:

77. 10. A system according to any preceding claim, capable of stimulating or inhibiting an auditory pathway in a subject.

78. 10. The system of any preceding claim, capable of stimulating or inhibiting the peripheral auditory pathway in a subject.

79. The system of any one of the preceding claims, wherein the system is implanted at, near, or in the cochlea of ​​a subject; at, near, or in the inferior colliculus (IC) of a subject; at, near, or in the cochlear nucleus of a subject; and / or at, near, or in the auditory cortex of a subject.

80. The system of claim 79, wherein the implanted system contacts at least one cell within the subject or contacts at least one cell at, near, or within the area of ​​the auditory cortex within the subject.

81. The system of claim 80, wherein the at least one cell in the subject is an endogenous cell.

82. 82. The system of claim 81 , wherein the at least one cell comprises a neuron.

83. 83. The system of claim 82, wherein the neurons comprise neurons present in the cochlea, auditory nerve, auditory brainstem nuclei, cortical brainstem nuclei, spiral ganglion, IC, or combinations thereof.

84. 81. The system of claim 80, wherein contacting the system with the at least one cell in the subject is determined to create a synapse between at least one neuron of the plurality of auditory neurons of the system and the at least one cell in the subject.

85. 85. The system of claim 84, wherein the synapse is or includes a neuronal synapse.

86. The system of any one of claims 79-85, wherein the implanted system provides an accessible interface for modulating the at least one neuron in the plurality of auditory neurons within the system, or the at least one cell within a subject.

87. The system of any one of the preceding claims, wherein the biocompatible construct has a non-linear shape.

88. 1. A method for manufacturing a system, comprising the steps of: (a) providing a biocompatible construct comprising a first end, a second end, and a body; and (b) associating the biocompatible construct with a plurality of auditory neurons.

89. 90. The method of claim 88, comprising maintaining the system under conditions that promote growth of at least one neuron in said plurality of auditory neurons.

90. 90. The method of claim 88 or 89, comprising maintaining the system under conditions that maintain viability of said at least one neuron in said plurality of auditory neurons.

91. 91. The method of any one of claims 88-90, comprising forming an aggregate of at least a portion of said plurality of auditory neurons.

92. 92. The method of claim 91, wherein the aggregates of at least a portion of the plurality of auditory neurons are formed prior to contacting the plurality of auditory neurons with a biocompatible construct.

93. 93. The method of any one of claims 88 to 92, wherein the system comprises a system provided in any one of claims 1 to 87.

94. (a) a biocompatible construct comprising a first end, a second end, and a body; and (b) a plurality of auditory neurons and instructions for using the same; A kit comprising a system including:

95. 95. The kit of claim 94, further comprising instructions for using the kit.

96. 96. The kit of claim 94 or 95, wherein the system comprises a system as provided in any one of claims 1 to 87.

97. A stimulator array; and a plurality of living electrodes, at least one of the plurality of living electrodes being in optical communication with an element of the stimulator array; a proximal end including a stimulator-complementary neuron; a distal end including an auditory neuron in neural communication with the optogenetic neuron within the living electrode; a plurality of living electrodes, each of the plurality of living electrodes including Including, A cochlear implant, wherein the plurality of living electrodes have staggered distal ends.

98. the stimulator array is an optrode array; and The stimulator complementary neuron is an optogenetic neuron; 98. The cochlear implant of claim 97.

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