Neural microphysiological system and method of using the same

Abstract

To provide a cell culturing system, specifically a three-dimensional cell culturing system for neuronal cells, which promotes both structural and functional characteristics that mimic those of in vivo peripheral fibers, including cell myelination.SOLUTION: Using a dual hydrogel construct and explants from neuronal cells, the present disclosure provides methods, devices, and systems for in vitro spatially-controlled three-dimensional models that permit intra- and extra-cellular electrophysiological measurements and recordings. The three-dimensional hydrogel constructs allow for flexibility in incorporated cell types, geometric fabrication, and electrical manipulation, providing viable systems for culture, perturbation, and testing of biomimetic neural growth with physiologically-relevant results.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 049,692, filed September 12, 2014, and U.S. Provisional Application No. 62 / 138,258, filed March 25, 2015, each of which is incorporated by reference in its entirety.

[0002] The present disclosure relates generally to cell culture systems, and specifically to a three-dimensional cell culture system for neural cells that promotes both structural and functional properties that mimic those of in vivo nerve fibers, including cell myelination and compound action potential propagation. [Background technology]

[0003] Replicating functional aspects of physiology for benchtop evaluation is particularly challenging for peripheral nerve tissue, where bioelectrical conduction over long distances is one of the most relevant physiological outcomes. For this reason, three-dimensional tissue models of peripheral nerves have lagged behind models of epithelial, metabolic, and tumor tissues, where soluble analytes serve as appropriate metrics. The application of electrophysiological techniques has only recently been made possible through multi-electrode array technology for screening environmental toxins, as well as for disease modeling and therapeutic testing. This application has pioneered research into both peripheral nervous system (PNS) and central nervous system (CNS) applications, but the dissociated nature of the cultures does not allow replicating the population-level environment and metrics important to peripheral tissues. Instead, clinical methods to investigate peripheral neuropathy and neuroprotection include nerve conduction studies using morphometric analysis of skin biopsies, with measurements of compound action potentials (CAPs) and nerve fiber density (NFD). Summary of the Invention [Means for solving the problem]

[0004] The present disclosure addresses the need for the creation and use of 3D hydrogel systems that enable in vitro physiological measurements of clinical nerve conduction and neural tissue that mimic NFD.

[0005] The present disclosure relates to a method of generating a three-dimensional culture of one or more neural cells in a culture vessel comprising a solid substrate, the method comprising: (a) contacting one or more isolated Schwann cells and / or oligodendrocytes with a solid substrate comprising at least one exterior surface, at least one interior surface, and at least one internal chamber defined by the at least one interior surface and accessible from a point outside the solid substrate through at least one opening; (b) seeding one or more isolated neural cells or tissue explants comprising neural cells into the at least one internal chamber; and (c) applying a volume of cell culture medium to the culture vessel sufficient to cover the at least one internal chamber, wherein at least a portion of the interior surface comprises a first cell-impermeable polymer and a first cell-permeable polymer. In some embodiments, step (a) is preceded by disposing a solution comprising a first cell-impermeable polymer and a first cell-permeable polymer in the culture vessel and inducing the first cell-impermeable polymer and the first cell-permeable polymer to physically adhere or chemically bond onto at least a portion of the inner surface. In some embodiments, the solid substrate comprises a base having a predetermined shape that defines the shape of the outer surface and the inner surface.

[0006] In some embodiments, the base comprises one or a combination of silica, plastic, ceramic, or metal, and the base has a first cell impermeable polymer and a second cell permeable polymer coating an interior surface of the base, forming a cylindrical or approximately cylindrical interior chamber. or a shape substantially similar to a cylinder so as to define a compartment, and the opening is located at one end of the cylinder. In some embodiments, the base comprises one or more pores of a size and shape sufficient to allow diffusion of proteins, nutrients, and oxygen through the solid substrate in the presence of cell culture medium.

[0007] In some embodiments, the step of inducing the first cell-impermeable polymer and the first cell-permeable polymer to crosslink on the solid substrate comprises exposing the solution to ultraviolet or visible light. In some embodiments, the first cell-impermeable polymer is polyethylene glycol (PEG) at a concentration of about 20% by weight or less of the solution. In some embodiments, the first cell-impermeable polymer is at a concentration of about 0.1% by weight to about 3.0% by weight of the solution.

[0008] In some embodiments, the method further includes exposing the culture vessel to 37 degrees Celsius and a carbon dioxide concentration of about 5.0% or less for a time sufficient to grow axons in the inner chamber. In some embodiments, at least a portion of the solid substrate is cylindrical or approximately cylindrical such that at least a portion of an inner surface of the solid substrate defines a cylindrical or approximately cylindrical inner chamber into which the one or more Schwann cells are seeded and into which the one or more neurons are seeded.

[0009] In some embodiments, step (c) comprises seeding a tissue explant selected from one or a combination of isolated dorsal root ganglia, spinal cord explants, retinal explants, and cortical explants. In some embodiments, step (c) comprises seeding a suspension of neural cells selected from one or a combination of motor neurons, cortical neurons, spinal neurons, peripheral neurons.

[0010] In some embodiments, the solid substrate comprises a plastic base crosslinked with a first cell-impermeable polymer and a mixture of a first cell-permeable polymer, the plastic base comprising a plurality of pores having diameters of about 1 micron or less.

[0011] In some embodiments, the method further comprises forming a solid substrate and positioning the solid substrate within the culture vessel, hi some embodiments, forming the solid substrate comprises curing the solution comprising the first cell-impermeable polymer and the first cell-permeable polymer by photolithography.

[0012] In some embodiments, the method further comprises, after step (c), allowing the neural cells to grow neurites and / or axons for a period of about 1 day to about 1 year.

[0013] In some embodiments, the method further comprises, prior to step (a), isolating one or more Schwann cells and / or one or more oligodendrocytes from the sample.

[0014] In some embodiments, the method further comprises isolating dorsal root ganglia (DRG) from the one or more mammals prior to step (b).

[0015] In some embodiments, the culture vessel does not include a sponge.

[0016] In some embodiments, the solid substrate comprises about 15% or less PEG and about 0.05% to about 1.00% of one or a combination of self-assembling peptides selected from RAD16-I, RAD16-II, EAK16-I, EAK16-II, and dEAK16.

[0017] In some embodiments, the culture vessel comprises about 1 to about 1200 wells in which steps (a)-(c) can be carried out sequentially or simultaneously.

[0018] In some embodiments, at least a portion of the substrate is formed in the shape of a cylinder or rectangular prism with an interior chamber defined by an interior surface and accessible by one or more openings.

[0019] In some embodiments, the solid substrate polymer does not include PEG.

[0020] In some embodiments, the cell culture medium comprises nerve growth factor (NGF) at a concentration of about 5 to about 20 picograms per milliliter, and / or ascorbic acid at a concentration ranging from about 0.001% to about 0.01% by weight / volume.

[0021] In some embodiments, the method further includes positioning at least one stimulating electrode at or near a neuronal cell body of one or more neuronal cells or tissue explants, and positioning at least one recording electrode at or near an axon at a point most distal from the neuronal cell body, such that upon introduction of a current to the stimulating electrode, the recording electrode can receive a signal corresponding to one or more electrophysiological metrics measurable at the recording electrode. In some embodiments, the one or more electrophysiological metrics are one or a combination of electrical conduction velocity, action potential, wave amplitude associated with the passage of an electrical impulse along a membrane of one or more neuronal cells, width of an electrical impulse along a membrane of one or more neuronal cells, latency of an electrical impulse along a membrane of one or more neuronal cells, and envelope of an electrical impulse along a membrane of one or more neuronal cells.

[0022] The present disclosure also relates to a composition comprising: (i) a culture vessel, a hydrogel matrix comprising a first cell-impermeable polymer and a first cell-permeable polymer, and one or more isolated Schwann cells and / or one or more oligodendrocytes, and one or more tissue explants or fragments thereof; or (ii) a suspension of cells comprising a culture vessel, a hydrogel matrix comprising a first cell-impermeable polymer and a first cell-permeable polymer, and one or more isolated Schwann cells and / or one or more oligodendrocytes, and one or more neural cells.

[0023] In some embodiments, the composition further comprises a solid substrate onto which the hydrogel matrix is ​​crosslinked, the solid substrate comprising at least one primarily plastic surface having pores with diameters of about 1 micron to about 5 microns. In some embodiments, the composition further comprises a solid substrate onto which the hydrogel matrix is ​​crosslinked, the solid substrate comprising at least one exterior surface, at least one interior surface, and at least one internal chamber defined by the at least one interior surface and accessible from a point outside the solid substrate through at least one opening. In some embodiments, the composition further comprises cell culture medium and / or cerebrospinal fluid.

[0024] In some embodiments, the cell explant or fragment thereof is one or a combination of a DRG explant, a retinal tissue explant, a cortical explant, a spinal cord explant, and a peripheral nerve explant.

[0025] In some embodiments, the composition further comprises a solid substrate having adjacent exterior and interior surfaces, the solid substrate comprising at least a portion of a cylindrical or generally cylindrical shape and at least one hollow interior defined by at least a portion of the interior surface at an edge, the interior being The surface comprises one or more pores having a diameter of about 0.1 microns to about 1.0 microns, the hollow interior of the solid substrate is accessible from a point outside the solid substrate through at least one opening, the hollow interior portion comprises a first portion proximate the opening and at least a second portion distal to the opening, the one or more neural cells and / or one or more tissue explants are positioned at or proximate to the first portion of the hollow interior and are in physical contact with the hydrogel matrix, and the at least one second portion of the hollow interior is in fluid communication with the first portion such that axons can grow from the one or more neural cells and / or one or more tissue explants into the second interior portion of the hollow interior.

[0026] In some embodiments, the composition does not include a sponge.

[0027] In some embodiments, the at least one cell-impermeable polymer comprises about 15% or less PEG, and the at least one cell-permeable polymer comprises about 0.05% to about 1.00% of one or a combination of self-assembling peptides selected from RAD16-I, RAD16-II, EAK16-I, EAK16-II, and dEAK16.

[0028] In some embodiments, the culture vessel comprises 96, 192, 384 or more chambers in which one or more isolated Schwann cells and / or one or more oligodendrocytes are in sufficient proximity to tissue explants and / or neural cells such that the Schwann cells or oligodendrocytes establish myelin-to-axonal growth from one or more isolated tissue explants and / or one or more neural cells.

[0029] In some embodiments, the solid substrate does not include PEG.

[0030] In some embodiments, at least a portion of the substrate is formed in the shape of a cylinder or rectangular prism with a space defined by an interior surface and accessible by one or more openings.

[0031] The composition further comprises a cell culture medium containing nerve growth factor (NGF) at a concentration of about 5 to about 20 picograms / milliliter, and / or ascorbic acid at a concentration in the range of about 0.001% by weight to about 0.01% by weight.

[0032] In some embodiments, the one or more neural cells comprise at least one cell selected from the group including glial cells, embryonic cells, mesenchymal stem cells, and cells derived from induced pluripotent stem cells. In some embodiments, the composition further comprises one or more stem cells or pluripotent cells. In some embodiments, the one or more neural cells comprise primary mammalian cells, the primary cells being derived from the peripheral nervous system of a mammal.

[0033] In some embodiments, the hydrogel matrix comprises at least 1% polyethylene glycol (PEG).

[0034] In some embodiments, the neural cells and / or tissue explants are cultured for 3, 30, 90, or 365 days or more.

[0035] In some embodiments, at least a portion of the solid substrate is cylindrical or approximately cylindrical, such that at least a portion of the interior surface of the solid substrate defines a cylindrical or approximately cylindrical hollow interior chamber in which the one or more Schwann cells and the one or more neurons are in contact.

[0036] In some embodiments, the one or more tissue explants comprise one or more DRGs having axonal growth from about 100 microns to about 500 microns in width and from about 0.11 to about 10,000 microns in length.

[0037] In some embodiments, the composition further comprises at least two electrodes interfaced with an electrochemical cell and a voltmeter, a first stimulating electrode positioned at or adjacent to a neural cell body of the tissue explant and a second recording electrode positioned at or adjacent to the distal end of the axon, such that the electrodes create a potential difference along the membrane distance of at least one cell in the tissue explant.

[0038] The present disclosure also relates to a method for evaluating a response from one or more neural cells, the method comprising: growing one or more neural cells in a culture vessel; introducing one or more stimuli to the one or more neural cells; and measuring one or more responses from the one or more neural cells to the one or more stimuli. In some embodiments, the one or more neural cells comprise peripheral sensory neurons. In some embodiments, the one or more neural cells comprise at least one or a combination of cells selected from spinal motor neurons, sympathetic neurons, and central nervous system (CNS) neurons.

[0039] In some embodiments, the culture vessel comprises a hydrogel matrix crosslinked to a solid substrate having a predetermined shape, the hydrogel matrix comprising at least one cell impermeable polymer and at least one cell permeable polymer, In some embodiments, the hydrogel matrix comprises one or a combination of compounds selected from PuraMatrix, methacrylated hyaluronic acid, agarose, methacrylated heparin, and methacrylated dextran.

[0040] In some embodiments, the one or more stimuli comprise an electrical current and the one or more responses comprise an electrophysiological metric, hi some embodiments, the response is measured by an optical recording technique.

[0041] In some embodiments, the one or more stimuli include one or a combination of one or more optogenetic actuators, one or more caged neurotransmitters, one or more infrared lasers, or one or more light-gated ion channels.

[0042] In some embodiments, the measuring step comprises monitoring the movement of a voltage-sensitive dye, a calcium dye, or using label-free photonic imaging. In some embodiments, the one or more neurons comprise isolated primary ganglion tissue.

[0043] In some embodiments, at least a portion of the solid substrate is micropatterned by photolithography and comprises an outer surface, an inner surface, and at least one inner chamber defined by the at least one inner surface, and the method further comprises seeding one or more neural cells onto the micropatterned solid substrate such that the growth of the one or more neural cells is confined to a specific geometric shape defined by the at least one inner chamber. In some embodiments, the inner chamber separates the cell body from the axonal processes in different locations. In some embodiments, the shape of the inner chamber allows for the matching of either morphometric or electrophysiological metrics to be detected and used at separate locations within the chamber. Typically, the inner chamber or interior compartment of the solid substrate, for example of a hydrogel matrix, allows one or more locations within the matrix or substrate to address the cell body and axonal processes in different locations when a solid substrate is not used.

[0044] In some embodiments, one or more nerve cells are derived from human primary tissue or human stem cell.In some embodiments, one or more nerve cells are mammalian primary neurons.In some embodiments, at least one nerve cell comprises isolated DRG or its fragment, and inducing stimulation from one or more nerve cells comprises placing a stimulating electrode on or adjacent to the cell neuronal soma of DRG or its fragment, and placing a recording electrode on or adjacent to the axonal process most distal from the neuronal soma.

[0045] In some embodiments, the one or more stimuli include an electrical or chemical stimulus. In some embodiments, the one or more stimuli include contacting the one or more neural cells and / or one or more tissue explants with at least one pharmacologically active compound.

[0046] The present disclosure also relates to a method of assessing toxicity of an agent comprising: (a) culturing one or more neuronal cells and / or one or more tissue explants in any of the compositions disclosed herein; (b) exposing at least one agent to the one or more neuronal cells and / or one or more tissue explants; (c) measuring and / or observing one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants; and (d) correlating the one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants to toxicity of the agent such that if the morphometric changes indicate decreased cell viability, the agent is characterized as toxic, and if the morphometric changes indicate no change or increased cell viability, the agent is characterized as non-toxic.

[0047] The present disclosure also provides a method for assessing the relative toxicity of a first agent compared to a second agent, comprising: (a) culturing one or more neuronal cells and / or one or more tissue explants in any of the compositions disclosed herein; (b) exposing the one or more neuronal cells and / or one or more tissue explants to the first agent and the second agent, either sequentially or in parallel over a period of time (sequentially in the case of the same set of cells, or in parallel in the case of a second set of cells, e.g., in a multiplexed system); and (c) exposing the one or more neuronal cells and / or one or more tissue explants to the first agent and the second agent. (d) correlating the one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants to a toxicity of a first agent; (e) correlating the one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants to a toxicity of a second agent; (f) comparing the toxicity of the first and second agents; and (g) characterizing the first or second agent as being more or less toxic than the second agent. In some embodiments, when characterizing a first or second agent as more or less toxic than a second agent, a first agent is more toxic than a second agent if the first agent induced morphometric changes that are more severe than the second compound and exhibit significantly reduced cell viability, and a second agent is more toxic than a first agent if the first agent induced morphometric changes that are less severe than the second compound and / or exhibits increased cell viability. The same characterization can be applied in embodiments where electrophysiological metrics are observed and / or measured.

[0048] In some embodiments, the degree of toxicity is determined by repeating any one or more of the steps described herein using one or a series of doses or amounts of the drug. Rather than comparing or contrasting the relative toxicity between two different drugs, one skilled in the art can thus add different doses of the same drug to characterize when and at what doses the drug may be toxic to one or more neurons.

[0049] The present disclosure also provides a method for assessing the toxicity of a drug, comprising: (a) culturing one or more neuronal cells and / or one or more tissue explants in any of the compositions disclosed herein; (b) exposing the one or more neuronal cells and / or one or more tissue explants to at least one agent; (c) measuring and / or observing one or more electrophysiological metrics of the one or more neuronal cells and / or one or more tissue explants; and (d) measuring and / or observing one or more electrophysiological metrics of the one or more neuronal cells and / or one or more tissue explants such that the agent is characterized as toxic if the electrophysiological metrics indicate decreased cell viability and the agent is characterized as non-toxic if the electrophysiological metrics indicate no change or increased cell viability. and correlating the morphometric metrics with toxicity of the agent, step (c) optionally comprising observing one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants, and step (d) optionally comprising correlating the one or more morphometric changes in the one or more neuronal cells and / or tissue explants with toxicity of the agent such that if the morphometric changes indicate reduced cell viability, the agent is characterized as toxic, and if the morphometric changes indicate no change or increased cell viability, the agent is characterized as non-toxic.

[0050] In some embodiments, the at least one agent comprises a small molecule chemical. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small molecule chemicals selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents such as bacterial antibiotics. In some embodiments, the at least one agent comprises a therapeutically effective amount of an antibody, such as a clinically relevant monoclonal antibody such as Tysabri.

[0051] In some embodiments, the one or more electrophysiological metrics are one or a combination of electrical conduction velocity, action potential, wave amplitude associated with the passage of an electrical impulse along the membrane of one or more neuronal cells, width of an electrical impulse along the membrane of one or more neuronal cells, latency of an electrical impulse along the membrane of one or more neuronal cells, and envelope of an electrical impulse along the membrane of one or more neuronal cells. In some embodiments, the one or more electrophysiological metrics include compound action potentials across the tissue explant.

[0052] The present disclosure also relates to a method of measuring the amount or degree of myelination or demyelination of one or more axons of one or more neuronal cells and / or one or more tissue explants, the method comprising: (a) culturing the one or more neuronal cells and / or one or more tissue explants in any of the compositions disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants; and (c) correlating the one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants with a quantitative or qualitative change in myelination of the neuronal cells or tissue explants.

[0053] The present disclosure also provides a method of measuring myelination or demyelination of one or more axons of one or more neuronal cells and / or one or more tissue explants, the method comprising: (a) culturing the one or more neuronal cells and / or one or more tissue explants in any of the compositions disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more electrophysiological metrics of the one or more neuronal cells and / or one or more tissue explants; and (c) correlating the one or more electrophysiological metrics of the one or more neuronal cells and / or one or more tissue explants with a quantitative or qualitative change in myelination of the neuronal cells or tissue explants, wherein step (b) optionally comprises: and / or observing one or more morphometric changes in the tissue explants above, and step (c) optionally comprises correlating the one or more morphometric changes in the one or more neuronal cells and / or tissue explants with a quantitative or qualitative change in myelination of the neuronal cells or tissue explants.

[0054] The present disclosure also relates to a method of measuring myelination or demyelination of one or more axons of one or more neuronal cells and / or one or more tissue explants, the method comprising: (a) culturing the one or more neuronal cells and / or one or more tissue explants in any of the compositions disclosed herein for a time and under conditions sufficient to grow at least one axon; and (b) detecting the amount of myelination in one or more axons of the one or more neuronal cells and / or one or more tissue explants.

[0055] In some embodiments, detecting the amount of myelination in one or more axons of one or more neural cells and / or one or more tissue explants comprises exposing the cells to an antibody that binds to myelin.

[0056] In some embodiments, the method further comprises: (i) exposing the one or more neuronal cells and / or one or more tissue explants to at least one agent after steps (a) and (b); (ii) measuring and / or observing one or more electrophysiological metrics, measuring and / or observing one or more morphometric changes, and / or detecting a quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants; (iii) calculating a change in the measurements, observations, and / or quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants in the presence and absence of the agent; and (iv) relating the changes in the measurements, observations, and / or quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants to the presence or absence of the agent.

[0057] In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small molecule chemicals selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents.

[0058] In some embodiments, the one or more electrophysiological metrics are one or a combination of electrical conduction velocity, action potential, wave amplitude associated with the passage of an electrical impulse along the membrane of one or more neuronal cells, width of an electrical impulse along the membrane of one or more neuronal cells, latency of an electrical impulse along the membrane of one or more neuronal cells, and envelope of an electrical impulse along the membrane of one or more neuronal cells. In some embodiments, the one or more electrophysiological metrics include compound action potentials across the tissue explant.

[0059] The present disclosure also provides a method for measuring myelination or demyelination of one or more axons of one or more neuronal cells and / or one or more tissue explants, the method comprising: (a) culturing one or more neuronal cells and / or one or more tissue explants in any of the compositions disclosed herein for a time and under conditions sufficient to grow at least one axon; (b) inducing compound action potentials in such one or more neuronal cells and / or one or more tissue explants; (c) measuring the compound action potentials; and (d) quantifying the level of myelination of such one or more neuronal cells based on the compound action potentials. and exposing the one or more neural cells and / or one or more tissue explants to an agent. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant.

[0060] In some embodiments, the at least one agent comprises one or a combination of small molecule chemicals selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents.

[0061] In some embodiments, the method further comprises measuring one or more electrophysiological metrics other than the compound action potential selected from one or a combination of electrical conduction velocity, individual action potentials, wave amplitude associated with the passage of an electrical impulse along the membrane of the one or more neural cells and / or tissue explants, width of an electrical impulse along the membrane of the one or more neural cells and / or tissue explants, latency of an electrical impulse along the membrane of the one or more neural cells and / or tissue explants, and envelope of an electrical impulse along the membrane of the one or more neural cells and / or tissue explants. In some embodiments, the method further comprises measuring one or more morphometric changes associated with the one or more neural cells and / or one or more tissue explants.

[0062] The present disclosure also relates to a method of inducing growth of one or more neural cells in a three-dimensional culture vessel comprising a solid substrate, the method comprising: (a) seeding one or more isolated Schwann cells with a solid substrate; (b) seeding one or more isolated neural cells in suspension or isolated neural cells in an explant into at least one internal chamber; and (c) introducing a volume of cell culture medium into the culture vessel sufficient to at least cover the cells, wherein the solid substrate comprises a first cell-impermeable polymer and a first cell-permeable polymer.

[0063] In some embodiments, the method further includes positioning at least one electrode at one or both ends of the solid substrate, the electrodes being positioned such that they can be used to stimulate or record action potentials (AP) and / or compound action potentials (cAP) and allow for measurement of AP / cAP propagation.

[0064] In some embodiments, the composition further comprises the arrangement of at least one electrode providing means for electrical stimulation, the electrode(s) being positioned at or distal to the neuronal cell body of the DRG neuron so as to create a potential difference between two points on the neurite / axon to induce a propagating AP / cAP.

[0065] The present disclosure also relates to methods of assessing the response of neurons in a culture vessel following the introduction of one or more stimuli to one or more neurons, and measuring AP or cAP responses from the one or more neurons to the one or more stimuli using local field potentials (LFPs) or other single cell recording methods.

[0066] In some embodiments, a solid substrate comprises an exterior surface and an interior surface, the solid substrate comprising at least a portion of a cylindrical or approximately cylindrical shape and at least one hollow interior defined by at least a portion of the interior surface at an edge, the interior surface comprising one or more pores having a diameter of about 0.1 microns to about 1.0 microns, the hollow interior of the solid substrate being accessible from a point outside the solid substrate through at least one opening, the hollow interior portion comprising a first portion proximate the opening and at least a second portion distal to the opening, the one or more neural cells and / or one or more tissue explants being positioned in or proximate the first portion of the hollow interior and in physical contact with at least one of the first cell impermeable polymer or the first cell permeable polymer, the second portion of the at least one hollow interior being in fluid communication with the first portion such that axons can grow from the one or more neural cells and / or one or more tissue explants into the second interior portion of the hollow interior.

[0067] In some embodiments, the method further comprises contacting the one or more neural cells with at least one agent. In some embodiments, the at least one agent is one or more stem cells or modified T cells. In some embodiments, the modified T cells express a chimeric antigen receptor specific for the cancer cells. In some embodiments, the cell culture medium comprises one or a combination of laminin, insulin, transferrin, selenium, BSA, FBS, ascorbic acid, type I collagen, and type III collagen.

[0068] The present disclosure also relates to a method of detecting and / or quantifying neuronal cell growth, comprising: (a) quantifying one or more neuronal cells; (b) culturing the one or more neuronal cells in any of the compositions disclosed herein; and (c) calculating the number of neuronal cells in the composition after culturing for a period of time sufficient to allow the one or more cells to grow. In some embodiments, step (c) comprises detecting internal and / or external recordings of the one or more neuronal cells after culturing such one or more neuronal cells and correlating the recordings with measurements of the same recordings corresponding to a known or control number of cells.

[0069] In some embodiments, the method further comprises contacting the one or more neurons with one or more agents. In some embodiments, the method further comprises (i) measuring intracellular and / or extracellular recordings before and after contacting the one or more neurons with the one or more agents, and (ii) relating a difference in a recording before contacting the one or more neurons with the one or more agents to a recording after contacting the one or more neurons with the one or more agents to a change in cell number.

[0070] The present disclosure also relates to a method of detecting or quantifying axonal degeneration of one or more neuronal cells, comprising: (a) seeding one or more neuronal cells on any of the compositions disclosed herein; (b) culturing the one or more neuronal cells for a period of time and under conditions sufficient to grow at least one or more axons from the one or more neuronal cells; (c) quantifying the number or density of axons growing from the neuronal cells; (d) contacting the one or more neuronal cells with one or more agents; (e) quantifying the number and / or density of axons growing from the neuronal cells after contacting the one or more cells with the one or more agents; and (f) calculating the difference in the number or density of axons cultured in the presence or absence of the agents.

[0071] In some embodiments, the step of determining the density of axons and / or axons growing from one or more neuronal cells comprises staining one or more neuronal cells with a dye, fluorophore, or labeled antibody.

[0072] In some embodiments, steps (c), (e), and / or (f) are performed by microscopy or digital imaging.

[0073] In some embodiments, steps (c) and (e) include taking measurements from a portion of one or more axons proximal to one or more neuronal cell bodies and taking measurements from a portion of one or more axons distal to the one or more neuronal cell bodies.

[0074] In some embodiments, the difference in the number or density of cultured axons in the presence or absence of an agent is a difference between a portion of the axon(s) proximal to the cell body of one or more neurons and a portion of the axons distal to the cell body of one or more neurons.

[0075] In some embodiments, taking the measurements includes measuring any one of a combination of morphometric or electrophysiological metrics, and calculating the difference in the number or density of cultured axons includes relating any one of the measurements or combinations thereof to the number or density of axons. In some embodiments, taking the measurements includes measuring any one of a combination of electrophysiological metrics, and calculating the difference in the number or density of cultured axons includes relating any one of the electrophysiological metrics or combinations thereof to the number or density of axons.

[0076] In some embodiments, the method further comprises (g) correlating the neurodegenerative effect of the agent to the electrophysiological metrics taken in steps (c) and (e).

[0077] The present disclosure also relates to a method of measuring intracellular or extracellular recordings, comprising: (a) culturing one or more neuronal cells in any of the compositions disclosed herein; (b) applying an electrical potential across the one or more neuronal cells; and (c) measuring one or more electrophysiological metrics from the one or more neuronal cells. In some embodiments, the one or more electrophysiological metrics are selected from one or a combination of electrical conduction velocity, intracellular action potential, compound action potential, amplitude of a wave associated with the passage of an electrical impulse along a membrane of one or more neuronal cells and / or tissue explants, width of an electrical impulse along a membrane of one or more neuronal cells and / or tissue explants, latency of an electrical impulse along a membrane of one or more neuronal cells and / or tissue explants, and envelope of an electrical impulse along a membrane of one or more neuronal cells and / or tissue explants.

[0078] The present disclosure also relates to a method of measuring or quantifying any neuroprotective effect of an agent comprising: (a) seeding one or more neuronal cells or tissue explants in any of the compositions disclosed herein, in the presence and absence of the agent; (b) applying an electrical potential across the one or more neuronal cells or tissue explants, in the presence and absence of the agent; (c) measuring one or more electrophysiological metrics from the one or more neuronal cells or tissue explants, in the presence and absence of the agent; and (d) relating the difference in the one or more electrophysiological metrics across the one or more neuronal cells or tissue explants to the neuroprotective effect of the agent, such that a decrease in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates a poor neuroprotective effect, and a no change or an increase in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates that the agent confers a neuroprotective effect.

[0079] The present disclosure also relates to a method of measuring or quantifying any neuromodulatory effect of an agent comprising: (a) seeding one or more neuronal cells or tissue explants in any of the compositions disclosed herein, in the presence and absence of the agent; (b) applying an electrical potential across the one or more neuronal cells or tissue explants, in the presence and absence of the agent; (c) measuring one or more electrophysiological metrics from the one or more neuronal cells or tissue explants, in the presence and absence of the agent; and (d) relating the difference in the one or more electrophysiological metrics across the one or more neuronal cells or tissue explants to the neuromodulatory effect of the agent, such that a change in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates a neuromodulatory effect, and a no change in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates that the agent does not impart a neuromodulatory effect.

[0080] The present disclosure also relates to a method of detecting or quantifying axonal myelination or demyelination in vitro, comprising: (a) culturing one or more neuronal cells in any of the compositions disclosed herein for a time and under conditions sufficient to cause the one or more neuronal cells to grow one or more axons; (b) applying an electrical potential across the one or more neuronal cells; (c) measuring a field potential or compound action potential through the one or more neuronal cells; (d) calculating the conduction velocity through the one or more neuronal cells; and (e) relating the one or more values ​​or conduction velocities to the amount of myelination of one or more axons.

[0081] In some embodiments, the method further comprises relating the conduction velocity of step (d) to values ​​of the conduction velocity of a known or predetermined number of healthy myelinated nerve cells.

[0082] In some embodiments, the method further comprises exposing one or more neural cells to an agent, and steps (a)-(e) are performed in the presence of the agent, and the method further comprises assessing a difference in the amount of myelination due to the presence of the agent, wherein the conduction velocity of the cell in the presence of the agent is compared to the conduction velocity of the cell in the absence of the agent.

[0083] In some embodiments, the method further comprises imaging the one or more neural cells and / or tissue explants using a microscope and / or a digital camera.

[0084] The present disclosure also relates to a method of culturing stem cells or immune cells, the method comprising: (a) culturing one or more neural cells and / or cell explants in any of the compositions disclosed herein; and (b) exposing isolated stem cells or immune cells to the composition.

[0085] The present disclosure also relates to a system comprising: (i) a cell culture vessel comprising a hydrogel; (ii) one or more neural cells in suspension or as a component of a tissue explant; (iii) an amplifier comprising a current generator; (iv) a voltmeter and / or ammeter; and (v) at least a first stimulating electrode and at least a first recording electrode, wherein the amplifier, voltmeter and / or ammeter, and the electrodes are electrically connected to each other via a circuit in which current is supplied from the amplifier to at least one stimulating electrode and received at the recording electrode and supplied to the voltmeter and / or ammeter, the stimulating electrode being positioned at or proximate to one or more neural somas of the neural cells, and the recording electrode being positioned at a predetermined distance distal to the neural somas such that an electric field is established across the cell culture vessel. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for generating a three-dimensional culture of one or more neural cells in a culture vessel comprising a solid substrate, the method comprising: (a) contacting one or more isolated Schwann cells and / or oligodendrocytes with said solid substrate, said substrate comprising at least one outer surface, at least one inner surface, and at least one internal chamber defined by said at least one inner surface and accessible from a point outside said solid substrate through at least one opening; (b) seeding one or more isolated neuronal cells or tissue explants comprising neuronal cells into said at least one internal chamber; (c) applying a volume of cell culture medium to the culture vessel sufficient to cover the at least one internal chamber; The method, wherein at least a portion of the inner surface comprises a first cell-impermeable polymer and a first cell-permeable polymer. (Item 2) 2. The method according to claim 1, wherein step (a) is preceded by disposing a solution containing the first cell-impermeable polymer and the first cell-permeable polymer in the culture vessel, and inducing the first cell-impermeable polymer and the first cell-permeable polymer to physically adhere or chemically bond onto at least a portion of the inner surface. (Item 3) 3. The method of claim 1 or 2, wherein the solid substrate comprises a base having a predetermined shape that defines the shape of the outer and inner surfaces. (Item 4) 4. The method of any one of items 1 to 3, wherein the base comprises one or a combination of silica, plastic, ceramic, or metal, the base is cylindrical in shape or substantially similar in shape to a cylinder such that the first cell-impermeable polymer and the first cell-permeable polymer coat the interior surface of the base and define a cylindrical or approximately cylindrical interior chamber or compartment, and the opening is located at one end of the cylinder. (Item 5) 5. The method according to any one of items 1 to 4, wherein the base comprises one or more pores of a size and shape sufficient to allow diffusion of proteins, nutrients, and oxygen through the solid substrate in the presence of cell culture medium. (Item 6) 6. The method according to any one of items 2 to 5, wherein the step of inducing the first cell-impermeable polymer and the second cell-permeable polymer to crosslink on the solid substrate comprises exposing the solution to ultraviolet or visible light. (Item 7) 7. The method according to any one of items 1 to 6, wherein the first cell-impermeable polymer is polyethylene glycol (PEG) at a concentration of about 20% by weight or less of the solution. (Item 8) 8. The method according to any one of items 1 to 7, wherein the first cell-permeable polymer is at a concentration of about 0.1% by weight to about 3.0% by volume of the solution. (Item 9) 9. The method of any one of items 1 to 8, further comprising exposing the culture vessel to 37° Celsius and a carbon dioxide concentration of about 5.0% or less for a time sufficient to grow axons in the inner chamber. (Item 10) 10. The method of any one of items 1 to 9, wherein at least a portion of the solid substrate is cylindrical or approximately cylindrical, such that at least a portion of the inner surface of the solid substrate defines a cylindrical or approximately cylindrical interior chamber into which the one or more Schwann cells are seeded and into which the one or more neurons are seeded. (Item 11) 11. The method of any one of items 1 to 10, wherein step (c) comprises seeding a tissue explant selected from one or a combination of an isolated dorsal root ganglion, a spinal cord explant, a retinal explant, and a cortical explant. (Item 12) 12. The method according to any one of items 1 to 11, wherein step (c) comprises seeding a suspension of neural cells selected from one of motor neurons, cortical neurons, spinal neurons, peripheral neurons or a combination thereof. (Item 13) The method according to any one of items 1 to 12, wherein the solid substrate comprises a plastic base crosslinked with the first cell-impermeable polymer and a mixture of the first cell-permeable polymer, and the plastic base comprises a plurality of pores having a diameter of about 1 micron or less. 14. The method according to any one of items 1 to 13, further comprising forming a solid substrate and positioning the solid substrate within a culture vessel. (Item 15) Item 15. The method of item 14, wherein the step of forming a solid substrate comprises curing a solution comprising the first cell-impermeable polymer and the first cell-permeable polymer by photolithography. (Item 16) 16. The method of any one of items 1 to 15, further comprising, after step (c), allowing the neural cells to grow neurites and / or axons for a period of about 1 day to about 1 year. (Item 17) 17. The method according to any one of items 1 to 16, further comprising a step of isolating one or more Schwann cells and / or one or more oligodendrocytes from the sample prior to step (a). (Item 18) 18. The method of any one of items 1 to 17, further comprising isolating dorsal root ganglia (DRG) from one or more mammals prior to step (b). (Item 19) 19. The method according to any one of items 1 to 18, wherein the culture vessel does not comprise a sponge. (Item 20) 20. The method of any one of items 1 to 19, wherein the solid substrate comprises about 15% or less of PEG and about 0.05% to about 1.00% of one or a combination of self-assembling peptides selected from RAD16-I, RAD16-II, EAK16-I, EAK16-II, and dEAK16. (Item 21) 21. The method according to any one of items 1 to 20, wherein the culture vessel comprises about 1 to about 1200 wells in which steps (a) to (c) can be performed sequentially or simultaneously. (Item 22) 22. The method of any one of items 1 to 21, wherein the solid substrate polymer does not comprise PEG. (Item 23) 23. The method according to any one of items 1 to 22, wherein at least a portion of the substrate is formed in the shape of a cylinder or a rectangular prism, the chamber being defined by the inner surface and accessible by one or more openings. (Item 24) 24. The method according to any one of items 1 to 23, wherein the cell culture medium comprises nerve growth factor (NGF) at a concentration of about 5 to about 20 picograms / milliliter, and / or ascorbic acid at a concentration in the range of about 0.001% by weight to about 0.01% by weight by volume. (Item 25) 25. The method of any one of items 1 to 24, further comprising positioning at least one stimulating electrode at or proximate to a neuronal cell body of the one or more neuronal cells or tissue explants, and positioning at least one recording electrode at or proximate to an axon at a point most distal from the neuronal cell body, such that upon introduction of current to the stimulating electrode, the recording electrode is capable of receiving a signal corresponding to one or more electrophysiological metrics measurable at the recording electrode. (Item 26) 26. The method of any one of items 1 to 25, wherein the one or more electrophysiological metrics are one or a combination of electrical conduction velocity, action potential, wave amplitude associated with the passage of an electrical impulse along the membrane of one or more neuronal cells, width of an electrical impulse along the membrane of one or more neuronal cells, latency of the electrical impulse along the membrane of one or more neuronal cells, and envelope of the electrical impulse along the membrane of one or more neuronal cells. (Item 27) (i) culture container, a hydrogel matrix comprising at least a first cell-impermeable polymer and a first cell-permeable polymer; one or more isolated Schwann cells and / or one or more oligodendrocytes, and one or more tissue explants or fragments thereof; or (ii) culture container, a hydrogel matrix comprising at least a first cell-impermeable polymer and a first cell-permeable polymer; one or more isolated Schwann cells and / or one or more oligodendrocytes, and A composition comprising a suspension of cells comprising one or more neural cells. (Item 28) 28. The composition of claim 27, further comprising a solid substrate onto which the hydrogel matrix is ​​crosslinked, the solid substrate comprising at least one primarily plastic surface having pores having diameters of about 1 micron to about 5 microns. (Item 29) 28. The composition of claim 27, further comprising a solid substrate on which the hydrogel matrix is ​​crosslinked, the solid substrate comprising at least one exterior surface, at least one interior surface, and at least one internal chamber defined by the at least one interior surface and accessible from a point outside the solid substrate through at least one opening. (Item 30) 30. The composition according to any one of items 27 to 29, further comprising a cell culture medium and / or cerebrospinal fluid. (Item 31) 31. The composition according to any one of items 27 to 30, wherein the cell explant or a fragment thereof is one or a combination of a DRG explant, a retinal tissue explant, a cortical explant, a spinal cord explant, and a peripheral nerve explant. (Item 32) The method further comprises a solid substrate having adjacent exterior and interior surfaces, the solid substrate comprising at least a portion of a cylindrical or approximately cylindrical shape and at least one hollow interior defined by at least a portion of the interior surface at an edge, the interior surface comprising one or more pores having a diameter of about 0.1 microns to about 1.0 microns, the hollow interior of the solid substrate being accessible from a point outside the solid substrate through at least one opening, the hollow interior portion being defined by a first portion proximate the opening and at least a second portion distal to the opening. 32. The composition of any one of items 27 to 31, comprising a first portion of the hollow interior and a second portion, wherein the one or more neural cells and / or the one or more tissue explants are positioned at or adjacent to the first portion of the hollow interior and are in physical contact with the hydrogel matrix, and the second portion of the at least one hollow interior is in fluid communication with the first portion such that axons can grow from the one or more neural cells and / or the one or more tissue explants into the second internal portion of the hollow interior. (Item 33) 33. The composition according to any one of items 27 to 32, which does not comprise a sponge. (Item 34) 34. The composition of any one of items 27 to 33, wherein the at least one cell-impermeable polymer comprises about 15% or less of PEG, and the at least one cell-permeable polymer comprises about 0.05% to about 1.00% of one or a combination of self-assembling peptides selected from RAD16-I, RAD16-II, EAK16-I, EAK16-II, and dEAK16. (Item 35) 35. The composition of any one of items 27 to 34, wherein the culture vessel comprises 96, 192, 384 or more chambers in which one or more isolated Schwann cells and / or one or more oligodendrocytes are in sufficient proximity to the tissue explants and / or neuronal cells such that the Schwann cells or oligodendrocytes establish myelin-to-axonal growth from the one or more isolated tissue explants and / or the one or more neuronal cells. (Item 36) The composition according to any one of items 27 to 35, wherein the solid substrate does not contain PEG. 37. The composition of any one of claims 27 to 36, wherein at least a portion of the substrate is formed in the shape of a cylinder or a rectangular prism, the space being defined by the inner surface and accessible by one or more openings. (Item 38) 38. The composition according to any one of items 27 to 37, further comprising a cell culture medium comprising nerve growth factor (NGF) at a concentration of about 5 to about 20 picograms / milliliter, and / or ascorbic acid at a concentration in the range of about 0.001% by weight to about 0.01% by weight by volume. (Item 39) 39. The composition according to any one of items 27 to 38, wherein the one or more neural cells comprise at least one cell selected from the group comprising glial cells, embryonic cells, mesenchymal stem cells, and cells derived from induced pluripotent stem cells. (Item 40) 40. The composition of any one of items 27 to 39, further comprising one or more stem cells or pluripotent cells. (Item 41) 41. The composition of any one of items 27 to 40, wherein the one or more neural cells comprise primary cells of a mammal, the primary cells being derived from the peripheral nervous system of the mammal. (Item 42) 42. The composition according to any one of items 27 to 41, wherein the hydrogel matrix comprises at least 1% polyethylene glycol (PEG). (Item 43) 43. The composition of any one of items 27 to 42, wherein the neuronal cell and / or tissue explant is cultured for 3, 30, 90, or 365 days or more. (Item 44) 44. The composition of any one of items 27 to 43, wherein at least a portion of the solid substrate is cylindrical or approximately cylindrical, such that at least a portion of the inner surface of the solid substrate defines a cylindrical or approximately cylindrical hollow interior chamber in which the one or more Schwann cells and one or more neurons are in contact. (Item 45) 45. The composition of any one of items 27 to 44, wherein the one or more tissue explants comprise one or more DRGs having axonal growth from about 100 microns to about 500 microns in width and from about 0.11 to about 10000 microns in length. (Item 46) 46. ​​The composition of any one of items 27 to 45, further comprising at least two electrodes interfaced with an electrochemical cell and a voltmeter, a first stimulating electrode being positioned at or adjacent to a neuronal cell body of the tissue explant and a second recording electrode being positioned at or adjacent to a distal end of an axon, such that the electrodes form a potential difference along the membrane distance of at least one cell in the tissue explant. (Item 47) 1. A method for assessing a response from one or more neurons, comprising: Growing one or more neural cells in a culture vessel; introducing one or more stimuli to the one or more neural cells; and measuring one or more responses from the one or more neurons to the one or more stimuli. (Item 48) 48. The method of claim 47, wherein the one or more neural cells comprise a peripheral sensory neuron. (Item 49) 49. The method of claim 47 or 48, wherein the one or more neural cells comprise at least one of cells selected from spinal motor neurons, sympathetic neurons, and central nervous system (CNS) neurons, or a combination thereof. (Item 50) 50. The method according to any one of items 47 to 49, wherein the culture vessel comprises a hydrogel matrix crosslinked to a solid substrate having a predetermined shape, the hydrogel matrix comprising at least one cell-impermeable polymer and at least one cell-permeable polymer. (Item 51) 51. The method according to any one of items 47 to 50, wherein the hydrogel matrix comprises one or a combination of compounds selected from the group consisting of PuraMatrix, methacrylated hyaluronic acid, agarose, methacrylated heparin, and methacrylated dextran. (Item 52) 52. The method of any one of items 47 to 51, wherein the one or more stimuli comprise an electrical current and the one or more responses comprise electrophysiological metrics. (Item 53) 53. The method according to any one of items 47 to 52, wherein the response is measured by optical recording techniques. (Item 54) 54. The method of any one of items 47 to 53, wherein the one or more stimuli comprise one or a combination of one or more optogenetic actuators, one or more caged neurotransmitters, one or more infrared lasers, or one or more light-gated ion channels. (Item 55) 55. The method of any one of items 47 to 54, wherein the measuring step comprises monitoring the movement of a voltage-sensitive dye, a calcium dye, or using label-free photonic imaging. (Item 56) 56. The method of any one of items 47 to 55, wherein the one or more neuronal cells comprise isolated primary ganglion tissue. (Item 57) 57. The method according to any one of items 47 to 56, wherein at least a portion of the solid substrate is micropatterned by photolithography and comprises an outer surface, an inner surface, and at least one internal chamber defined by the at least one inner surface, the method further comprising seeding the one or more neural cells onto the micropatterned solid substrate such that growth of the one or more neural cells is confined to a specific geometric shape defined by the at least one internal chamber. (Item 58) 58. The method of claim 57, wherein the inner chamber separates the cell body from the axon at different locations. (Item 59) 59. The method according to any one of items 47 to 58, wherein the one or more neural cells are derived from human primary tissue or human stem cells. (Item 60) 60. The method according to any one of items 47 to 59, wherein the one or more nerve cells are mammalian primary neurons. (Item 61) 61. The method of any one of items 47 to 60, wherein at least one neuronal cell comprises an isolated DRG or fragment thereof, and inducing stimulation from the one or more neuronal cells comprises placing a stimulating electrode at or adjacent to a cellular neuronal soma of the DRG or fragment thereof, and placing a recording electrode at or adjacent to an axon most distal from the neuronal soma. (Item 62) 62. The method according to any one of items 47 to 61, wherein the one or more stimuli comprise an electrical stimulus or a chemical stimulus. (Item 63) 63. The method according to any one of items 47 to 62, wherein the one or more stimuli comprise contacting the one or more neuronal cells and / or the one or more tissue explants with at least one pharmacologically active compound. (Item 64) 1. A method for assessing the toxicity of a drug, comprising: (a) culturing one or more neuronal cells and / or one or more tissue explants in a composition according to any one of items 27 to 46; (b) exposing said one or more neural cells and / or one or more tissue explants to at least one agent; (c) measuring and / or observing one or more morphometric changes in said one or more neural cells and / or one or more tissue explants; and (d) correlating one or more morphometric changes in the one or more neural cells and / or one or more tissue explants with toxicity of the agent, such that if the morphometric changes indicate reduced cell viability, the agent is characterized as toxic, and if the morphometric changes indicate no change or increased cell viability, the agent is characterized as non-toxic. (Item 65) 1. A method for assessing the toxicity of a drug, comprising: (a) culturing one or more neuronal cells and / or one or more tissue explants in a composition according to any one of items 27 to 46; (b) exposing said one or more neural cells and / or one or more tissue explants to at least one agent; (c) measuring and / or observing one or more electrophysiological metrics of said one or more neural cells and / or one or more tissue explants; and (d) correlating one or more electrophysiological metrics of the one or more neural cells and / or one or more tissue explants with toxicity of the agent, such that if the electrophysiological metrics indicate decreased cell viability, the agent is characterized as toxic, and if the electrophysiological metrics indicate unchanged or increased cell viability, the agent is characterized as non-toxic; step (c) optionally comprises observing one or more morphometric changes in said one or more neural cells and / or one or more tissue explants; The method, wherein step (d) optionally includes correlating one or more morphometric changes in the one or more neural cells and / or tissue explants with toxicity of the agent, such that if the morphometric changes indicate reduced cell viability, the agent is characterized as toxic, and if the morphometric changes indicate no change or increased cell viability, the agent is characterized as non-toxic. (Item 66) 66. The method of claim 64 or 65, wherein the at least one agent comprises a small molecule chemical. (Item 67) 67. The method according to any one of items 64 to 66, wherein the at least one agent comprises at least one environmental or industrial pollutant. (Item 68) 67. The method according to any one of items 64 to 66, wherein the at least one agent comprises one or a combination of small molecule chemicals selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents. (Item 69) 69. The method of any one of items 65 to 68, wherein the one or more electrophysiological metrics are one or a combination of electrical conduction velocity, action potential, wave amplitude associated with the passage of an electrical impulse along the membrane of one or more neuronal cells, width of an electrical impulse along the membrane of one or more neuronal cells, latency of the electrical impulse along the membrane of one or more neuronal cells, and envelope of the electrical impulse along the membrane of one or more neuronal cells. (Item 70) 70. The method of any one of items 65-69, wherein the one or more electrophysiological metrics comprises compound action potentials across the tissue explant. (Item 71) 1. A method for measuring myelination or demyelination of one or more axons of one or more neural cells and / or one or more tissue explants, said method comprising: (a) culturing one or more neural cells and / or one or more tissue explants in the composition according to any one of items 27 to 46 for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more morphometric changes in said one or more neural cells and / or one or more tissue explants; (c) correlating one or more morphometric changes in the one or more neuronal cells and / or one or more tissue explants with a quantitative or qualitative change in myelination of the neuronal cells or tissue explants. (Item 72) 1. A method for measuring myelination or demyelination of one or more axons of one or more neural cells and / or one or more tissue explants, said method comprising: (a) culturing one or more neural cells and / or one or more tissue explants in the composition according to any one of items 27 to 46 for a time and under conditions sufficient to grow at least one axon; (b) measuring and / or observing one or more electrophysiological metrics of said one or more neural cells and / or one or more tissue explants; and (c) correlating one or more electrophysiological metrics of the one or more neuronal cells and / or one or more tissue explants with a quantitative or qualitative change in myelination of the neuronal cells or tissue explants; step (b) optionally comprises observing one or more morphometric changes in said one or more neural cells and / or one or more tissue explants; The method, wherein step (c) optionally comprises correlating one or more morphometric changes in said one or more neuronal cells and / or tissue explants with a quantitative or qualitative change in myelination of said neuronal cells or tissue explants. (Item 73) 1. A method for measuring myelination or demyelination of one or more axons of one or more neural cells and / or one or more tissue explants, said method comprising: (a) culturing one or more neural cells and / or one or more tissue explants in the composition according to any one of items 27 to 46 for a time and under conditions sufficient to grow at least one axon; (b) detecting the amount of myelination in one or more axons of said one or more neural cells and / or one or more tissue explants. (Item 74) 74. The method of claim 73, wherein the step of detecting the amount of myelination in one or more axons of the one or more neural cells and / or one or more tissue explants comprises exposing the cells to an antibody that binds to myelin. (Item 75) 75. The method of any one of items 71 to 74, further comprising: (i) exposing the one or more neuronal cells and / or one or more tissue explants to at least one agent after steps (a) and (b); (ii) measuring and / or observing one or more electrophysiological metrics, measuring and / or observing one or more morphometric changes, and / or detecting a quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants; (iii) calculating a change in the measurements, observations, and / or quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants in the presence and absence of the agent; and (iv) relating the change in the measurements, observations, and / or quantitative amount of myelin from the one or more neuronal cells and / or one or more tissue explants to the presence or absence of the agent. (Item 76) 76. The method of claim 75, wherein the at least one agent comprises at least one environmental or industrial pollutant. (Item 77) 77. The method of claim 75 or 76, wherein the at least one agent comprises one or a combination of small molecule chemicals selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents. (Item 78) 73. The method of claim 72, wherein the one or more electrophysiological metrics are one or a combination of electrical conduction velocity, action potential, wave amplitude associated with the passage of an electrical impulse along the membrane of one or more neuronal cells, width of an electrical impulse along the membrane of one or more neuronal cells, latency of the electrical impulse along the membrane of one or more neuronal cells, and envelope of the electrical impulse along the membrane of one or more neuronal cells. (Item 79) 80. The method of claim 72 or 78, wherein the one or more electrophysiological metrics comprises compound action potentials across a tissue explant. (Item 80) 1. A method for measuring myelination or demyelination of one or more axons of one or more neural cells and / or one or more tissue explants, said method comprising: (a) culturing one or more neural cells and / or one or more tissue explants in the composition according to any one of items 27 to 46 for a time and under conditions sufficient to grow at least one axon; (b) inducing compound action potentials in such one or more neural cells and / or one or more tissue explants; and (c) measuring said compound action potential; (d) quantifying a level of myelination of said one or more neurons based on said compound action potentials. (Item 81) 81. The method of claim 80, further comprising exposing the one or more neural cells and / or one or more tissue explants to an agent. (Item 82) 82. The method of claim 81, wherein the at least one agent comprises at least one environmental or industrial pollutant. (Item 83) 83. The method of claim 81 or 82, wherein the at least one agent comprises one or a combination of small molecule chemicals selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol level regulators, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents. (Item 84) 84. The method of any one of items 80-83, further comprising measuring one or more electrophysiological metrics other than the compound action potential selected from one or a combination of: electrical conduction velocity, individual action potentials, wave amplitude associated with the passage of an electrical impulse along the membrane of the one or more neuronal cells and / or tissue explants, width of the electrical impulse along the membrane of the one or more neuronal cells and / or tissue explants, latency of the electrical impulse along the membrane of the one or more neuronal cells and / or tissue explants, and envelope of the electrical impulse along the membrane of the one or more neuronal cells and / or tissue explants. (Item 85) 85. The method of any one of items 80 to 84, further comprising measuring one or more morphometric changes associated with said one or more neural cells and / or said one or more tissue explants. (Item 86) 1. A method of inducing the growth of one or more neural cells in a three-dimensional culture vessel comprising a solid substrate, the method comprising: (a) seeding one or more isolated Schwann cells with said solid substrate; (b) seeding one or more isolated neuronal cells in suspension or isolated neuronal cells in an explant into at least one inner chamber; (c) introducing a volume of cell culture medium into the culture vessel sufficient to cover at least the cells; The method, wherein the solid substrate comprises a first cell-impermeable polymer and a first cell-permeable polymer. (Item 87) the solid substrate having an exterior surface and an interior surface, the solid substrate having at least a portion of a cylindrical or approximately cylindrical shape and at least one hollow interior defined by at least a portion of the interior surface at an edge, the interior surface comprising one or more pores having a diameter of from about 0.1 microns to about 1.0 microns, the hollow interior of the solid substrate being accessible from a point outside the solid substrate through at least one opening; a hollow interior portion comprising a first portion proximate the opening and at least a second portion distal to the opening, the one or more neural cells and / or one or more tissue explants being positioned at or proximate to the first portion of the hollow interior and in physical contact with at least one of the first cell impermeable polymer or the first cell permeable polymer; 87. The method of claim 86, wherein the second portion of the at least one hollow interior is in fluid communication with the first portion such that axons can grow from the one or more neural cells and / or the one or more tissue explants into the second interior portion of the hollow interior. (Item 88) 88. The method of claim 86 or 87, further comprising contacting the one or more neuronal cells with at least one agent. (Item 89) 89. The method of any one of items 86 to 88, wherein the at least one agent is one or more stem cells or modified T cells. (Item 90) 90. The method of claim 89, wherein the modified T cells express a chimeric antigen receptor specific for a cancer cell. (Item 91) 91. The method of any one of items 86 to 90, wherein the cell culture medium comprises one or a combination of laminin, insulin, transferrin, selenium, BSA, FBS, ascorbic acid, type I collagen, and type III collagen. (Item 92) 1. A method for detecting and / or quantifying neural cell growth, comprising: (a) quantifying one or more neurons; (b) culturing the one or more nerve cells in a composition according to any one of items 27 to 46; and (c) counting the number of neural cells in the construct after culturing for a period of time sufficient to allow growth of the one or more cells. (Item 93) 93. The method of claim 92, wherein step (c) comprises detecting internal and / or external recordings of one or more neurons after culturing such one or more neurons and correlating said recordings with measurements of the same recordings corresponding to a known or control number of cells. (Item 94) 94. The method of claim 92 or 93, further comprising contacting the one or more neural cells with one or more agents. (Item 95) 95. The method of any one of items 92 to 94, further comprising: (i) measuring intracellular and / or extracellular recordings before and after contacting the one or more neurons with the one or more agents; and (ii) relating the difference in the recordings before contacting the one or more neurons with the one or more agents to the recordings after contacting the one or more neurons with the one or more agents to a change in cell number. (Item 96) 1. A method for detecting or quantifying axonal degeneration in one or more neural cells, comprising: (a) seeding one or more neuronal cells onto the composition according to any one of items 27 to 46; (b) culturing the one or more neural cells for a period of time and under conditions sufficient to grow at least one or more axons from the one or more neural cells; (c) quantifying the number or density of axons growing from said neuronal cells; and (d) contacting the one or more neural cells with one or more agents; (e) quantifying the number and / or density of axons growing from the neural cells after contacting the one or more cells with one or more agents; and (f) calculating the difference in the number or density of cultured axons in the presence or absence of the agent. (Item 97) 97. The method of claim 96, wherein the step of determining the density of the axons growing from the one or more axons and / or neurons comprises staining the one or more neurons with a dye, fluorophore, or labeled antibody. (Item 98) 98. The method according to item 96 or 97, wherein steps (c), (e) and / or (f) are carried out by microscopy or digital imaging. (Item 99) 99. The method of any one of items 96 to 98, wherein steps (c) and (e) comprise taking measurements from a portion of one or more axons proximal to one or more neuronal cell bodies and taking measurements from a portion of one or more axons distal to the one or more neuronal cell bodies. (Item 100) 99. The method of any one of items 96 to 99, wherein the difference in the number or density of cultured axons in the presence or absence of the agent is a difference between a portion of axon(s) proximal to a cell body of the one or more neurons and a portion of axons distal to the cell body of the one or more neurons. (Item 101) 99. The method of claim 99, wherein taking measurements includes measuring any one of a combination of morphometric or electrophysiological metrics, and wherein the step of calculating the difference in the number or density of cultured axons includes relating any one or combination of the measurements to the number or density of axons. (Item 102) 100. The method of claim 99, wherein taking measurements includes measuring any one of a combination of electrophysiological metrics, and wherein the step of calculating differences in the number or density of cultured axons includes relating any one or combination of electrophysiological metrics to the number or density of axons. (Item 103) (g) correlating the neurodegenerative effect of the agent to the electrophysiological metrics collected in steps (c) and (e), (Item 104) 1. A method for measuring intracellular or extracellular recordings, comprising: (a) culturing one or more neuronal cells in a composition according to any one of items 27 to 46; (b) applying an electrical potential across the one or more neural cells; and (c) measuring one or more electrophysiological metrics from the one or more neural cells. (Item 105) 105. The method of claim 104, wherein the one or more other electrophysiological metrics are selected from one or a combination of electrical conduction velocity, intracellular action potential, compound action potential, wave amplitude associated with the passage of an electrical impulse along the membrane of one or more neuronal cells and / or tissue explants, width of an electrical impulse along the membrane of one or more neuronal cells and / or tissue explants, latency of the electrical impulse along the membrane of one or more neuronal cells and / or tissue explants, and envelope of the electrical impulse along the membrane of one or more neuronal cells and / or tissue explants. (Item 106) 1. A method for measuring or quantifying any neuroprotective effect of an agent, comprising: (a) culturing one or more neuronal cells or tissue explants in the composition according to any one of items 27 to 46, in the presence and absence of said agent; (b) applying an electrical potential across the one or more neuronal cells or tissue explants in the presence and absence of the agent; (c) measuring one or more electrophysiological metrics from the one or more neural cells or tissue explants in the presence and absence of the agent; and (d) relating the difference in one or more electrophysiological metrics across the one or more neural cells or tissue explants to a neuroprotective effect of the agent, such that a decrease in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates a poor neuroprotective effect, and a no change or increase in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates that the agent confers a neuroprotective effect. (Item 107) 1. A method for measuring or quantifying any neuromodulatory effect of an agent, comprising: (a) culturing one or more neuronal cells or tissue explants in the composition according to any one of items 27 to 46, in the presence and absence of said agent; (b) applying an electrical potential across the one or more neuronal cells or tissue explants in the presence and absence of the agent; (c) measuring one or more electrophysiological metrics from the one or more neural cells or tissue explants in the presence and absence of the agent; and (d) relating the difference in one or more electrophysiological metrics across the one or more neural cells or tissue explants to a neuromodulatory effect of the agent, such that a change in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates a neuromodulatory effect, and no change in the electrophysiological metric in the presence of the agent compared to the electrophysiological metric measured in the absence of the agent indicates that the agent does not impart a neuromodulatory effect. (Item 108) 1. A method for detecting or quantifying axonal myelination or demyelination in vitro, comprising: (a) culturing one or more neuronal cells in the composition according to any one of items 27 to 46 for a time and under conditions sufficient for said one or more neuronal cells to grow one or more axons; (b) applying an electrical potential across the one or more neural cells; and (c) measuring a field potential or a compound action potential through the one or more neurons; and (d) calculating a conduction velocity through the one or more neurons; and (e) relating said one or more values ​​or conduction velocity to an amount of myelination of one or more axons. (Item 109) 109. The method of claim 108, further comprising relating the conduction velocity of step (d) to values ​​of conduction velocity of a known or predetermined number of healthy myelinated nerve cells. (Item 110) 110. The method of claim 108 or 109, further comprising exposing the one or more neural cells to an agent, and wherein steps (a)-(e) are performed in the presence of the agent, and the method further comprises assessing a difference in the amount of myelination due to the presence of the agent, wherein the conduction velocity of the cell in the presence of the agent is compared to the conduction velocity of the cell in the absence of the agent. (Item 111) 111. The method according to any one of items 47 to 110, further comprising imaging the one or more neural cells and / or tissue explants using a microscope and / or a digital camera. (Item 112) 1. A method for culturing stem cells or immune cells, comprising: (a) culturing one or more neuronal cells and / or tissue explants in a composition according to any one of items 27 to 46; (b) exposing isolated stem cells or immune cells to the composition. (Item 113) (i) a cell culture vessel comprising a hydrogel; (ii) one or more neural cells, either in suspension or as a component of a tissue explant; (iii) an amplifier having a current generator; (iv) a voltmeter and / or an ammeter; (v) a system comprising at least a first stimulating electrode and at least a first recording electrode, The amplifier, voltmeter and / or ammeter, and electrodes are electrically connected to each other via a circuit in which current is supplied from the amplifier to the at least one stimulation electrode, current is received at the recording electrode and supplied to the voltmeter and / or ammeter, the stimulation electrode is positioned at or proximate to one or more neuronal cell bodies of the neuronal cells, and the recording electrode is positioned at a predetermined distance distal to the neuronal cell bodies such that an electric field is established across the cell culture vessel. [Brief description of the drawings]

[0086] [Figure 1AB]FIG. 1A shows an exemplary micropatterning of PEG constructs using dynamic mask projection photolithography. FIG. 1A shows an exemplary schematic of a digital micromirror device (DMD) dynamic mask photolithography method. FIG. 1B shows a macroscopic view of an exemplary PEG construct in a 6-well cell culture insert. FIG. 1C shows a close-up view of an exemplary PEG construct in a cell culture insert. FIG. 1D shows an exemplary DMD photomask. FIG. 1E shows an exemplary PEG construct crosslinked around an attached DRG. [Figure 1C] FIG. 1A shows an exemplary micropatterning of PEG constructs using dynamic mask projection photolithography. FIG. 1A shows an exemplary schematic of a digital micromirror device (DMD) dynamic mask photolithography method. FIG. 1B shows a macroscopic view of an exemplary PEG construct in a 6-well cell culture insert. FIG. 1C shows a close-up view of an exemplary PEG construct in a cell culture insert. FIG. 1D shows an exemplary DMD photomask. FIG. 1E shows an exemplary PEG construct crosslinked around an attached DRG. [Figure 1DE] FIG. 1A shows an exemplary micropatterning of PEG constructs using dynamic mask projection photolithography. FIG. 1A shows an exemplary schematic of a digital micromirror device (DMD) dynamic mask photolithography method. FIG. 1B shows a macroscopic view of an exemplary PEG construct in a 6-well cell culture insert. FIG. 1C shows a close-up view of an exemplary PEG construct in a cell culture insert. FIG. 1D shows an exemplary DMD photomask. FIG. 1E shows an exemplary PEG construct crosslinked around an attached DRG. [Diagram 2] 1 shows the stability of PuraMatrix within an exemplary PEG construct versus amount of PBS added. A representative image of a fluorescence micrograph of PuraMatrix labeled Fluosphere 48 hours after gelation in PEG (broken white outlines indicate PEG voids) and a schematic of the dual hydrogel construct are shown above a bar plot of PuraMatrix stability versus amount of PBS added (n=18 for each of 3 experiments, bars represent standard error of the mean). [Figure 3ABC]Exemplary DRG neurite outgrowth and cell migration in a dual hydrogel construct. Figure 3A shows the live / dead stained construct (live cells and cell structures, dead cells, bright areas) after 5 days in culture. Figures 3B and 3C show DRG explants cultured for 7 days in a dual hydrogel construct, indicated by β-III tubulin positive neurites and DAPI stained nuclei. Figure 3D shows a close-up of the leading outgrowth (β-III tubulin) in the channel after 5 days. Figure 3E shows a DRG explant cultured for 7 days with stained MAP2-positive dendrites and β-III tubulin positive neurites. Figure 3F shows the branched portion of the construct focused on the surface of the cell culture insert (β-III tubulin). [Figure 3DEF] Exemplary DRG neurite outgrowth and cell migration in a dual hydrogel construct. Figure 3A shows the live / dead stained construct (live cells and cell structures, dead cells, bright areas) after 5 days in culture. Figures 3B and 3C show DRG explants cultured for 7 days in a dual hydrogel construct, indicated by β-III tubulin positive neurites and DAPI stained nuclei. Figure 3D shows a close-up of the leading outgrowth (β-III tubulin) in the channel after 5 days. Figure 3E shows a DRG explant cultured for 7 days with stained MAP2-positive dendrites and β-III tubulin positive neurites. Figure 3F shows the branched portion of the construct focused on the surface of the cell culture insert (β-III tubulin). [Figure 4ABC] Confocal micrographs of constructs stained for β-III tubulin and DAPI (4A only) are shown. FIG. 4A shows a three-dimensional view of the growth near the branch point, showing both orthogonal and side views to demonstrate thickness. Image slices are interpolated to account for the distance between slices. FIG. 4B shows merged z-stack projections of neurite outgrowth in the dual hydrogel construct. FIG. 4C shows merged z-stack projections of neurite outgrowth in the PEG construct without pure matrix. FIG. 4D shows depth-coded z-stack projections of neurite outgrowth in the PEG construct without pure matrix. FIG. 4E shows depth-coded z-stack projections of neurite outgrowth in the dual hydrogel construct. Standard deviation projections were used in FIGS. 4B-4E. [Figure 4DE] Confocal micrographs of constructs stained for β-III tubulin and DAPI (4A only) are shown. FIG. 4A shows a three-dimensional view of the growth near the branch point, showing both orthogonal and side views to demonstrate thickness. Image slices are interpolated to account for the distance between slices. FIG. 4B shows merged z-stack projections of neurite outgrowth in the dual hydrogel construct. FIG. 4C shows merged z-stack projections of neurite outgrowth in the PEG construct without pure matrix. FIG. 4D shows depth-coded z-stack projections of neurite outgrowth in the PEG construct without pure matrix. FIG. 4E shows depth-coded z-stack projections of neurite outgrowth in the dual hydrogel construct. Standard deviation projections were used in FIGS. 4B-4E. [Figure 5A] Fluorescence microscopy of DRG neurite outgrowth and cell migration within 3D dual hydrogel constructs after 7 days in vitro; β-III tubulin-positive neurites, DAPI-stained nuclei, and S100-positive glial cells confined within channels filled with PuraMatrix; supporting cells near the end of the channel, approximately 1.875 mm from the ganglion as measured from the end of the circular area containing the ganglion and the beginning of the straight channel (CD). [Figure 5B] Fluorescence microscopy of DRG neurite outgrowth and cell migration within 3D dual hydrogel constructs after 7 days in vitro; β-III tubulin-positive neurites, DAPI-stained nuclei, and S100-positive glial cells confined within channels filled with PuraMatrix; supporting cells near the end of the channel, approximately 1.875 mm from the ganglion as measured from the end of the circular area containing the ganglion and the beginning of the straight channel (CD). [Figure 5C]Fluorescence microscopy of DRG neurite outgrowth and cell migration within 3D dual hydrogel constructs after 7 days in vitro; β-III tubulin-positive neurites, DAPI-stained nuclei, and S100-positive glial cells confined within channels filled with PuraMatrix; supporting cells near the end of the channel, approximately 1.875 mm from the ganglion as measured from the end of the circular area containing the ganglion and the beginning of the straight channel (CD). [Figure 5D] Fluorescence microscopy of DRG neurite outgrowth and cell migration within 3D dual hydrogel constructs after 7 days in vitro; β-III tubulin-positive neurites, DAPI-stained nuclei, and S100-positive glial cells confined within channels filled with PuraMatrix; supporting cells near the end of the channel, approximately 1.875 mm from the ganglion as measured from the end of the circular area containing the ganglion and the beginning of the straight channel (CD). [Figure 6AB] Three-dimensional representations of confocal images are shown of the beginning (FIG. 6A), middle (FIG. 6B), and end (FIG. 6C) of the channel with β-III tubulin-positive neurites, DAPI-stained nuclei, and S100-positive glial cells shown in 3D, and corresponding cross sections in the z-plane are shown below. [Figure 6C] Three-dimensional representations of confocal images are shown of the beginning (FIG. 6A), middle (FIG. 6B), and end (FIG. 6C) of the channel with β-III tubulin-positive neurites, DAPI-stained nuclei, and S100-positive glial cells shown in 3D, and corresponding cross sections in the z-plane are shown below. [Figure 7A] Transmission electron microscopy of neuronal culture cross sections is shown. Figure 7A shows dense parallel bundles of unmyelinated neurites in the channel about 1.875 mm from the ganglion, and the inset in Figure 7B shows a zoomed view. Figure 7C shows a focus centered on an axon (Ax) encapsulated by a Schwann cell (SC) about 1 mm from the ganglion. Figure 7D shows a Schwann cell nucleus (SN) found in the ganglion, with all measurements taken from the end of a circular area containing the ganglion at the beginning of a straight channel. [Figure 7B]Transmission electron microscopy of neuronal culture cross sections is shown. Figure 7A shows dense parallel bundles of unmyelinated neurites in the channel about 1.875 mm from the ganglion, and the inset in Figure 7B shows a zoomed view. Figure 7C shows a focus centered on an axon (Ax) encapsulated by a Schwann cell (SC) about 1 mm from the ganglion. Figure 7D shows a Schwann cell nucleus (SN) found in the ganglion, with all measurements taken from the end of a circular area containing the ganglion at the beginning of a straight channel. [Figure 7C] Transmission electron microscopy of neuronal culture cross sections is shown. Figure 7A shows dense parallel bundles of unmyelinated neurites in the channel about 1.875 mm from the ganglion, and the inset in Figure 7B shows a zoomed view. Figure 7C shows a focus centered on an axon (Ax) encapsulated by a Schwann cell (SC) about 1 mm from the ganglion. Figure 7D shows a Schwann cell nucleus (SN) found in the ganglion, with all measurements taken from the end of a circular area containing the ganglion at the beginning of a straight channel. [Figure 7D] Transmission electron microscopy of neuronal culture cross sections is shown. Figure 7A shows dense parallel bundles of unmyelinated neurites in the channel about 1.875 mm from the ganglion, and the inset in Figure 7B shows a zoomed view. Figure 7C shows a focus centered on an axon (Ax) encapsulated by a Schwann cell (SC) about 1 mm from the ganglion. Figure 7D shows a Schwann cell nucleus (SN) found in the ganglion, with all measurements taken from the end of a circular area containing the ganglion at the beginning of a straight channel. [Figure 8AB]The construct is shown stained with bromophenol blue with the placement of recording (left) and stimulating (right) electrodes placed within the ganglion and tract within the channel for field recording, respectively. Figure 8B shows an example trace of the collective response demonstrating good field potential recording in the three-dimensional neural construct, and waveform characteristics characteristic of compound action potentials (CAPs). Figure 8C shows field potentials evoked within the ganglion of the three-dimensional neural culture from proximal (1.5 mm) and distal (2.25 mm) locations (n=4) (marked with dotted lines). The average trace highlights the increase in latency to onset when stimulating distally. Figure 8D shows distal stimulation caused a significant increase in latency to onset (p=0.02), increasing the mean latency from 0.82 ms to 2.88 ms, as well as a 29.46% decrease in the mean response amplitude. Stimulation distance was measured from the beginning of the straight channel to the stimulation point. Onset latency was measured as the time for the stimulation artifact to return to baseline relative to the positive peak of the response. FIG. 8E shows blockade of Na+ channel activity using 0.5 μM tetrodotoxin (TTX) in three-dimensional neural constructs. The average trace demonstrates the abolition of the population response by TTX (n=3). FIG. 8F shows that the response amplitudes were significantly different (p=0.029), with the average amplitude decreasing from 448.75 μV to 0.04 μV after TTX washout. Amplitudes were measured peak-to-peak. [Figure 8C]The construct is shown stained with bromophenol blue with the placement of recording (left) and stimulating (right) electrodes placed within the ganglion and tract within the channel for field recording, respectively. Figure 8B shows an example trace of the collective response demonstrating good field potential recording in the three-dimensional neural construct, and waveform characteristics characteristic of compound action potentials (CAPs). Figure 8C shows field potentials evoked within the ganglion of the three-dimensional neural culture from proximal (1.5 mm) and distal (2.25 mm) locations (n=4) (marked with dotted lines). The average trace highlights the increase in latency to onset when stimulating distally. Figure 8D shows distal stimulation caused a significant increase in latency to onset (p=0.02), increasing the mean latency from 0.82 ms to 2.88 ms, as well as a 29.46% decrease in the mean response amplitude. Stimulation distance was measured from the beginning of the straight channel to the stimulation point. Onset latency was measured as the time for the stimulation artifact to return to baseline relative to the positive peak of the response. FIG. 8E shows blockade of Na+ channel activity using 0.5 μM tetrodotoxin (TTX) in three-dimensional neural constructs. The average trace demonstrates the abolition of the population response by TTX (n=3). FIG. 8F shows that the response amplitudes were significantly different (p=0.029), with the average amplitude decreasing from 448.75 μV to 0.04 μV after TTX washout. Amplitudes were measured peak-to-peak. [Figure 8DE]The construct is shown stained with bromophenol blue with the placement of recording (left) and stimulating (right) electrodes placed within the ganglion and tract within the channel for field recording, respectively. Figure 8B shows an example trace of the collective response demonstrating good field potential recording in the three-dimensional neural construct, and waveform characteristics characteristic of compound action potentials (CAPs). Figure 8C shows field potentials evoked within the ganglion of the three-dimensional neural culture from proximal (1.5 mm) and distal (2.25 mm) locations (n=4) (marked with dotted lines). The average trace highlights the increase in latency to onset when stimulating distally. Figure 8D shows distal stimulation caused a significant increase in latency to onset (p=0.02), increasing the mean latency from 0.82 ms to 2.88 ms, as well as a 29.46% decrease in the mean response amplitude. Stimulation distance was measured from the beginning of the straight channel to the stimulation point. Onset latency was measured as the time for the stimulation artifact to return to baseline relative to the positive peak of the response. FIG. 8E shows blockade of Na+ channel activity using 0.5 μM tetrodotoxin (TTX) in three-dimensional neural constructs. The average trace demonstrates the abolition of the population response by TTX (n=3). FIG. 8F shows that the response amplitudes were significantly different (p=0.029), with the average amplitude decreasing from 448.75 μV to 0.04 μV after TTX washout. Amplitudes were measured peak-to-peak. [Figure 8F]The construct is shown stained with bromophenol blue with the placement of recording (left) and stimulating (right) electrodes placed within the ganglion and tract within the channel for field recording, respectively. Figure 8B shows an example trace of the collective response demonstrating good field potential recording in the three-dimensional neural construct, and waveform characteristics characteristic of compound action potentials (CAPs). Figure 8C shows field potentials evoked within the ganglion of the three-dimensional neural culture from proximal (1.5 mm) and distal (2.25 mm) locations (n=4) (marked with dotted lines). The average trace highlights the increase in latency to onset when stimulating distally. Figure 8D shows distal stimulation caused a significant increase in latency to onset (p=0.02), increasing the mean latency from 0.82 ms to 2.88 ms, as well as a 29.46% decrease in the mean response amplitude. Stimulation distance was measured from the beginning of the straight channel to the stimulation point. Onset latency was measured as the time for the stimulation artifact to return to baseline relative to the positive peak of the response. FIG. 8E shows blockade of Na+ channel activity using 0.5 μM tetrodotoxin (TTX) in three-dimensional neural constructs. The average trace demonstrates the abolition of the population response by TTX (n=3). FIG. 8F shows that the response amplitudes were significantly different (p=0.029), with the average amplitude decreasing from 448.75 μV to 0.04 μV after TTX washout. Amplitudes were measured peak-to-peak. [Figure 9AB]Figure 9D shows the absence of effects of the excitatory glutamate blockers DNQX and APV in 3D neuroconstructs (n=4). Average traces of responses before (t1-t5) and after (t16-t20) drug washout demonstrate no significant changes in response amplitude (Figure 9B) or duration (Figure 9C) from the drug. Response amplitude and duration were not statistically different after DNQX and APV. Amplitudes were measured at peak-to-peak and half-peak durations to minimize deviations between measurements. Figure 9D shows the consistent occurrence of responses during high-frequency stimulation in 3D neuroconstructs (n=3). Example traces demonstrate the consistency of electrically evoked population spikes during a 50 Hz train, zooming in on the first and last traces for comparison. The amplitude (Figure 9E) and duration (Figure 9F) of the response at the end of the 50 Hz pulse train are not significantly different from the first. Amplitudes were measured at peak-to-peak and half-peak durations to minimize deviations between measurements. [Figure 9C] Figure 9D shows the absence of effects of the excitatory glutamate blockers DNQX and APV in 3D neuroconstructs (n=4). Average traces of responses before (t1-t5) and after (t16-t20) drug washout demonstrate no significant changes in response amplitude (Figure 9B) or duration (Figure 9C) from the drug. Response amplitude and duration were not statistically different after DNQX and APV. Amplitudes were measured at peak-to-peak and half-peak durations to minimize deviations between measurements. Figure 9D shows the consistent occurrence of responses during high-frequency stimulation in 3D neuroconstructs (n=3). Example traces demonstrate the consistency of electrically evoked population spikes during a 50 Hz train, zooming in on the first and last traces for comparison. The amplitude (Figure 9E) and duration (Figure 9F) of the response at the end of the 50 Hz pulse train are not significantly different from the first. Amplitudes were measured at peak-to-peak and half-peak durations to minimize deviations between measurements. [Fig. 9DEF]Figure 9D shows the absence of effects of the excitatory glutamate blockers DNQX and APV in 3D neuroconstructs (n=4). Average traces of responses before (t1-t5) and after (t16-t20) drug washout demonstrate no significant changes in response amplitude (Figure 9B) or duration (Figure 9C) from the drug. Response amplitude and duration were not statistically different after DNQX and APV. Amplitudes were measured at peak-to-peak and half-peak durations to minimize deviations between measurements. Figure 9D shows the consistent occurrence of responses during high-frequency stimulation in 3D neuroconstructs (n=3). Example traces demonstrate the consistency of electrically evoked population spikes during a 50 Hz train, zooming in on the first and last traces for comparison. The amplitude (Figure 9E) and duration (Figure 9F) of the response at the end of the 50 Hz pulse train are not significantly different from the first. Amplitudes were measured at peak-to-peak and half-peak durations to minimize deviations between measurements. [Figure 10ABC] Electrophysiological experiments on cultured neurons are shown. Figure 10A shows the placement of recording (left) and stimulating (right) electrodes for whole-cell patch clamp. Figure 10B shows a successful whole-cell patch clamp of a primary sensory neuron in a 3D neural construct. Figure 10C shows a successful whole-cell patch clamp recording in a 3D neural culture that shows no evidence of synaptic activity (n=3). The example trace shows an electrically evoked action potential recorded from a cell within the ganglion. Figure 10D shows a zoomed-in trace that demonstrates the rapid, non-graded development of the response. Figure 10E shows a voltage clamp trace with no spontaneous currents. Figure 10F shows a current clamp trace with no spontaneous changes in potential. [Fig. 10DEF]Electrophysiological experiments on cultured neurons are shown. Figure 10A shows the placement of recording (left) and stimulating (right) electrodes for whole-cell patch clamp. Figure 10B shows a successful whole-cell patch clamp of a primary sensory neuron in a 3D neural construct. Figure 10C shows a successful whole-cell patch clamp recording in a 3D neural culture that shows no evidence of synaptic activity (n=3). The example trace shows an electrically evoked action potential recorded from a cell within the ganglion. Figure 10D shows a zoomed-in trace that demonstrates the rapid, non-graded development of the response. Figure 10E shows a voltage clamp trace with no spontaneous currents. Figure 10F shows a current clamp trace with no spontaneous changes in potential. [Figure 11A] Analysis of the depth of neurite outgrowth within the constructs is shown. Figure 11A shows the average height of β-III labeled neurites within constructs both with and without PuraMatrix (p<0.005). Figure 11B shows neurite outgrowth throughout the depth of the PuraMatrix as a percentage of total neurite outgrowth. [Figure 11B] Analysis of the depth of neurite outgrowth within the constructs is shown. Figure 11A shows the average height of β-III labeled neurites within constructs both with and without PuraMatrix (p<0.005). Figure 11B shows neurite outgrowth throughout the depth of the PuraMatrix as a percentage of total neurite outgrowth. [Figure 12AB] Fluorescence microscopy of neurite outgrowth after 7 days in vitro. Figure 12A shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the top focal plane. Figure 12B shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the bottom focal plane. Figure 12C shows restricted neurite outgrowth along the surface of the insert membrane in channels that do not contain Pura Matrix. Figure 12D shows preferential growth along the PEG boundary. Figure 12E shows the absence of myelin before Fluoromyelin™ red fluorescent myelin staining. Figure 12F shows the absence of myelin after Fluoromyelin™ red fluorescent myelin staining. [Figure 12C]Fluorescence microscopy of neurite outgrowth after 7 days in vitro. Figure 12A shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the top focal plane. Figure 12B shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the bottom focal plane. Figure 12C shows restricted neurite outgrowth along the surface of the insert membrane in channels that do not contain Pura Matrix. Figure 12D shows preferential growth along the PEG boundary. Figure 12E shows the absence of myelin before Fluoromyelin™ red fluorescent myelin staining. Figure 12F shows the absence of myelin after Fluoromyelin™ red fluorescent myelin staining. [Figure 12DE] Fluorescence microscopy of neurite outgrowth after 7 days in vitro. Figure 12A shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the top focal plane. Figure 12B shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the bottom focal plane. Figure 12C shows restricted neurite outgrowth along the surface of the insert membrane in channels that do not contain Pura Matrix. Figure 12D shows preferential growth along the PEG boundary. Figure 12E shows the absence of myelin before Fluoromyelin™ red fluorescent myelin staining. Figure 12F shows the absence of myelin after Fluoromyelin™ red fluorescent myelin staining. [Figure 12F] Fluorescence microscopy of neurite outgrowth after 7 days in vitro. Figure 12A shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the top focal plane. Figure 12B shows branching and random orientation of leading neurite outgrowth in Pura Matrix, shown from the bottom focal plane. Figure 12C shows restricted neurite outgrowth along the surface of the insert membrane in channels that do not contain Pura Matrix. Figure 12D shows preferential growth along the PEG boundary. Figure 12E shows the absence of myelin before Fluoromyelin™ red fluorescent myelin staining. Figure 12F shows the absence of myelin after Fluoromyelin™ red fluorescent myelin staining. [Figure 13]The methodology for co-culturing SCs and DRGs is shown: step 1 is the formation of a PEG mold, step 2 is the insertion of DRGs, step 3 is mixing SCs with gel solution at a specific cell number and adding the gel solution to the void, and step 4 is irradiation and gel formation using a negative mask. [Figure 14] Quantification (three-dimensional) of the amount of neuronal growth in each of the four culture models is shown. More neuronal growth was observed in the two systems using collagen. No significant effects on neuronal proliferation due to changes in media regimen were detected. [Figure 15] Development of myelin protein (MBP) after 25 days. DRG / SCs were co-cultured with fixed neurons and immunolabeled for compact myelin and neurofilaments using anti-MBP and β-III tubulin antibodies. Objective 20x, scale bar represents 25 μm. SCs completely envelop axons after 25 days and form MBP-positive axons in all experimental groups. [Figure 16A] Three-dimensional representations of confocal images are shown. Figure 16A shows immunohistochemistry for MBP protein. Figure 16B shows immunohistochemistry for MAG. Both culture thicknesses are 190 μm, confirming the three-dimensional myelination capacity of the in vitro system. [Figure 16B] Three-dimensional representations of confocal images are shown. Figure 16A shows immunohistochemistry for MBP protein. Figure 16B shows immunohistochemistry for MAG. Both culture thicknesses are 190 μm, confirming the three-dimensional myelination capacity of the in vitro system. [Figure 17]Immunohistochemistry of neurofilament β-III and MBP are shown. FIG. 17A visually shows immunohistochemistry in various media. These figures are acquired using z-stack acquisition with a confocal microscope. Maximum projections were then obtained. Dense bundled growth can be observed after 25 days. Scale bar=500 μm. FIG. 17B shows a graph of the volume of myelination. The amount of MBP-positive myelin was increased in the presence of collagen. NCol-15 with less AA exposure has the least amount of myelin. FIG. 17C shows a graph of the ratio of the amount of MBP-positive myelin to the amount of neurofilament, showing that cultures with longer AA exposure form more compact myelin. In all experimental groups, the percentage of myelination was dramatically reduced in the control group, demonstrating that exogenous SC has a major role in the myelination process. [Figure 18]Immunohistochemistry of neurofilament β-III and PO are shown. Figure 18A visually shows the immunohistochemistry in various media. Scale bar = 500 fill. Figure 18B shows a graph of the volume of myelination. The amount of PO-positive myelin increased in the presence of collagen. PO is present in PNS compact myelin, therefore PO-positive myelin represents PNS compact myelin. Col-25 with higher AA exposure and collagen incorporation has the greatest amount of compact myelin. The decreasing trend shows that removing both factors, the presence of collagen and longer exposure to AA, results in minimal myelination in 3D cultures after 25 days. Figure 18C shows a graph of the percentage of PO-positive myelin relative to neurofilament, showing the productivity of the system in myelination alone, despite the amount of neuronal output. By excluding the volume of neuronal growth, it is shown that exposure to AA plays a key role in 3D myelination in the presence or absence of collagen (Col or N-Col). However, Col-15 is statistically equivalent to NCol-25, indicating that the efficiency of the constructs after 25 days of AA exposure in the absence of collagen is similar to that after 15 days of AA exposure in the presence of collagen. Note that these quantities are substantially different from those shown in Figure 18B. [Figure 19] Immunohistochemistry of neurofilament β-III and MAG are shown. FIG. 19A visually shows the immunohistochemistry in various media. MAG is one of the major proteins present in non-compact myelin. Scale bar=500 μm. FIG. 19B shows a graph of the amount of compact myelin in all four experimental groups. Col-25 with higher AA exposure and incorporation of collagen has the highest amount of non-compact myelin. FIG. 19C shows a graph of the ratio of the volume of MAG positive myelin to neurofilament, with the shortest time of AA exposure and NCol-15 in the absence of collagen has the lowest non-compact myelination efficiency, regardless of the volume of nerve fibers in the system. [Figure 20A]Transmission electron micrographs of cross-sections of neural cultures are shown, demonstrating myelin sheaths around individual nerve fibers in 25-day cultures, i.e., (Fig. 20A) NCol-25, (Fig. 20B) NCol-15, (Fig. 20D) Col-25, (Fig. 20E) Col-15. Fig. 20C shows dense parallel fasciculated neurites within the channel. Either neurons have myelinated or SCs have started to encapsulate around nerve fibers, illustrating the abundance of myelin proteins positive in immunohistochemical staining. Fig. 20F shows a magnified view of the thick myelin sheath. A=axon, M=myelin, S=Schwann cell. [Figure 20B] Transmission electron micrographs of cross-sections of neural cultures are shown, demonstrating myelin sheaths around individual nerve fibers in 25-day cultures, i.e., (Fig. 20A) NCol-25, (Fig. 20B) NCol-15, (Fig. 20D) Col-25, (Fig. 20E) Col-15. Fig. 20C shows dense parallel fasciculated neurites within the channel. Either neurons have myelinated or SCs have started to encapsulate around nerve fibers, illustrating the abundance of myelin proteins positive in immunohistochemical staining. Fig. 20F shows a magnified view of the thick myelin sheath. A=axon, M=myelin, S=Schwann cell. [Figure 20CD] Transmission electron micrographs of cross-sections of neural cultures are shown, demonstrating myelin sheaths around individual nerve fibers in 25-day cultures, i.e., (Fig. 20A) NCol-25, (Fig. 20B) NCol-15, (Fig. 20D) Col-25, (Fig. 20E) Col-15. Fig. 20C shows dense parallel fasciculated neurites within the channel. Either neurons have myelinated or SCs have started to encapsulate around nerve fibers, illustrating the abundance of myelin proteins positive in immunohistochemical staining. Fig. 20F shows a magnified view of the thick myelin sheath. A=axon, M=myelin, S=Schwann cell. [Figure 20E]Transmission electron micrographs of cross-sections of neural cultures are shown, demonstrating myelin sheaths around individual nerve fibers in 25-day cultures, i.e., (Fig. 20A) NCol-25, (Fig. 20B) NCol-15, (Fig. 20D) Col-25, (Fig. 20E) Col-15. Fig. 20C shows dense parallel fasciculated neurites within the channel. Either neurons have myelinated or SCs have started to encapsulate around nerve fibers, illustrating the abundance of myelin proteins positive in immunohistochemical staining. Fig. 20F shows a magnified view of the thick myelin sheath. A=axon, M=myelin, S=Schwann cell. [Figure 20F] Transmission electron micrographs of cross-sections of neural cultures are shown, demonstrating myelin sheaths around individual nerve fibers in 25-day cultures, i.e., (Fig. 20A) NCol-25, (Fig. 20B) NCol-15, (Fig. 20D) Col-25, (Fig. 20E) Col-15. Fig. 20C shows dense parallel fasciculated neurites within the channel. Either neurons have myelinated or SCs have started to encapsulate around nerve fibers, illustrating the abundance of myelin proteins positive in immunohistochemical staining. Fig. 20F shows a magnified view of the thick myelin sheath. A=axon, M=myelin, S=Schwann cell. [Figure 21] Structure-function correlations are shown. Figure 21A shows confocal image stacks of unmyelinated nerve fiber tracts proximal to the dorsal root ganglion, midway through the ganglion, and distal to the ganglion, stained for β-III tubulin neurites, DAPI nuclei, and S100 Schwann cells. Figure 21B shows data showing that proximally stimulated recorded cAPs exhibit higher amplitudes and shorter latencies than those stimulated distally. [Fig. 22AB] 22 shows the physiological evaluation of neuronal cultures under toxic stress in high glucose conditions. Figure 22A shows electrophysiological traces of cell cultures in the presence of 25 mM and 60 mM glucose for 48 hours. Figure 22B shows a graph showing that exposure to 60 mM glucose conditions leads to a reduction in the amplitude of compound action potentials. Figure 22B shows a graph showing that exposure to 60 mM glucose conditions leads to an increase in the latency of compound action potentials. [Figure 22C] 22 shows the physiological evaluation of neuronal cultures under toxic stress in high glucose conditions. Figure 22A shows electrophysiological traces of cell cultures in the presence of 25 mM and 60 mM glucose for 48 hours. Figure 22B shows a graph showing that exposure to 60 mM glucose conditions leads to a reduction in the amplitude of compound action potentials. Figure 22B shows a graph showing that exposure to 60 mM glucose conditions leads to an increase in the latency of compound action potentials. [Diagram 23AB] Figure 23 shows physiological evaluation of neuronal cultures under toxic stress using 0.1 μM paclitaxel. Figure 23A shows electrophysiological traces of cell cultures before and after application of paclitaxel. Figure 23B shows a graph showing that exposure to paclitaxel reduces the amplitude of compound action potentials. Figure 23C shows a graph showing that exposure to paclitaxel increases the latency of compound action potentials. [Figure 23C] Figure 23 shows physiological evaluation of neuronal cultures under toxic stress using 0.1 μM paclitaxel. Figure 23A shows electrophysiological traces of cell cultures before and after application of paclitaxel. Figure 23B shows a graph showing that exposure to paclitaxel reduces the amplitude of compound action potentials. Figure 23C shows a graph showing that exposure to paclitaxel increases the latency of compound action potentials. [Figure 24] A list of morphological and physiological measurements that can be taken at the ganglion, at the proximal tract of the dorsal root ganglion, at the midpoint of the tract, and at the distal tract is provided. [Diagram 25] A list of candidate targets of chemotherapy-induced peripheral neurotoxicity in the dorsal root ganglion, microtubules, ion channels, myelin, mitochondria, and small nerve fibers is presented. [Figure 26] The experimental design is shown in which baseline physiological recordings are taken after growth and myelination in culture. The experiment is limited to acute (48 h) application of each drug followed by immediate or delayed (7 days) assessment by physiological recording (Rec) and imaging (CFM and TEM). The control group consisted of drug-free vehicle administration. [Figure 27A]Figure 23 shows retinal (CNS) tissue culture. Retinal explants from fetal rats were cultured within 3D micropatterned hydrogels in "neurobasal Sato" medium supplemented with either CNTF (Figure 23A) or BDNF (Figure 23B). Visible retinal ganglion cell axonal outgrowth, stained with β-III tubulin, was visualized after 1 week of culture. [Figure 27B] Figure 23 shows retinal (CNS) tissue culture. Retinal explants from fetal rats were cultured within 3D micropatterned hydrogels in "neurobasal Sato" medium supplemented with either CNTF (Figure 23A) or BDNF (Figure 23B). Visible retinal ganglion cell axonal outgrowth, stained with β-III tubulin, was visualized after 1 week of culture. [Figure 28] Experiments are presented showing that DRG neurites preferentially grow towards NGF, as opposed to BSA, diffusing from a reservoir within the hydrogel construct. [Figure 29] We present a microphysiological culture system and non-invasive electrophysiological analysis featuring selective illumination and simultaneous activation of individual cortical neurons as well as individual dendrites in cells expressing GFP and ChR2. This application of DLP microscopy and optogenetics for optical neural activation is combined with voltage-sensitive dye imaging such as VF. [Diagram 30] A multi-well format using fluorescence microscopy and electrophysiology is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] With respect to the methods and other aspects of the present disclosure, various terms are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted consistently with the definitions provided herein.

[0088] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0089] As used herein, the term "greater than two" is defined as any whole integer greater than two, for example, three, four, or five.

[0090] The term "plurality," as used herein, is defined as any amount or number greater than one.

[0091] The term "bioreactor" refers to an enclosure or subenclosure in which cells are cultured, optionally in suspension. In some embodiments, bioreactor refers to an enclosure or subenclosure in which cells are cultured, which may be in liquid suspension or may be in contact with, grown on, or in another non-liquid substrate, including, but not limited to, a solid growth support material. In some embodiments, the solid growth support material or solid substrate comprises at least one of silica, plastic, metal, hydrocarbon, or gel, or a combination thereof. The present disclosure relates to a system comprising a bioreactor, which includes one or more culture vessels in which neural cells can be cultured in the presence of a cell growth medium.

[0092] As used herein, the term "culture vessel" is defined as any vessel suitable for growing, culturing, cultivating, propagating, propagating, or otherwise manipulating cells. The culture vessel may also be referred to herein as a "culture insert." In some embodiments, the culture vessel is made of biocompatible plastic and / or glass. In some embodiments, the plastic is a thin layer of plastic with one or more pores that allow the diffusion of proteins, nucleic acids, nutrients (such as heavy metals and hormones), antibiotics, and other components of cell culture media through the pores. In some embodiments, the width of the pores is about 0.1, 0.5, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 microns or less. In some embodiments, the culture vessel does not include a base or any other structure within a hydrogel matrix. In some embodiments, the culture vessel is designed to contain a hydrogel or hydrogel matrix and various culture media. In some embodiments, the culture vessel consists or consists essentially of a hydrogel or hydrogel matrix. In some embodiments, the only plastic components of the culture vessel are those components of the culture vessel that form the sidewalls and / or base of the culture vessel that separate the volume of the cell growth well or zone from points outside the culture vessel. In some embodiments, the culture vessel comprises a hydrogel and one or more isolated glial cells. In some embodiments, the culture vessel comprises a hydrogel and one or more isolated glial cells onto which one or more neural cells are seeded.

[0093] The term "electrical stimulation" refers to a process in which cells are exposed to electrical current, either alternating current (AC) or direct current (DC). This current may be introduced into a solid substrate or applied via the cell culture medium or other suitable components of the cell culture system. In some embodiments, electrical stimulation is provided to a device or system by positioning one or more electrodes at different locations within the device or system to create an electrical potential across the cell culture vessel. The electrodes are operably connected by one or more wires to one or more amplifiers, voltmeters, ammeters, and / or electrochemical systems (batteries or generators). Such devices and wires create a circuit through which an electrical current is generated and which generates an electrical potential across the tissue culture system.

[0094] As used herein, the term "hydrogel" is defined as any water-insoluble, crosslinked, three-dimensional network of polymer chains that is water-filled or has voids between the polymer chains that can be water-filled. As used herein, the term "hydrogel matrix" is defined as any three-dimensional hydrogel construct, system, device, or similar structure. Hydrogels and hydrogel matrices are known in the art, and various types are described, for example, in U.S. Pat. Nos. 5,700,289 and 6,129,761, as well as Curley and Moore, 2011, Curley et al., 2011, Irons et al., 2008, and Tibbitt and Anseth, 2009, each of which is incorporated by reference in its entirety. In some embodiments, the hydrogel or hydrogel matrix can be solidified by subjecting the liquefied pregel solution to ultraviolet light, visible light, or any light with a wavelength greater than about 300 nm, 400 nm, 450 nm, or 500 nm. In some embodiments, the hydrogel or hydrogel matrix may be solidified into various shapes, for example, branched shapes designed to mimic nerve tracts. In some embodiments, the hydrogel or hydrogel matrix comprises poly(ethylene glycol) dimethacrylate (PEG). In some embodiments, the hydrogel or hydrogel matrix comprises PuraMatrix. In some embodiments, the hydrogel or hydrogel matrix comprises glycidyl methacrylate-dextran (MeDex). In some embodiments, neural cells are incorporated into the hydrogel or hydrogel matrix. In some embodiments, cells from the nervous system are incorporated into the hydrogel or hydrogel matrix. In some embodiments, the cells from the nervous system are Schwann cells and / or oligodendrocytes. In some embodiments, the hydrogel or hydrogel matrix comprises tissue explants from the nervous system of an animal (such as a mammal) and a complement of cells derived from the nervous system but isolated and cultured to enrich for that population in culture.In some embodiments, the hydrogel or hydrogel matrix comprises an explant, such as a retinal tissue explant, DRG, or spinal cord tissue explant, as well as a population of isolated and cultured Schwann cells, oligodendrocytes, and / or microglial cells. In some embodiments, two or more hydrogels or hydrogel matrices are used simultaneously in a cell culture vessel. In some embodiments, two or more hydrogels or hydrogel matrices are used simultaneously in the same cell culture vessel, but the hydrogels are separated by walls that create independently addressable microenvironments, such as wells, in the tissue culture vessel. In a multiplexed tissue culture vessel, some embodiments can include any number of the aforementioned wells or independently addressable locations in the cell culture vessel, such that the hydrogel matrix in one well or location can be different or identical to the hydrogel matrix in another well or location of the cell culture vessel.

[0095] In some embodiments, the two or more hydrogels may contain different amounts of PEG and / or pure matrix. In some embodiments, the two or more hydrogels may have different densities. In some embodiments, the two or more hydrogels may have different permeabilities that may allow cells to grow within the hydrogel. In some embodiments, the two or more hydrogels may have different flexibility.

[0096] The term "cell-permeable polymer" refers to a hydrophilic polymer having identical or mixed monomer subunits in a sufficient concentration and / or density to create spaces upon crosslinking in a solid or semi-solid state on a solid substrate, such spaces being sufficiently biocompatible to allow cells or portions of cells to grow in culture.

[0097] The term "cell-impermeable polymer" refers to a hydrophilic polymer having identical or mixed monomer subunits at a sufficient concentration and / or density so as not to create biocompatible spaces or compartments upon crosslinking in a solid or semi-solid state on a solid substrate. In other words, a cell-impermeable polymer is one that cannot support the growth of a cell or a portion of a cell in culture after crosslinking at a particular concentration and / or density.

[0098] One of skill in the art will appreciate that the cell-impermeable polymer and the cell-permeable polymer may comprise the same or substantially the same polymer, but differences in concentration or density after crosslinking create a hydrogel matrix having some portions that support the growth of cells or portions of cells in culture.

[0099] In some embodiments, the hydrogel or hydrogel matrix may have a variety of thicknesses. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 100 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 150 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 200 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 250 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 300 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 350 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 400 μm to about 800 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 450 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 500 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 550 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 600 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 650 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 700 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 750 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 650 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 600 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 550 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 450 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 300 μm to about 600 μm, In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 400 μm to about 500 μm.

[0100] In some embodiments, the hydrogel or hydrogel matrix may have a variety of thicknesses. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 10 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 150 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 200 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 250 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 300 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 350 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 400 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix may have a thickness of about 450 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 550 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 600 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 650 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 700 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 750 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 800 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 850 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 900 μm to about 3000 μm, In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 950 μm to about 3000 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 1000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 1500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 2000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 2500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 2500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 2000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 1500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 1000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 950 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 900 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 850 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 650 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 550 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 500 μm, In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 450 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 300 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 400 μm to about 500 μm.

[0101] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic polymers, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following synthetic polymers: polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly-2-hydroxyethyl methacrylate, polyacrylamide, silicone, and any derivatives or combinations thereof.

[0102] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic and / or natural polysaccharides, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following polysaccharides, hyaluronic acid, heparin sulfate, heparin, dextran, agarose, chitosan, alginate, and any derivative or combination thereof.

[0103] In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following proteins: collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, and any derivatives or combinations thereof.

[0104] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic and / or natural polypeptides, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following polypeptides: polylysine, polyglutamate, or polyglycine.

[0105] In some embodiments, the hydrogel comprises one or a combination of polymers selected from those published in Khoshakhlagh et al., “Photoreactive interpenetrating network of hyaluronic acid and Puramatrix as a selectively tunable scaffold for neurite growth” Acta Biomaterialia, January 21, 2015.

[0106] Any hydrogel suitable for cell growth can be formed by placing any one or combination of the polymers disclosed herein in sufficient concentrations, under conditions, and for a sufficient period of time to create two distinct densities of crosslinked polymer, one cell-permeable and one cell-impermeable. These polymers may be synthetic polymers, polysaccharides, natural proteins or glycoproteins, and / or polypeptides, such as those selected from the following:

[0107] Synthetic Polymers Polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly-2-hydroxyethyl methacrylate, polyacrylamide, silicone, combinations thereof, and derivatives thereof.

[0108] Polysaccharides (derived from synthetic or natural sources) Hyaluronic acid, heparan sulfate, heparin, dextran, agarose, chitosan, alginate, combinations thereof, and derivatives thereof.

[0109] Natural proteins or glycoproteins Collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, combinations thereof, and derivatives thereof.

[0110] Polypeptides (synthetic or natural origin) Polylysine, etc., as well as all of the RAD and EAK peptides already listed.

[0111] The term "isolated neurons" refers to neural cells that have been removed or dissociated from the organism or culture in which they originally grew. In some embodiments, the isolated neurons are neurons in suspension. In some embodiments, the isolated neurons are components of a larger mixture of cells, including tissue samples or suspensions that include non-neuronal cells. In some embodiments, neural cells are isolated when they are removed from the animal from which they originate, for example in the case of tissue explants. In some embodiments, the isolated neurons are neurons in a DRG that have been excised from an animal. In some embodiments, the isolated neurons include at least one or more cells that are derived from one or a combination of species selected from sheep cells, goat cells, horse cells, bovine cells, human cells, monkey cells, mouse cells, rat cells, rabbit cells, dog cells, cat cells, pig cells, or other non-human mammals. In some embodiments, the isolated neurons are human cells. In some embodiments, the isolated neurons are stem cells that are preconditioned to have a differentiation phenotype similar or substantially similar to a human neural cell. In some embodiments, the isolated neurons are human cells. In some embodiments, the isolated neurons are stem cells that are preconditioned to have a differentiation phenotype similar or substantially similar to non-human neural cells. In some embodiments, the stem cells are selected from mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, hematopoietic stem cells, epidermal stem cells, stem cells isolated from mammalian umbilical cords, or endodermal stem cells.

[0112] The term "neurodegenerative disease" is used throughout the present specification to describe diseases caused by damage to the central and / or peripheral nervous system. Exemplary neurodegenerative diseases, which may be examples of diseases that may be studied using the disclosed models, systems, or devices, include, for example, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, lysosomal storage diseases ("white matter diseases" or glial / demyelinating diseases, e.g., as described in Folkerth, J. Neuropath. Exp. Neuro., 58, 9, Sep., 1999), Tay-Sachs disease (beta-hexosaminidase deficiency), other genetic diseases, multiple sclerosis, brain injury or trauma caused by ischemia, accidents, environmental insults, etc., spinal cord injury, ataxia, and alcoholism. In addition, the present invention can be used to test the efficacy, toxicity, or neurodegenerative effects of drugs on neurons in culture for studies regarding the treatment of neurodegenerative diseases. The term neurodegenerative disease also includes neurodevelopmental disorders, including, for example, autism and related neurological disorders such as schizophrenia, among others.

[0113] As used herein, the term "neuronal cell" is defined as a cell that includes at least one or a combination of dendrites, axons, and cell bodies, or any cell or group of cells isolated from nervous system tissue. In some embodiments, a neuronal cell is any cell that includes or can form an axon. In some embodiments, the neuronal cell is a Schwann cell, a glial cell, a neuroglia, a cortical neuron, an embryonic cell isolated from or derived from nervous tissue, or differentiated into a cell having a phenotype substantially similar to a neuronal phenotype, an induced pluripotent stem cell (iPS) differentiated into a neuronal phenotype, or a mesenchymal stem cell derived from nervous tissue or differentiated into a neuronal phenotype. In some embodiments, the neuronal cell is a neuron from dorsal root ganglion (DRG) tissue, retinal tissue, spinal cord tissue, or brain tissue from an adult, adolescent, child, or fetal subject. In some embodiments, the neuronal cell is any one or more of the cells isolated from the nervous tissue of a subject. In some embodiments, the neuronal cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cells are non-human mammalian cells or are derived from cells isolated from a non-human mammal. When isolated or dissociated from the original animal from which they are derived, the neural cells may include neurons isolated from multiple species.

[0114] In some embodiments, the neural cell is one or more of the following neurons: sympathetic neurons, spinal motor neurons, central nervous system neurons, motor neurons, sensory neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, serotonergic neurons, interneurons, adrenergic neurons, and trigeminal ganglion neurons. In some embodiments, the neural cell is one or more of the following glial cells, astrocytes, oligodendrocytes, Schwann cells, microglia, ependymal cells, radial glia, satellite cells, enteric glial cells, and pituitary cells. In some embodiments, the neural cell is one or more of the following immune cells, macrophages, T cells, B cells, leukocytes, lymphocytes, monocytes, mast cells, neutrophils, natural killer cells, and basophils. In some embodiments, the neural cells are one or more of the following stem cells, hematopoietic stem cells, neural stem cells, adipose-derived stem cells, bone marrow-derived stem cells, induced pluripotent stem cells, astrocyte-derived induced pluripotent stem cells, fibroblast-derived induced pluripotent stem cells, renal epithelial-derived induced pluripotent stem cells, keratinocyte-derived induced pluripotent stem cells, peripheral blood-derived induced pluripotent stem cells, hepatocyte-derived induced pluripotent stem cells, mesenchymal-derived induced pluripotent stem cells, neural stem cell-derived induced pluripotent stem cells, adipose stem cell-derived induced pluripotent stem cells, preadipocyte-derived induced pluripotent stem cells, chondrocyte-derived induced pluripotent stem cells, and skeletal muscle-derived induced pluripotent stem cells. In some embodiments, the neural cells are carcinocytes. In some embodiments, the neural cells are endothelial cells.

[0115] The term "neuronal cell culture medium" or simply "culture medium", as used herein, is defined as any nutrient substance suitable for supporting, cultivating, expanding, propagating, or otherwise manipulating the growth, culture of neural cells. In some embodiments, the medium comprises a neurobasal medium supplemented with nerve growth factor (NGF). In some embodiments, the medium comprises fetal bovine serum (FBS). In some embodiments, the medium comprises L-glutamine. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.001% to about 0.01% by weight volume. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.001% to about 0.008% by weight volume. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.001% to about 0.006% by weight volume. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.001% to about 0.004% by weight volume. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.002% by weight to about 0.01% by weight. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.003% by weight to about 0.01% by weight. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.004% by weight to about 0.01% by weight. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.006% by weight to about 0.01% by weight. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.008% by weight to about 0.01% by weight. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.002% by weight to about 0.006% by weight. In some embodiments, the medium comprises ascorbic acid at a concentration ranging from about 0.003% by weight to about 0.005% by weight.

[0116] In some embodiments, the hydrogel, hydrogel matrix, and / or neuronal culture medium may contain any of the following components: artemin, ascorbic acid, ATP, β-endorphin, BDNF, fetal bovine serum, bovine serum albumin, calcitonin gene-related peptide, capsaicin, carrageenan, CCL2, ciliary neurotrophic factor, CX3CL1, CXCL1, CXCL2, D-serine, fetal bovine serum, fluorocitrate, formalin, glial cell line-derived neurotrophic factor, glial fibrillary acidic protein, glutamate, IL-1, IL-1α, IL-1β, IL-6, The therapeutic agent may include any one or more of IL-10, IL-12, IL-17, IL-18, insulin, laminin, lipoxin, mac-1-saporin, methionine sulfoximine, minocycline, neuregulin-1, neuroprotectin, neurturin, NGF, nitric oxide, NT-3, NT-4, persephin, platelet lysate, PMX53, poly-D-lysine (PLL), poly-L-lysine (PLL), propentofylline, resolvin, S100 calcium binding protein B, selenium, matrix P, TNF-α, type I-V collagen, and zymosan.

[0117] As described herein, the term "optogenetics" refers to a biological technique that involves the use of light to control cells, usually neurons, in living tissue that have been genetically modified to express light-sensitive ion channels. It is a neuromodulation method used in neuroscience that uses a combination of techniques from optics and genetics to control and monitor the activity of individual neurons in living tissue (and even in freely moving animals) and precisely measure the effects of such manipulation in real time. The key reagents used in optogenetics are light-sensitive proteins. Spatially precise neuronal control is achieved using optogenetic actuators such as channelrhodopsin, halorhodopsin, and archaerhodopsin, while temporally precise recording can be performed with the help of optogenetic sensors of calcium (Aequorin, Cameleon, GCaMP), chloride (Clomeleon), or membrane voltage (Mermaid). In some embodiments, neural cells modified with optogenetic actuators and / or sensors are used in the culture systems described herein.

[0118] The term "plastic" refers to a biocompatible polymer that includes a hydrocarbon. In some embodiments, the plastic is selected from the group consisting of polystyrene (PS), polyacrylonitrile (PAN), polycarbonate (PC), polyvinylpyrrolidone, polybutadiene (PVP), polyvinylbutyral (PVB), polyvinyl chloride (PVC), polyvinyl methyl ether (PVME), polylactic-co-glycolic acid (PLGA), poly(I-lactic acid), polyester, polycaprolactone (PCL), polyethylene oxide (PEO), polyaniline (PANI), polyfluorene, polypyrrole (PPY), polyethylenedioxythiophene (PEDOT), and mixtures of two or any of the aforementioned polymers.

[0119] As used herein, the term "seeding" is defined as the transfer of a certain amount of cells into a new culture vessel. This amount may be defined, or the amount or number of cells may be used as the basis for the defined amount. The cells may be part of a suspension.

[0120] As used herein, the term "solid substrate" refers to any material that is a solid support that is free or substantially free of cytotoxins. In some embodiments, the solid substrate comprises one or a combination of silica, plastic, and metal. In some embodiments, the solid substrate comprises pores of a size and shape sufficient to allow diffusion or non-active transport of proteins, nutrients, and gases through the solid substrate in the presence of cell culture medium. In some embodiments, the pore size is about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 microns in diameter or less. One skilled in the art would be able to determine how large a pore size is needed based on the contents of the cell culture medium and the exposure of cells growing on the solid substrate in a particular microenvironment. For example, one skilled in the art would be able to observe whether any cultured cells in the system or device are viable under conditions in which the solid substrate comprises pores of various diameters. In some embodiments, the solid substrate comprises a base having a predetermined shape that defines the shape of the outer and inner surfaces. In some embodiments, the base comprises one or a combination of silica, plastic, ceramic, or metal, the base is cylindrical in shape or substantially similar in shape to a cylinder such that the first cell impermeable polymer and the first cell permeable polymer coat an interior surface of the base and define a cylindrical or approximately cylindrical interior chamber, and the opening is located at one end of the cylinder, In some embodiments, the base comprises one or more pores of a size and shape sufficient to allow diffusion of proteins, nutrients, and oxygen through the solid substrate in the presence of cell culture medium.In some embodiments, the solid substrate comprises a plastic base having a pore size of 1 micron or less in diameter and at least one layer of a hydrogel matrix, the hydrogel matrix comprising at least a first cell impermeable polymer and at least a first cell permeable polymer, the base comprising a predetermined shape around which the first cell impermeable polymer and at least a first cell permeable polymer are physically attached or chemically bonded, the solid substrate comprising at least one compartment defined at least in part by the shape of an inner surface of the solid substrate and accessible from a point outside the solid substrate by an opening, optionally located at one end of the solid substrate. In embodiments where the solid substrate comprises a hollow interior portion defined by at least one inner surface, cells in suspension or tissue explants may be seeded by placing the cells in or adjacent to the opening, allowing the cells to adhere to at least a portion of the inner surface of the solid substrate prior to growth. At least one compartment or hollow interior of the solid substrate allows for storage of the cells in a specific three-dimensional shape defined by the shape of the inner surface of the solid substrate, promoting directional growth of the cells away from the opening. In the case of nerve cells, the degree of containment and shape of the at least one compartment is conducive to axonal growth from the nerve cell body located within the at least one compartment and at or adjacent to the opening. In some embodiments, the solid substrate is tubular or approximately tubular, whereby the interior compartment is cylindrical or partially cylindrical in shape. In some embodiments, the solid substrate comprises one or more branched tubular interior compartments. In some embodiments, the branched or multi-branched shape of the solid hollow interior portion configures or allows the axon to grow in a multi-branched pattern. When electrodes are placed at or near the distal end of the axon and at or adjacent to the nerve cell body of the nerve cell, electrophysiological metrics such as intracellular action potentials can be measured within the device or system.

[0121] The present disclosure also provides (i) a hydrogel matrix; (ii) one or more neural cells, either in suspension or as a component of a tissue explant; (iii) a device for generating an electric current; (iv) a voltmeter and / or an ammeter; (v) a system comprising at least a first stimulating electrode and at least a first recording electrode, The system relates to a generator, a voltmeter and / or ammeter, and electrodes electrically connected to each other via a circuit in which current is supplied from the generator to at least one stimulating electrode, current is received at a recording electrode and supplied to the voltmeter and / or ammeter, the stimulating electrode is positioned at or proximate to one or more neuronal somas of neuronal cells, and the recording electrode is positioned at a predetermined distance distal to the neuronal somas such that an electrical potential is established across the cell culture vessel.

[0122] In some embodiments, the solid substrate is made of a hydrogel or a hydrogel matrix. In some embodiments, the solid substrate is made of a hydrogel or a hydrogel matrix and does not include glass, metal, or ceramic. In some embodiments, the solid substrate is molded into a shape or form that is predetermined for seeding cells of a particular size suitable for axonal growth. In some embodiments, the solid substrate or at least one base portion is molded with at least one branched internal tube-like structure, with a taper of any diameter the more distal the location of the tube is from the location where the seeding of tissue explants or neural cells is performed. For example, the present disclosure contemplates a focal point at one end of a semi-cylindrical or cylindrical portion of the solid substrate that is accessible to a point outside the solid substrate by an opening or hole in the outer surface. The opening or hole can be used to place or seed cells (either neural cells and / or glial cells) at the focal point. When the cells are grown in culture for several days, the cells are exposed to a culture medium that includes any of the components disclosed herein at a concentration and for a period of time sufficient for axons to grow from the neural cells. If the cells are myelinating or myelination is desired for study, glial cells may be introduced through the same holes or seeded prior to the addition of the neural cells or explants. When the axons grow in semi-cylindrical or tube-like structures, the growth of the axonal projections may occur progressively distal to the focal point. Access points or openings in the solid substrate at points progressively distal to the focal point (or seeding point) can be used to address or observe the axonal growth in the axonal state. The present disclosure contemplates that the structure of the solid substrate may take any form to promote axonal growth. In some embodiments, the internal chamber or compartment that houses the axonal projections comprises a semicircular or roughly cylindrical diameter. In some embodiments, the solid substrate is branched into two or more internal compartments at points distal to the focal point. In some embodiments, the branches may resemble a keyhole shape or tree, with two, three, four, five, six, seven, or eight or more tube-like or roughly cylindrical chambers in fluid communication with one another, such that axonal growth originates at one or more cell body seeding points and extends longitudinally along the chambers to any one or more branches.In some embodiments, one or more electrodes can be placed at or adjacent to one or more openings, allowing recordings to be taken across one or more locations along the axon length, which can also be used to interrogate one or more locations along the length of the axon.

[0123] As used herein, the term "recording" is defined as measuring the response of one or more neurons. Such responses may be electrophysiological responses, such as patch clamp electrophysiological recordings or field potential recordings.

[0124] The present disclosure discloses methods and devices for obtaining physiological measurements of microscale organotypic models of in vitro neural tissue that mimic clinical nerve conduction and NFD testing. Results obtained from the use of these methods and devices are better predictive of clinical outcomes, allowing for a more cost-effective approach to selecting promising lead compounds with higher late-stage success. The present disclosure includes the fabrication and utilization of a three-dimensional microengineered system that allows for the growth of unique dense, highly parallel nerve fiber tracts. Due to the localized nature of the tracts, this in vitro model is capable of measuring both CAPs and intracellular patch clamp recordings. In addition, subsequent confocal and transmission electron microscopy (TEM) analysis allows for quantitative structural analysis, including NFD. Taken together, this in vitro model system has the novel ability to assess tissue morphology and collective electrophysiology, similar to clinical histopathology and nerve conduction testing.

[0125] The present disclosure also provides a method for measuring the myelination of axons created using the in vitro model described herein. Similar to the structure of human peripheral afferents, dorsal root ganglion (DRG) neurons in these in vitro constructs project long, parallel, fasciculated axons to the periphery. In native tissues, axons of different diameters and degrees of myelination return sensory information to the central nervous system at different rates. Schwann cells support sensory relays by myelining axons and providing insulation for faster conduction. Similarly, the three-dimensional growth induced by this in vitro construct contains axons of various diameters in a tightly packed, parallel orientation, spanning distances of up to 3 mm. The presence and sheath formation of Schwann cells was observed in confocal and TEM imaging.

[0126] Although neuronal morphology is a useful indicator of phenotypic maturity, a more clear sign of healthy neurons is their ability to conduct action potentials. Apoptosis alone is not a complete measure of neuronal health, as many pathological changes can occur before cell death manifests. Electrophysiological studies of action potential generation can determine whether the observed structure supports the predicted function, and the ability to measure clinically relevant endpoints can provide more predictive results. Similarly, information gleaned from imaging can determine quantitative metrics for the degree of myelination, but CAP measurements reveal the overall health of myelin and add further insight into the toxicity and neuroprotective mechanisms of various drugs or compounds of interest.

[0127] In some embodiments, the at least one agent comprises a small molecule chemical. In some embodiments, the at least one agent comprises at least one environmental or industrial pollutant. In some embodiments, the at least one agent comprises one or a combination of small molecule chemicals selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatory agents, and antimicrobial agents.

[0128] In some embodiments, the at least one agent is actinomycin, alitretinoin, all-trans retinoic acid, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, fluorouracil, gefitinib, gemcitabine, hydroxyurea, idarubicin. , imatinib, irinotecan, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosoureas, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tafluposide, temozolomide (oral dacarbazine), teniposide, thioguanine (formerly thioguanine), topotecan, tretinoin, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, vismodegib, and vorinostat.

[0129] In some embodiments, the at least one medication comprises one or a combination of analgesics selected from paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, opioids, flupirtine, tricyclic antidepressants, carbamazepine, gabapentin, and pregabalin.

[0130] In some embodiments, the at least one agent comprises one or a combination of cardiovascular modulators selected from nepicastat, cholesterol, niacin, scutellaria, prenylamine, dehydroepiandrosterone, monatepir, esketamine, niguldipine, asenapine, atomoxetine, flunarizine, milnacipran, mexiletine, amphetamine, thiopental sodium, flavonoids, bretylium, oxazepam, and honokirol.

[0131] In some embodiments, the at least one agent comprises one or a combination of neuroprotective and / or neuromodulatory agents selected from tryptamine, galanin receptor 2, phenylalanine, phenethylamine, N-methylphenethylamine, adenosine, kiptorphin, substance P, 3-methoxytyramine, catecholamines, dopamine, GABA, calcium, acetylcholine, epinephrine, norepinephrine, and serotonin.

[0132] In some embodiments, the at least one agent comprises one or a combination of immune modulating agents selected from clenolizimab, enoticumab, ligelizumab, simtuzumab, batelizumab, palsatuzumab, imgatuzumab, tregalizaumb, pateclizumab, namulumab, perakizumab, faralimomab, patritumab, atinumab, ublituximab, futuximab, and zurigotumab.

[0133] In some embodiments, the at least one agent comprises one or a combination of anti-inflammatory agents selected from ibuprofen, aspirin, ketoprofen, sulindac, naproxen, etodolac, fenoprofen, diclofenac, flurbiprofen, ketorolac, piroxicam, indomethacin, mefenamic acid, meloxicam, nabumetone, oxaprozin, ketoprofen, famotidine, meclofenamate, tolmetin, and salsalate.

[0134] In some embodiments, the at least one agent comprises one or a combination of antimicrobial agents selected from antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, heat, radiation, and ozone.

[0135] The present disclosure additionally discloses a method for measuring both intracellular and extracellular recordings of biomimetic neural tissues in a three-dimensional culture platform. Previously, electrophysiological experiments have been performed in either dissociated surface-plated cultures or organotypic slice preparations, with limitations inherent to each method. Investigations in dissociated cell cultures are typically limited to single-cell recordings due to the lack of organized multicellular neurite architecture as found in organotypic preparations. Organotypic preparations have intact neural circuits, allowing for both intracellular and extracellular studies. However, acute brain slices present a complex simultaneous array of variables without a means of controlling individual factors, and thus inherently limited throughput probability.

[0136] Intracellular recordings in in vitro three-dimensional cultures have been demonstrated previously. However, neuronal outgrowths were not spatially confined to anatomically relevant structures that support extracellular ensemble investigations. More biomimetic three-dimensional neural cultures are needed to allow examination of ensemble-level electrophysiological behavior. This disclosure supports the use of whole-cell patch clamp techniques and synchronous ensemble-level events in extracellular field recordings resulting from localized neurite outgrowth in three-dimensional geometries. Prior to this disclosure, measurements of these endpoints that are directly analogous to clinical nerve conduction studies have not been demonstrated for purely cellular in vitro studies.

[0137] Using the methods and devices disclosed herein, field recordings are used to measure the composite extracellular changes in potentials caused by signal conduction in all recruited fibers. The collective response induced by electrical stimulation is a CAP. Electrically evoked collective spikes are graded in nature and contain the combined effect of action potentials in slow and fast fibers. The spikes are single aggregate events with fast onset and short duration characteristic of CAPs, or in the absence of synaptic inputs, responses that consist purely of action potentials with rapid signal conduction. The three-dimensional neural constructs disclosed by the present disclosure also support CAPs stimulated from greater distances along neurite tracts or channels, demonstrating the ability of the neural cultures to carry signals from distal stimuli as quickly as afferent peripheral nerves. The three-dimensional neural cultures of the present disclosure support proximal and distal stimulation techniques useful for measuring conduction properties.

[0138] The present disclosure may be used with one or more growth factors that induce recruitment of many fiber types as is normal in nerve fiber tracts. Specifically, nerve growth factor (NGF) preferentially recruits small diameter fibers, often associated with pain signaling, as evidenced by the data presented herein. Brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT-3) have been shown to preferentially support larger diameter proprioceptive fibers. Factors that affect growth, such as bioactive molecules and drugs, can be incorporated into electrophysiological investigations, allowing for systematic manipulation of conditions for mechanistic studies.

[0139] The three-dimensional neural cultures created using the present disclosure can be used as a platform to study the mechanisms underlying myelin-damaging diseases and peripheral neuropathy by investigating the effects of known hypomyelination drugs on neural cultures, culture conditions that induce neuropathy, and compounds that induce toxic neuropathy. The present disclosure allows conduction velocity to be used as a functional measure of myelin and nerve fiber integrity under toxic and therapeutic conditions, facilitating research into drug safety and efficacy. The incorporation of genetic mutations and drugs into neural cultures generated using the techniques disclosed herein can allow for the recapitulation of disease phenomena under controlled conditions, leading to a better understanding of neurodegeneration and possible therapeutic therapies.

[0140] The present disclosure provides devices, methods, and systems involved in the generation, maintenance, and physiological interrogation of neural cells in microengineered configurations designed to mimic the anatomy of native neural tissue. In some embodiments, these devices and systems include one or more of cultured or isolated Schwann cells and / or one or more of cultured or isolated oligodendrocytes in contact with one or more of neural cells in a cell culture vessel comprising a solid substrate, the substrate comprising at least one outer surface, at least one inner surface, and at least one inner chamber, the shape of the inner chamber being defined, at least in part, by the at least one inner surface and accessible from a point outside the solid substrate through at least one opening in the outer surface, the soma of one or more of the neural cells being positioned at one end of the inner chamber, and the axon being capable of growing within the inner chamber along at least one length of the inner chamber such that the location of the tip of the axon extends distally from the soma. In some embodiments, the inner surface of the solid substrate is cylindrical in shape or approximately cylindrical, whereby a neuronal cell body from a neuronal cell is positioned proximal to an opening at one end of the cylindrical or approximately cylindrical inner surface, and an axon of the neuronal cell includes a length of cytoplasm that extends from a point at the edge of the neuronal cell body along the length of the inner surface to a point distal from the cell body. In some embodiments, the inner surface of the solid substrate is cylindrical in shape or approximately cylindrical, whereby a neuronal cell body from a neuronal cell is positioned proximal to an opening at one end of the cylindrical or approximately cylindrical inner surface, and an axon of the neuronal cell includes a length of cytoplasm that extends from a point at the edge of the neuronal cell body along the length of the inner surface to a point distal from the cell body.In some embodiments, the inner surface of the solid substrate is cylindrical in shape or approximately cylindrical, whereby the neuronal cell body from the neuronal cell is positioned proximal to an opening at one end of the cylindrical or approximately cylindrical inner surface, the axon of the neuronal cell includes a length of cytoplasm that extends from a point at the edge of the neuronal cell body along the length of the inner surface to a point distal from the neuronal cell body, and when the cell culture vessel includes a plurality of neuronal cells, the axons extend from the plurality of cell bodies (or neuronal cell bodies), whereby the axons define an axonal bundle that can grow distal from the neuronal cell body along the length of the inner surface. In some embodiments, the neuronal cells are grown on and within the permeable polymer. In some embodiments, one or more of the electrodes are positioned at or near the tip of at least one axon, and one or more of the electrodes are positioned at or near the neuronal cell body such that an electrical potential is established across the length of one or more of the neuronal cells.

[0141] Another object of the present disclosure is to provide a medium for high throughput assays of neurological function for screening of pharmacological and / or toxicological properties of chemical and biological agents. In some embodiments, these agents are cells, such as any type of cells disclosed herein, or antibodies, such as antibodies used to treat clinical diseases. In some embodiments, these agents are any drugs or drugs used to treat human diseases, so that toxicity, efficacy, or neuromodulation can be compared between the new agent, which is the proposed mammalian treatment, and an existing treatment from the human disease. In some embodiments, the new agent for the treatment of the human disease is a treatment for a neurodegenerative disease and is compared to an existing treatment for the neurodegenerative disease. As a non-limiting example, in the case of multiple sclerosis, the effect of the new agent (modified cells, antibodies, or small chemical compounds) may be compared to that of an existing treatment for multiple sclerosis, such as Copaxone, Rebif, other interferon therapies, Tysabri, dimethyl fumarate, fingolimod, teriflunomide, mitoxantrone, prednisone, tizanidine, baclofen, etc.

[0142] Another object of the present disclosure is to use a unique assembly of techniques such as two-dimensional and three-dimensional micro-engineered nerve bundles in conjunction with electrophysiological stimulation and recording of neuronal ensembles.

[0143] Another object of the present disclosure is to provide a novel approach for assessing neurophysiology in vitro using compound action potentials (CAPs) as a clinically similar metric, yielding results that are more sensitive and predictive of human physiology than those obtained by current methods.

[0144] Another object of the present disclosure is to provide microengineered neural tissue that mimics native anatomical and physiological characteristics and is amenable to evaluation using high-throughput electrophysiological stimulation and recording methods.

[0145] Another object of the present disclosure is to provide methods to replicate, manipulate, modify, and evaluate the mechanisms underlying myelin-damaging diseases and peripheral neuropathy.

[0146] Another object of the present disclosure is to enable the medium for high throughput assays of neuromodulation in human neuronal cells for screening of pharmacological and / or toxicological activity of chemical and biological agents.

[0147] Another object of the present disclosure is to use a unique assembly of techniques such as 2D and 3D microengineered nerve bundles in conjunction with optical and electrochemical stimulation of human neuronal ensembles.

[0148] Another objective of the present disclosure is to quantify evoked postsynaptic potentials in biomimetic engineered thalamocortical circuits. Our observations of antidromically generated population spikes in neural tracts suggest that they are capable of population-level physiological functions such as the conduction of compound action potentials and postsynaptic potentials.

[0149] Another object of the present disclosure is to enable non-invasive stimulation and recording of multi-unit physiological responses to evoked potentials in neural circuits using optogenetic methods, hardware and software control of illumination, and fluorescence imaging.

[0150] Another goal of this disclosure is to use the microengineered circuits in testing selective 5-HT reuptake inhibitors (SSRIs) and second-generation antipsychotics to see whether they alter developmental maturation.

[0151] In one embodiment, projection photolithography using a digital micromirror device (DMD) is used to micropattern a combination of polyethylene glycol dimethacrylate and PuraMatrix hydrogels, as shown in FIG. 1. This method allows for rapid micropatterning of one or more hydrogels directly on top of traditional cell culture materials. Because the photomask never comes into contact with the gel material, multiple hydrogels can be rapidly cured in succession, allowing large quantities of gel constructs to be fabricated in less than an hour without automation. This approach allows for the use of polyethylene glycol (PEG), a mechanically robust cell growth-restricting gel, to constrain neurite outgrowth within a biomimetic growth-conducting gel. In some embodiments, this growth-conducting gel can be PuraMatrix, agarose, or methacrylated dextran. When embryonic dorsal root ganglion (DRG) explants are grown within this constrained three-dimensional environment, axons grow out of the ganglia with high density and fasciculation, as shown in FIG. 5 and FIG. 6. The majority of axons emerge as small diameter unmyelinated fibers and grow to lengths of approximately 1em over 2-4 weeks. The structure of this culture model, with dense, highly parallel three-dimensional nerve fiber tracts extending from the ganglion, roughly resembles peripheral nerve architecture. Its morphology may be assessed using neuromorphometry, allowing for clinically similar assessments unavailable to traditional cellular assays.

[0152] In a preferred embodiment, this culture model provides the ability to record electrically evoked collective field potentials resulting from compound action potentials (CAPs). The illustrated traces show a characteristically uniform fast and short latency collective spike response that is still consistent with high frequency (100 Hz) stimulation, as seen in FIG. 8B. The CAPs are reversibly abolished by tetrodotoxin (TTX), as shown in FIG. 8E and 8F, demonstrating that drugs can be applied and shown to have an effect. There is a measurable increase in the latency to onset associated with distal pathway stimulation, as seen in FIG. 8C and 8D. The response is insensitive to neurotransmitter blocking agents, indicating that the evoked response is primarily a CAP other than synaptic potentials, as shown in FIG. 10. Embryonic DRG cultures have been effectively used as a model of peripheral neurobiology for many years. Although extremely useful as a model system, conventional DRG cultures are known to be poor predictors of clinical toxicity when assessed with traditional cell viability assays. Although single cell patch clamp recordings can be performed in DRG cultures, there have been no reports of CAPs for recording due to the lack of tissue architecture. In preferred embodiments, the present disclosure provides the ability to assess histomorphometry and collective electrophysiology, similar to clinical histopathology and nerve conduction studies.

[0153] In some embodiments, the present disclosure uses human neural cells to grow neural tissue in a three-dimensional environment in which neural cell bodies are bundled together and located separately from axonal fiber tracts, mimicking natural neural architecture and allowing for the measurement of morphometric and electrophysiological data, including CAPs. In some embodiments, the present disclosure uses neural and glial cells derived from primary human tissue. In other embodiments, the neural and glial cells may be derived from human stem cells, including induced pluripotent stem cells.

[0154] In another embodiment, the present disclosure uses conduction velocity as a functional measure of neural tissue condition under toxic and therapeutic conditions. Information regarding the degree of myelination, myelin health, axonal transport, mRNA transcription, and neuronal damage may be determined from electrophysiological analysis. In combination with morphometric analysis of neural density, percentage of myelination, and nerve fiber type, the mechanism of action for compounds of interest can be determined. In some embodiments, the devices, methods, and systems disclosed herein incorporate genetic mutations and drugs to recapitulate disease phenomena in a controlled manner, leading to a better understanding of neurodegeneration and possible therapeutic therapies.

[0155] The following examples are meant to be non-limiting examples of how to make and use the embodiments disclosed in this application. These examples and any publications disclosed in the body of the specification are incorporated by reference in their entirety. EXAMPLES

[0156] Example 1: Growth of neural tissue within hydrogel constructs and physiological evaluation (non-predictive) A. Materials and Methods Dynamic Mask Projection Photolithography Hydrogel micropatterns were formed via projection photolithography. A DMD development kit (Discovery™ 3000, Texas Instruments, Dallas, TX) with a USB computer interface (ALP3Basic) served as a dynamic mask by converting a digital black-and-white image into a micromirror pattern on the DMD array, where individual mirrors could be turned "on" or "off" by rotating their angle of reflection from +12° to -12°, respectively. Ultraviolet (UV) light filtered from 320-500 nm from an OmniCure 1000 (EXFO, Quebec, Canada) Hg vapor light source was collimated with an adjustable collimating adapter (EXPO) and projected onto the DMD array. The reflected light was projected through a 4x Plan Fluor objective with a numerical aperture of 0.13 (Nikon Instruments, Melville, NY) and focused directly onto the photocrosslinkable hydrogel solution, as shown by Figure 1A. The iris of the UV light source was adjusted to maintain an irradiation power of 5.0 watts / cm2 as measured by a radiometer (EXPO). The hydrogel solution was cured for approximately 55 seconds to induce crosslinking through a free radical chain reaction. Unlike previous reports, this method initiates crosslinking throughout the bulk with a single irradiation, eliminating the need for a layer-by-layer approach.

[0157] Formation of dual hydrogel constructs Hydrogel polymerization was performed as previously described for dynamic mask projection photolithography. Photocrosslinkable solutions were made by diluting polyethylene glycol dimethacrylate (PEG) with an average molecular weight (MW) of 1000 Da (Polysciences, Warrington, PA) to 10% (w / v) in either PBS or growth medium containing 0.5% (w / v) Irgacure 2959 (1-2959) (Ciba Specialty Chemicals, Basel, Switzerland) as a photoinitiator. The concentration and molecular weight of PEG were chosen based on previously published data to minimize cell adhesion to the polymerization surface and maximize hydrogel adhesion. Micropatterned PEG constructs were crosslinked directly to one of three types of permeable cell culture inserts, polyester, polycarbonate, and collagen-coated PTFE Transwell® permeable supports (Coming, Coming, NY) with a 24 mm diameter membrane and 0.411 m pores. The inner walls of the culture inserts, but not the membrane itself, were treated with Rain-X® (SO PUS Products, Houston, TX) to reduce the meniscus effect of the PEG solution. Each support was placed on the stage of an inverted microscope, which was positioned directly under the lithography projection lens. After crosslinking, the support was rinsed to remove excess uncrosslinked PEG solution, and the micropatterned PEG remained attached to the surface. Hydration of the PEG gels was maintained in a buffered saline solution (4° C.) if not used immediately.

[0158] A self-assembling peptide gel, PuraMatrix (BD Biosciences, Bedford, MA), was diluted to 0.15% (w / v) in deionized H2O prior to use and, when used for neurite outgrowth experiments, was pregelled and supplemented with 1 μg / mL soluble laminin (Invitrogen, Carlsbad, CA). This second gel has also been substituted with agarose, as well as methacrylated versions of hyaluronic acid, heparin, and dextran. Both the concentration of PuraMatrix and the addition of laminin followed the manufacturer's instructions for neural applications. Using a pipette, the solution was carefully added into the voids within the micropatterned PEG hydrogel. Contact with the salt solution, which hydrated the PEG gel, induced self-assembly of PuraMatrix, which remained localized within the PEG geometry. Gelation of PuraMatrix was maintained by incubation at 37°C and 5% CO2.

[0159] Tissue harvesting and culture The NIH guidelines for the care and use of laboratory animals (NIH publication #85-23 revised 1985) were observed. Embryonic day 15 (E-15) pups were removed from timed-pregnant Long Evans rats (Charles River, Wilmington, MA) and placed in Hank's balanced salt solution. Spinal columns were isolated from the fetuses from which dorsal root ganglia (DRGs) were harvested and placed in Neurobasal medium supplemented with nerve growth factor (NGF), 10% fetal bovine serum (FBS), and penicillin / streptomycin (P / S) (Invitrogen) to promote attachment. After attachment, DRGs were placed on collagen-coated cell culture inserts and maintained in an incubator at 37°C and 5% CO2 with B-27 and L-glutamine substituting for FBS for the growth medium.

[0160] Primary DRG neurons were obtained through dissociation of DRGs by trypsinization and trituration followed by subsequent removal of supporting cells using fluorodeoxyuridine and uridine (treatment for 3 days). Cells were then cultured at 3 × 10 5Cells were suspended in Pura Matrix at a concentration of 1000 cells / mL. The cell suspension was added to either 24-well cell culture inserts or 24-well tissue culture plates (Coming) in sufficient volume to obtain a hydrogel approximately 480 μm thick, and self-assembly was initiated upon addition of growth medium (n=4). Constructs were incubated for approximately 48 hours before testing for cell viability.

[0161] Neurite outgrowth in dual hydrogels. Collagen-coated PTFE cell culture inserts were soaked overnight in adhesion medium to hydrate the membrane. Four DRGs were then placed on the surface of the insert and allowed to adhere for approximately 2 hours before the medium was replaced with 500 μL of 10% PEG in growth medium as previously described without FBS. This amount can be adjusted to vary the thickness of the PEG constructs. The DMD was illuminated with a visible light source to aid in the alignment of the projected mask with each adhered DRG. The visible light source was then replaced with a UV source and the PEG hydrogel was crosslinked around the tissue explants. The PEG constructs containing DRGs were washed three times with PBS to remove any uncrosslinked PEG solution. When applicable, modified PuraMatrix was added to the void inside the PEG and 1.5 mL of growth medium was introduced underneath the insert to induce self-assembly of the PuraMatrix. Constructs referred to as No PuraMatrix were made as above except without the addition of PuraMatrix, thereby restricting the DRGs to the two-dimensional environment of the collagen-coated PTFE membrane. The constructs were maintained in an incubator at 37° C. and 5% CO 2 for 7 days, with medium changes after 2 and 5 days.

[0162] Constructs were prepared and visualized for morphology, viability, neurite outgrowth, and retraction. Samples were considered to have contained growth if no neurites were visualized growing on or outside of the PEG voids. This was done within each PEG construct both with and without added PuraMatrix, with 12 trials for each condition attempted for the five different PEG heights listed above. Trials were discarded in the case of incomplete PEG polymerization or lack of DRG attachment.

[0163] Specimen preparation and visualization Live specimens were assessed for viability using the Live / Dead® assay (Invitrogen) according to the manufacturer's instructions. For cell suspensions in Pura Matrix, wide-field fluorescent images were captured at multiple focal planes across the depth of the gel in three different regions of each hydrogel specimen. Standard deviation projections were then analyzed for cell viability by counting calcein AM (live marker) and ethidium homodimer-1 (dead marker) in both cell culture inserts and tissue culture plates, resulting in a total of 12 samples per condition. Specimens assessed by immunohistochemistry were fixed in 4% paraformaldehyde for approximately 2 hours. Cell nuclei were stained with DAPI nucleic acid stain (Molecular Probes) according to the manufacturer's instructions. Neurites were stained using a mouse monoclonal against neuron-specific βIII tubulin primary antibody and a goat anti-mouse IgG-H&L (CY2) secondary antibody [2G10], and dendrite staining was performed using a rabbit polyclonal against MAP2 primary antibody and a donkey anti-rabbit IgG Dylight 594 secondary antibody (AbCam, Cambridge, MA). Each step was performed overnight in PBS containing 0.1% saponin and 2.0% BSA, followed by three washes in PBS containing 0.1% saponin. Brightfield and conventional fluorescence images were acquired using a Nikon AZ100 stereo zoom microscope equipped with a fluorescence cube (Nikon, Melville, NY), while confocal images were acquired using a Zeiss LSM 510 meta microscope (Zeiss, Oberkocken, Germany). The average depth of β-III labeled structures was calculated from the confocal images, measuring the distance between the first and last focal planes containing fluorescence (n=7). Image processing was performed using Image J (National Institutes of Health, Bethesda, MD) and V3D software (Howard Hughes Medical Institute, Ashburn, VA), used for 3D visualization of confocal image stacks.

[0164] The percentage of neurite outgrowth across the gel depth was quantified from pixel counts of manually thresholded confocal slices. Confocal slices were binned at 10% increments of the total depth, and the measured fluorescence of the binned slices was compared to the total measured for each z-stack to obtain the percentage of neurite outgrowth throughout the depth of the construct (n=3). A z-stack projection was created using the VolumeJ plugin, which encoded the depth of neurite outgrowth. Confocal z-stacks were acquired through the maximum depth of visible neurite outgrowth (186 μm) using 3.0 μm thick slices (1024×1024×63) for both constructs containing and not containing PuraMatrix. Color was added using the Z-code stack function with a spectral depth-encoded LUT, and stacks were merged using standard deviation z-projection. Finally, the z-stacks were despeckled to remove background noise. Cryo-scanning electron microscopy (Cryo-SEM) was performed by freezing specimens in poured liquid nitrogen and imaging using a Hitachi S4800 field emission SEM (Hitachi, Krefeld, Germany) and a Gatan Alto 2500 cryosystem (Gatan, Warrendale, PA) at 3 kV and −130 °C.

[0165] Incorporation of Dorsal Root Ganglion Explants All animal handling and tissue harvesting procedures were performed under the guidelines set by the NIH (NIH publication #85-23 revised 1985) and under the observation of the Institutional Animal Care and Use Committee (IACUC) of Tulane University. Nerve explants were incorporated into the dual hydrogel constructs as described above. Briefly, 6-well collagen-coated PTFE cell culture inserts were immersed overnight in attachment medium consisting of Neurobasal medium supplemented with penicillin / streptomycin, nerve growth factor (NGF), 10% fetal bovine serum (FBS), and L-glutamine (Gibco-Invitrogen, Carlsbad, CA). Embryonic day 15 Long-Evans rat pups (Charles Four dorsal root ganglia (DRGs) isolated from mice (Hepatitis B, Wilmington, Mass.) were placed on hydrated cell culture inserts and allowed to adhere by incubating in adhesion medium for approximately 2 hours at 37° C. and 5% CO2. The adhesion medium was then replaced with 500 μL of 10% PEG / 0.5% Irgacure 2959 in PBS for construct polymerization.

[0166] The projected photomask pattern of the PEG construct was aligned around the adhered DRG using visible light and an inverted microscope. The photomask was projected as above using UV light for 55 seconds, effectively localizing the DRG within the polymerized PEG construct. The time the tissue cultures spent outside the biosafety cabinet was kept to a minimum to help prevent contamination, and the uncrosslinked hydrogel solution was rinsed three times with PBS containing 1% penicillin / streptomycin (Gibco-Invitrogen, Carlsbad, CA) to remove unpolymerized PEG solution and improve sterility of the cultures. Excess PBS was removed from the patterned voids inside the PEG, and PuraMatrix was carefully pipetted into the remaining space. The inserts containing the dual hydrogel constructs, each with a viable DRG explant, were immediately placed in 1.5 mL of growth medium (Neurobasal medium supplemented with NGF, penicillin / streptomycin, L-glutamine, and B27, Gibco-Invitrogen, Carlsbad, Calif.) to initiate self-assembly of the pure matrix and maintained at 37° C. and 5% CO2 with medium changes approximately every 48 hours. Experiments were initiated 7 days later to allow for neurite outgrowth and neuronal maturation.

[0167] Immunocytochemistry Specimens evaluated for immunohistochemistry were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for approximately 2 hours at 37°C. Cell nuclei were stained with DAPI nucleic acid stain according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Neurites were tagged using a monoclonal [2G 10] neuron-specific β-III tubulin primary antibody (1:200) followed by fluorescent tagging with a Cy3.5-conjugated goat anti-mouse immunoglobulin G (H+L) secondary antibody (1:100, Abeam, Cambridge, MA). Glial cells were stained using a rabbit polyclonal S100-specific primary antibody (1:500, Abeam, Cambridge, MA) and a Cy2-conjugated goat anti-rabbit immunoglobulin G (H+L) secondary antibody (1:100, Jackson ImmunoResearch Laboratories, Westgrove, PA). The antibody tagging step was carried out overnight at 4°C in PBS containing 0.1% saponin and 2% bovine serum albumin (Sigma-Aldrich, St. Louis, MO), followed by three 10 min washes at room temperature in PBS containing 0.1% saponin.

[0168] For constructs stained for myelin, neurites were stained with mouse monoclonal [2G 10] were tagged using a neuron-specific β-III tubulin primary antibody (1:200), followed by fluorescent tagging with a Cy2-conjugated goat anti-mouse immunoglobulin G (H&L) secondary antibody (1:500, Abeam, Cambridge, MA). Myelin was stained for 40 min using Fluoromyelin™ red fluorescent myelin stain (Molecular Probes, Eugene, OR) following the manufacturer's recommended preparation.

[0169] Fluorescence Microscopy and Image Processing Brightfield and conventional fluorescence images were acquired using a Nikon AZ100 stereo zoom microscope (Nikon, Melville, NY) using 1× and 2× objectives, while confocal images were taken using a Leica TCS SP2 laser scanning microscope and a 20× objective (Leica Microsystems, Buffalo Grove, IL). Confocal z-stacks were acquired through the maximum depth of visible neurite outgrowth, with thicknesses ranging from 55 to 65 μm, and imaged across 20 slices, each 512 × 512. Image processing was performed using ImageJ (National Institutes of Health, Bethesda, MA). For color-coding depth in confocal z-stacks, the Z-coded stack function using Rainbow LUI was applied using the MacBiophotonics plugin package in ImageJ. Projections of the z-stacks were taken as maximum intensity projections. V3D-viewer software (Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA) allowed 3D rendering and visualization of confocal z-stack images.

[0170] Transmission electron microscopy Transmission electron microscopy was used to qualitatively evaluate the morphology, spatial distribution, and nanoscale features of the neuronal cultures. After 7 days in vitro, constructs were fixed in 4% paraformaldehyde for approximately 2 hours at 37°C, washed three times for 10 minutes in PBS, and sectioned to expose areas of interest. Post-fixation using 1% OsO4 for approximately 1 hour and 2% uranyl acetate for approximately 30 minutes was performed at restricted light settings, with three 10-minute PBS washes in between. Samples were dehydrated in ethanol (50, 70, 95, and 100% (twice), approximately 30 minutes each) and embedded in 1:1 propylene oxide-Sparr resin for approximately 45 minutes and 100% Sparr resin overnight (low viscosity embedding kit, Electron Microscopy Sciences, Hatfield, PA). Polymerization of the specimens occurred at 70°C for 24 hours.

[0171] The embedded specimens were trimmed and sliced ​​to different thicknesses between 80 nm and 100 nm using a Reichert Ultracut S ultratome (Leica Microsystems, Buffalo Grove, IL) and an Ultra 45° diamond knife (Diatome, Fort Washington, PA). The slices were placed on copper grids coated with Formvar carbon with 200 mesh and stained with 2% uranyl acetate and 0.1% lead citrate (approximately 20 min each). The specimens were mounted on a single tilting stage and examined with an FEI Tecnai G2 F30 Twin transmission electron microscope (FEI, Hillsboro, OR) using an accelerator voltage of 200 kV. Images were taken at 4000 × 4000 pixel resolution at magnifications between 3,000 × and 20,000 ×. All materials and reagents used for sample preparation were obtained from Electron Microscopy Sciences (Hatfield, PA).

[0172] After 7 days in vitro, the double hydrogel constructs containing live DRG explants were transferred to an interface chamber held at room temperature and perfused with bicarbonate-buffered artificial cerebrospinal fluid (ACSF) made up of 124 NaCl, 5 KCl, 26 NaHCO3, 1.23 NaHCO4, 4 MgSO4, 2 CaClz, and 10 glucose (in mM). ACSF was constantly bubbled with 95% Oz, 5% COz to maintain constant oxygenation and pH. Constructs were stained with 1% bromophenol blue (Sigma-Aldrich, St. Louis, MO) for contrast and visualized using a SMZ 745 stereomicroscope (Nikon, Melville, NY). Thin-walled borosilicate glass pipettes (OD=1.5, ID=1.6, Warner Instruments, Hamden, CT) were pulled to a resistance of approximately 3 to approximately 7 MQ using a P-97 Flaming / Brown micropipette puller (Sutter Instrument Co., Novato, CA) and backfilled with ACSF.

[0173] Recording electrodes were placed near the cellular neuronal soma in the vicinity of each ganglion, and constructs were stimulated with concentric bipolar electrodes (CBARB75, FHC, Bowdoin, ME) at various distances from the ganglion along the neurite tract. An isolated pulse stimulator (Model 2100, AM Systems, Sequim, WA), a PowerLab 26T digitizer (AD Instruments, Colorado Springs, CO), and an Axopatch-1C amplifier (Molecular Devices, Sunnyvale, CA) coupled with LabChart software (AD Instruments, Colorado Springs, CO) were used for recording, stimulation, and data acquisition. Recordings were filtered at 5 kHz, displayed on a Tektronix oscilloscope, and analyzed offline using custom-written routines in Igor Pro (WaveMetrics, Portland, OR). Standard deviations were calculated where appropriate. Statistics were calculated using two-tailed paired tests, and p values ​​<0.05 were considered significant. All values ​​are reported with error as standard error of the mean (SEM).

[0174] 20 μM DNQX (6,7-dinitroquinoxaline-2,3-dione) and 50 μM APV(2R)-amino-5-phosphonopentanoate) were used to specifically block synaptic activity. 0.5 μM tetrodotoxin (TTX) was used as a complete blockade of Na+ channel activity. All drugs and salts used in the experimental solutions were obtained from Tocris (Minneapolis, MN) and Sigma-Aldrich (St. Louis, MO), respectively.

[0175] Whole-cell patch clamp recordings. After 7 days in vitro, constructs were transferred to a submerged recording chamber at room temperature and allowed to equilibrate for 20 min. Bicarbonate-buffered ACSF solution (containing, in mM, 124 NaCl, 5 KCl, 26 NaHCO3, 1.23 NaH2PO4, 1.5 MgCh, 2 CaCh, and 10 glucose) was constantly bubbled with 95% 02, 5% CO2 to maintain consistent oxygenation and pH. For voltage clamp recordings, borosilicate glass pipettes were filled with cesium-substituted intracellular solution containing, in mM, 120 CsMeSO3, 1 NaCl, 0.1 CaCh, 2 ATP, 0.3 GTP, 10 HEPES, and 10 EGTA. For current decay recordings, pipettes were filled with a potassium gluconate-based internal solution containing, in mM, 120 K gluconate, 1 KCl, 10 Hepes, 10 D-sorbitol, 1 MgCh*6H20, 1 NaCl, 1 CaCh, 10 EGTA, 2 ATP. Pipette resistance ranged from about 4 to about 7 MQ. Series access resistance ranged from about 7 to about 15 MQ and was monitored for consistency. For evoked action potential recordings, a concentric bipolar stimulating electrode (CBARC75, FHC, Bowdoin, ME) was placed within the afferent fiber of the DRG and, after obtaining a whole-cell patch, action potentials were evoked using the minimum necessary stimulation, typically <0.01 mA. The placement of the recording and stimulating electrodes is shown in Figure 10A below.

[0176] DRGs were visualized using a BX61 WI Olympus upright microscope (Olympus, Center Valley, PA) along with live differential interference contrast (DIC) imaging. Whole-cell recordings were made using a PC-505B patch clamp amplifier (Warner Instruments, Harnden, CT). Signals were digitized using a PowerLab 26T digitizer and collected by Lab Chart acquisition software (AD Instruments, Colorado Springs, CO). Signals were amplified, sampled at 20 kHz, filtered to 2 kHz, and analyzed using custom-written routines in Igor Pro (WaveMetrics, Portland, OR).

[0177] Rat Ganglion Explants and Electrophysiology Evaluation Rat E-15 dorsal root ganglion explants were cultured within the dual hydrogel constructs micropatterned by the dynamic mask projection lithography method described above. The neural constructs were incubated for 1 or 2 weeks, and neurite outgrowth was localized to narrow tracts filled with Pura matrix measuring approximately 200 μm in diameter, approximately 400 μm in thickness, and up to approximately 2 mm in length. The constructs were placed in an interface chamber perfused with bicarbonate-buffered ACSF solution, and electrophysiology was assessed using extracellular field potential electrodes. Recording electrodes were placed near the cellular neuronal soma in the vicinity of each ganglion, and the constructs were stimulated with bipolar electrodes along the neurite tracts at different distances from the ganglion.

[0178] B. Results Neurite growth in dual hydrogels The PEG thickness required to constrain neurite growth was investigated by culturing DRG explants in constructs with increasing thicknesses. Retention was measured here because the ability of this system to function reliably as an in vitro model is important. Unbalanced polymerization often occurs at 233 μm thick PEG, leading to unusable constructs. In addition, throughout the polymerization process, some DRGs detach from the surface of the membrane, resulting in fewer attempts than expected for analysis. For constructs containing PuraMatrix, a clear increase in neurite retention was seen as the gel thickness increased, as shown in Table 1. At a thickness of 233 μm, none of the constructs restricted neurite growth. Retention rates for posterior heights of 368, 433, 481, and 543 μm were 10%, 22.2%, 63.6%, and 87.5%. Overall, higher retention rates were seen in constructs lacking PuraMatrix. Although neurites appear to be able to grow across the sloping PEG wall at a certain thickness in both groups, there was more efficient containment at similar heights other than 534 μm in constructs without PuraMatrix, and at lower heights than PEG with PuraMatrix, as listed in Table 1. [Table 1-1]

[0179] In an attempt to balance neurite containment with void size resolution and pattern fidelity, subsequent neurite outgrowth experiments were performed in constructs with an average PEG thickness of 481 μm (500 μL solution). Constructs monitored for cell viability after 5 days showed an overwhelming amount of live cells, with very few dead cells located in the DRG itself, as shown in Figure 3A. After fixation and staining on day 7, neurites and migrating cells were constrained by the geometry of the PEG hydrogel, as suggested by double labeling with β-III tubulin and DAPI, as seen in Fig. 3B. Neurite outgrowth was consistently robust, with all labeled structures concentrated inside the PuraMatrix portion of the double hydrogel, as shown in Figs. 1A-1E and 5A-5D. In addition, MAP2 antibody labeling suggests that the majority of neurite outgrowth within the construct appeared to be dendritic, as seen in Fig. 3E. Growth initially appeared to occur along the boundary between the two gels, as evidenced in Fig. 3A. However, behind the neurites extending along the channels, growth was seen to fill the interior space between the PEG, also shown in Fig. 3A. Images of the leading neurite outgrowth within the three-dimensional bulk of the PuraMatrix showed a tendency to grow in random directions, as shown in Fig. 3D.

[0180] In direct contrast, neurites growing along the surface of the cell culture insert orient themselves obliquely and clearly follow the fibers of the insert membrane, as shown in Figure 3F. Growth was observed to spread out at the branching points and with no apparent preferred direction. A significant amount of branching and fasciculation was observed, which was particularly evident at the leading edge of the growth, as seen in Figure 3D. Within approximately 7 days, growth was seen throughout the entire length of the channel. As shown in Figures 4B and 4C, significantly more growth was observed in the constructs filled with PuraMatrix, compared to the apparently immature and limited growth seen in the constructs that did not contain PuraMatrix. Confocal imaging confirmed that neurite growth occurred in three dimensions, as shown in Figures 7A-7D. The average thickness of β-III labeled structures was 159.8 ± 23.9 μm in constructs containing pure matrix, while the average thickness in constructs not containing pure matrix was 85.4 ± 38.6 μm, a difference found to be statistically significant and shown in Figure 11A (p < 0.005).

[0181] FIG. 4D shows an example of growth in a construct lacking PuraMatrix, where growth appeared to be dense, with neurites growing to a maximum height of 54.0 μm. Neurite growth in constructs without PuraMatrix was visualized growing along the collagen-coated PTFE membrane, with no growth occurring on the PEG itself. Alternatively, FIG. 4E demonstrates neurite growth in a dual hydrogel construct, where significantly less neurite density was observed, with individual neurites growing through the PuraMatrix in multiple focal planes, reaching a maximum height of 120.0 μm. FIG. 11B further demonstrates that neurite growth was not localized to either the PuraMatrix membrane or the top surface, as only 7.3±2.9% and 4.9±1.3% of the total growth was seen on the bottom and top 10% of the slice, respectively. Unlike neurites, DAPI staining indicates that the migrating cells were not affected to migrate into the pure matrix but remained localized near the supporting surface, as shown in Figure 4A, although previous studies suggest that glial cell migration and neurite outgrowth often occur together.

[0182] Spatial and Morphological Properties of Three-Dimensional Neural Cultures This disclosure describes in vitro three-dimensional neural cultures that approximate the cellular and macroscale architecture of native afferent peripheral nerve tissue. The three-dimensional neural constructs consist of DRG tissue explants cultured on the surface of cell culture inserts, contained by PEG constructs that allow growth within patterned voids filled with a pure matrix. The narrow tracts that direct neurite outgrowth from the ganglion along the x-axis measure approximately 490 μm in diameter, up to approximately 400 μm thick, and approximately 3 mm in length. The three-dimensional dual hydrogel constructs (after 7 days in vitro) containing DRG neurons, glia, and neurite outgrowth are shown in Figures 12A-12D.

[0183] Neurites and supporting glial cells were efficiently constrained by the geometry of the PEG hydrogel. Co-labeling with anti-β-III tubulin, anti-S100, and DAPI confirmed that proliferation was consistently robust after 7 days in vitro, and all labeled structures were within the pure matrix portion of the construct, as shown in Figures 5A and 5B. The presence and migration of supporting cells, including glial cells, extended up to three-quarters of the length of the channel, approximately 1.875 mm away from the ganglion, measured from the beginning of the straight channel, as shown in Figures 5C and 5D.

[0184] Leading neurite outgrowth throughout the depth of the PuraMatrix occurred randomly within the channel, with substantial branching and fasciculation in multiple focal planes, as seen in Figures 6A-6C. Conversely, growth in channels depleted of PuraMatrix appears to be restricted and aligned along the insert's fibers at the membrane surface. Antibody labeling in the images suggests more dense neurite outgrowth along the edges of the channel, as shown in Figure 12D. Consistent with the literature suggesting that myelination begins after 14 days in vitro, the 3D neural cultures showed an absence of myelin after staining with Fluoromyelin™ red fluorescent myelin stain (Molecular Probes, Eugene, OR) at 7 days in vitro, as shown in Figures 12E and F. Confocal imaging confirmed that β-III tubulin-positive neurites occurred in 3D at the beginning, middle, and end of the channel, as previously demonstrated. DAPI stained nuclei and S100 positive glial cells occurred throughout the z-stack, as shown in Figures 6A-6C.

[0185] Using techniques from the sample preparation protocol for transmission electron microscopy (TEM) on embedded biological samples, several iterations of the post-fixation procedure were attempted on the neural constructs before obtaining TEM images with recognizable structures. Additional modifications to the staining process can provide the structures with higher resolution for clearer visualization. Cross-sectional images from the TEM support the evidence shown by the fluorescent microscope. Slices taken within the ganglion and within the tract show a high density of parallel, highly fasciculated unmyelinated neurites, as seen in Figures 7A and 7B, the presence of Schwann cells, as seen in Figure 7D, and the beginning of Schwann cell encapsulation of the neurites, as seen in Figure 7C.

[0186] Electrophysiological Properties of Neurons in Three-Dimensional Constructs To test the functional properties of the three-dimensional neural cultures and determine whether they serve as a physiologically active and relevant model of afferent peripheral nerve tissue, intracellular and extracellular electrophysiological experiments were performed after 7 days in vitro. Using techniques adapted from traditional field potential recordings in acute rodent brain slices, these constructs were studied on an interface chamber, allowing the use of custom rigs for extracellular recordings. For each experiment, recording electrodes were placed in the ganglion or neuronal soma area of ​​the construct, as shown in Figure 8A, and stimulating electrodes in channels were inserted along the neurite tract. Following stimulation, compound action potentials (CAPs) propagated retrogradely to the neuronal soma area and were recorded as the resulting extracellular potential changes in the ganglion for each construct (n=19). The three-dimensional neural constructs supported field recordings for over an hour and consistently showed coherent population spikes upon stimulation. An example trace of the population response or CAP is shown in Figure 8B. Similar to compound action potentials recorded from intact nerves, responses consistently exhibit a short latency to onset followed by a single aggregate event with a graded nature representing the summation of the effects of each action potential on the recruited axon and corresponding cell. The consistent short envelope and delayed onset of the response are also characteristic of CAPs, suggesting a fast event purely driven by the action potential. Similar to nerve stimulation, more fibers with higher stimulation intensity are recruited, resulting in a stronger response, until maximal excitation occurs.

[0187] When the distance between the recording and stimulating electrodes was enlarged, the latency to onset of the response also increased, as shown in Figures 8C and 8D, confirming the ability of the geometrically localized nucleus cultures to conduct signals at various distances along their nerve-like pathways. On average, responses showed an onset latency of 0.82 ms when stimulated proximally, or within 1.5 mm, from the ganglion area, measured from the beginning of the straight channel. However, when the stimulating electrode was moved 2.25 mm from the ganglion, distal stimulation resulted in an onset latency of 2.88 ms on average, which is statistically significant (p<0.05) from that observed with proximal stimulation [p=0.02, Figure 8D]. As seen in the fluorescence microscope and shown in Figures 6A-6C and Figures 12A and 12B, 7 days of in vitro growth did not allow the neurites to completely fill the channel, and distal stimulation exhibited a 29.46% amplitude reduction. Furthermore, by inhibiting Na+ channel activity, action potentials were no longer generated upon stimulation. The response from the construct could be completely abolished within 2 minutes of introduction of 0.5 μM TTX, confirming the origin and biological nature of the response. The response before and after TTX washout is statistically significant (p=0.029, n=3), as shown in Figure 8E and F.

[0188] To investigate whether the responses were synaptic in nature, the glutamate receptor inhibitors DNQX and APV were introduced at 20 and 50 μM, respectively, to block excitatory synaptic transmission. The experiment continued for 35 min, with recordings taken every minute and at time points designated t1-t35 for simple reference. Drug washout occurred 5 min into the experiment, at t6, and washout occurred 20 min or 25 min into the experiment, at t26. Responses before drug washout, t1-t5, were compared to responses recorded after a 10 min drug washout, t16-t20, allowing sufficient time for the drug to perfuse and exert its effect. As shown in Figures 9A-9C, no statistically significant differences were observed in response amplitude or duration before and after drug washout, suggesting that there was no synaptic component of the response to block.

[0189] High frequency trains of pulses were also induced to assess the properties of the response. When 20 pulses at 50 Hz were applied to the cultures, the population spikes maintained consistent onset latency, envelope, and amplitude, suggesting that robust responses can be repeatedly generated without attenuation or facilitation caused by synaptic input. Response amplitudes and durations at half peak before and after high frequency stimulation were not statistically significant, as shown in Figures 9D-9F.

[0190] Intracellular recordings were also used to allow whole-cell patch clamp access inside three-dimensional neural constructs for over an hour. A technique modified from whole-cell patch clamp in acute rodent brain slices allowed voltage and current clamp recordings. Pura matrix gel is more adhesive than native brain tissue, and the dense DRG explants contain connective tissue. As with field recordings, these characteristics made electrode movement, replacement, and continuous use difficult. Cells within the DRG were densely packed together, with less contrast and more difficult to visualize than more sparsely dispersed cells in brain slices that are usually surrounded by features with different diffraction indices. As shown in Figures 10A-10F, good whole-cell patch clamp recordings were possible through repeated visualization in multiple focal planes, positive pressure while passing through the gel, and tilted electrode approach angles.

[0191] A bipolar stimulating electrode was placed in the neurite tract within the channel, as shown in Figures 10A and 10B, and recordings were taken from cells within the neuronal soma area of ​​the construct. The cells supported electrically evoked action potentials driven from neurite outgrowth within the channel, as shown in Figure 10C. As is characteristic of responses lacking synaptic input, the intracellular response had a fast rise time averaging 2 ms from baseline to peak, as shown in Figure 10D, and had a clear non-phasic onset. There was no rise in potential leading to a threshold prior to the onset of the response, as seen in Figure 10D, nor any smaller phasic events following the response, as seen in Figure 10C, providing no evidence of synaptic input. Furthermore, if synaptic activity was contributing to the onset of the response, the threshold for the initiation of an action potential would be difficult to reach under hyperpolarization. However, cells were still able to support action potentials when hyperpolarized from the resting membrane potential (RMP) to -100 mV (on average 1.95-fold less than the RMP), showing responses that were not different from those at the RMP. Furthermore, no spontaneous activity evoked by synaptic activation was observed in baseline recordings under voltage or current clamp, as shown in Figures 10E and 10F.

[0192] Example 2. Evaluation of neural tissue growth and myelination and demyelination by Schwann cells within hydrogel constructs (non-predictive) Successful axonal regeneration within the peripheral nervous system (PNS) depends on properly targeting neuronal growth towards selected locations and forming functional synapses for signal propagation. Schwann cells (SCs), which are indigenous to the PNS, play a key role in this process. SCs wrap around developing axons within myelin and produce extracellular matrix (ECM) components, cell adhesion molecules, and neurotrophic factors. These events depend on a complex network of signals, including SC-neuron, SC-SC, and SC-ECM communication, from the local microenvironment. Experiments housing SC / neuron cocultures have provided insight into these processes and led to new clinical approaches for diseases of the nervous system.

[0193] Primary neurons and SCs have been previously co-cultured in two- and three-dimensional systems to study the mechanisms involved in SC / neuron integration. It has been demonstrated that SCs play a key role in orienting developing axons towards their desired targets, leading to functional reinnervation in these models, regardless of the number of dimensions. However, many properties involved in SC / neuron integration, such as morphology and gene expression, are dramatically affected by the system architecture. Three-dimensional systems provide a better understanding of cell-cell and cell-ECM mechanisms, along with a more accurate representation of the structure and function of the neuronal microenvironment. It has been shown that resting potentials, action potential propagation, and voltage-gated channel functions are significantly different in two dimensions compared to three-dimensional models. Although the importance of using three-dimensional biomimetic nervous system microenvironments has been demonstrated, few studies have investigated SC / neuron interactions in co-cultures and their impact on myelination.

[0194] Here, we use the facile and fast technique described above, which uses a digital micromirror device (DMD) integrated into a simple microscope with the aim of photopatterning the desired three-dimensional hydrogels. The DMD is capable of structural and molecular three-dimensional micropatterning. This in vitro model provides a setting that mimics the support and three-dimensional architecture of the ECM with the ability to independently introduce fixed or soluble chemical biomolecules, mechanical cues, and drugs to evaluate the effect of each on neuronal behavior. This system provides a platform to three-dimensionally co-culture different cell types in one specimen to study them within a more biomimetic environment. Using this approach, we photomicropatterned functionalized dextran and encapsulated DRG and SC in a three-dimensional co-culture system in conditions close to their native environment to investigate the factors that lead to the formation of myelin.

[0195] A. Materials and Methods Fabrication of dual hydrogel systems Dual hydrogel culture systems were fabricated using digital projection photolithography, as described above. A schematic of this process can be seen in Figure 13. Briefly, a photolithography setup consisting of a collimated UV light source (OmniCure 1000 with a 320-500 nm filter, EXFO, Quebec, Canada) and a visible light source (SOLA light engine with a 375-650 run filter, Lumencor, OR, USA), a digital micromirror device (DMD) (Discovery™ 3000, Texas Instruments, Dallas, TX) as a dynamic photomask, and a 2x Plan Fluor objective lens (Nikon Instruments, Tokyo, Japan) was used to irradiate a photocurable hydrogel solution housed in a permeable cell culture insert with a 0.4 μm pore size. The inserts were either collagen-coated PTFE Transwell® permeable supports or Transwell™ clear polyester membrane inserts (Corning Inc., Corning, NY, USA) to investigate the effect of collagenized substrates on SC / neuron integration. The dual hydrogel system consists of two compartments, a cell-permeable compartment that houses neurons, and a cell-restrictive compartment that acts as a hydrogel mold. To create the cell-restrictive compartment, a solution of 10% (w / v) PEG-diacrylate (Mn 1000, Polysciences Inc., Warrington, PA) and 0.5% (w / v) Irgacure 2959 in PBS was irradiated with 85 mW / cm2 UV light for 38 seconds as measured by a radiometer (306 UV Powermeter, Optical Associates, San Jose, CA) to create a PEG micromold, as shown in Figure 13. To avoid meniscus behavior, cell culture inserts were treated with filtered Rain-X® Original Glass Treatment (RainX, Houston, TX) prior to addition of gel solution. 0.5 mL of solution was added to each 6-well plate insert. Addition of 0.5 mL solution results in a gel thickness of 480 μm.Hydrogel constructs were washed in DPBS containing 1% antibiotic-antimycotic additive to prevent contamination.

[0196] Synthesis and Characterization of Dextran and Gel Constituents Dextran (MW=70 kDa) was grafted with glycidyl methacrylate (GMA) based on published protocols. First, 1 g of dextran was weighed and added to 9 mL of dimethyl sulfoxide (DMSO) under nitrogen. 0.2 g of 4-dimethylaminopyridine (DMAP) was dissolved in 1 mL of DMSO. Then, the DMAP solution was added dropwise to the dextran solution, followed by 232 μL of GMA under nitrogen. The final solution was stirred at room temperature for 48 hours. To quench the reaction after 48 hours, 280 μL of 37% hydrochloric acid (HCl) was added to the solution, and the resulting product was dialyzed against deionized water for about 3 days and lyophilized for about 2 days. The resulting product was glycidyl methacrylate-dextran (MeDex), and the addition of methacrylate groups to dextran was 1 The degree of substitution was confirmed to be 42% using H NMR [(D2O) δ 6.1-5.7 (m, 2H, CH2), δ 5.2 (m, 1H, CH), δ 4.9 (m, 1H, CH), δ 1.9 (s, 3H, CH3)]. Gel compositions were prepared with a final solution of MeDex 50% (w / v), MeDex with Arg 0.1% (w / w), MeDex with RF 0.001% (w / w), and TEMED 0.2% (v / v).

[0197] Primary tissue culture within the dual hydrogel system As the first step of co-culture, primary tissue culture was performed. PEG constructs were prepared, immersed in adhesion medium, and incubated overnight (37°C, 5% CO2) before tissue culture. The adhesion medium consisted of Neurobasal medium supplemented with B27 (2% v / v), L-glutamine (0.25% v / v), nerve growth factor (NGF) (0.02 μg / mL), fetal bovine serum (FBS) (10% v / v), and penicillin / streptomycin (1% v / v) (all from Life Technologies, CA). The constructs were then cultured with Long-Evans rat embryonic dorsal root ganglion (DRG) tissue in accordance with the guidelines of the Institutional Animal Care and Use Committee. DRG were isolated from embryonic day 15 rat embryos and trimmed before culture. A single DRG explant was placed within each construct. The DRGs were then incubated overnight in fresh adhesion medium to allow the tissue to adhere to the insert.

[0198] Schwann Cell Cultures A SC cell line isolated from neonatal rat sciatic nerves was purchased (ScienCell Research Laboratories, CA). A cryopreserved vial containing >5×105 cells / mL was thawed in a 37°C water bath. The contents of the vial were then gently resuspended and dispensed into culture vessels coated with equilibrated poly-L-lysine to give a concentration of 2:10,000 cells / cm. 2 A seeding density of 1000 μg / ml was used to promote cell attachment. Cultures were not disturbed until at least 16 h later. Culture medium was changed first after 24 h and then every other day to remove residual DMSO and unattached cells. Culture medium consisted of SC medium containing FBS (5% v / v), penicillin / streptomycin (1% v / v), and SC medium supplement (1% v / v) (all from ScienCell Research Laboratories, CA). Cultures were subcultured whenever they reached 90% confluence and were not used after the third passage.

[0199] SC encapsulation and incorporation in dual hydrogel systems SCs were dispersed in a 50% MeDex solution in SC medium as described above at 20 × 106 A cell number of 1000 cells / mL was reached. The gel mixture was vigorously dispensed up and down to obtain a uniformly distributed single-cell solution. The adhesion medium was gently aspirated from the channels to avoid disturbing the attached DRGs, and 2 μL of MeDex single-cell solution was added to each PEG micromold. A negative photomask was loaded onto the DMD, and the gel solution in the channels was illuminated with a 30-s pulsed IR (IR) intensity of 85 mW / cm2 as measured by a radiometer (306 UV Powermeter, Optical Associates, San Jose, CA) after 30 s of irradiation using a visible light source (SOLA light engine with 375-650 nm filter, Lumencor, OR, USA). 2 Crosslinking was performed with visible light. The constructs were gently washed three times using the above wash buffer.

[0200] Media regimes for DRG / SC co-culture in 3D hydrogel systems To understand the effect of different media regimes on the behavior of DRG and SC in three-dimensional co-cultures, two different culture systems were applied. The culture systems are described in Table 2. Culture system 1 has two phases, where media 1 (10 days) and 2 (15 days) are applied in that order. This media regime was previously used to promote growth and neurite outgrowth as well as to promote the incorporation of endogenous SCs of the DRG bulk into the myelination process. Culture system 2 applies only medium 2, specialized to induce myelination. For each specimen in each experimental group, the medium was changed every other day. [Table 2]

[0201] Immunohistochemistry Immunohistochemistry techniques were used to assess neurite outgrowth and myelination. Tissues were first fixed with 4% paraformaldehyde (PFA) for 2 hours at 37°C, followed by three washing steps before each staining procedure. All reagents were provided by AbCam (Cambridge, MA) unless otherwise stated.

[0202] Neurites were labeled with mouse monoclonal [2G10] neuron-specific β-III tubulin primary antibody and Cy3.5-conjugated goat anti-mouse immunoglobulin G (H+L) secondary antibody (AbCam, Cambridge, MA). Labeling steps were completed overnight at 4°C in 2% bovine serum albumin (BSA) and 0.1% saponin in PBS, followed by three washing steps with PBS after each step.

[0203] To assess myelination, constructs were labeled with three myelin proteins, myelin basic protein (MBP), protein zero (PO), and myelin associated glycoprotein (MAG). Primary antibodies chicken polyclonal anti-myelin basic protein, mouse monoclonal anti-myelin associated glycoprotein, and rabbit polyclonal anti-myelin protein zero were used. The dyes were diluted in a 2% BSA / PBS solution at a concentration of 1:500. To avoid any non-specific protein binding, constructs were immersed in 5% goat serum at room temperature for 30 minutes. Constructs were kept in the primary antibody solution overnight at 4°C and washed three times with PBS. After three washing cycles, the hydrogel system was incubated in the secondary antibody solution at 4°C. The secondary solutions were prepared as follows: 1:500 antibody solutions in 2% BSA solution of anti-chicken IgY H&L, goat anti-mouse IgG H&L, and goat anti-rabbit IgG, respectively.

[0204] Image processing, neurite outgrowth, and myelination. The volume of growth into the three-dimensional hydrogel was measured using a confocal microscope (Nikon AI, Tokyo, Japan). Due to the tangled and dense neurites in this model, it is difficult to count the number of individual neurons as they extend along their length. Therefore, it is optimal to take the volume of the cell mass in the dual hydrogel culture system to measure the growth of the system in three dimensions. Each sample was imaged in three dimensions using a 10x objective lens with a resolution of 1024 × 1024 pixels, using optical slices at a depth of 11 μm or less with an average of 20 slices per sample. Preprocessing steps, including thresholding and conversion to a binary representation, were applied uniformly to all images. Data analysis was performed using custom algorithms in ImageJ and Matlab (Mathworks, Natick, MA). Neurite outgrowth was quantified using the pixel count of the threshold slice throughout the depth of the gel. After 25 days, the myelin was densely partitioned, and the same image processing procedure was used to evaluate the volume of myelin throughout the depth. This process allows for the measurement of the volume throughout the depth, given the three-dimensional nature of the cultures. For both imaging processes, large image z-stacks were taken (1x5) since the size of the constructs was too large to image all at once. For photo documentation, the samples were imaged in three dimensions using optical slices below 11 μm in depth with an average of 20 slices per sample, with a resolution of 1024x1024 pixels and a 20x objective. Maximum projection acquisition was used to form a two-dimensional image of the total growth. The same procedure was used for the volume of growth, and a three-dimensional volume acquisition was used to confirm that growth and myelination occurred throughout the depth.

[0205] Transmission electron microscopy (TEM) was used to investigate the nanoscale structure of neurites, SCs, and their spatial crosstalk, distribution, and morphology in hydrogel cultures. All of the reagents used in this procedure were provided by Electron Microscopy Sciences (Hatfield, PA) unless otherwise stated. The hydrogel constructs were fixed after submersion in 4% PFA solution for approximately 2 hours at 37°C. The samples were then washed with PBS three times at 15-minute intervals. The post-fixation step included staining with 1% osmium tetroxide (OsO4) in 100 mM acetic acid phosphate for approximately 2 hours, followed by four washing steps with PBS. The tissues were then stained with 2% aqueous uranyl acetate for approximately 30 minutes at room temperature in the dark. This procedure was followed by a dehydration step, which included immersing the samples in 50% and 70% ethanol for 10 minutes each, and then in 95% ethanol overnight. The samples were then immersed in 100% ethanol, which was filtered through molecular sieves, 4 Å (Sigma-Aldrich, St. Louis, MO) for two 30 min intervals. Resin embedding was performed after cutting the constructs to keep only the area of ​​interest. The infiltration step was performed for 45 min using 1:1 propylene oxide-Sparr resin. The samples were then embedded in 100% Sparr resin at 70° C. for approximately 48 h to complete the resin polymerization.

[0206] The embedded samples were trimmed and sliced ​​to different thicknesses ranging from 80 nm to 100 nm using a Reichert Ultracut S ultratome (Leica Microsystems, Buffalo Grove, IL) and an Ultra 45° diamond knife (Diatome, Fort Washington, PA). The slices were mounted on copper grids (Formvar carbon-coated, 200 mesh) and the grids were floated on a drop of 2% uranyl acetate for approximately 20 min and rinsed by floating on deionized water drops three times at 1 min intervals. After the grids were mounted on a single tilted stage, they were imaged using an FEI Tecnai G2 F30 Twin transmission electron microscope (FEI, Hillsboro, OR) with an accelerator voltage of 100 to 200 kV. Rows were taken at 4000 × 4000 pixel resolution at 3,000 × 20,000 × magnification.

[0207] B. Results Three-dimensional dual hydrogel system and DRG / SC co-culture This disclosure provides a three-dimensional model to investigate the use of dual hydrogel platform for co-culture applications and a three-dimensional hydrogel system using DMD as a dynamic photolithography tool. The model was used to investigate the in vitro effects of mechanical stimuli and chemical cues, including repulsive and attractive biomolecules, on neuronal growth. The model mimics the three-dimensional structure of ECM and more accurately translates the neuronal microenvironment. The ability of the system to handle two cell types in a single culture and to investigate cell behavior was evaluated. SCs and neurons were co-cultured to investigate the myelination process in conditions close to their native environment. The model allows for myelination as a result of three-dimensional SC-neuron co-culture. The methodology for the dual hydrogel system is shown in Figure 13.

[0208] Effects of Collagen on Neurite Outgrowth in Three-Dimensional Co-Cultures We demonstrated the formation of three-dimensional cultures within hydrogels formed within permeable inserts with or without a collagen coating. The outgrowth in both cultures was robust, bundled, and aligned. This characteristic distinguishes this system from previously developed in vitro models because the outgrowth is oriented within the channels. The outgrowth becomes highly confluent after 25 days, but is largely contained within the cell-permeable compartment of the three-dimensional hydrogel system. β-III tubulin positive neuronal filaments are shown in Figures 17A, 18A, and 19A. The volume of neuronal outgrowth is significantly higher in cultures with collagen compared to cultures without collagen (n=15-18 constructs). The amount of outgrowth was not significantly different between the two media regimens.

[0209] Myelin Development in Three-Dimensional Co-Culture Model in Dual Hydrogel System The co-culture system of the present disclosure promotes three-dimensional myelination. Immunohistochemistry and TEM were used to demonstrate myelin formation. Cultures were stained with three antibodies, MBP, MAG, and PO. Constructs were positive for MAG, MBIP, and PO, confirming the formation of compact and non-compact myelin. Both Figure 17B and Figure 18B show neurofilaments stained for β-III tubulin, and the merged images confirm the formation of MBP and PO segments along the axonal extension, with Figure 17B showing MBP-positive mature myelin sheath and Figure 18B showing PO-positive mature myelin sheath.

[0210] All images were taken through z-stack acquisition, as described above. Confocal imaging confirmed that neurite outgrowth occurred in three dimensions throughout the channel. The depth of outgrowth and myelination of these constructs was 88 ± 15 μm. TEM images confirmed myelination, as seen in Figures 20A-20F. Slices taken within the nerve tract show a high density of parallel, highly fasciculated and myelinated neurites, the presence of Schwann cells, and Schwann cell encapsulation of the neurites. Myelin segments were consistently identified in the TEM images, confirming compact myelination. These findings demonstrate that this three-dimensional in vitro model allows SCs to form a mature myelin layer around the neurites.

[0211] Effect of Ascorbic Acid (AA) on Three-Dimensional Myelination Two media regimes were used for the cultures. For NCol-25 and Col-25, the 25 day media containing AA resulted in a significant increase in the amount of myelin. The amount of myelin demonstrates the ability of the cultures to form myelin sheaths regardless of the amount of neuronal outgrowth. The ratio of myelin to neuronal outgrowth was measured, showing that the percentage of myelin within the constructs increases with longer exposure to AA. This was confirmed through staining with three immunohistochemical antibodies: MBP, MAG, and PO, thus demonstrating that this is accurate for both compact and non-compact myelin.

[0212] The effect of collagen on myelin development The effect of collagen I and III on compact and non-compact myelin development was evaluated in the system. Myelin proteins followed similar trends as shown in Figures 17A-17C, 18A-18C, and 19A-19C. The addition of collagen increased the amount of myelin formation in the system. The ratio of myelin to neurite outgrowth was similar for Col-15 and NCol-25. This demonstrates that the increased amount of myelin in Col-15 compared to NCol-25 is due to an increased amount of neuronal outgrowth. The efficiency of the two systems in developing myelin depends on AA exposure. Figures 16A and 16B show that collagen dramatically increases neuronal outgrowth. NCol-15 shows that in the absence of collagen, minimal myelin forms with short exposure to AA.

[0213] C. Consideration The myelin sheath is a specialized cell membrane with a multi-membrane spiral structure that surrounds axons and reduces the capacitance of the nervous system. A well-myelinated nerve is completely surrounded by the myelin sheath, except for small periodic gaps known as nodes of Ranvier that are exposed to the extracellular environment. Myelin exists in two forms: compact and non-compact. The compact myelin ultrastructure consists of a spiral cell sheath that lacks cytoplasm as well as extracellular space but contains two plasma membranes. Non-compact myelin is a channel-like segment of myelin that is non-aggregated and forms at Schmidt-Lantelmann notches, periodic interruptions in the myelin layer, and paranodal areas.

[0214] Compact and non-compact myelin each contain various proteins, such as myelin basic protein (MBP), which is an essential component of CNS and PNS compact myelin. MBP is located on the cytoplasmic surface of the myelin sheath and is highly charged. Another important myelin protein in the PNS is PO, a transmembrane glycoprotein that plays an important role in influencing cell adhesion, maintaining the main dense line of PNS compact myelin, and maintaining the space between compact myelin constructs. One of the main components of non-compact myelin is myelin-associated glycoprotein (MAG), which is not present on the outer layer of myelin but is present in the inner layer. It contacts and binds axons to compact myelin. These three proteins are essential for the formation and maintenance of myelin and have been widely used to detect myelin in culture.

[0215] Co-culture systems of SCs and neurons derived from either primary tissue sources or cell lines can accurately depict events in the native PNS and the complex myelin architecture. PC12 cell lines and SCs have been used previously to establish motor neuron / SC co-culture models to study motor neuron diseases. In vitro models of sensory neurons and SCs have been used previously to understand the mechanisms behind myelination. Many previous studies have used DRGs because they are well-studied and recognized as a powerful in vitro model using the development of neuron / SC co-cultures to evaluate the myelination process in the PNS.

[0216] These previous in vitro co-culture models have mostly been performed in 2D cell cultures and 3D tissue slices, with few studies investigating the incorporation of neuron / SC co-cultures and their effects on myelination in 3D cultures.

[0217] A photomicropatterning setup was used to design a three-dimensional biomimetic polymer model to study myelination in neuron / SC co-cultures. Photopatterning has been used to study the nervous system, as it allows for the proper translation of a three-dimensional biomimetic neuronal microenvironment. The dynamic mask projection photolithography device used in this study provided a facile fabrication technique with the aim of generating micropatterned hydrogels. These hydrogels were created on permeable cell culture inserts, providing the basis for nerve regeneration experiments.

[0218] To generate these constructs, a dynamic photomask was created using a DMD device. This mask was used with an irradiated PEG solution to create a mold onto which the DRGs were first attached, followed by the addition of a light-curable single-cell MeDex solution. A negative dynamic photomask was used to encapsulate the SCs in three dimensions and integrate them with the DRGs. Using visible light with a short (30 s) exposure length was used in this design because it is the most practical for hydrogel formation and cell encapsulation to reduce cytotoxicity. This model provides a long-term (25 days) in vitro platform to ensure the survival of neurons, their extension, and their myelination in a three-dimensional environment.

[0219] These models used two different cell culture media, described in Table 2. Medium 1 is composed of well-characterized factors and is known to support the growth of DRGs and SCs. This medium contains BSA and has been shown to support migration of SCs. However, this system is not specialized to promote myelination. Medium 2 contains FBS along with ascorbic acid, which has been shown to promote myelination in two-dimensional cultures. Previous studies of SCs in the presence of neurons show that they can create a complete ECM with basement membrane and collagen fibrils in vitro. SC / DRG co-cultures have shown that ascorbic acid promotes SCs to generate myelin by allowing them to form a basement membrane. Medium 2 also contains ITS (insulin, transferrin, and selenium) and has been shown to promote myelination in rat cell lines.

[0220] Laminin was used in all experimental groups because it was shown to be essential for myelination in neuron / SC co-cultures. Absence of laminin in vivo has been shown to lead to peripheral neuropathy in both mice and humans. Mutant mice lacking laminin will have a breakdown of the endoneurial basement membrane, which subsequently reduces nerve conduction velocity.

[0221] The system disclosed herein also examines the effect of the presence of collagen on neuronal growth in this three-dimensional model through the use of collagen-coated substrates. Type I and III collagen were used in these studies. Type III collagen binds to and activates a receptor coupled to adhesion g-protein on Schwann cells, Gpr56, which can lead to activation of Gpr125 and initiate myelination. Type I and III collagen are important components of the endoneurium, which is the outermost layer of dense tissue that supports and surrounds peripheral nerves and myelin.

[0222] To investigate the ability of neural cells to form myelination in a three-dimensional model, the effects of two different media and the effect of collagen were evaluated. The four culture systems are differentiated by the presence of collagen and the media regime to which the co-cultures were exposed. Two media regimes were used. One regimen included medium 1 for 10 days followed by medium 2 for 15 days (culture system 1), and in the second regimen, the cells were exposed to medium 2 alone for 25 days (culture system 2). Table 3 describes the groups. To determine whether myelination was affected by exogenous SCs, the above experiments were performed without the addition of encapsulated SCs to the dual hydrogel system, keeping all other variables constant. [Table 3]

[0223] The formation of myelin was confirmed using immunohistochemistry and confocal imaging and further verified by TEM. Two-dimensional images at 20x magnification show the formation of myelin segments wrapping around neuronal projections in MBP / β-III tubulin positive cultures as shown in FIG. 15. Three-dimensional development of myelin with both MBP and MAG staining due to the formation of compact and non-compact myelin is shown in FIGS. 16A and 16B. TEM images also confirmed the occurrence and abundance of a mature myelin layer in all of the experimental groups as shown in FIGS. 20A-20D. A magnified image of the myelin layer is shown in FIG. 20F. FIG. 20E shows that after 25 days in culture, SCs had formed myelin sheaths around many of the neurites and some SCs had begun to surround a cytoplasmic layer around the nerve fibers. This image clearly demonstrates that the amount of myelin is significant and that the cultures can be used for long-term studies including three-dimensional long-term drug evaluation. 20A-20F also show that neurons in the cultures are dense, aligned, and highly fasciculated.

[0224] The first set of analyses performed quantified neuronal growth in each of the four three-dimensional culture systems, as described in Figure 14. It is well established that collagens and their receptors promote neurite outgrowth. The data presented herein demonstrate that there is significantly more neuronal growth in the two systems in which collagen is present. However, there is no significant effect on growth due to the media regimen used, which has little effect on the amount of neurite outgrowth after 25 days in the systems involved.

[0225] The amount of myelin was measured by two different approaches. The first approach was to look at myelination as an independent variable, closely examining the total amount of myelination regardless of the amount of neuronal development in the system. The second approach was to calculate the ratio of myelin to neurite outgrowth and normalize the amount of myelin development. This provides an understanding of the efficiency of myelination and accounts for the percentage of neuronal protrusions that have a myelin sheath wrapped around them. Staining for MBP, MAG, and PO was used to investigate the amount of myelin produced by the four experimental groups.

[0226] FIG. 17C illustrates the percentage of myelin formed in the culture system. MBP antibody was used for these data. All four samples were positive for MBP after 25 days of culture, but there was a significant difference between the groups. MBP is a protein present in compact myelin, and its expression in cultures demonstrates the formation of compacted membrane segments of mature myelin sheaths. When AA exposure is increased, increased myelination occurs in these systems. These results were obtained in a three-dimensional in vitro model that mimics the nervous system environment more closely than regular two-dimensional cultures or tissue sections. The data show that the ratio of myelin to neuronal proliferation in these systems was significantly increased when exposed to myelin-forming medium for 25 days. The medium regimen results in increased myelination when cultures are in the presence of collagen for the same length of exposure.

[0227] Based on these data, two factors play a role in these cultures: the presence of collagen and longer AA exposure. Constructs lacking both of these factors (NCol-15) are minimally myelinated. The percentage of myelin versus neuronal outgrowth for cultures showed that the same AA exposure had a similar effect regardless of the number of neurons generated. However, FIG. 17B shows that when both factors were present in the experiment (Col-25), a synergistic response was observed, resulting in a significant increase in myelin magnitude. Maximal projections of the z-stack planes are included to support these data.

[0228] To confirm that exogenous SC significantly alters myelination, a control group without additional SC was used. The data in Figure 17C show that all experimental groups had a significant increase in myelination over their corresponding controls, demonstrating that exogenous SC had a significant effect on the system. These results indicate that collagen significantly increases myelination in the control group, while the duration of AA exposure has a lesser effect.

[0229] Myelination was measured in three-dimensional cultures using a PO protein antibody. 70% of the total protein in PNS myelin consists of PO, and loss of this protein demonstrates loss of non-compact myelin. The ratio of PO expression to β-III tubulin-positive neurofilaments was evaluated. The results shown in Figure 18C show that NCol-15 presents the least amount of PO from all cultures. The percentage of PO expression is significantly higher in 25-day cultures in the presence of AA, which is consistent with the results from MBP staining, which shows the greatest expression of MBP in the Col-25 group. This is interesting because both PO and MBP are characteristic proteins of compact myelin in the PNS, but have different roles. PO maintains an organized reappearance of both the ECM and the cytoplasmic space of the myelin membrane, while MBP plays a role in cytoplasmic fusion. This value is comparable to the NCol-25 group, indicating that the efficiency of the cultures after 25 days in medium 2 was the same regardless of whether collagen was present in the cultures or not.

[0230] FIG. 18B shows the amount of myelin in the cultures labeled with PO. Col-25 shows the greatest amount of compact myelin PO development, regardless of the amount of neuronal growth. These results show that samples with collagen in the culture lead to more neurons and a greater amount of myelin. Between the two collagen-containing samples, AA exposure increases the amount of PO generation. This remains after normalizing the amount of myelin value in the collagen-containing samples by calculating the ratio of the amount of myelin to neurofilament, as shown in FIG. 18C. These images clearly show that the amount of PO was dramatically reduced in the collagen-free, NCol-15, and NCol-25 constructs. The volume of neuronal growth was also reduced, and as a result, the percentage of compact myelination did not show any significant deviation from Col-15. In such long-term three-dimensional constructs, the percentage of compact myelin that expressed PO after cultures were exposed to AA for 25 days was not substantially different from the percentage of compact myelin that expressed PO in cultures with collagen for 15 days in the presence of AA. AA is essential for myelination in serum-containing medium for two-dimensional cultures. The duration of AA exposure plays an important role in the efficiency of the formation of myelin. Collagen I and III can support neuronal growth and help initiate the myelination process. The presence of collagen in this system increases the three-dimensional extension of neurons, resulting in an increased amount of myelination in a three-dimensional setting.

[0231] A different measure of myelin is MAG, a protein abundant in noncompact myelin. The ability of rat DRG / SC cocultures to form myelin in three-dimensional constructs was assessed by MAG immunostaining. All of the constructs were MAG positive, following the same pattern as PO and MBP. High levels of myelin synthesis were evident by confocal microscopy analysis of MAG, as well as PO and MBP. MAG shows Schmidt-Lantelman notches and paranodal areas characteristic of noncompact myelin. The amount of noncompact myelin was higher in the Col-25 group exposed to AA for longer in the presence of collagen, regardless of the amount of neuronal outgrowth. AA helps the system form a basement membrane and promotes myelination. The percentage of MAG-labeled structures does not differ substantially between cultures (Col-25 and N-Col25) with the same exposure to AA. However, the amount of outgrowth is greatly reduced when collagen is not added to the system.

[0232] This disclosure describes a novel three-dimensional in vitro co-culture model that allows the incorporation of SCs and neurons. A facile high-throughput photolithography method provided a three-dimensional setup to recapitulate neuronal phenomena in a controlled microenvironment and introduce mechanical and chemical cues with highly resolved spatiotemporal precision. Here, data clearly demonstrate that this co-culture setup provided aligned and highly fasciculated neuronal growth with myelin sheaths neatly wrapped along them. Myelination was confirmed through immunohistochemistry and TEM. The two culture systems were used to investigate the effects of collagen on neuronal growth and myelination. This platform provides devices, methods, and systems useful for drug discovery and evaluation.

[0233] Example 3. Model calibration and feasibility (non-predictive and predictive) Drug development pipelines suffer from unacceptable attrition rates, due in large part to toxicities that go unidentified during the preclinical stages of development. Chemotherapeutic drugs, in particular, are effective against a broad range of cancers but are commonly associated with dose-limiting systemic toxicities. The peripheral nervous system often bears the brunt of these adverse effects, and such toxicities are often only initially identified in animal studies or overlooked until clinical trials. Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of cancer treatment, forcing many patients to modify their dose regimens and some to discontinue treatment altogether due to severe neurotoxic damage. The ability to screen drug candidates for peripheral neurotoxicity in cell models would accelerate the drug discovery process by helping companies identify promising lead compounds before undergoing costly and time-consuming animal studies.

[0234] "Organoid-on-a-chip" technology shows great promise as a modern cellular model that can provide medium throughput and high content data useful for late-stage drug development, insofar as it provides predictive information for human physiology or pathology. Many contract research organizations have seen commercial success in providing such assays for various organ systems. However, the development of peripheral nerve-on-a-chip assays has been slow. Commonly used peripheral nerve culture preparations are not predictive with respect to clinical toxicity, in part because they typically use apoptosis or neurite outgrowth as measurable endpoints, whereas adult peripheral nerves are fully grown and known to be resistant to apoptosis. Nerve conduction studies and tissue morphology of tissue biopsies are the most clinically relevant measures of neuropathy. Nevertheless, no culture models currently exist that provide such metrics.

[0235] We developed novel sensory nerve-on-a-chip models by culturing dorsal root ganglia within micropatterned hydrogel constructs to constrain axonal growth in a 3D arrangement that resembles peripheral nerve anatomy. Furthermore, electrically evoked collective field potentials resulting from compound action potentials (cAPs) can be reproducibly recorded in these model systems. These early results demonstrate the feasibility of using micromanipulated nerve tissue that is subject to morphological and physiological measurements similar to those in clinical trials. We hypothesize that chemotherapy-induced neurotoxicity will manifest in these measurements in a way that mimics clinical neuropathy. The goal of this proposal is to demonstrate the feasibility of using compound action potential waveforms as a measure of peripheral neurotoxicity in vitro. To this end, we will apply a chemotherapeutic drug with known peripheral neurotoxicity and measure changes in cAPs and compare them to morphological changes as well as record clinical pathophysiology. The following specific objectives will enable us to achieve this goal.

[0236] Aim 1: Calibrate the nerve-on-chip model by quantifying key morphological metrics and correlating them with compound action potential (cAP) metrics. Confocal and transmission electron microscopy will be used to quantify cell body size and density, as well as neurite density, diameter, and % of myelinated neurites at three lengths along the tract over a 4 week period in vitro. Determine the consistency of evoked population action potential responses over a 4-week period. ·Correlate cAP waveforms with morphometric parameters to determine baseline structure-function relationships.

[0237] Aim 2: Demonstrate the feasibility of using cAP waveforms to measure toxicity induced by acute application of four chemotherapeutic drugs known to cause clinical neuropathy. Determine the appropriate dosages and incubation times of oxaliplatin, paclitaxel, vincristine, and bortezomib for the nerve-on-a-chip model in a pilot study. ·Measure cAP conduction velocity, amplitude, latency, and integral following drug administration at endpoints determined in pilot studies. - Quantify morphometric changes and determine correlation with changes in cAP waveform.

[0238] It is widely recognized that the current attrition rate of experimental drugs as they progress from discovery to clinical use is unacceptably high, costing up to $2.6 billion to market a single drug (DiMasi et al. 2014). Dose-limiting toxicities undetected during drug development are estimated to be the second leading cause of post-marketing drug withdrawals, and these late-stage deficiencies are commonly associated with the lack of reliable screening methods for toxicity of drug candidates (Kola & Landis 2004, Li 2004, Schuster et al. 2005). Despite this, recent guidelines from the FDA on in vitro-in vivo correlation (IVIVC) emphasize the relationship between drug dissolution and bioavailability (Emami 2006), and there are no defined IVIVC guidelines for correlating clinical toxicity with in vitro toxicity tests. Cell-based toxicity screening assays can help companies identify less toxic lead compounds, but unfortunately there are no reliably predictive in vitro assays for clinical toxicology and it is clear that there is an urgent need for one (Astashkina & Grainger 2014).

[0239] Chemotherapeutic drugs are a special class of drugs because they are truly cytotoxic in nature. Therefore, toxic side effects are inevitable, and the level of clinically tolerable systemic toxicity limits drug dosage. The nervous system is particularly vulnerable to adverse effects, with chemotherapy-associated neurotoxicity second only to hematological toxicity in incidence (Malik&Stillman 2008, Windebank&Grisold 2008). Peripheral nerves are particularly susceptible, likely due to being outside the protective blood-brain barrier and having very long axons that reach far from their cell bodies. Chemotherapy-induced peripheral neuropathy (CIPN) is estimated to occur in 30-40% of treated patients, with sensory nerves consistently more severely affected than motor nerves (Windebank&Grisold 2008). Symptoms range from chronic pain in the limbs to tingling, loss of sensation or joint position sense, and movement disorders. The National Cancer Institute has identified CIPN as the most dose-limiting side effect and one of the most common reasons why patients choose to reduce dosage or stop treatment altogether (Moya del Pino 2010). In some cases, symptoms resolve after treatment is stopped, but in many cases, CIPN is only partially reversible and some symptoms remain permanent. Unlike hematological toxicities, which can be easily treated, there is currently no standard of care clinical treatment for CIPN (Windebank & Grisold 2008).

[0240] The classes of chemotherapy drugs known to pose the greatest risk for peripheral neurotoxicity are platinum derivatives; tubulin-binding compounds including vinca alkaloids, taxanes, and epothilones; the proteasome inhibitor bortezomib; and thalidomide. These drugs are also standard treatment for the six most common malignancies (Argyriou et al. 2012, Cavaletti & Marmiroli 2010, Wang et al. 2012). The exact neurotoxic molecular mechanisms leading to the reported constellation of symptoms vary and remain unclear in some cases. In general, platinum compounds bind to DNA and cause apoptosis, while antitubulins interfere with tubulin dynamics including axonal transport (Malik & Stillman 2008), bortezomib is thought to disrupt mRNA transcription and processing within ganglia, and the mechanism of thalidomide is unclear but may involve interactions with the vasculature and / or inflammatory cells (Argyriou et al. 2012). The specific presentation and severity of CIPN can be most objectively and reliably diagnosed by nerve conduction studies and / or skin or nerve biopsies (Dyck & Thomas 2005). These measurements are currently available only from safety studies in animals and humans. Therefore, most pharmaceutical companies simply do not specifically screen for peripheral neurotoxicity until after lead chemistry has been identified, even though peripheral neurotoxicity is one of the most likely causes of failure in the later stages of development.

[0241] The use of 3D “organoid-on-a-chip” models has gained acceptance as the best hope for developing predictive cell-based assays suitable for drug development and toxicity screening (Ghaemmaghami et al. 2012, Kimlin et al. 2013). However, it is critical that such model systems go beyond 3D versions of traditional cell viability assays to models that truly recapitulate functional aspects of organ physiology that can be evaluated to identify toxicity pathways (Astashkina & Grainger 2014). Such physiological assessments are particularly challenging for peripheral nerve tissue, where bioelectrical conduction over long distances may arguably be the most relevant physiological endpoint. For this reason, 3D tissue models of peripheral nerves have lagged behind models of epithelial, metabolic, and tumor tissues, where soluble analytes serve as appropriate metrics. Nerve-on-a-chip models utilizing clinically relevant toxicity metrics would be invaluable for preclinical drug development by enabling the selection of promising lead compounds with low likelihood of late-stage damage due to peripheral neurotoxicity. Furthermore, the high-content information provided by such models will be valuable for investigative toxicology by providing insight into possible mechanisms of toxicity, thus guiding reformulation. By demonstrating the feasibility of our model system, we expect to maintain our position as a commercial pioneer with a pre-market technology in predictive screening for peripheral neurotoxicity.

[0242] We have developed a simple yet unique method of digital projection lithography to rapidly micropattern one or more hydrogels directly onto traditional cell culture materials (Curley et al. 2011, Curley & Moore 2011). Our simple and rapid approach uses two gels, polyethylene glycol (PEG) as the restrictive mold, and cross-linked methacrylated heparin (Me-Hep (previously we used PuraMatrix)) as the permissive matrix. These dual gels effectively constrain neurite outgrowth from embryonic dorsal root ganglion (DRG) explants within a specific 3D geometry, resulting in axonal growth with high density and fasciculation. When cultured in myelin-inducing medium, we observed abundant myelin staining positive for myelin basic protein (MBP), with characteristic helical structures evident from TEM images, indicating compact myelin. We demonstrate the robustness of this culture model with dense, highly parallel myelinated 3D nerve fiber tracts extending from the ganglion. The structure of the nerve corresponds to peripheral nerve architecture and may be evaluated using neuromorphometry, allowing for clinically similar assessments unavailable to traditional cellular assays. The ability to record electrically evoked collective field potentials resulting from compound action potentials (cAPs) is most unique to our nerve-on-a-chip culture model. Traces show the characteristic uniform short latency collective response, which remains consistent even at high frequency (100 Hz) stimulation, show a measurable increase in latency associated with distal tract stimulation (Figures 21A and 21B), which can be reversibly abolished by tetrodotoxin (TTX), and the response is insensitive to neurotransmitter blockers, indicative of a cAP rather than a synaptic potential (Huval et al. 2014). 2015). Preliminary evidence indicates that high levels of glucose (60 mM) result in significantly reduced cAP amplitude with increased latency compared to moderate glucose levels (20 mM) (Figures 22A-22C). Preliminary evidence indicates that acute (48 h) administration of 0.1 μM paclitaxel (PTX) results in significantly reduced cAP amplitude with increased latency (Figures 23A-23C).This concentration has previously resulted in 50% cell death in conventional DRG cultures compared to significant measurable cAP changes in our model, suggesting a potentially informative metric of toxicity. Embryonic DRG cultures have been effectively used as models of peripheral neurobiology for many years (Melli & Hoke 2009). Although useful as a model system, conventional DRG cultures are known to be poor predictors of clinical toxicity when assessed with traditional cell death assays. Single cell recordings can be obtained from DRGs, but due to the lack of tissue architecture, we are not aware of any reports on recording cAP. What makes our model system innovative is the inherent ability to assess histomorphometry and collective electrophysiology, analogous to clinical histopathology and nerve conduction studies.

[0243] The goal of this project is to demonstrate that certain peripherally neurotoxic chemotherapeutic agents will induce toxicity in microengineered neural tissue that can be quantified using morphological and physiological measures similar to clinical metrics. We will approach this objective by first calibrating the model system to determine baseline variability and characterize structure-function relationships. We will then quantify changes induced by acute application of specific chemotherapeutic agents known to cause clinical neuropathy to demonstrate the technical merit of using compound action potential (cAP) waveforms as a preclinical assay of neurotoxicity.

[0244] Aim 1 Rationale and Justification: Traditional assays of neuronal viability have not proven useful as preclinical screens for neurotoxicity. This is not surprising, as it is well known that embryonic dorsal root ganglion (DRG) neurons are much more susceptible to apoptosis than mature neurons (Kole et al. 2013). Assays of DRG neurite outgrowth may be highly relevant as early high-throughput screens for toxicity (Melli & Hoke 2009). However, high-content assays useful for distinguishing potential neuropathic symptoms and informing lead compound selection remain elusive. Neurons cultured in 3D have been shown to exhibit more biomimetic, morphological, and electrophysiological behaviors compared to 2D cultures (Desai et al. 2006, Irons et al. 2008, Lai et al. 2012, Paivalainen et al. 2008). Thus, functional measurements in 3D cultures may be the most promising candidates for such high-content analysis, insofar as they correspond to clinically relevant organ physiology. Nerve conduction studies have been shown to predict the type and severity of clinical neuropathology even before symptoms are fully manifested (Velasco et al. 2014). We propose similar electrophysiological metrics in an in vitro setting, and to interpret results, it is first necessary to establish baseline measurements and determine structure-function correlations.

[0245] Aim 1 Study Design: Myelinated and non-myelinated neural tissue constructs will be fabricated with improvements on published studies (Curley & Moore 2011, Huval et al. 2015). Dual hydrogel constructs will be fabricated from PEG gel micromolds filled with Me-Hep gel supplemented with collagen and laminin. Neurite outgrowth constructs will be fabricated to approximately 400 μm in width and up to 5 mm in length. Dorsal root ganglia (DRG) will be harvested from the thoracic level of spinal cords dissected from embryonic day 15 (E15) rat embryos and incorporated within the medullary region of the dual hydrogel constructs. Myelinated tissue constructs are cultured in Eagle's basal medium containing ITS supplement and 0.2% BSA for 10 days to promote Schwann cell migration and neurite outgrowth, followed by up to an additional 4 weeks of culture in the same medium supplemented with additional 15% FBS and 50 μg / mL ascorbic acid to induce myelination (Eshed et al. 2005). Unmyelinated constructs are formed by culturing in the same medium regimen (induction of proliferation followed by myelin induction) but omitting ascorbic acid. At least 2 weeks of culture in myelin induction medium containing ascorbic acid is required for substantial formation of compact myelin. To assess tissue morphology at various maturation stages, approximately 12 each of myelinated and unmyelinated tissue constructs are fixed in 4% paraformaldehyde at 1, 2, 3, and 4 weeks (or a total of 17, 24, 31, and 38 days in vitro, DIV) in myelin-inducing medium and stained for nuclei (Hoechst), neurites (βIII-tubulin), Schwann cells (S-100), myelin basic protein (MBP), and apoptosis (annexin-V and TUNEL). Samples are imaged by confocal microscopy in segments within the DRG, proximal to the ganglion, near the midpoint of the fiber tract, and within the fiber tract distal to the ganglion, with the precise distance proportional to the average maximum neurite outgrowth in each group. After confocal imaging, samples are post-fixed in 2% osmium tetroxide, dehydrated, and embedded in epoxy resin.Approximately ten ultrathin sections are cut from each specimen at each defined region (ie, ganglion, proximal, midpoint, distal) and stained with lead citrate and uranyl acetate for TEM imaging.

[0246] Physiological analysis is performed as previously described (Huval et al. 2015). Both myelinated and unmyelinated constructs are removed from culture and placed on a field recording rig perfused with artificial cerebrospinal fluid (aCSF). As shown in Figure 24, field potential electrodes are placed within the neuronal cell body area of ​​the DRG explant, and bipolar stimulating electrodes are inserted into the channel to a depth of approximately 300 μm, at a distance proximal to the ganglion, near its midpoint, and distal to it, the distance being informed by morphometry. For each specimen at each stimulation location, stimulation intensity is increased until a characteristically fast (<5 ms), short latency, negative deflection potential is recorded. DRG spike recordings from each stimulation location are taken from approximately 5-10 specimens at 17, 24, 31, and 38 DIV. These same specimens are fixed immediately after electrophysiological recordings and processed for confocal and TEM analysis.

[0247] Morphological analysis is assessed as summarized in FIG. 24. The density and diameter distribution of cell bodies are measured within the ganglion. In the nerve fiber tracts, measurements include axon density and diameter distribution, % axons with myelin, and myelin sheath thickness distribution. This analysis provides important quantitative metrics of morphological variability and correlation with physiology. Physiological metrics are also summarized in FIG. 24. cAPs are recorded at three time points along the length of the tract, and measurements include the distribution of cAP amplitudes (and number of peaks), envelope (width), integral (area under the curve), and conduction velocity (from latency). Morphometric parameters of the recorded constructs are compared to a larger pool of morphometric data to ensure that they are within expected variability. Statistical cross-correlation is performed to determine which morphological measurements best correlate with physiological measures (Manoli et al. 2014). Additionally, these experiments provide measures of variability that will be used in statistical power analyses to determine appropriate sample sizes for Objective 2 and will be used to define exclusion criteria, e.g., samples with neurite outgrowth 2 standard deviations greater / less than the mean will be excluded.

[0248] Aim 1 Expected Results: We assume that the recorded cAP waveforms will reflect morphological observations. For example, our preliminary data suggests that after 2 weeks of culture, neurite outgrowth within the hydrogel channels was much more dense proximal to the ganglion than distally (Figures 21A and 21B). Thus, the recorded cAPs showed larger amplitudes and integrals when stimulated proximally and distally. The latency of the cAPs was expected to be longer when stimulated distally, reflecting conduction times. The calculated conduction velocity was approximately 0.5 m / s, which is slow as expected in constructs containing primarily small diameter unmyelinated axons.

[0249] In the proposed experiments, we expect to see cAP conduction velocity correlate with % myelination and / or axon diameter, while cAP amplitude should correlate with axon density at the stimulation location. We will also perform correlation analysis using morphological metrics, looking at the number of peaks, envelopes, and integrals.

[0250] Aim 1 Potential Issues and Alternative Strategies: Preliminary findings strongly demonstrate the technical feasibility of the study proposed for this objective. The most likely anticipated pitfall is that as more cultures are measured, one may find that morphological and / or physiological variability may be too high for many of the strong correlations to be identified. If this occurs, increase sample size as necessary and / or focus efforts on those metrics that represent the strongest correlations. One can also attempt to reduce variability by refining culture conditions, for example, by using defined media or using dissociated cells pooled from multiple animals.

[0251] Aim 2 Rationale and Justification: The most commonly administered chemotherapeutic drugs for which the most severe neurotoxicity has been documented are platinum derivatives; tubulin-binding compounds including vinca alkaloids, taxanes, and epothilones; the proteasome inhibitor bortezomib; and thalidomide (Argyriou et al. 2012, Cavaletti & Marmiroli 2010). All of these agents appear to be more toxic to sensory neurons than to motor or sympathetic neurons, but they each target different parts of the nerve and lead to a different set of clinically measurable histological and physiological changes, as summarized in Figure 25. A high-content functional assay for toxicity should be able to detect the range of in vivo effects associated with these compounds. To allow for a manageable range, we limit our experiments to oxaliplatin, vincristine, paclitaxel, and bortezomib. This list includes one compound from each family, except for the epothilones, which, like the taxanes except for thalidomide, bind to tubulin and therefore potentially involve interactions with other cell types and cytokines, ensuring an appropriately diverse range of responses (Argyriou et al. 2012). We further restrict our experiments to acute application of neurotoxic doses confirmed to be neurotoxic in vitro; chronic low-dose administration is reserved for further detailed studies.

[0252] We propose to demonstrate the feasibility of using cAP as a measure of toxicity by quantifying morphological and physiological responses to four chemotherapeutics. The proposed experiments are designed to establish a model with assessments that are directly analogous to nerve conduction studies as well as clinical histology. Molecular mechanistic studies are beyond the scope of this proposal, but the pseudo-3D nature of the micropatterned cultures is amenable to traditional cellular and molecular assays.

[0253] Aim 2 Study Design: A small pilot study will be performed first to ensure the use of an effective dose. Starting with a dose proven to induce statistically significant neuronal cell death in vitro after acute application (48 hours), and to demonstrate that morphological and physiological changes are measurable at these concentrations in the model. The overall experimental design is summarized in Figure 26. DRG explants (n=20) will be cultured in micropatterned gels (as described in Aim 1) following a myelination induction regimen. At the time points identified from Aim 1 to generate fully myelinated constructs, specimens will be checked for sufficient neurite outgrowth (Cell Tracker Green) and myelination (FluoroMyelin Red) and specimens without sufficient neurite outgrowth and / or myelination at this time will be removed from the experiment. Electrophysiological recordings will be taken of healthy tissue constructs and neurotoxic concentrations of the four drugs will be acutely applied the next day for 48 hours, as summarized in Table 4. Controls will receive vehicle without drugs. Electrophysiology was performed on half of the explants (n=10) at the end of the 48-hour treatment period and on the remaining half 7 days after the treatment period. All specimens were fixed, stained, and evaluated immediately after the final recordings as summarized in Figure 24. In addition, quantitative observations were made of neuronal cell body and axonal damage, including chromatin condensation, blebbing, and axonal disruption. [Table 1-2]

[0254] The results of this pilot study will be used to assess the adequacy of dose administration and to adjust doses as needed for the full study (below). The pilot study results will also be used to determine the most strongly correlated morphological and physiological measures and perform a statistical power analysis to estimate the sample size required to detect approximately 10% differences in those measures. In the larger scale study, we assume that the in vitro morphological and physiological changes following acute drug administration will closely parallel the in vivo neuropathy as reported in the literature. The goal of this experiment is to classify quantifiable neurotoxicity signatures for each of the drugs in the nerve-on-a-chip model. The full-scale experimental design will reflect the pilot study, as shown in Figure 26, but the sample sizes and doses for all four drugs (oxaliplatin, vincristine, paclitaxel, bortezomib) will reflect any changes determined from the pilot study.

[0255] Objective 2 Expected Results: We hypothesize that acute administration of each drug will induce toxicity that can be detected by measuring changes in cAP relative to baseline. We expect most of these changes to correlate with any morphological damage, as quantified by our morphometric analysis. For example, with reference to FIG. 25, with the tubulin-binding drugs vincristine and paclitaxel, we expect to see axonal atrophy, as measured by reduced axonal diameter and density, which we expect to be accompanied by a decrease in cAP amplitude. We may also see a decrease in myelin thickness and % of axons myelinated, which may be accompanied by a decrease in cAP conduction velocity. With oxaliplatin, we expect to see higher levels of apoptosis, but less axonal atrophy and myelin damage. Thus, although cAP amplitude may still be decreased due to the effect of oxaliplatin on Na+ channels, we do not expect to frequently see a decrease in conduction velocity without myelin toxicity. We further expect that the physiological and morphological changes will resemble the clinical pathology recorded, as measured by nerve conduction studies and histomorphometry.

[0256] Aim 2 Potential issues and alternative strategies: Although the neurotoxicity of the four compounds tested was already observed in vitro, the biological effects may be unpredictably affected by the 3D preparation. It is possible that the type of expected morphological and physiological pathology does not appear in the pilot study or that cell death otherwise overwhelms the functional measures. In that case, the dose can be increased / decreased and / or switched to a chronic application (7 days). Another plausible scenario is that neurological damage becomes evident, but the quantitative measures also vary, making a 10% detectable difference impractical. In that case, design a larger scale study to detect a 20%-30% detectable difference, as would be practical. References: Argyriou AA, Bruna J, Marmiroli P, Cavaletti G.2012. 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[0257] Example 4: Retinal explant model (non-predictive) Research and experimental data on dorsal root ganglion models are presented in this disclosure. For central nervous system models, the growth of retinal explants has also been explored. Figures 27A-27B show retinal (CNS) tissue cultures. Retinal explants from fetal rats were cultured within 3D micropatterned hydrogels in "neurobasal Sato" medium supplemented with either ciliary neurotrophic factor CNTF (Figure 23A) or brain-derived neurotrophic factor BDNF (Figure 23B). Observable retinal ganglion cell axonal outgrowth, stained with β-III tubulin, was visualized after one week of culture.

[0258] Example 5: Thalamocortical Model (Prophetic) One embodiment of the present invention quantifies postsynaptic potentials evoked within biomimetic engineered thalamocortical circuits. DLP lithography is used to cure 10% polyethylene glycol diacrylate (PEG) micromolds approximately 500 μm thick. These molds contain two reservoirs approximately 500 μm in diameter separated by a tract approximately 200 μm wide and 1 mm long. Thalamic and cortical neurons are isolated from E18 rat embryos, dissociated with trypsin / papain, and pelleted using a general procedure. A concentrated cell suspension (approximately 5E6 cells / mL) in PuraMatrix gel is formed by resuspending the pellet in a 10% sucrose solution and combining with equal volumes of 0.3% PuraMatrix and 10% sucrose. Respective thalamic and cortical cell suspensions are placed by micropipette into individual reservoirs within each mold, and cell-free PuraMatrix is ​​placed into the space between them. The micropatterned co-culture constructs are cultured for up to two weeks to allow circuits to form spontaneously. At approximately 3-day intervals, constructs are fixed and stained for cell nuclei (DAPI), neurites (β3-tubulin), dendrites (MAP2), and synapses (synapsin) to determine the time course required for circuit generation. Next, a paste of lipophilic tracing dyes Di-I and Di-O is placed on either end of the construct and fixed before synapse formation occurs to determine the spread and organization of neurite outgrowth from either cell population. These morphological parameters are quantified by confocal analysis and used to finalize the design of microengineered circuits. This procedure results in a high and reproducibly uniform density of thalamic axon synapsis onto a defined population of postsynaptic cortical neurons, while minimizing corticothalamic reinnervation (<10% of synapses).

[0259] We then determine the electrophysiological properties of the circuits. We activate both antidromically propagating action potentials (APs) and orthodromically evoked excitatory synaptic potentials (EPSPs) in these TC circuits using a single bipolar stimulating electrode. We measure responses by both field potential and whole-cell voltage clamp recordings. We record antidromic action potentials to confirm the induction and propagation of active currents in these axons. Consistent with the results from our DRG constructs, we expect to be able to record antidromic APs using field potential electrodes in the thalamic neuronal pool. This is seen as short, consistent latency, TTX-sensitive, negatively deflecting, short-lived field potentials. We use whole-cell voltage recordings to validate these antidromic APs based on their kinetics, direct generation from baseline, and insensitivity to hyperpolarization. We then confirm glutamatergic EPSPs and excitatory postsynaptic potentials (EPSCs) in the cortical neuronal ensemble following bipolar stimulation of thalamic axons. EPSCs are confirmed using 1) kinetic analysis of field potential responses recorded in cortical neuronal pools, 2) whole-cell current recordings using a voltage clamp strategy to isolate AMPAR-mediated (at hyperpolarized holding potentials) and AMPAR+NMDAR-mediated currents (at positive holding potentials), and 3) standard glutamatergic synaptic pharmacology, including DNQX (20 μM) to selectively block AMPAR-mediated currents and d-APV (50 μM) to antagonize NMDAR-mediated currents. AMPAR- and NMDAR-mediated postsynaptic currents then occur in response to thalamic axon stimulation. The relative ratio of AMPAR- to NMDAR-mediated currents is increased in vitro for 2 weeks, mimicking the in vivo situation.

[0260] In these embodiments, corticothalamic reinnervation is consistently greater than 10% or dendritic arbors connect between the two cell populations. If these scenarios are observed to an undesirable extent, stagger the timing of introduction of each cell type, allowing time for thalamic neurons to be introduced and generate axons to extend toward the cortical neuron reservoir before the addition of cortical target neurons. Alternatively, or in conjunction, use the micropatterning capabilities of hydrogels to introduce artificial trophic signaling during culture. We have shown that DRG neurites preferentially grow toward NGF, as opposed to BSA, and diffuse from the reservoir within the hydrogel construct, as shown in Figure 28. Potential chemoattractant molecules for TC axons include netrin-1 and neurotrophin 3. Similarly, we use semaphorin 3A, as it has been shown to polarize cortical neurons by attracting dendrites and repelling axons. If these approaches are not effective, use a photodegradable version of the PEG hydrogel, which we have been able to synthesize. This gel allows for the placement of a PEG barrier between the cortical and thalamic pools, which can be degraded with UV light to allow synapse formation when desired.

[0261] Example 6: Combination of a microphysiological culture system and non-invasive electrophysiological analysis (Prophetic) One embodiment of the present invention is to use a unique combination of microphysiological culture systems and non-invasive electrophysiological analysis. This has the potential to change the paradigm for performing population-level functional assays in vitro in a biomimetic configuration. We manually configured the DLP device on a fluorescence microscope recording rig and demonstrated selective illumination and simultaneous activation of individual cortical neurons and individual dendrites in cells expressing GFP and ChR2. We also developed custom software for flexible user control of illumination by allowing designation of areas of interest directly on the microscope camera live feed as viewed by the user. This powerful and versatile application of DLP microscopy and optogenetics for optical neural activation is combined with a new form of voltage-sensitive dye imaging, e.g., VF. This unique and timely combination of optogenetics and VF imaging with DLP microscopy represents a powerful, fully optical method for non-invasive stimulation of microengineered circuits in FIG. 29.

[0262] In one embodiment, DLP optical stimulation and recording protocols are performed in traditional flat dissociated cultures of thalamic and cortical neurons, respectively. Cortical and thalamic cultures are generated using the methods described above. ChR2 plasmid and lentivirus-based DNA constructs obtained from Optogenetics, Inc. are used, which contain red fluorescent protein (mCherry) as a transfected / infected reporter. Neurons are plated and infected with ChR2, followed by staining with VF dye (2 μM). Whole-cell patch recordings are then established on the transfected / infected cells, followed by DLP illumination at about 475 nm (blue-green). Step potentials and action potentials are recorded in current clamp mode, varying illumination intensity and magnification (4-40x). Alternatively, voltage is clamped to variable potentials, while VF fluorescence is monitored at about 535 nm (yellow-green, VF is relatively insensitive to excitation wavelength), again varying excitation intensity and magnification. These tests are repeated to determine the range and limits of illumination and voltage sensitivity. In addition, in this example we determine the timing requirements for simultaneous illumination (or near simultaneous) for optical stimulation and recording. We generate low density cortical cultures to evoke and record synaptic potentials. This manipulation (approximately 10-100k cells / mL) is necessary to maximize connectivity and couple neurons in individual fields of view in these cortical cultures. After establishing whole cell patches, transfected / infected neighboring cells are then illuminated with DLP and postsynaptic potentials are recorded in current clamp mode. We use these experiments to determine the precise optical settings, illumination, and timing of optical sampling required to detect ChR2 / light-evoked postsynaptic potentials.

[0263] Optical stimulation and recording protocols are then performed within the 3D cell assembly. Stimulation and recording are performed at relatively low magnification (10x) so that the thalamic and cortical pools are visible in the field of view at once. The TC circuit is micromanipulated according to the method described above. However, thalamic cells are infected with the ChR2 virus by adding particles to the cell suspension in a purified matrix solution prior to injection into the PEG micromold, and the gel is then washed several times to remove particles before addition of the cortical neurons. The stimulating field electrode is placed within the thalamic neuron pool and the recording electrode within the cortical neuron pool to confirm the ability to evoke EPSPs. Shortly after, DLP illumination of ChR2 is used to stimulate the thalamic neurons while recording the response in the cortical pool. EPSP responses to various different presynaptic ChR2 illumination intensities at different magnifications (4x-40x) are investigated. In some embodiments, EPSPs are confirmed by field recording in the TC circuit with VF stained cortical neuro...

Claims

1. A method for generating a three-dimensional culture of a plurality of neural cells in a culture vessel comprising a solid substrate, the method comprising: (a) contacting one or more isolated Schwann cells and / or oligodendrocytes with the solid substrate, the substrate comprising at least one exterior surface, at least one interior surface, and at least one internal chamber defined by the at least one interior surface and accessible from a point outside the solid substrate through at least one opening, at least a portion of the interior surface comprising a cell-impermeable polymer having one or more voids in the cell-impermeable polymer; (b) seeding one or more isolated neural cells or tissue explants containing neural cells into the at least one internal chamber and allowing the isolated neural cells or tissue explants to adhere to one or more portions of the internal surface that are voids in the cell-impermeable polymer; (c) applying cell culture medium containing a cell-permeable polymer to the culture vessel in a volume of cell culture medium sufficient to fill the voids and provide a channel in the cell-impermeable polymer, whereby the isolated neural cells or tissue explants can project bundled axons in a parallel orientation into the cell-permeable polymer channel and are capable of conducting action potentials; A method comprising:

2. The method of claim 1, wherein step (b) comprises seeding a tissue explant selected from one or a combination of isolated dorsal root ganglia, spinal cord explants, retinal explants, and cortical explants.

3. The method of claim 1 or 2, wherein step (b) comprises seeding a suspension of spinal cord cells selected from one or a combination of motor neurons, cortical neurons, spinal neurons, and peripheral neurons.

4. A method according to any one of claims 1 to 3, further comprising (d) after step (c), allowing the neural cells to grow into neurites and / or axons for a period of from one day to one year. (i) prior to step (a), isolating one or more Schwann cells and / or one or more oligodendrocytes from the sample; and / or (ii) prior to step (b), isolating dorsal root ganglia (DRGs) from one or more mammals; The method according to any one of claims 1 to 4, further comprising the steps of:

6. The method further includes the steps of positioning at least one stimulating electrode at or adjacent to a neuronal cell body of one or more neuronal cells or tissue explants, and positioning at least one recording electrode at or adjacent to an axon at a point most distal from the neuronal cell body, such that upon introduction of current to the stimulating electrode, the recording electrode is capable of receiving a signal corresponding to one or more electrophysiological metrics measurable at the recording electrode; 6. The method of any of claims 1 to 5, wherein the one or more electrophysiological metrics are one or a combination of electrical conduction velocity, action potential, wave amplitude associated with the passage of an electrical impulse along the membrane of one or more neuronal cells, width of the electrical impulse along the membrane of one or more neuronal cells, latency of the electrical impulse along the membrane of one or more neuronal cells, and envelope of the electrical impulse along the membrane of one or more neuronal cells.

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