Cell systems using spheroids, and methods for producing and using the same

Abstract

The present disclosure relates generally to cell culture systems, and more particularly to a three-dimensional neuronal cell culture system that promotes both structural and functional properties that mimic those of in vivo peripheral fibers, including cell myelination. The present disclosure provides methods, devices, and systems for spatially controlled in vitro three-dimensional models capable of intracellular and extracellular electrophysiological measurements and recordings using dual hydrogel constructs and neuronal spheroids. The three-dimensional hydrogel constructs allow for cell type incorporation, geometric fabrication, and electrical manipulation, providing a system capable of culturing, perturbing, and testing biomimetic neuronal outgrowth with physiologically relevant results. [Selection diagram] Figure 12

Description

Technical Field

[0001] Indication of Research Supported by the Federal Government This invention was made with government support under grant number R42-TR001270 from the NIH STTR. The United States government has certain rights in this invention.

[0002] Cross - Reference to Related Applications This application is an international application designating the United States, filed under 35 U.S.C. § 120, claiming priority to U.S. Provisional Application No. 62 / 594,525, filed on December 4, 2017, which is hereby incorporated by reference in its entirety.

[0003] This disclosure generally relates to cell culture systems, and more particularly to a three - dimensional cell culture system of spheroids that promotes both structural and functional properties that mimic the structural and functional properties of in - vivo nerve fibers, including cell myelination and propagation of compound action potentials.

Background Art

[0004] Regarding peripheral nerve tissue, which is one of the most relevant physiological results for long - distance bioelectrical conduction, it is particularly difficult to reproduce the functional aspects of physiology as an evaluation using a desktop device. For this reason, three - dimensional tissue models of peripheral nerves lag behind models of epithelial tissue, metabolic tissue, and tumor tissue where soluble analytes function as appropriate evaluation metrics (metrics). Recently, multi - electrode array technology has enabled the use of electrophysiological techniques for screening environmental toxins and for testing in disease modeling and treatment. The use of this technique is revolutionary for research in peripheral nervous system (PNS) applications and central nervous system (CNS) applications, but since the above - mentioned cultures have a dissociative nature, they cannot reproduce the population - level environment and important metrics as peripheral tissue. Instead, clinical methods for examining peripheral neuropathy and neuroprotection include nerve conduction examination by measuring compound action potential (CAP) and nerve fiber density (NFD) using morphometric analysis of skin biopsies.

Summary of the Invention

Means for Solving the Problems

[0005] The present disclosure relates to a microphysiological model of the nervous system that provides not only specific tissues but also three-dimensional constructs. Other model systems tend to be able to provide only one or the other. Organotypic tissue pieces can provide not only natural tissues but also three-dimensional constructs, but these models are not suitable for very high-throughput analysis.

[0006] The present disclosure relates to a composition comprising a spheroid of cells comprising one or a combination of cells and / or tissues selected from neurons, ganglia, stem cells, and immune cells. In some embodiments, the spheroid comprises a tissue selected from dorsal root ganglia and trigeminal ganglia. In some embodiments, the spheroid comprises one or more cells selected from glial cells, embryonic cells, mesenchymal stem cells, cells derived from induced pluripotent stem cells, sympathetic neurons, parasympathetic neurons, spinal motor neurons, central nervous system neurons, peripheral nervous system neurons, enteric nervous system neurons, motor neurons, sensory neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, serotonergic neurons, interneurons, adrenergic neurons, trigeminal ganglia, astrocytes, oligodendrocytes, Schwann cells, microglial cells, ependymal cells, radial glial cells, satellite cells, enteric glial cells, and pituitary cells. In some embodiments, the spheroid comprises one or more glial cells. In some embodiments, the spheroid comprises one or more embryonic cells. In some embodiments, the spheroid comprises one or more mesenchymal stem cells. In some embodiments, the spheroid comprises one or more cells derived from induced pluripotent stem cells. In some embodiments, the spheroid comprises one or more parasympathetic neurons. In some embodiments, the spheroid comprises one or more spinal motor neurons. In some embodiments, the spheroid comprises one or more central nervous system neurons. In some embodiments, the spheroid comprises one or more peripheral nervous system neurons. In some embodiments, the spheroid comprises one or more enteric nervous system neurons. In some embodiments, the spheroid comprises one or more motor neurons. In some embodiments, the spheroid comprises one or more sensory neurons. In some embodiments, the spheroid comprises one or more interneurons. In some embodiments, the spheroid comprises one or more cholinergic neurons. The spheroid comprises one or more GABAergic neurons.In some embodiments, the spheroid comprises one or more glutamatergic neurons. In some embodiments, the spheroid comprises one or more dopaminergic neurons. In some embodiments, the spheroid comprises one or more serotonergic neurons. In some embodiments, the spheroid comprises one or more trigeminal ganglion cells. In some embodiments, the spheroid comprises one or more astrocytes. In some embodiments, the spheroid comprises one or more oligodendrocytes. In some embodiments, the spheroid comprises one or more Schwann cells. In some embodiments, the spheroid comprises one or more microglial cells. In some embodiments, the spheroid comprises one or more ependymal cells. In some embodiments, the spheroid comprises one or more radial glial cells. In some embodiments, the spheroid comprises one or more satellite cells. In some embodiments, the spheroid comprises one or more enteric glial cells. In some embodiments, the spheroid comprises one or more pituitary cells.

[0007] In some embodiments, the spheroid comprises one or more of one or a combination of immune cells selected from T cells, B cells, macrophages, and astrocytes. In some embodiments, the spheroid comprises one or more of one or a combination of stem cells selected from embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells. In some embodiments, the nerve cells are derived from stem cells selected from embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells. Embodiments include each of the cell types described above, individually or in combination with each other.

[0008] In some embodiments, the diameter of the spheroid is from about 200 microns to about 700 microns. In some embodiments, the diameter of the spheroid is from about 150 microns to about 800 microns. In some embodiments, the diameter of the spheroid is about 200 microns. In some embodiments, the diameter of the spheroid is about 300 microns. In some embodiments, the diameter of the spheroid is about 400 microns. In some embodiments, the diameter of the spheroid is about 500 microns. In some embodiments, the diameter of the spheroid is about 600 microns. In some embodiments, the diameter of the spheroid is about 700 microns. In some embodiments, the diameter of the spheroid is about 800 microns. In some embodiments, the diameter of the spheroid is about 900 microns. In some embodiments, the diameter of the spheroid is about 350 microns. In some embodiments, the diameter of the spheroid is about 450 microns. In some embodiments, the diameter of the spheroid is about 550 microns. In some embodiments, the diameter of the spheroid is about 650 microns.

[0009] In some embodiments, the spheroid comprises one or more types of nerve cells and one or more types of Schwann cells in a cell type ratio equal to about 4 nerve cells per 1 Schwann cell. In some embodiments, the spheroid comprises one or more types of nerve cells and one or more types of astrocytes in a ratio of about 4 nerve cells per 1 astrocyte. In some embodiments, the spheroid comprises one or more types of nerve cells and one or more types of astrocytes in a ratio of about 1 nerve cell per 1 astrocyte. In some embodiments, the spheroid comprises one or more types of nerve cells and one or more types of Schwann cells in a ratio of about 10 nerve cells per 1 Schwann cell. In some embodiments, the spheroid comprises one or more types of nerve cells and one or more types of glial cells in a ratio equal to about 4 nerve cells per 1 glial cell.

[0010] In some embodiments, any one or more of the cells described herein are differentiated from induced pluripotent stem cells. In some embodiments, the spheroid does not contain induced pluripotent stem cells and / or immune cells. In some embodiments, the spheroid does not contain undifferentiated stem cells.

[0011] In some embodiments, the spheroid contains about 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, or 75,000 or more cells. In some embodiments, the spheroid contains about 75,000 or more cells. In some embodiments, the spheroid contains about 65,000 or more cells. In some embodiments, the spheroid contains about 60,000 or more cells. In some embodiments, the spheroid contains about 100,000 or more cells. In some embodiments, the spheroid contains about 125,000 or more cells. In some embodiments, the spheroid contains about 150,000 or more cells. In some embodiments, the spheroid contains about 175,000 or more cells. In some embodiments, the spheroid contains about 200,000 or more cells. In some embodiments, the spheroid contains about 225,000 or more cells. In some embodiments, the spheroid contains about 250,000 or more cells. In some embodiments, the spheroid contains about 12,500 or more cells. In some embodiments, the spheroid contains from about 12,500 cells to about 250,000 cells. In some embodiments, the spheroid contains from about 12,500 cells to about 100,000 cells. In some embodiments, the spheroid contains from about 12,500 cells to about 75,000 cells.

[0012] In some embodiments, the spheroid further comprises one or more magnetic particles. In some embodiments, the magnetic particles comprise one or more hollow interiors. In some embodiments, the magnetic particles comprise a layer of one or more polymers on which the cells form spheroids.

[0013] The present disclosure also provides (i) a cell culture vessel comprising a hydrogel; and (ii) one or more spheroids comprising one or more neural cells and / or isolated tissue explants; and (iii) an amplifier comprising a current generator; and (iv) a voltmeter and / or an ammeter; and (v) at least a first stimulating electrode and at least a first recording electrode A system comprising: The amplifier, the voltmeter and / or ammeter, and the electrodes are electrically connected to each other via a circuit through which current is supplied from the amplifier to the at least one stimulating electrode, received at the recording electrode, and supplied to the voltmeter and / or ammeter. The stimulating electrode is disposed at or near one or more cell bodies of the neural cells and / or isolated tissue explants, and the recording electrode is disposed at a predetermined distance distal to the cell bodies, such that an electric field is established across the cell culture vessel. In some embodiments, the spheroid is any of the spheroids described herein.

[0014] In some embodiments, the culture vessel comprises 96, 192, 384, or more internal chambers. In some embodiments, the 96, 192, 384, or more internal chambers comprise Schwann cells and / or oligodendrocytes that are sufficiently close to the tissue explants and / or neurons such that one or more isolated Schwann cells or one or more oligodendrocytes accumulate myelin on axonal outgrowths from the one or more isolated tissue explants and / or the one or more neurons.

[0015] In some embodiments, the system further comprises a solid substrate on which the hydrogel matrix is crosslinked, the solid substrate comprising at least one plastic surface having pores with a diameter of from about 1 micron to about 5 microns. In some embodiments, the solid substrate comprises a continuous outer surface and an inner surface, the solid substrate comprising at least one cylindrical or substantially cylindrical portion and at least one hollow interior, at the end of the hollow interior, the hollow interior defined by at least one portion of the inner surface, the inner surface comprising one or more pores having a diameter of from about 0.1 micron to about 1.0 micron. The hollow interior of the solid substrate is accessible through at least one opening from a point external to the solid substrate. The hollow interior portion comprises a first portion proximate to the opening and at least a second portion distal to the opening. The one or more neurons and / or the one or more tissue explants are disposed in or proximate to the first portion of the hollow interior and are in physical contact with the hydrogel matrix. 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 neurons and / or the one or more tissue explants into the second interior portion of the hollow interior.

[0016] In some embodiments, the above system or composition does not include or does not contain a sponge respectively. In some embodiments, the above hydrogel includes at least a polymer that is impenetrable to the first cells and a polymer that is penetrable to the first cells. In some embodiments, the PEG contained in the at least one polymer that is impenetrable to cells is about 15% or less, and the at least one polymer that is penetrable to cells includes one or a combination of self-assembling peptides selected from RAD 16-I, RAD 16-II, EAK 16-I, EAK 16-II, and dEAK 16 in an amount of about 0.05% to about 1.00%. In some embodiments, the above composition does not contain polyethylene glycol (PEG). In some embodiments, the above hydrogel includes a first region and a second region. The first region is formed in the shape of a cylinder or a rectangular parallelepiped whose longitudinal axis is oriented in a direction penetrating the upper and lower surfaces of the cell culture vessel. Each of the cylinder or the rectangular parallelepiped has a space defined by the inner surface of the cylinder or the rectangular parallelepiped, and is accessible through the space and one or more openings penetrating the upper surface of the cell culture vessel; The second region is adjacent to the first region and has a space formed in the shape of the inner wall of the second region with an opening on its side, which is in fluid communication with the first region. In some embodiments, the above composition contains at least 1% polyethylene glycol (PEG).

[0017] In some embodiments, the above system further includes a cell culture medium containing nerve growth factor (NGF) at a concentration of about 5 to about 20 picograms per milliliter and / or ascorbic acid at a concentration in the range of about 0.001 wt / vol% to about 0.01 wt / vol%.

[0018] In some embodiments, the system comprises one or more spheroids comprising at least one or a combination of cells selected from glial cells, embryonic cells, mesenchymal stem cells, cells derived from induced pluripotent stem cells, sympathetic neurons, parasympathetic neurons, spinal motor neurons, central nervous system neurons, peripheral nervous system neurons, enteric nervous system neurons, motor neurons, sensory neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, serotonergic neurons, interneurons, adrenergic neurons, trigeminal ganglion neurons, astrocytes, oligodendrocytes, Schwann cells, microglial cells, ependymal cells, radial glial cells, satellite cells, enteric glial cells, and pituitary cells. In some embodiments, the system further comprises one or more of stem cells, pluripotent cells, myoblasts, and osteoblasts. In some embodiments, the one or more nerve cells include primary mammalian cells derived from the peripheral nervous system of a mammal.

[0019] In some embodiments, the spheroid has been cultured for about 3, 30, 90, or more than 365 days.

[0020] In some embodiments, at least a portion of the solid substrate is cylindrical or substantially cylindrical such that at least a portion of the inner surface of the solid substrate defines a cylindrical or substantially cylindrical hollow inner chamber in which the spheroid is disposed. In some embodiments, the hydrogel comprises a series of two or more cavities in fluid communication with each other by a series of channels, at least one cavity comprising a spheroid, at least a second cavity comprising a second spheroid, a cell suspension, or a DRG, and the spheroid and the second spheroid, cell suspension, or DRG are connected by three-dimensional axons. In some embodiments, the cavity is a well having a U-shaped or circular well disposed on a horizontal or substantially horizontal surface of the solid substrate, and each channel comprises one or more axons connected to one or more spheroids.

[0021] In some embodiments, the one or more spheroids comprise one or more types of nerve cells having axonal growth with a width of about 100 microns to about 500 microns and a length of about 0.11 to about 10,000 microns. In some embodiments, the height of the three-dimensional axon is at least about 10 microns at its lowest point or at least three cell monolayers.

[0022] In some embodiments, the system (i) one or more types of nerve cells, and / or (ii) one or more types of Schwann cells or oligodendrocytes comprising a first spheroid, (i) one or more types of peripheral neurons comprising a second spheroid and each spheroid is disposed within the cavity. In some embodiments, the system comprises first, second, and third cavities, each configured to hold a spheroid and at least 50 microliters of cell culture medium, and the cavities are aligned such that the first cavity is proximal to the second cavity and distal to the third cavity. In some embodiments, the system comprises at least a fourth cavity, and the cavities are arranged in a pattern such that each cavity defines a corner of a square. In some embodiments, the cavities are aligned in a row such that an axon originating from the first spheroid in the first cavity extends to the second cavity, and an axon from the spheroid in the second cavity extends to an axon in the third cavity.

[0023] The present disclosure also relates to a method for manufacturing a three-dimensional culture of one or more spheroids in a culture vessel. In some embodiments, the method (a) contacting one or more nerve cells with the solid substrate, the solid substrate comprising at least one outer surface, at least one inner surface, and at least one internal chamber defined by the at least one inner surface and accessible from a point external to the solid substrate through at least one opening; (b) disposing one or more spheroids comprising nerve cells in the at least one internal chamber; (c) applying a cell culture medium into the culture vessel with an amount of the cell culture medium sufficient to cover the at least one spheroid comprising, at least a portion of the inner surface comprises a polymer impenetrable by a first cell and a polymer penetrable by the first cell. In some embodiments, step (b) comprises disposing a spheroid comprising a tissue explant selected from one or a combination of isolated dorsal root ganglia, spinal cord explants, retinal explants, and cortical explants.

[0024] In some embodiments, the spheroid is formed as a suspension of nerve cells selected from one or a combination of motor neurons, sensory neurons, sympathetic neurons, parasympathetic neurons, cortical neurons, spinal cord neurons, and peripheral neurons, optionally derived from stem cells. In some embodiments, the spheroid is formed from a suspension of nerve cells selected from one or a combination of motor neurons, sensory neurons, sympathetic neurons, parasympathetic neurons, cortical neurons, spinal cord neurons, and peripheral neurons, optionally derived from stem cells. In some embodiments, the spheroid further comprises isolated Schwann cells and / or oligodendrocytes.

[0025] In some embodiments, the method further comprises (d) growing neurites and / or axons on the spheroid for about 12 hours to about 1 year after step (c). In some embodiments, the method comprises isolating one or more nerve cells from a sample before step (a), and / or, if the one or more spheroids comprise dorsal root ganglia (DRG), isolating DRG from one or more mammals before step (b), and / or, if the one or more spheroids comprise Schwann cells or oligodendrocytes, further comprising isolating one or more Schwann cells and / or one or more oligodendrocytes.

[0026] In some embodiments, the method is such that when a current is introduced into the stimulating electrode, the recording electrode can receive a signal corresponding to one or more electrophysiological metrics measurable at the recording electrode. placing at least one stimulating electrode in or proximate to the cell bodies of the one or more nerve cells or tissue explants; placing at least one recording electrode in or proximate to an axon at the point most distal from the cell bodies; and further comprising wherein the one or more electrophysiological metrics are one or a combination of conduction velocity, action potential, amplitude of the wave associated with the passage of an electrical impulse along the membrane of one or more nerve cells, width of the electrical impulse along the membrane of one or more nerve cells, latency of the electrical impulse along the membrane of one or more nerve cells, and envelope of the electrical impulse along the membrane of one or more nerve cells.

[0027] The present disclosure also provides (a) culturing one or more spheroids in any of the compositions disclosed herein; (b) exposing the one or more spheroids to at least one agent; (c) measuring and / or observing one or more morphometric changes and / or one or more electrophysiological metrics of the one or more spheroids; (d) Correlate one or more morphological changes and / or one or more electrophysiological metrics of the above-mentioned one or more spheroids with the toxicity of the above-mentioned drug. As a result, if the above-mentioned morphological changes and / or electrophysiological metrics indicate a decrease in cell viability, the above-mentioned drug is characterized as toxic. If the above-mentioned morphological changes and / or electrophysiological metrics indicate that the cell viability does not change or increases, the above-mentioned drug is characterized as non-toxic and / or having a neuroprotective effect. Also relates to a method for evaluating the toxicity and / or neuroprotective effect of a drug, including the above.

[0028] The present disclosure also provides a method for determining myelination or demyelination of one or more axons of one or more spheroids, comprising: (a) Culturing one or more spheroids in any of the compositions disclosed herein in the presence or absence of a drug for a time and under conditions sufficient to grow at least one axon; (b) Detecting the amount of myelination in one or more axons derived from the above-mentioned one or more spheroids. Including Detecting is optional, (i) Measuring and / or observing one or more morphological changes and / or one or more electrophysiological metrics of the above-mentioned one or more spheroids in the presence or absence of a drug; (ii) Correlating one or more morphological changes and / or one or more electrophysiological metrics of the above-mentioned one or more spheroids in the presence or absence of a drug with the quantitative or qualitative changes in myelination of the above-mentioned spheroids. Also relates to the above method including the above.

[0029] The present disclosure also provides (a) Quantifying the number or density of one or more spheroids and / or axons grown from spheroids; (b) culturing one or more spheroids in any of the compositions disclosed herein; (c) after culturing the spheroid(s) for a period sufficient to grow the one or more axons or cells in the spheroid(s), calculating the number of cells within the spheroid(s) and / or the number or density of axons grown from the spheroid(s) in the composition; relates to a method for detecting and / or quantifying nerve cell growth and / or axonal degeneration. In some embodiments, step (b) is optional and includes contacting the one or more spheroids with one or more agents. In some embodiments, step (c) is optional and includes, after culturing the one or more spheroids, detecting recordings inside and / or outside of such one or more spheroids and correlating the recordings with measurements of similar recordings corresponding to a known or control number of cells. In some embodiments, step (c) is optional and (i) a further step of measuring intracellular and / or extracellular recordings and / or morphometric changes before and after the step of contacting the one or more spheroids with one or more agents; (ii) correlating the difference in the recordings and / or morphometric changes after contacting the one or more spheroids with the one or more agents with the recordings and / or morphometric changes before contacting the one or more spheroids with the one or more agents with the changes in the number of cells and / or the number or density of axons; including.

[0030] The present disclosure also relates to a method for measuring or quantifying the neuromodulatory effect of an agent, comprising: (a) culturing one or more spheroids in any of the compositions disclosed herein in the presence and absence of the agent; (b) applying a potential across the one or more spheroids in the presence and absence of the agent; (c) Measuring one or more electrophysiological metrics from the one or more spheroids in the presence and absence of the agent; (d) Correlating the difference in the one or more electrophysiological metrics by the one or more spheroids with the neuromodulatory effect of the agent, such that a change in the electrophysiological metric in the presence of the agent as 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 as compared to the electrophysiological metric measured in the absence of the agent indicates that the agent does not confer a neuromodulatory effect; also relates to the method comprising the above.

[0031] The present disclosure also relates to a method for measuring or quantifying the neuromodulatory effect of an agent, comprising: (a) Culturing one or more spheroids in any of the compositions disclosed herein in the presence and absence of the agent; (b) Measuring and / or observing one or more morphological changes of the one or more spheroids in the presence and absence of the agent; (c) Correlating the one or more morphological changes with the neuromodulatory effect of the agent, such that a change in the Form metric in the presence of the agent as compared to the Form metric measured and / or observed in the absence of the agent indicates a neuromodulatory effect, and no change in the Form metric in the presence of the agent as compared to the Form metric measured and / or observed in the absence of the agent indicates that the agent does not confer a neuromodulatory effect; also relates to the method comprising the above. In certain embodiments, for example, the following items are provided. (Item 1) A composition comprising a spheroid of cells comprising one or a combination of cells selected from neurons, ganglia, stem cells, and immune cells and / or tissues. (Item 2) The composition according to item 1, wherein the spheroid comprises a tissue selected from dorsal root ganglia and trigeminal ganglia. (Item 3) The composition according to item 1 or 2, wherein the spheroid comprises one or more cells selected from glial cells, embryonic cells, mesenchymal stem cells, cells derived from induced pluripotent stem cells, sympathetic neurons, parasympathetic neurons, spinal motor neurons, central nervous system neurons, peripheral nervous system neurons, enteric nervous system neurons, motor neurons, sensory neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, serotonergic neurons, interneurons, adrenergic neurons, trigeminal ganglia, astrocytes, oligodendrocytes, Schwann cells, microglial cells, ependymal cells, radial glial cells, satellite cells, enteric glial cells, pituitary cells, and combinations thereof. (Item 4) The composition according to any one of items 1 to 3, wherein the spheroid comprises one or more immune cells selected from T cells, B cells, macrophages, astrocytes, and combinations thereof. (Item 5) The composition according to any one of items 1 to 4, wherein the spheroid comprises one or more stem cells selected from embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, and combinations thereof. (Item 6) The composition according to any one of items 1 to 5, wherein the neuron is derived from a stem cell selected from embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells. (Item 7) The composition according to any one of items 1 to 6, wherein the diameter of the spheroid is about 200 microns to about 700 microns. (Item 8) The composition according to any one of items 1 to 7, wherein the spheroid comprises one or more neurons and one or more Schwann cells in a cell type ratio equal to about 4 neurons per one Schwann cell. (Item 9) The composition according to any one of items 1 to 8, wherein the spheroid comprises one or more neurons and one or more astrocytes in a ratio of about 4 neurons per one astrocyte. (Item 10) The composition according to any one of Items 1 to 9, wherein the spheroid contains one or more types of nerve cells and one or more types of astrocytes at a ratio of about one nerve cell per one astrocyte. (Item 11) The composition according to any one of Items 1 to 10, wherein the spheroid contains one or more types of nerve cells and one or more types of Schwann cells at a ratio of about ten nerve cells per one Schwann cell. (Item 12) The composition according to any one of Items 1 to 11, wherein the spheroid contains one or more types of nerve cells and one or more types of glial cells at a ratio equal to about four nerve cells per one glial cell. (Item 13) The composition according to any one of Items 1 to 12, wherein one or more of the cells are differentiated from induced pluripotent stem cells. (Item 14) The composition according to any one of Items 1 to 13, wherein the spheroid does not contain induced pluripotent stem cells and / or immune cells. (Item 15) The composition according to any one of Items 1 to 14, wherein the spheroid does not contain undifferentiated stem cells. (Item 16) The composition according to any one of Items 1 to 15, wherein the spheroid contains about 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 90,000, 100,000, 150,000, 200,000, 225,000, or 250,000 or more cells. (Item 17) The composition according to any one of Items 1 to 16, wherein the spheroid contains 75,000 or more cells. (Item 18) The composition according to any one of Items 1 to 17, wherein the spheroid further contains one or more types of magnetic particles. (Item 19) (i) A cell culture container comprising a hydrogel, (ii) One or more spheroids containing one or more types of nerve cells and / or isolated tissue explants, (iii) An amplifier equipped with a current generator, (iv) A voltmeter and / or an ammeter, (v) At least a first stimulating electrode and at least a first recording electrode A system comprising: The amplifier, the voltmeter and / or the ammeter, and the electrodes are electrically connected to each other through a circuit in which current is supplied from the amplifier to the at least one stimulating electrode, received at the recording electrode, and supplied to the voltmeter and / or the ammeter. The stimulating electrode is disposed at or in proximity to one or more cell bodies of the nerve cells and / or isolated tissue explants such that an electric field is established across the entire cell culture vessel, and the recording electrode is disposed at a predetermined distance distal to the cell body. The system. (Item 20) The system according to item 19, wherein the spheroid is the spheroid according to any one of items 1 to 18. (Item 21) The system according to item 19 or 20, wherein the culture vessel comprises 96, 192, 384 or more internal chambers. (Item 22) The system according to item 21, wherein the 96, 192, 384 or more internal chambers comprise Schwann cells of one or more types or oligodendrocytes of one or more types that accumulate myelin with respect to axonal outgrowth from the one or more isolated tissue explants and / or the one or more nerve cells, and the Schwann cells and / or the oligodendrocytes are in sufficient proximity to the tissue explants and / or nerve cells. (Item 23) The system according to any one of items 19 to 22, further comprising a solid substrate on which the hydrogel matrix is crosslinked, and the solid substrate comprises at least one plastic surface having pores with a diameter of about 1 micron to about 5 microns. (Item 24) The solid substrate comprises a continuous outer surface and an inner surface, and such solid substrate comprises at least one cylindrical or substantially cylindrical portion and at least one hollow interior, and at the end of the hollow interior, the hollow interior defined by at least one portion of the inner surface, and the inner surface comprises one or more pores having a diameter of about 0.1 micron to about 1.0 micron. The hollow interior of the solid substrate is accessible through at least one opening from a point outside the solid substrate. The interior hollow portion comprises a first portion proximate to the opening and at least a second portion distal to the opening. The one or more nerve cells and / or the one or more tissue explants are disposed in or in proximity to the first portion of the hollow interior and are in physical contact with the hydrogel matrix. 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 nerve cells and / or the one or more tissue explants into the second interior portion of the hollow interior. The system according to item 23. (Item 25) The system according to any one of items 19 to 24, wherein the composition does not contain a sponge. (Item 26) The system according to any one of items 19 to 25, wherein the hydrogel comprises at least a polymer impenetrable to a first cell and a polymer penetrable to the first cell. (Item 27) The system according to item 26, wherein the at least one polymer impenetrable to cells contains about 15% or less of PEG, and the at least one polymer penetrable to cells contains one or a combination of self-assembling peptides selected from RAD 16-I, RAD 16-II, EAK 16-I, EAK 16-II, and dEAK 16 in an amount of about 0.05% to about 1.00%. (Item 28) The system according to any one of items 19 to 24, wherein the composition does not contain polyethylene glycol (PEG). (Item 29) The hydrogel comprises a first region and a second region, the first region being in the shape of a cylinder or a rectangular parallelepiped, the longitudinal axis of which is oriented in a direction penetrating the upper and lower surfaces of the cell culture vessel, each of the cylinder or the rectangular parallelepiped having a space defined by the inner surface thereof, the space being accessible through one or more openings penetrating the upper surface of the cell culture vessel; The second region is adjacent to the first region and in fluid communication with the first region, and has a space formed in the shape of the inner wall of the second region having an opening in its side portion. The system according to any one of items 19 to 28. (Item 30) The system according to any one of items 19 to 29, further comprising a cell culture medium containing nerve growth factor (NGF) at a concentration of about 5 to about 20 picograms per milliliter and / or ascorbic acid at a concentration in the range of about 0.001 wt / vol% to about 0.01 wt / vol%. (Item 31) The system according to any one of items 19 to 30, wherein the one or more spheroids include at least one or a combination of cells selected from glial cells, embryonic cells, mesenchymal stem cells, cells derived from induced pluripotent stem cells, sympathetic neurons, parasympathetic neurons, spinal motor neurons, central nervous system neurons, peripheral nervous system neurons, enteric nervous system neurons, motor neurons, sensory neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, serotonergic neurons, interneurons, adrenergic neurons, trigeminal ganglion neurons, astrocytes, oligodendrocytes, Schwann cells, microglial cells, ependymal cells, radial glial cells, satellite cells, enteric glial cells, and pituitary cells. (Item 32) The system according to any one of items 19 to 31, further comprising one or more of stem cells, pluripotent cells, myoblasts, and osteoblasts. (Item 33) The system according to any one of items 19 to 32, wherein the one or more nerve cells include primary mammalian cells derived from the peripheral nervous system of a mammal. (Item 34) The system according to any one of items 19 to 33, wherein the hydrogel contains at least 1% polyethylene glycol (PEG). (Item 35) The system according to any one of items 19 to 34, wherein the spheroid has been cultured for about 3, 30, 90, or 365 days or more. (Item 36) The system according to any one of items 19 to 35, wherein at least a part of the solid substrate is cylindrical or substantially cylindrical so as to define a cylindrical or substantially cylindrical hollow internal chamber in which the spheroid is disposed, and at least a part of the inner surface of the solid substrate is cylindrical or substantially cylindrical. (Item 37) The system according to any one of items 19 to 36, wherein the one or more spheroids include one or more nerve cells having axonal growth with a width of about 100 microns to about 500 microns and a length of about 0.11 to about 10,000 microns. (Item 38) The system according to any one of items 19 to 37, wherein the hydrogel includes a series of two or more cavities that are in fluid communication with each other through a series of channels, at least one cavity includes a spheroid, at least a second cavity includes a second spheroid, a cell suspension, or a DRG, and the spheroid and the second spheroid, cell suspension, or DRG are connected by three-dimensional axons. (Item 39) The system according to item 38, wherein the height of the three-dimensional axon is at least about 10 microns at its lowest point or at least three cell monolayers. (Item 40) The system according to item 38, wherein the cavity is a well having a U-shaped or circular well disposed on a horizontal or substantially horizontal surface of the solid substrate, and each channel comprises one or more axons connected to one or more spheroids. (Item 41) (i) one or more nerve cells, and / or (ii) one or more Schwann cells or oligodendrocytes comprising a first spheroid, (i) one or more peripheral neurons comprising a second spheroid and The system according to item 40, wherein each spheroid is disposed in the cavity. (Item 42) The first, second, and third cavities, each comprising a cavity configured to hold a spheroid and at least 50 microliters of cell culture medium, The cavities are aligned such that the first cavity is proximal to the second cavity and distal to the third cavity. The system according to item 40. (Item 43) The system according to item 42, comprising at least a fourth cavity, wherein the cavities are arranged in a pattern such that each cavity defines a corner of a square. (Item 44) The system according to item 42, wherein the cavities are aligned in a row such that axons originating from the first spheroid in the first cavity extend to the second cavity, and axons from the spheroid in the second cavity extend to the axons in the third cavity. (Item 45) A method for producing a three-dimensional culture of one or more spheroids in a culture vessel comprising a solid substrate, comprising: (a) contacting one or more nerve cells with the solid substrate, the solid substrate comprising at least one outer surface, at least one inner surface, and at least one inner chamber defined by the at least one inner surface and accessible from a point outside the solid substrate through at least one opening; (b) disposing one or more spheroids containing nerve cells in the at least one inner chamber; (c) applying to the culture vessel a sufficient amount of cell culture medium to cover the at least one spheroid. and The method, wherein at least a part of the inner surface comprises a polymer impenetrable to the first cells and a polymer penetrable to the first cells. (Item 46) The method according to item 45, wherein step (b) comprises placing a spheroid comprising a tissue explant selected from one or a combination of isolated dorsal root ganglia, spinal cord explants, retinal explants, and cortical explants. (Item 47) The method according to item 45 or item 46, wherein the spheroid is optionally formed as a suspension of nerve cells selected from one or a combination of motor neurons, sensory neurons, sympathetic neurons, parasympathetic neurons, cortical neurons, spinal cord neurons, and peripheral neurons derived from stem cells. (Item 48) The method according to any one of items 45 to 47, wherein the spheroid further comprises isolated Schwann cells and / or oligodendrocytes. (Item 49) (d) The method according to any one of items 45 to 48, further comprising a step of growing neurites and / or axons on the spheroid for about 12 hours to about 1 year after step (c). (Item 50) (i) A step of isolating one or more nerve cells from a sample before step (a), and / or (ii) When the one or more spheroids comprise dorsal root ganglia (DRG), a step of isolating DRG from one or more mammals before step (b), and / or (iii) When the one or more spheroids comprise Schwann cells or oligodendrocytes, a step of isolating one or more Schwann cells and / or one or more oligodendrocytes The method according to any one of items 45 to 49, further comprising. (Item 51) When an electric current is introduced into the stimulating electrode, so that the recording electrode can receive a signal corresponding to one or more electrophysiological metrics measurable at the recording electrode, placing at least one stimulating electrode in or near the cell bodies of the one or more nerve cells or tissue explants, and placing at least one recording electrode in or near the axon at the point most distal from the cell bodies, further comprising, The one or more electrophysiological metrics are one or a combination of conduction velocity, action potential, amplitude of the wave associated with the passage of an electrical impulse along the membrane of one or more nerve cells, width of the electrical impulse along the membrane of one or more nerve cells, latency of the electrical impulse along the membrane of one or more nerve cells, and envelope of the electrical impulse along the membrane of one or more nerve cells. The method according to any one of items 45 to 50. (Item 52) (a) Culturing one or more spheroids in the composition according to any one of items 1 to 18; (b) Exposing at least one agent to the one or more spheroids; (c) Measuring and / or observing one or more morphological changes and / or one or more electrophysiological metrics of the one or more spheroids A method for evaluating the toxicity and / or neuroprotective effect of a drug, comprising: (Item 53) A method for determining myelination or demyelination of one or more axons of one or more spheroids, comprising: (a) Culturing one or more spheroids in the composition according to any one of items 1 to 18 in the presence or absence of a drug for a time and under conditions sufficient to grow at least one axon; (b) Detecting the amount of myelination in one or more axons derived from the one or more spheroids Including: Optionally, detecting: (i) Measuring and / or observing one or more morphological changes and / or one or more electrophysiological metrics of the one or more spheroids in the presence or absence of a drug; (ii) Correlating one or more morphological changes and / or one or more electrophysiological metrics of the one or more spheroids in the presence or absence of a drug with quantitative or qualitative changes in myelination of the spheroids The method as described above. (Item 54) (a) Quantifying the number or density of one or more spheroids and / or axons grown from spheroids; (b) Culturing one or more spheroids in the composition according to any one of items 1 to 18; (c) culturing the spheroid for a period sufficient to grow the one or more axons or cells within the spheroid, and then calculating the number of cells within the spheroid and / or the number or density of axons grown from the spheroid in the composition comprising step (b) being optional and comprising contacting the one or more spheroids with one or more agents step (c) being optional and comprising, after culturing the one or more spheroids, detecting a record inside and / or outside of such one or more spheroids and correlating the record with a measurement of a similar record corresponding to a known or control number of cells, or step (c) being optional (i) a further step of measuring intracellular and / or extracellular records and / or morphometric changes before and after the step of contacting the one or more spheroids with one or more agents, and (ii) correlating the difference in the records and / or morphometric changes before and after contacting the one or more spheroids with the one or more agents with the change in the number of cells and / or the number or density of axons A method for detecting and / or quantifying neuronal cell growth and / or axonal degeneration, comprising (Item 55) A method for measuring or quantifying the neuromodulatory effect of an agent, comprising (a) culturing one or more spheroids in the composition according to any one of Items 1 to 18 in the presence and absence of the agent, and (b) applying a potential across the one or more spheroids in the presence and absence of the agent, and (c) measuring one or more electrophysiological metrics from the one or more spheroids in the presence and absence of the agent (d) correlating the difference in one or more electrophysiological metrics by the one or more spheroids with the neuromodulatory effect of the agent, such that a change in the electrophysiological metric in the presence of the agent as 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 as compared to the electrophysiological metric measured in the absence of the agent indicates that the agent does not confer a neuromodulatory effect; and comprising, or (a) culturing one or more spheroids in the composition according to any one of items 1 to 18 in the presence and absence of the agent; (b) measuring and / or observing one or more morphometric changes of the one or more spheroids in the presence and absence of the agent; (c) correlating the one or more morphometric changes with the neuromodulatory effect of the agent, such that a change in the morphological metric in the presence of the agent as compared to the morphological metric measured and / or observed in the absence of the agent indicates a neuromodulatory effect, and no change in the morphological metric in the presence of the agent as compared to the morphological metric measured and / or observed in the absence of the agent indicates that the agent does not confer a neuromodulatory effect; the method comprising the above. (Item 56) A method for manufacturing the system according to any one of items 19 to 37, comprising: (a) culturing nerve cells in a cell culture medium for a period sufficient for the cells to form spheroids; (b) placing the spheroids into the hydrogel; (c) exposing the spheroids to the cell culture medium for a period sufficient for growing neurites or axons. The method comprising the above. (Item 57) The method according to item 56, wherein step (a) comprises mixing the nerve cells with one or more magnetic particles. (Item 58) The method according to item 57, wherein step (b) comprises using a magnetic force to place the spheroids into the cavities of the hydrogel. (Item 59) The method according to item 56, wherein step (b) comprises using a force by ultrasound, mechanical force, or fluid to place the spheroids into the cavities of the hydrogel. (Item 60) The method according to item 56, wherein the spheroid is the spheroid disclosed in any of the compositions according to items 1 to 18. (Item 61) The method according to item 52, further comprising correlating one or more morphometric changes and / or one or more electrophysiological metrics of the one or more spheroids with the toxicity of the agent, such that if the morphometric changes and / or electrophysiological metrics indicate a decrease in cell viability, the agent is characterized as toxic, and if the morphometric changes and / or electrophysiological metrics indicate no change or an increase in cell viability, the agent is characterized as non-toxic and / or having a neuroprotective effect.

Brief Description of the Drawings

[0032]

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Modes for Carrying Out the Invention

[0033] Throughout this specification and the claims, various terms are used with respect to the methods and other aspects of the present disclosure. Such terms shall have their ordinary meanings in the art, unless otherwise indicated. Other specifically defined terms shall be construed in a manner consistent with the definitions made herein.

[0034] In this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents.

[0035] As used herein, the term "greater than 2" is defined as any and all integers greater than the number 2, such as 3, 4, or 5.

[0036] As used herein, the term "about", when referring to a measurable value such as an amount, duration, etc., means an inclusion of a variation of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, or ±0.1% from the explicitly stated value, which is due to such variation being reasonable for carrying out the disclosed method.

[0037] In this specification and the claims, the phrase "and / or" is to be understood to mean "either or both" of the elements joined by that phrase, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Other elements other than those specifically identified by the "and / or" clause may optionally be present, whether or not they are related to those specifically identified elements, unless it is expressly stated to the contrary. Thus, by way of non-limiting example, a reference to "A and / or B" when used in combination with an open-ended word such as "comprising" may, in one embodiment, refer to A without B (optionally including elements other than B), in another embodiment, refer to B without A (optionally including elements other than A), in yet another embodiment, refer to both A and B (optionally including other elements), in yet another embodiment, refer to B without A (optionally including elements other than A), and so on.

[0038] In this specification and the claims, "or" is to be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is inclusive, i.e., it should be interpreted to include not only at least one of a list of numbers or elements, but also a plurality of them, and optionally, additional items not listed. Terms such as "only one of" or "only one" or "consisting of" in the claims that are expressly stated to the contrary shall refer to including only one of the elements of a list of numbers or elements. In this specification, generally, the term "or" should be interpreted as indicating an exclusive alternative (i.e., "one or the other but not both") only when preceded by the exclusive terms "either", "one of", "only one of", or "only one". When "consisting essentially of" is used in the claims, it should have its ordinary meaning as used in the field of patent law.

[0039] As used herein, the terms "comprising" (and any form of "comprising" such as "comprise", "comprises", and "comprised"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "includes" and "include"), or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0040] As used herein, the phrase "an integer from X to Y" means any integer including the endpoints. That is, when a range is disclosed, each integer within the range including the endpoints is disclosed. For example, the phrase "an integer from X to Y" discloses 1, 2, 3, 4, or 5, as well as the range from 1 to 5.

[0041] As used herein, the term "a plurality" is defined as any amount greater than one or any number greater than 1.

[0042] As used herein, "substantially equal" may mean, for example, within a range known to correlate with the abnormal or normal range in a given measured metric. For example, if the control sample is from a patient suffering from a disease, substantially equal means within the abnormal range. If the control sample is from a patient known not to be suffering from the disease being tested, substantially equal means within the normal range of the given metric.

[0043] The present disclosure generally relates to a system capable of accommodating and culturing one or more spheroids in three-dimensional culture. In some embodiments, the system uses a solid substrate, such as plastic or a similar polymer, having pores, on which a hydrogel of any shape or size can be placed. The hydrogel of the present system, in some embodiments, functions as a support for the cells of the present disclosure to extend and grow neurites and / or form axons, under conditions sufficient for mature cells of the nervous system to extend, divide, and / or grow axons, whether in the form of spheroids or in suspension. In some embodiments, the system comprises a cavity forming a first region having at least two regions, namely, a first region, similar to a well, having a flat or curved bottom and a diameter across the longitudinal plane of the support sold, an opening on the upper surface of the region accessible from the outside of the present system, and an opening on at least one side of the first region in fluid communication with the second region, and a hydrogel comprising the first region. In some embodiments, the diameter of the first region is about 1 mm or less. The second region is in the form of a channel extending laterally from the first region, with the sides defining the height of the channel. In some embodiments, the width of the channel is from about 10 to about 750 microns. In some embodiments, the length of the channel is from about 100 to about 10,000 microns. After growing spheroids from any one or combination of the cells specified in the present disclosure, these spheroids may be placed in the first region containing the cell culture medium. After placement, neurites may spontaneously extend or their extension may be promoted by exposure to one or more growth-stimulating molecules. The extension of neurites and / or axons may originate in the first region of the present system, pass through the at least one opening on the side surface of the hydrogel, enter the second region, and occur in the second region. After the neurites or axons have extended to a desired length, a drug may be exposed to the cell culture to determine the situation of the effect of the drug on the extension, morphology, or action potential of the axons or neurites.

[0044] In some embodiments, the cavity or well that holds the spheroid and defines the first region may be a pattern or network connected by a corresponding second region such that the spheroids are connected by axonal channels extending from one or more of the spheroids. In some embodiments, the spheroids are in a square or rectangular pattern connected by channels disposed between each spheroid. In some embodiments, the pattern is in an "L" shape, with the spheroids defining the ends and corners of the "L" shape. In some embodiments, the spheroids may be arranged in a pattern having a triangle, i.e., a corner with three channels between each of the three cavities housing the spheroids. At one end of the hydrogel network, a spheroid having characteristics of the central nervous system may be placed in the first cavity. In these embodiments, cells normally present in the central nervous system constitute the spheroid. Such cells can be selected from any combination or composition including individual neurons and may include astrocytes or immune cells. In the same embodiment, the cavity most distal from the first cavity may hold a spheroid having sensory characteristics, such as a spheroid containing sensory neurons. Thus, the axonal connection between the first spheroid and the spheroid most distal from the first spheroid models a sensory nerve fiber in which the axon extends from a spheroid having characteristics of the central nervous system to a spheroid containing peripheral sensory neurons. Electrophysiological measurements between such spheroids can be made by placing electrodes at both ends of the circuit and measuring the recordings.

[0045] In some embodiments, the spheroid comprises a mixture of nerve cells and non-nerve cells. Examples of non-nerve cells include skeletal muscle cells, cardiomyocytes, and smooth muscle cells. Non-nerve cells also include cells derived from organ tissues such as kidney cells, liver cells, and pancreatic cells. Examples of non-nerve cells also include endothelial cells, epithelial cells of the skin, and corneal cells of the eye. In some embodiments, the cells are mammalian cells, non-human animal cells, or human cells. In some embodiments, one or more of the cells of the spheroid are primary human cells. In some embodiments, the cells are collected from a human subject. In some embodiments, the cells are rat or mouse cells. In some embodiments, the cells are non-human primate cells, pig cells, dog cells, or bovine cells. In some embodiments, any of the disclosed systems may comprise a spheroid of nerve cells, either mixed or not mixed with non-nerve cells.

[0046] The method of the present disclosure is a method for culturing spheroids disclosed herein, and a method for measuring the toxicity or biological effects of toxins, drugs, therapeutic agents, biomolecules, or contaminants, including the method in this system when such molecules, drugs, or therapeutic agents are exposed to the spheroids and cultures of axons or neurites extending from such spheroids. In some embodiments, the method includes a method for generating unidirectional extension of axons and / or neurites in culture from a first spheroid to a second spheroid. In some embodiments, any of the systems of the present disclosure includes an agent that stimulates, accelerates, decelerates, or halts the extension of neurites and / or axons in culture. In some embodiments, any method of the present disclosure includes stimulating the directional extension of axons in culture. In some embodiments, an agent is used to either attract the guidance of axon and / or neurite extension or repel axon and / or neuron extension. In some embodiments, an attractive guidance protein selected from netrin, neurotrophin, adhesive extracellular matrix protein, cell adhesion receptors (such as cadherin, Ig-CAM, or integrin, etc.) is added to the system, and peptides that mimic the putative binding sites of these proteins can also be used. In some embodiments, a protein that repels axon and / or neurite extension is a component of this system. Examples of repulsive proteins include ephrin (sometimes), semaphorin (most cases), slit, chondroitin sulfate proteoglycan, and the like.

[0047] A method for producing spheroids, with or without magnetic particles or magnetic beads, is also disclosed. When the magnetic particles are a component of the spheroid, one or more spheroids can be placed using any device equipped with a magnet at a position within one of the disclosed cavities formed by the hydrogel walls. Generally speaking, the present disclosure relates to a device with a movable frame, where the movable frame is movable in any lateral direction parallel to the horizontal plane on which the device operates. The frame is attached to one or more magnets with a magnetic force sufficient to attract the spheroids containing magnetic particles. In some embodiments, the frame, which is movable in the x and y directions in the longitudinal plane of the device, is mechanically attached to the magnet by an adhesive, polymer, or fastener such that moving the frame causes the magnet, which has a magnetic force sufficient to move the spheroid in any direction when the spheroid is within the magnetic field of the magnet, to move. In some embodiments, the device comprises a first frame and a second frame, and at least one of the first or second frames is movable in a lateral direction parallel to the longitudinal plane of the device and is horizontal or substantially horizontal on the device.

[0048] The term "bioreactor" refers to a container or a partial container in which cells are cultured, optionally in suspension. In some embodiments, the bioreactor is a container or a partial container in which cells are cultured, where the cells are present in suspension or in contact with another non-liquid substrate, including but not limited to, a solid growth support material, and may grow on or within the substrate. In some embodiments, the solid growth support material or solid substrate comprises at least one or a combination of silica, plastic, metal, hydrocarbon, or gel. The present disclosure relates to a system comprising a bioreactor comprising one or more culture containers in which nerve cells may be cultured in the presence or in a cell growth medium.

[0049] As used herein, the term "culture vessel" may be any vessel suitable for growing, culturing, cultivating, proliferating, propagating, or otherwise manipulating cells. The culture vessel may sometimes 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 having one or more pores through which proteins, nucleic acids, nutrients (such as heavy metals and hormones), antibiotics, and other cell culture media components can diffuse. 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 is in a hydrogel matrix and contains no bases or any other structures. In some embodiments, the culture vessel is designed to accommodate a hydrogel or hydrogel matrix and various culture media. In some embodiments, the culture vessel consists of or consists essentially of a hydrogel or hydrogel matrix. In some embodiments, the only plastic component of the culture vessel is the component of the culture vessel that constitutes the side wall and / or bottom of the culture vessel that separates the volume of the cell growth well or zone from an external point. 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, wherein the hydrogel and glial cells are seeded with one or more neurons.

[0050] The term "electrical stimulation" refers to the process in which the above cells are exposed to either alternating current (AC) or direct current (DC). The current may be introduced into the solid substrate or applied through the cell culture medium or other suitable components of the present cell culture system. In some embodiments, the electrical stimulation is provided to the present apparatus or system by arranging one or more electrodes at different positions within the apparatus or system and generating a potential across the entire cell culture vessel. The electrodes are operably connected by one or more wires to one or more of an amplifier, a voltmeter, an ammeter, and / or an electrochemical system (such as a battery or a generator). Such devices and wires form a circuit through which a current is generated and a potential is generated across the entire tissue culture system by the circuit.

[0051] As used herein, the term "hydrogel" may be, for example, a cross-linked three-dimensional network of polymer chains that has voids between the polymer chains that are filled with or can be filled with water and is insoluble in any water. As used herein, the term "hydrogel matrix" refers to, for example, any three-dimensional hydrogel construct, system, device, or similar structure. Hydrogels and hydrogel matrices are known in the art and are described in various types in, for example, U.S. Pat. Nos. 5,700,289 and 6,129,761, and 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 exposing a liquefied pregel solution to ultraviolet light, visible light, or any light having 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, a bifurcated shape designed to mimic a neural pathway. 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, nerve cells are incorporated into the hydrogel or hydrogel matrix. In some embodiments, cells derived from the nervous system are incorporated into the hydrogel or hydrogel matrix. In some embodiments, the cells derived from the nervous system are Schwann cells and / or oligodendrocytes. In some embodiments, the hydrogel or hydrogel matrix comprises an explant of tissue derived from the nervous system of an animal (such as a mammal) and an auxiliary population of the cells that are derived from the nervous system but are isolated and cultured to enrich the population of the cells in the culture.In some embodiments, the hydrogel or hydrogel matrix includes tissue explants such as retinal tissue explants, DRGs, or spinal cord tissue explants, as well as populations 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 in the tissue culture vessel by walls that form microenvironments such as independently addressable wells. In a multiplexed tissue culture vessel, some embodiments can comprise any number of the above-described wells or independently addressable locations within the cell culture vessel such that the hydrogel matrix in one well or location is different from or the same as the hydrogel matrix in another well or location of the cell culture vessel.

[0052] As used herein, the term "immune cell" may be any cell involved in the immune activity of a subject, including, for example, defending the subject from an infection or symptoms of an infection, or attacking, removing, or otherwise eliminating dysfunctional cells or pathogens from the subject's cells, or improving the symptoms of a disease caused by a pathogen. In some embodiments, the immune cells include one or more of B cells; T cells; antigen-presenting cells such as stellate cells, dendritic cells, and macrophages; astrocytes; granulocytes; monocytes; basophils; eosinophils; and / or mast cells. In some embodiments, the immune cells express CD4 or CD8 and one or more immune regulatory molecules. In some embodiments, the immune regulatory molecules are selected from the following, namely, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIR-Iα, MIP-Iβ, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, a variant of IL-18, CD40, CD40L, a vascular growth factor, a fibroblast growth factor, IL-7, a nerve growth factor, a vascular endothelial growth factor, Fas, a TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, an interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPl, TAP2 and their functional fragments, or combinations thereof.The immunomodulatory proteins are exemplified in U.S. Patent No. 8,008,265.

[0053] The term "immunomodulatory" refers to a substance having a regulatory effect on the immune system. Such substances can be readily identified using standard assays that measure various aspects of the immune response, such as cytokine secretion, antibody production, NK cell activation, and T cell proliferation. See, for example, WO97 / 28259, WO98 / 16247, WO99 / 11275, Krieg et al. (1995) Nature 374:546-549; Yamamoto et al. (1992) J. Immunol. 148:4072-76; Ballas et al. (1996) J. Immunol. 157:1840-45; Klinman et al. (1997) J. Immunol. 158:3635-39; Sato et al. (1996) Science 273:352-354; Pisetsky (1996) J. Immunol. 156:421-423; Shimada et al. (1986) Jpn. J. Cancer Res. 77:808-816; Cowdery et al. (1996) J. Immunol. 156:4570-75; Roman et al. (1997) Nat. Med. 3:849-854; Lipford et al. (1997a) Eur. J. Immunol. 27:2340-44, WO98 / 55495, and WO00 / 61151. Thus, using these and other methods, immunostimulatory substances, such as immunostimulatory nucleotides and isolated nucleic acids with immunostimulatory properties, can be identified, tested, and / or confirmed.

[0054] In some embodiments, the above two or more hydrogels may contain different amounts of PEG and / or Puramatrix. In some embodiments, the densities of the above two or more hydrogels may vary. In some embodiments, the above two or more hydrogels may have various penetrabilities that allow cells to elongate within the hydrogel. In some embodiments, the flexibilities of the above two or more hydrogels may vary. In some embodiments, the bioreactor, cell culture device, or composition disclosed herein comprises a hydrogel having two layers of polymers, i.e., a polymer that is penetrable by cells and a polymer that is non-penetrable by cells. In some embodiments, the layer penetrable by the cells is laminated on at least one region of the upper surface of the layer non-penetrable by the cells.

[0055] The term "cell-penetrable polymer" refers to a hydrophilic polymer having the same or mixed monomer units at a concentration and / or density sufficient to create spaces when crosslinking in a solid or semi-solid state on a solid substrate, and such spaces are sufficiently biocompatible to allow cells or parts of cells to elongate in culture.

[0056] The term "cell-non-penetrable polymer" refers to a hydrophilic polymer having the same or mixed monomer units at a concentration and / or density sufficient to prevent spaces or compartments from being created when crosslinking in a solid or semi-solid state on a solid substrate. In other words, a cell-non-penetrable polymer is a polymer that, after crosslinking, cannot support the elongation of cells or parts of cells in culture at a specific concentration and / or density.

[0057] The term "functional fragment" refers to any portion of a polypeptide or nucleic acid sequence that is related to the respective full-length polypeptide or nucleic acid and has a length and structure sufficient to provide at least a similar or substantially similar biological effect as the full-length polypeptide or nucleic acid on which the fragment is based. In some embodiments, the functional fragment is part of a full-length or wild-type nucleic acid sequence encoding any one of the nucleic acid sequences disclosed herein, and the portion encodes a polypeptide of a certain length and / or structure that is shorter than the full-length but still encodes a biologically functional domain compared to the full-length or wild-type protein. In some embodiments, the functional fragment may have a reduced biological activity, a substantially equivalent biological activity, or an improved biological activity compared to the wild-type or full-length polypeptide sequence on which the fragment is based. In some embodiments, the functional fragment is derived from a sequence of an organism such as a human. In such embodiments, the functional fragment may retain 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the wild-type human sequence from which the sequence is derived. In some embodiments, the functional fragment may retain 87%, 85%, 80%, 75%, 70%, 65%, or 60% sequence homology to the wild-type human sequence from which the sequence is derived.

[0058] One of ordinary skill in the art will appreciate that polymers that are non-cell penetrating and polymers that are cell penetrating may contain the same or substantially the same polymers, but due to differences in concentration or density after crosslinking, a hydrogel matrix is formed that has some portion that promotes the elongation of cells or portions of cells in culture.

[0059] In some embodiments, the thickness of the hydrogel or hydrogel matrix may vary. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 150 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 200 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 250 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 300 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 350 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 400 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 450 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 500 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 550 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 600 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 650 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 700 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 750 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from 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 from 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 from about 100 μm to about 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 300 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 400 μm to about 500 μm.

[0060] In some embodiments, the thickness of the hydrogel or hydrogel matrix may vary. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 10 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 150 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 200 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 250 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 300 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 350 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 400 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 450 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 550 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 600 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 650 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 700 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 750 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 800 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from 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 from about 1000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 1500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 2000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 2500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 2500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 2000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 1500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 1000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 950 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 900 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 850 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from 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 from 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 from about 100 μm to about 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 100 μm to about 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 300 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is from about 400 μm to about 500 μm.

[0061] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic polymers. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following synthetic polymers, namely, polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), polyacrylamide, silicone, and any derivatives or combinations thereof.

[0062] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic and / or natural polysaccharides. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following polysaccharides, namely, hyaluronic acid, heparin sulfate, heparin, dextran, agarose, chitosan, alginate, and any derivatives or combinations thereof.

[0063] In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following proteins, namely, collagen, gelatin, elastin, chitin, laminin, fibronectin, fibrin, keratin, silk fibroin, and any derivatives or combinations thereof.

[0064] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic and / or natural polypeptides. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following polypeptides, namely, polylysine, polyglutamate, or polyglycine.

[0065] In some embodiments, the hydrogel comprises one or a combination of polymers selected from the hydrogels 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.

[0066] Any hydrogel suitable for cell growth can be formed by placing any one or combination of the polymers disclosed herein under conditions of sufficient time, sufficient concentration, and sufficient conditions to produce two distinct densities of cross-linked polymers, namely, a cross-linked polymer penetrable by one type of cell and a cross-linked polymer impenetrable by one type of cell. The polymers may be synthetic polymers, polysaccharides, natural proteins, or glycoproteins, and / or polypeptides such as those selected from the following. Synthetic polymers Polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), polyacrylamide, silicone, combinations thereof, and derivatives thereof, etc. Polysaccharides (either from synthetic or natural sources) Hyaluronic acid, heparan sulfate, heparin, dextran, agarose, chitosan, alginate, combinations thereof, and derivatives thereof, etc. Natural proteins or glycoproteins Collagen, gelatin, elastin, chitin, laminin, fibronectin, fibrin, keratin, silk fibroin, combinations thereof, and derivatives thereof, etc. Polypeptides (from synthetic or natural sources) Polylysine, and all RAD and EAK peptides already listed, etc.

[0067] As used herein, the term "three-dimensional" or "3D" means, for example, the thickness of a cell culture such that there are at least three layers of cells growing adjacent to each other. In some embodiments, the term three-dimensional means, in the context of the systems of the present disclosure, that the thickness or height of neurites and / or axons is from about 10 to about 1000 microns. In some embodiments, the term three-dimensional means, in the context of the systems of the present disclosure, that the thickness or height of neurites and / or axons is from about 10 to about 100 microns.

[0068] The term "isolated neuron" refers to a nerve cell that is a living organism or a culture, and the nerve cell is removed or detached from the living organism or culture from which the nerve cell originally grew. In some embodiments, the isolated neuron is a neuron in suspension. In some embodiments, the isolated neuron is a component of a larger mixture of cells that includes a tissue sample or a suspension with non-neuronal cells. In some embodiments, the nerve cell is isolated at the stage when it is removed from the animal from which it is derived, as in the case of a tissue explant. In some embodiments, the isolated neuron is a neuron in a DRG excised from an animal. In some embodiments, the isolated neuron includes at least one or a plurality of cells derived from one species or a combination of species selected from ovine cells, caprine cells, equine cells, bovine cells, human cells, simian cells, murine cells, rat cells, rabbit cells, canine cells, feline cells, porcine cells, or other non-human mammalian cells. In some embodiments, the isolated neuron is a human cell. In some embodiments, the isolated neuron is a stem cell pretreated to have a differentiated phenotype similar or substantially similar to that of a human nerve cell. In some embodiments, the isolated neuron is a human cell. In some embodiments, the isolated neuron is a stem cell pretreated to have a differentiated phenotype similar or substantially similar to that of a non-human nerve cell. In some embodiments, the stem cell is selected from mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, hematopoietic stem cells, epidermal stem cells, stem cells isolated from the umbilical cord of a mammal, or endodermal stem cells.

[0069] The term "neurodegenerative disease" is used throughout this specification to describe diseases caused by damage to the central nervous system and / or peripheral nervous system. Exemplary neurodegenerative diseases that can be examples of diseases that can 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 (e.g., the "white matter disease" or glial / demyelinating diseases described by Folkerth, J. Neuropath. Exp. Neuro., 58, 9, Sep., 1999); Tay-Sachs disease (β-hexosaminidase deficiency); other genetic diseases; multiple sclerosis; brain injury or trauma resulting from ischemia, accidents, environmental injuries, etc.; spinal cord injury; ataxia, and alcohol dependence. Further, for the study of the treatment of neurodegenerative diseases, the present invention may be used to test the efficacy, toxicity, or neurodegenerative effects of drugs on nerve cells in culture. The term neurodegenerative disease includes, inter alia, neurodevelopmental disorders such as, for example, autism and related neurological diseases (such as schizophrenia).

[0070] As used herein, the term "nerve cell" refers to, for example, a cell that includes at least one or a combination of dendrites, axons, and cell bodies, or any cell or cell group isolated from nervous tissue. In some embodiments, the nerve cell is any cell that can include or form an axon. In some embodiments, the nerve cell is a Schwann cell, glial cell, neuroglial cell, cortical neuron, embryonic cell isolated from or derived from nervous tissue, or an embryonic cell differentiated into a cell having a phenotype substantially similar to that of a neuron or a nerve cell, 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 nerve cell is a neuron derived from dorsal root ganglion (DRG) tissue, retinal tissue, spinal cord tissue, or brain tissue from a subject of adult, adolescent, juvenile, or fetal origin. In some embodiments, the nerve cell is any one or more cells isolated from the nerve tissue of a subject. In some embodiments, the nerve cell is a mammalian cell. In some embodiments, the cell is a human cell and / or a rat cell. In some embodiments, the cell is a non-human mammalian cell or is derived from a cell isolated from a non-human mammalian. The nerve cell may include neurons isolated from multiple species when the cell is isolated or separated from the original animal from which it is derived. In some embodiments, the spheroid does not include DRG tissue.

[0071] In some embodiments, the nerve cells are one or more of the following, namely, central nervous system neurons, peripheral nervous system neurons, sympathetic neurons, parasympathetic neurons, enteric nervous system neurons, spinal motor neurons, motor neurons, sensory neurons, autonomic neurons, somatic neurons, dorsal root ganglia, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, serotonergic neurons, interneurons, adrenergic neurons, and trigeminal ganglia. In some embodiments, the glial cells are one or more of the following, namely, astrocytes, oligodendrocytes, Schwann cells, microglial cells, ependymal cells, radial glial cells, satellite cells, enteric glial cells, and pituitary cells. In some embodiments, the immune cells are one or more of the following, namely, macrophages, T cells, B cells, white blood cells, lymphocytes, monocytes, mast cells, neutrophils, natural killer cells, and basophils. In some embodiments, the stem cells are one or more of the following, namely, hematopoietic stem cells, neural stem cells, embryonic 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 spheroids may also contain other cell types such as keratinocytes or endothelial cells.

[0072] As used herein, the term "neuronal cell culture medium" or simply "culture medium" may be any nutrient suitable for supporting the growth, culture (Culture), cultivation (cultivating), proliferation (proliferating), propagation (propagating), or other manipulations of neuronal cells. In some embodiments, the medium comprises 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 in the range of about 0.001 weight / volume % to about 0.01 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.001 weight / volume % to about 0.008 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.001 weight / volume % to about 0.006 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.001 weight / volume % to about 0.004 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.002 weight / volume % to about 0.01 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.003 weight / volume % to about 0.01 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.004 weight / volume % to about 0.01 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.006 weight / volume % to about 0.01 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.008 weight / volume % to about 0.01 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.002 weight / volume % to about 0.006 weight / volume %. In some embodiments, the medium comprises ascorbic acid at a concentration in the range of about 0.003 weight / volume % to about 0.005 weight / volume %.

[0073] In some embodiments, the hydrogel, hydrogel matrix, and / or neural cell culture medium comprises one or more of the following components: artemin, ascorbic acid, ATP, β-endorphin, BDNF, 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, IL-10, IL-12, IL-17, IL-18, insulin, laminin, lipoxin, mac-1-saporin, methionine sulfoximine, minocycline, neuregulin-1, neuroprotectin, neuritin, NGF, nitric oxide, NT-3, NT-4, persephin, platelet lysate, PMX53, poly-D-lysine (PLL), poly-L-lysine (PLL), prolyl endopeptidase, resolvin, S100 calcium-binding protein B, selenium, substance P, TNF-α, types I-V collagen, and zymosan.

[0074] As used herein, the term "optogenetics" refers to a biological technique that involves the use of light to control living tissue, typically neurons, that have been genetically engineered to express light-sensitive ion channels. Optogenetics is a method of neuromodulation used in neuroscience that combines optical and genetic techniques to control and monitor the activity of individual neurons in living tissue (even within freely moving animals) and accurately measure the effects of these genetic manipulations in real time. The primary reagents used in optogenetics are light-sensitive proteins. Spatial and precise control of neurons is achieved using optogenetic actuators such as channelrhodopsin, halorhodopsin, archaerhodopsin, etc., while temporal and accurate recordings can be made by utilizing optogenetic sensors for calcium (aequorin, cameleon, GCaMP), chloride (chromeleon), or membrane voltage (mermaid). In some embodiments, neurons that have been recombinantly engineered using optogenetic actuators and / or sensors are used in the culture systems described herein.

[0075] The term "plastic" refers to a biocompatible polymer containing hydrocarbons. In some embodiments, the plastic is selected from the group consisting of polystyrene (PS), polyacrylonitrile (PAN), polycarbonate (PC), polyvinylpyrrolidone (PVP), polybutadiene, polyvinyl butyral (PVB), polyvinyl chloride (PVC), polyvinyl methyl ether (PVME), poly(lactic-co-glycolic acid) (PLGA), poly(l-lactic acid), polyester, polycaprolactone (PCL), polyethylene oxide (PEO), polyaniline (PANI), polyfluorene, polypyrrole (PPY), polyethylenedioxythiophene (PEDOT), and mixtures of any two or more of the above-mentioned polymers. In some embodiments, the plastic is a mixture of 3, 4, 5, 6, 7, 8 or more polymers.

[0076] As used herein, the term "seeding" refers to, for example, transferring a certain amount of cells into a new culture vessel. The above amount may be defined, and as the amount, an amount based on the volume or number of cells may be used. The above cells may be part of a suspension.

[0077] As used herein, the term "sequence identity" refers to the specified percentage of residues that are identical over a specified region in the context of two or more nucleic acid or polypeptide sequences. This term is a synonym for "sequence homology" to another sequence or a sequence that is "homologous" to another sequence. The above percentage can be calculated by optimally aligning the two sequences in question, comparing the two sequences over the specified region, determining the number of positions where the same residues exist in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the lengths of the two sequences are different, or one or more attachment ends occur by sequence comparison and only a single sequence is included in the specified comparison region, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity can be performed manually or by using computer sequence algorithms such as BLAST or BLAST 2.0.

[0078] As used herein, the term "solid substrate" refers to any substance that is a solid support that does not contain or substantially does not contain cytotoxins. In some embodiments, the solid substrate includes one or a combination of silica, plastic, and metal. In some embodiments, the solid substrate has pores of a size and shape sufficient for proteins, nutrients, and gases to diffuse or be passively transported through the solid substrate in the presence of a cell culture medium. In some embodiments, the size of the pores is about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron or less in diameter. One of ordinary skill in the art can determine how large the pore size needs to be based on the contents of the cell culture medium and the exposure of the cells growing on the solid substrate in a particular microenvironment. For example, one of ordinary skill in the art can determine whether any cultured cells in the system or device can survive under conditions where the solid substrate has pores of various diameters. In some embodiments, the solid substrate includes a base having a predetermined shape that defines the shape of the outer and inner surfaces. In some embodiments, the base includes one or a combination of silica, plastic, ceramic, or metal, and the base has a cylindrical or substantially cylindrical shape similar such that a polymer that is impenetrable to a first cell and a polymer that is penetrable to the first cell coat the inner surface of the base, defining a cylindrical or substantially cylindrical inner chamber, and an opening is located at one end of the cylinder. In some embodiments, the base has one or more pores of a size and shape sufficient for proteins, nutrients, and oxygen to diffuse through the solid substrate in the presence of a cell culture medium.In some embodiments, the solid substrate comprises a plastic base having a pore size of 1 micron or less, and comprises at least one hydrogel matrix layer. The hydrogel matrix comprises at least a polymer impenetrable to at least a first cell and a polymer penetrable to at least a first cell. The base has a predetermined shape, and has a shape in which a polymer impenetrable to a first cell and a polymer penetrable to at least a first cell physically adhere or chemically bond around it. The solid substrate comprises at least one compartment at least partially defined by the shape of the inner surface of the solid substrate and accessible from a point outside the solid substrate by an opening optionally disposed at one end of the solid substrate. In some embodiments, when the solid substrate comprises a hollow interior defined by at least one inner surface, the cells may be seeded by placing the cells at or near the opening so that the cells can adhere to at least a portion of the inner surface of the solid substrate before growing, in a suspension or tissue explant. The at least one compartment or hollow interior of the solid substrate can confine the cells into a specific three-dimensional shape defined by the shape of the inner surface solid substrate, promoting directional growth of the cells out of the opening. In the case of nerve cells, the degree and shape of confinement of the at least one compartment promote axonal outgrowth from cell bodies disposed within the at least one compartment and at or near the opening. In some embodiments, the solid substrate is cylindrical, tubular, or substantially tubular or cylindrical such that the shape of the internal compartment is cylindrical or partially cylindrical. In some embodiments, the solid substrate comprises one or more branched tubular internal compartments. In some embodiments, the bifurcated or multiply bifurcated shape of the hollow interior of the solid is configured such that axons grow in a multi-branched pattern or the shape enables axons to grow in a multi-branched pattern. If electrodes are disposed at or near the distal end of an axon and at or near the cell body of a nerve cell, electrophysiological metrics such as intracellular action potentials can be measured within the device or system.In some embodiments, the electrode is a voltmeter, an ammeter, and / or a device that is operably coupled to a device capable of generating a current on a length of wire that physically connects the electrode to the voltmeter, ammeter, and / or device.

[0079] The present disclosure relates to a suitably filled hydrogel comprising a mixture of both a cell-permeable polymer and a cell-impermeable polymer. In some embodiments, the hydrogel comprises about 10% to about 20% PEG and has an overall elastic modulus of about 0.1 to about 200 Pa. In some embodiments, the elastic modulus of the hydrogel is about 0.5 Pa. In some embodiments, the elastic modulus of the hydrogel is about 10 Pa. In some embodiments, the elastic modulus of the hydrogel is about 50 Pa. In some embodiments, the elastic modulus of the hydrogel is about 75 Pa. In some embodiments, the elastic modulus of the hydrogel is about 90 Pa. In some embodiments, the elastic modulus of the hydrogel is about 100 Pa. In some embodiments, the elastic modulus of the hydrogel is about 125 Pa. In some embodiments, the elastic modulus of the hydrogel is about 150 Pa. In some embodiments, the elastic modulus of the hydrogel is about 175 Pa. In some embodiments, the elastic modulus of the hydrogel is about 200 Pa. In some embodiments, the elastic modulus of the hydrogel is about 230 Pa or less.

[0080] Sphroid As used herein, "sphroid" or "cell sphroid" may generally refer to any population of cells having a three-dimensional shape corresponding to an ellipse or circle or convex or concave circular arc that rotates about one of its major axes (major or minor axis), and examples include three-dimensional ovoids, oblate spheroids, and prolate spheroids, spheres, lens shapes, or substantially equivalent shapes.

[0081] The width, length, thickness, and / or diameter of the spheroid of the present invention may be arbitrary and appropriate. In some embodiments, the width, length, thickness, and / or diameter of the spheroid may be in the range of about 10 μm to about 50,000 μm, or about 10 μm to about 900 μm, about 100 μm to about 700 μm, about 300 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 1,000 μm, about 600 μm to about 1,000 μm, about 700 μm to about 1,000 μm, about 800 μm to about 1,000 μm, about 900 μm to about 1,000 μm, about 750 μm to about 1,500 μm, about 1,000 μm to about 5,000 μm, about 1,000 μm to about 10,000 μm, about 2,000 to about 50,000 μm, about 25,000 μm to about 40,000 μm, or about 3,000 μm to about 15,000 μm, etc., but not limited thereto, and may be any range within the above ranges. In some embodiments, the width, length, thickness, and / or diameter of the spheroid may be about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1,000 μm, 5,000 μm, 10,000 μm, 20,000 μm, 30,000 μm, 40,000 μm, or 50,000 μm. In some embodiments, a plurality of spheroids are generated, and the width, length, thickness, and / or diameter of each of the plurality of spheroids may vary by less than about 20%, for example, less than about 15%, 10%, or 5%. In some embodiments, the width, length, thickness, and / or diameter of each of the plurality of spheroids may be within any of the above ranges.

[0082] The cells in the spheroid may have a specific orientation. In some embodiments, the above spheroid may comprise an inner core and an outer surface. In some embodiments, the above spheroid may be hollow (i.e., it may not contain cells inside). In some embodiments, the cells of the inner core and the cells of the outer surface are different types of cells. In some embodiments, the above inner core contains magnetic nanoparticles.

[0083] The rigidity of the above-mentioned spheroid, measured, for example, by elastic modulus (Pascal, Pa), may vary. In certain embodiments, the elastic modulus of the above-mentioned spheroid ranges from about 100 Pa to about 10,000 Pa, for example, from about 100 Pa to about 12,000 Pa, or from about 100 Pa to about 4,800 Pa. In some embodiments, the elastic modulus of the above-mentioned spheroid may be about 1200 Pa. As another example, the elastic modulus of the above-mentioned spheroid may vary from at least about 10 Pa, at least about 100 Pa, at least about 150 Pa, at least about 200 Pa, or at least about 450 Pa. In some embodiments, the composition or device of the present disclosure comprises one or more wells, and each well contains / includes a population of one or more different spheroids, a first, a second, a third, a fourth, or a fifth spheroid, or more spheroids. In one embodiment, the first spheroid has an elastic modulus ranging from about 100 Pa to about 300 Pa, and the second spheroid has an elastic modulus ranging from about 400 Pa to about 800 Pa. In another example, the first spheroid is characterized by an elastic modulus of about 50 to about 200 Pa, and the second spheroid is characterized by an elastic modulus of about 250 Pa to about 500 Pa.

[0084] In some embodiments, the spheroid may be composed of one, two, three, or more different cell types, including one or more neuronal cell types and / or one or more stem cell types. In some embodiments, the cells of the above-mentioned inner core may be composed of one, two, three, or more different cell types. In some embodiments, the cells of the above-mentioned outer surface may be composed of one, two, three, or more different cell types.

[0085] In some embodiments, the spheroid comprises at least two types of cells. In some embodiments, the spheroid comprises neurons and non-neurons. In some embodiments, the spheroid comprises neurons and astrocytes at a ratio of neuronal cells to astrocyte cells of about 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the spheroid comprises neurons and non-neurons at a ratio of about 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the spheroid comprises neurons and non-neurons at a ratio of about 1:5:1:4, 1:3, or 1:2. Any combination of cell types disclosed herein may be used in the ratios specified above within the spheroids of the present disclosure.

[0086] Depending on the particular embodiment, a group of cells may be arranged according to any suitable shape, geometry, and / or pattern. In some embodiments, the cells are arranged on the surface of a sphere spanning the entire surface area of a bead or nanoparticle having a solid or hollow core. For example, a group of independent cells may be deposited as spheroids, and the spheroids may be arranged within a three-dimensional grid or any other suitable three-dimensional pattern. All of the independent spheroids may contain approximately the same number of cells and be approximately the same size, or different spheroids may have different numbers of cells and different sizes. In some embodiments, multiple spheroids are arranged in a shape such as an L-shape or a T-shape, radially from a single point or multiple points, in a single line or parallel lines, in a continuous spheroid, tube, cylinder, torus, hierarchically branched vascular network, high aspect ratio object, thin closed shell, organoid, or other complex shape corresponding to the geometry of a tissue, blood vessel, or other biological structure.

[0087] In the method of the present disclosure, any and appropriate physiological responses of the spheroid may be determined, evaluated, measured, and / or identified. In some embodiments, in the method of the present disclosure, one, two, three, four, or more types of physiological responses (plural) of the spheroid may be determined, evaluated, measured, and / or identified. In some embodiments, the physiological response of the spheroid may be a morphological change of the spheroid. The method may include measuring the morphological change of the spheroid, and the measuring may include evaluating at least one morphological parameter before contacting the spheroid with an agent such as a chemical and / or biological compound, evaluating the at least one morphological parameter after contacting the spheroid with the agent, and calculating the difference between the at least one morphological parameter before and after contacting the spheroid with the agent to obtain the morphological change of the spheroid. In some embodiments, the physiological response of the spheroid may be contraction or expansion of the spheroid in response to contact with an agent. The morphology of the spheroid may be measured using any method known to those skilled in the art, such as but not limited to quantification of eccentricity and / or cross-sectional area.

[0088] In some embodiments, the physiological response of the spheroid may be a change in the volume of the spheroid. The method may include measuring the change in the volume of the spheroid, and the measuring may include evaluating a first volume before contacting the spheroid with an agent, evaluating a second volume after contacting the spheroid with the agent, and calculating the difference between the first volume and the second volume to obtain the change in the volume of the spheroid. In some embodiments, the physiological response of the spheroid may be contraction or expansion of the spheroid in response to contact with an agent.

[0089] The above-mentioned agent may be any appropriate compound such as, for example, an organic compound, a small molecule compound (e.g., a small molecule organic compound), a protein, an antibody, an oligonucleotide (e.g., DNA and / or RNA), a gene therapy vehicle (e.g., a viral vector), and any combination thereof. In the method of the present invention, one or more (e.g., one, two, three, four, five, or more types of) agents may be used. For example, the method of the present invention may include contacting the spheroid of the present invention with two or more different agents. In some embodiments, the method of the present invention may indirectly regulate the activity of the spheroid, for example, by contacting the spheroid of the present invention with a gene therapy vehicle (e.g., a viral vector).

[0090] The method of the present invention may include culturing cells and / or spheroids. The culturing can be performed using methods known to those skilled in the art. In some embodiments, the cells and / or spheroids may be cultured for any desired period such as several hours, several days, several weeks, or several months, but not limited thereto. In some embodiments, the cells and / or spheroids may be cultured for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or 11 weeks or more.

[0091] Cell culture media suitable for the methods of the present invention are known in the art and include, but are not limited to, BEGM™ Bronchial Epithelial Cell Growth Medium, Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Modified Eagle Medium High Glucose (DMEM-H), McCoy's 5A Modified Medium, RPMI, Ham's Medium, Medium 199, mTeSR, and the like. The above cell culture media may be supplemented with additional components such as vitamins, minerals, salts, growth factors, carbohydrates, proteins, serum, amino acids, adhesion factors, cytokines, growth factors, hormones, antibiotics, therapeutic agents, buffers, etc., but are not limited thereto. The above cell culture components and / or conditions may be selected to enhance and / or stimulate the characteristics and / or properties of a particular cell and / or may be varied during the methods of the present invention. Examples of seeding and cell culture methods are described in U.S. Pat. Nos. 5,266,480, 5,770,417, 6,537,567, and 6,962,814, and Oberpenning et al., “De novo reconstitution of a functional mammalian urinary bladder by tissue engineering” Nature Biotechnology 17:149-155 (1999), which are hereby incorporated by reference in their entirety. In some embodiments, the cell culture medium is modified stepwise to promote myelination of axons in the culture. The pre-myelination medium and the myelination medium contain the following components.

Table 1-1

[0092] In some embodiments, the solid substrate, cell culture device, or nanoparticles comprise / contain spheroids comprising one or more cell types disclosed herein. In the present application, any of the above particles may contain any one or combination of one, two, three, four, five, six, seven, eight, or more than eight types of cell types.

[0093] The term "nanoparticle" or "nanoshuttle" (since the terms may be used synonymously), is a particle that includes at least one region. Magnetic particles in the range of about 0.7 to about 1.5 microns are described, for example, in patent documents including U.S. Patent Nos. 3,970,518, 4,018,886, 4,230,685, 4,267,234, 4,452,773, 4,554,088, 4,659,678, 6,623,982, 6,645,731, and U.S. Application No. 20110250146 (each of which is incorporated by reference in its entirety). Using the above nanoparticles, any cell or spheroid described herein can be magnetized, i.e., made responsive to a magnetic field. Some compositions and / or systems of the present disclosure include / comprise cells in contact with a magnetic-responsive element or spheroids comprising a magnetic-responsive element. In some embodiments, the compositions and / or systems of the present disclosure include / comprise cells in contact with one or more magnetic nanoparticles or spheroids comprising one or more magnetic nanoparticles. As used herein, a "magnetic-responsive element" may be any element or molecule that responds to a magnetic field. One or more of the above nanoparticles need to contain a magnetic-responsive element or be a magnetic-responsive element. In some embodiments, the nanoparticles may be taken up or adsorbed by any of the cells described herein. In some embodiments, a magnetic field can be used to manipulate the position, shape, pattern, or movement of a cell or spheroid.

[0094] In some embodiments, the size of the charged nanoparticles is on the nanoscale. In some embodiments, the size of the nanoparticles of the present disclosure is from about 5 nm to about 1000 nm, or any range therein. In some embodiments, the diameter of the nanoparticles is from about 5 nm to about 250 nm, from about 25 nm to about 225 nm, from about 50 nm to about 200 nm, from about 75 nm to about 150 nm. In some embodiments, the diameter of the nanoparticles is about 5 nm, about 10 nm, about 20 nm, about 40 nm, about 60 nm, about 80 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 250 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. In some embodiments, the diameter of the nanoparticles is about 5 nm or less, about 10 nm or less, about 20 nm or less, about 40 nm or less, about 60 nm or less, about 80 nm or less, about 100 nm or less, about 120 nm or less, about 140 nm or less, about 160 nm or less, 180 nm or less, about 200 nm or less, about 250 nm or less, about 300 nm or less, about 400 nm or less, about 500 nm or less, about 600 nm or less, about 700 nm or less, about 800 nm or less, about 900 nm or less, or about 1000 nm or less. In some embodiments, the size of the nanoparticles is substantially uniform. In some embodiments, the size of the nanoparticles varies. In some embodiments, the size of the nanoparticles will depend on the type of cells being used.

[0095] The above-mentioned "magnetic-responsive element" may be any element or molecule that responds to a magnetic field. In some embodiments, the magnetic-responsive element is a rare-earth magnet such as samarium cobalt (SmCo) or neodymium iron boron (NdFeB). In some embodiments, the magnetic-responsive element is a ceramic magnet material such as strontium ferrite. In some embodiments, the magnetic-responsive element is a magnetic element such as iron, cobalt, nickel, or any alloy or oxide thereof. In some embodiments, the magnetic-responsive element contains gold. In some embodiments, the magnetic-responsive element is a paramagnetic material that reacts to a magnetic field but is not a magnet itself, which facilitates the assembly of the material.

[0096] In some embodiments, the nanoparticles include one or more iron oxides such as iron(III) oxide, α-Fe2O3, γ-Fe2O3, β-Fe2O3, ε-Fe2O3, iron(II) oxide, or iron(II,III) oxide. In some embodiments, the nanoparticles include one or more of gold, iron oxide, and polylysine.

[0097] In some aspects of the present disclosure, there are provided coated magnetic particles comprising a nanoparticle core of a magnetic material and a base coating material on the magnetic core in an amount sufficient to prevent non-specific binding of a biopolymer to the magnetic core. These magnetic particles are characterized by extremely low non-specific binding and very efficient target capture, which are essential for achieving the enrichment levels necessary to effectively isolate very rare cells such as the neurons or other cell types disclosed herein. In an alternative embodiment, namely, i. a nanoparticle core of a magnetic material, and ii. a base coating material that forms a discontinuous coating on the magnetic core, the base coating material providing at least one discontinuous region that contributes to non-specific binding of the base-coated particles to a biopolymer when accessible. iii. A further coating material that hinders access to the discontinuous region by the biopolymer and Coated magnetic particles are provided that include it. The magnetic core material of the particles described immediately above may include at least one transition metal oxide, and suitable base coating materials include proteins. Examples of proteins suitable for coating the magnetic particles include, but are not limited to, bovine serum albumin and casein. The further coating material may be the initial coating protein or one of the components of a specific binding pair that binds to the base material on the magnetic core. Exemplary specific binding pairs include biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin. In one embodiment, the component of the specific binding pair binds to the base coating material via a bifunctional linking compound. Exemplary bifunctional linking compounds include succinimidyl-propiono-dithiopyridine (SPDP) and sulfo-succinimidyl-4-[maleimidomethyl]cyclohexane-1-carboxylate (SMCC). However, various other such heterobifunctional linker compounds are available from Pierce (Rockford, Ill.).

[0098] The magnetic mass of the coated magnetic particles of the present invention is preferably 70-90%. In some embodiments, the particle size of the main portion of the magnetic particles ranges from about 90 to about 150 nm. The particles may be synthesized such that they are more monodisperse, for example, in the range of about 90 to about 120 nm or about 120 to about 150 nm. The particles of the present invention are generally suspended in a biologically compatible medium.

[0099] In some embodiments, nanoparticles may be combined with a support molecule. A "support molecule" is generally a polymer or other long molecule that serves to hold the nanoparticles and cells together in a tight mixture. The support molecule may be positively charged, negatively charged, have a mixed charge, or be neutral, and may be a combination of multiple support molecules. In some embodiments, the support molecule is a natural polymer or a cell-derived polymer. Non-limiting examples of such polymers include peptides, polysaccharides, and nucleic acids. In other embodiments, the support molecule is a synthetic polymer. In some embodiments, the polymer is polylysine. In some embodiments, the support molecule may be one or more of polylysine, fibronectin, collagen, laminin, BSA, hyaluronic acid, glycosaminoglycan, anionic, non-sulfated glycosaminoglycan, gelatin, nucleic acid, extracellular matrix protein mixture, Matrigel, antibody, and mixtures and derivatives thereof. In some embodiments, the nanoparticles comprise Feridex, a material composed of dextran-coated superparamagnetic iron oxide nanoparticles (SPION).

[0100] The nanoparticles may be charged either positively or negatively. In some embodiments, the negatively charged nanoparticles contain a charge-stabilizing metal (e.g., silver, copper, platinum, palladium, gold). In some embodiments, the negatively charged nanoparticles contain gold.

[0101] In some embodiments, the positively charged nanoparticles contain an alloy and / or oxide (e.g., elemental iron, iron-cobalt, nickel oxide, iron oxide) stabilized or coated with a surfactant or polymer. In some embodiments, the positively charged nanoparticles contain iron oxide.

[0102] The present disclosure also relates to (i) a hydrogel matrix, (ii) one or more spheroids, (iii) a generator, (iv) A voltmeter and / or an ammeter, and (v) At least a first stimulating electrode and at least a first recording electrode A system comprising: The generator, the voltmeter and / or the ammeter, and the electrodes are electrically connected to each other via a circuit through which current is supplied from the generator to the at least one stimulating electrode, received at the recording electrode, and supplied to the voltmeter and / or the ammeter. The present invention also relates to the system in which the stimulating electrode is disposed at or near one or more cell bodies of the nerve cells, and the recording electrode is disposed at a predetermined distance distal to the cell body so that a potential is established across the entire cell culture vessel.

[0103] In some embodiments, the solid substrate consists of a hydrogel or a hydrogel matrix. In some embodiments, the solid substrate consists of a hydrogel or a hydrogel matrix and does not contain glass, metal, or ceramic. In some embodiments, the solid substrate is shaped into a predetermined form or mold for seeding specific-sized cells suitable for axon elongation. In some embodiments, the solid substrate or at least one base portion has a shape with at least one branched internal tubular structure where the position of the tube becomes smaller in diameter as it becomes more distal from the position where the tissue explant or nerve cells are seeded. For example, the present disclosure contemplates a focus 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 (any one or more of any one or a combination of the disclosed cells) at the focus. During the growth of the cells over several days during culture, the cells are exposed to a medium containing any of the components disclosed herein at a concentration sufficient for axons to extend from the nerve cells for a period sufficient for this. If the cells are to be myelinated or myelination is desired for research, glial cells may be introduced and seeded through the same pores before adding the nerve cells or explants. As the axons extend within the semi-cylindrical or tubular structure, the elongation of the axon projections can occur more distally from the focus. The state of the axons can be addressed or observed using an opening or access point in the solid substrate at a point that becomes more distal from the focus (or the point of seeding). This disclosure contemplates the structure of the solid substrate taking any form to promote axon elongation. In some embodiments, the internal chamber or compartment for housing the axon projections has a semi-circular or substantially cylindrical diameter. In some embodiments, the solid substrate branches into two or more internal compartments at a point distal from the focus.In some embodiments, this bifurcation may be keyhole-shaped or tree-like with two, three, four, five, six, seven, or eight or more tubular or substantially cylindrical internal chambers that are in fluid communication with each other, such that axonal outgrowth originates from a point seeded with one or more cell bodies and extends longitudinally along the internal chamber to any one or more of the branches. In some embodiments, one or more electrodes may be disposed in or proximate to one or more openings so as to be able to take recordings over one or more positions along the length of the axon. This can also be used to examine one or more positions along the length of the axon.

[0104] The present disclosure relates to a system for accurately measuring recordings between ganglia of the central nervous system and ganglia of the peripheral nervous system, the system comprising at least a first spheroid and a second spheroid, the first spheroid including dorsal root ganglia or neurons derived from the central nervous system or mammalian embryonic cells, and the second spheroid including at least one type of neuron or primary mammalian stem cells derived from the peripheral nervous system. The present disclosure relates to the manufacture of any of the systems disclosed herein by placing at least the first or second spheroid containing a magnetic material in a well or channel defined by a hydrogel and moving the first or second spheroid by aligning a magnet at or adjacent to the location of the well or channel. When the system mimics axons running between ganglia or groups of cells of the central nervous system and ganglia or groups of cells of the peripheral nervous system, in some embodiments, the first spheroid is placed in or near a first well or channel, and the second spheroid is a second well or channel, the second well or channel being at a distance sufficient to allow axonal outgrowth between the two spheroids after exposure to cell culture medium. The present disclosure relates to the measurement of recordings between ganglia or groups of cells of the central nervous system and ganglia or groups of cells of the peripheral nervous system, the method including placing electrodes on or near the first spheroid, placing electrodes on or near the second spheroid, and stimulating. Stimulating the system with an amplifier or generator equipped with a generator and passing a current through the system. In some embodiments, the method further includes measuring an electrophysiological response.

[0105] As used herein, the term "recording" refers to, for example, measuring the response of one or more neurons. Such responses may be, for example, electrophysiological responses such as patch clamp electrophysiological recordings or field potential recordings.

[0106] The present disclosure discloses methods and apparatus for obtaining physiological measurements of a microscale organotypic model of in vitro neural tissue that mimics clinical nerve conduction and NFD testing. The results obtained using these methods and apparatus can better predict clinical outcomes, enabling a more cost-effective approach for selecting promising lead compounds with a higher likelihood of success in later stages of development. The present disclosure includes the manufacture and utilization of a three-dimensional microengineering system that uniquely enables high-density and highly parallel neural fiber outgrowth. Due to the restricted nature of the neural fibers, this in vitro model can measure both CAP and intracellular patch-clamp recordings. Furthermore, subsequent confocal and transmission electron microscopy (TEM) analysis enables quantitative structural analysis, including NFD. In summary, this in vitro model system has novel functions similar to clinical histopathology and nerve conduction studies that evaluate histomorphometry and population electrophysiology.

[0107] The present disclosure also provides a method for measuring myelination of axons generated using the in vitro models described herein. Similar to the structure of human afferent peripheral nerves, dorsal root ganglion (DRG) neurons in these in vitro constructs project long, parallel, bundled axons peripherally. In native tissue, axons of various diameters and degrees of myelination conduct sensory information back to the central nervous system at various speeds. Schwann cells support sensory relay by insulating axons to myelinate them and enable more rapid conduction. Similarly, the three-dimensional outgrowth induced by this in vitro construct includes axons of various diameters with a dense, parallel orientation spanning distances of up to 3 mm. The presence and myelination of Schwann cells were observed in confocal and TEM imaging.

[0108] The morphology of neurons is a useful indicator of phenotypic maturity, but a clearer indication of a healthy neuron is its ability to conduct action potentials. Since many pathological changes can occur before cell death appears, cell death alone is not a complete measure of neuronal health. Electrophysiological examination of action potential generation can determine whether the observed structure supports the predicted function, and more predictive results can be obtained if clinically relevant evaluation items can be measured. Similarly, measurement of CAP indicates the overall health of myelin and provides further insight into the toxicity and neuroprotective mechanisms of various drugs or compounds of interest, while information gathered from imaging can determine quantitative metrics regarding the degree of myelination.

[0109] In some embodiments, the at least one agent comprises a small compound. In some embodiments, the at least one agent comprises at least one environmental or industrial contaminant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds selected from chemotherapeutic agents, analgesics, cardiovascular regulators, cholesterol, neuroprotective agents, neuromodulators, immunomodulators, anti-inflammatory agents, and antibacterial agents.

[0110] In some embodiments, the at least one agent comprises one or a combination of chemotherapeutic agents selected from 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, nitrosourea, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tafurposide, temozolomide (oral dacarbazine), teniposide, thioguanine (Tioguanine) (formerly Thioguanine), topotecan, tretinoin, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, visomodegib, and vorinostat.

[0111] In some embodiments, the at least one agent comprises one or a combination of analgesics selected from paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, opioids, flupirtine, tricyclic antidepressants, carbamazepine, gabapentin, and pregabalin.

[0112] In some embodiments, the at least one agent comprises one or a combination of cardiovascular regulators selected from nepicastat, cholesterol, niacin, scutellaria, prenylamine, dehydroepiandrosterone, monatepil, esketamine, niludipine, asenapine, atomoxetine, flunarizine, milnacipran, mexiletine, amphetamine, thiopental sodium, flavonoid, bretylium, oxazepam, and honokiol.

[0113] In some embodiments, the at least one agent comprises one or a combination of neuroprotective agents and / or neuromodulators selected from tryptamine, galanin receptor 2, phenylalanine, phenethylamine, N-methylphenethylamine, adenosine, kyotorphin, substance P, 3-methoxythyramine, catecholamines, dopamine, GABA, calcium, acetylcholine, epinephrine, norepinephrine, and serotonin.

[0114] In some embodiments, the at least one agent comprises one or a combination of immunomodulators selected from clenoliximab, enoticumab, rigerizumab, simtuzumab, batelizumab, palsatuzumab, imagatuzumab, tregalizumab, pateclizumab, namilumab, perakizumab, faralimomab, patritumab, atinumab, ublituximab, futuximab, and durigotumab.

[0115] 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.

[0116] In some embodiments, the at least one agent comprises one or a combination of antibacterial agents selected from antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, heat, radiation, and ozone.

[0117] The present disclosure further discloses a method for measuring both intracellular and extracellular recordings of biomimetic neural tissue in a three-dimensional culture platform. Heretofore, electrophysiological experiments have been conducted either in dissociated surface-seeded cultures or in organotypic slice specimens, each method having its own limitations. Studies in dissociated cell cultures are usually limited to single-cell recordings because there is no organized multicellular neurite structure that exists in organotypic specimens. Organotypic specimens have a complete neural circuit and allow for both intracellular and extracellular studies. However, in acute brain slices, complex and simultaneous variables are presented without means to control individual factors, and thus the processing speed may be essentially limited.

[0118] Intracellular recordings in in vitro three-dimensional cultures have been demonstrated heretofore. However, the growth of neurons has not been limited to anatomically valid structures that spatially assist the study of extracellular populations. A more biomimetic three-dimensional neural culture is required to enable testing of population-level electrophysiological behavior. The present disclosure supports synchronous population-level events in whole-cell patch-clamp techniques and extracellular field recordings resulting from limited neurite outgrowth in three-dimensional geometries. Prior to the present disclosure, measurements of these assessment items directly analogous to clinical nerve conduction tests have not been demonstrated in purely cell-based in vitro studies.

[0119] Using the methods and apparatus disclosed herein, field recordings are used to measure the complex extracellular changes in potential that occur by signal conduction in all recruited fibers. The population response elicited by electrical stimulation is the CAP. The electrically evoked population spikes are essentially graded and include the combined effects of action potentials in slow and fast fibers. The spike is a response composed only of action potentials with a rapid rise and short duration characteristic of the CAP or with rapid signal conduction in the absence of a single, focused event or synaptic input characteristic of the CAP. The three-dimensional nerve constructs disclosed by the present disclosure also support the CAPs that receive stimulation from more distant distances along neurite pathways or channels, demonstrating the ability of nerve cultures to rapidly carry signals from remote stimulation, such as in afferent peripheral nerves. The three-dimensional nerve cultures of the present disclosure support proximal and distal stimulation techniques useful for measuring conduction properties.

[0120] The present disclosure may also be used with one or more growth factors that induce recruitment of multiple fiber types, as is common in nerve fiber pathways. In particular, nerve growth factor (NGF) preferentially recruits small-diameter fibers that are often associated with pain signaling, as demonstrated by the data presented herein. Brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT-3) have been shown to preferentially support the outgrowth of larger-diameter proprioreceptive fibers. Electrophysiological studies may be incorporated with factors that affect growth, such as bioactive molecules and pharmacological agents, to systematically manipulate the conditions for studying mechanisms.

[0121] The three-dimensional neural cultures generated using the present disclosure can be used as a platform for investigating the mechanisms underlying myelin disorders and peripheral neuropathies by examining the effects of known myelin-disrupting substances, culture conditions that induce neuropathy, and toxic neuropathy-inducing compounds on these neural cultures. The present disclosure enables the use of conduction velocity as a functional measure of the integrity of myelin and nerve fibers under toxic and therapeutic conditions, thereby facilitating research on the safety and efficacy of drugs. Incorporating genetic mutations and drugs into the neural cultures generated using the techniques disclosed herein enables the reproduction of disease phenomena in a controlled manner, which may lead to a better understanding of neurodegeneration and potential therapies.

[0122] The present disclosure provides an apparatus, method, and system for the generation, maintenance, and physiological investigation of microengineered neurons and neural networks designed to mimic the anatomical structure of natural neural tissue. In some embodiments, the apparatus and system comprise one or more cultured or isolated Schwann cells and / or one or more cultured or isolated oligodendrocytes in contact with one or more neurons 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 internal chamber, the shape of the internal chamber being at least partially defined by the at least one inner surface and being accessible from a point external to the solid substrate through at least one opening in the at least one outer surface, the cell bodies of the one or more neurons being disposed at one end of the internal chamber and axons being capable of extending within the internal chamber along at least one length of the internal chamber such that the tip of the axon extends distally from the cell body. In some embodiments, the inner surface of the solid substrate is cylindrical or substantially cylindrical, such that the cell bodies of the neurons are disposed proximate to an opening at one end of the cylindrical or substantially cylindrical inner surface and the axons of the neurons comprise a length of cellular material extending along the length of the inner surface from a point at the end of the cell body to a point distal from the cell body. In some embodiments, the inner surface of the solid substrate is cylindrical or substantially cylindrical, such that the cell bodies of the neurons are disposed proximate to an opening at one end of the cylindrical or substantially cylindrical inner surface and the axons of the neurons comprise a length of cellular material extending along the length of the inner surface from a point at the end of the cell body to a point distal from the cell body.In some embodiments, the inner surface of the solid substrate is cylindrical or substantially cylindrical, such that the cell bodies of the nerve cells are disposed proximate to an opening at one end of the cylindrical or substantially cylindrical inner surface, and the axons of the nerve cells include a length of cellular material extending along the length of the inner surface from a point at the end of the cell body to a point distal from the cell body. When the cell culture vessel contains multiple types of nerve cells, the multiple axons extend from the cell bodies such that they define a bundle of axons that can extend distally along the length of the inner surface from the cell bodies. In some embodiments, the nerve cells extend on and within the penetrable polymer. In some embodiments, one or more electrodes are disposed at or proximate to the tip of at least one axon and one or more electrodes are disposed at or proximate to the cell body such that a potential is established across the entire length of one or more of the nerve cells.

[0123] Another object of the present disclosure is to provide a medium- to high-speed processing assay of neurological function for screening the pharmacological and / or toxicological properties of chemical and biological agents. In some embodiments, the agent is a cell such as any type of cell disclosed herein, or an antibody such as an antibody used to treat clinical diseases. In some embodiments, the agent is any drug or agent used to treat human diseases such that toxicity, effect, or neuromodulation can be compared between a novel agent that is a proposed mammalian therapeutic agent and a therapeutic agent from an existing human disease. In some embodiments, the novel agent for the treatment of human diseases is a therapeutic agent for neurodegenerative diseases and is compared with existing therapeutic agents for neurodegenerative diseases. In the case of multiple sclerosis as a non-limiting example, the effect of a novel agent (modified cell, antibody, or small compound) is compared and contrasted with the same effect of existing therapeutic agents for multiple sclerosis such as Copaxone, Rebif, other interferon therapeutic agents, Tysabri, dimethyl fumarate, fingolimod, teriflunomide, mitoxantrone, prednisone, tizanidine, baclofen.

[0124] Another object of the present disclosure is to use the assembly of unique technologies such as nerve bundles by two-dimensional and three-dimensional microengineering in conjunction with electrophysiological stimulation and recording of neuronal populations.

[0125] Another object of the present disclosure is to provide a novel method for evaluating neurophysiology in vitro by using compound action potential (CAP) as a clinically similar metric and obtaining results that are more sensitive and predictive of human physiology than the results obtained by current methods.

[0126] Another object of the present disclosure is to provide microengineered nerve tissue that mimics natural anatomical and physiological features and is sensitive to evaluations using high-speed processing electrophysiological stimulation and recording methods.

[0127] Another object of the present disclosure is to provide methods for replicating, manipulating, modifying, and evaluating the mechanisms underlying myelin disorders and peripheral neuropathies.

[0128] Another object of the present disclosure is to enable medium- to high-throughput assays of neural regulation in human neurons for screening the pharmacological and / or toxicological activities of chemical and biological agents.

[0129] Another object of the present disclosure is to use unique assemblies of techniques such as neural bundles by two-dimensional and three-dimensional microengineering in conjunction with optical and electrochemical stimulation and recording of human neuronal populations.

[0130] Another object of the present disclosure is to quantify the evoked postsynaptic potentials in a biomimetic, engineered thalamocortical circuit. The inventors' observation of retrogradely generated population spikes in neural pathways suggests that they enable population-level physiology such as the conduction of compound action potentials and postsynaptic potentials.

[0131] Another object of the present disclosure is to enable non-invasive stimulation of evoked potentials in neural circuits and recording of multi-unit physiological responses using optogenetic methods of illumination, hardware and software control of illumination, and fluorescence imaging.

[0132] Another object of the present disclosure is to examine whether the above agents change the developmental maturation of the above circuits using microengineered circuits in tests of selective serotonin reuptake inhibitors (SSRI) and second-generation antipsychotics.

[0133] In one embodiment, as shown in FIG. 1, a combination of polyethylene glycol dimethacrylate and Puramatrix hydrogel is micropatterned using projection photolithography with a digital micromirror device (DMD). By this method, one or more hydrogels can be directly and rapidly micropatterned onto conventional cell culture materials. Since the photomask never contacts the gel material, multiple hydrogels can be successively and rapidly cured, enabling the fabrication of dozens of gel constructs in one hour without automation. This technique can be used to confine neurite outgrowth within a biomimetic growth-promoting gel using mechanically robust, cell elongation-inhibiting polyethylene glycol (PEG) gels. In some embodiments, this growth-promoting gel may be Puramatrix, agarose, or methacrylated dextran. When embryonic dorsal root ganglion (DRG) explants grow in this confined three-dimensional environment, axons grow out from the ganglion in a dense and fasciculated manner, as shown in FIGS. 5 and 6. Most of the axons appear as small-diameter, unmyelinated fibers that grow to a length approaching 1 mm in 2 to 4 weeks. The structure of this culture model, with its dense and highly parallel three-dimensional nerve fiber tracts extending from the ganglion, is generally similar to the peripheral nerve structure. Its morphology can be evaluated using neuromorphometry, enabling clinically similar evaluations not available with conventional cell assays.

[0134] In some embodiments, the culture model provides the ability to record electrically induced population field potentials arising from compound action potentials (CAPs). Exemplary recording curves exhibit characteristic, uniform, fast, short latency, population spike responses that maintain consistency at high frequency (100 Hz) stimulation, as shown in B of FIG. 8. As shown in E of FIG. 8 and 8 of F, the CAP is reversibly abolished by tetrodotoxin (TTX), demonstrating that drugs can be applied and that the drugs can have an effect. The delay to rise associated with distal pathway stimulation, shown in C of FIG. 8 and 8 of D, is measurably increased. The response is insensitive to neurotransmitter blockers, indicating that the induced response is primarily CAP rather than the synaptic potential shown in FIG. 10. Embryonic DRG cultures have been effectively used as a model for peripheral neurobiology for decades. While conventional DRG cultures are very useful as a model system, they have been found to not adequately predict clinical toxicity when evaluated with conventional cell viability assays. While it is possible to perform single cell patch clamp recordings in DRG cultures, there are no reports of CAP recordings due to the lack of tissue architecture. In a preferred embodiment, the present disclosure provides the ability to evaluate histomorphometry and population electrophysiology, as well as clinical histopathology and nerve conduction tests.

[0135] In some embodiments, the present disclosure uses human neurons to grow neural tissue in a three-dimensional environment that mimics the natural neural structure, where the neuronal cell bodies are bundled together and located at a different position from the axonal fiber tracts, enabling the measurement of histomorphometric and electrophysiological data, including CAPs. In some embodiments, the present disclosure uses neurons and glial cells derived from primary human tissue. In other embodiments, the neurons and glial cells may be derived from human stem cells, including induced pluripotent stem cells.

[0136] In another embodiment, the present disclosure uses conduction velocity as a functional measure of the state of neural tissue under toxic and therapeutic conditions. Information regarding the degree of myelination, the integrity of myelin, axonal transport, mRNA transcription, and neuronal damage can be determined from electrophysiological analysis. In combination with neuropil density, myelination rate, and morphometric analysis of nerve fiber types, the mechanism of action of a compound of interest can be determined. In some embodiments, the devices, methods, and systems disclosed herein incorporate genetic mutations and drugs to reproduce disease phenomena in a controlled manner, leading to a better understanding of neurodegeneration and possible treatment methods.

[0137] The present disclosure relates to systems comprising any of the disclosed compositions and methods of using these systems to obtain ambient data that is more physiologically relevant than data collected using a 2D tissue culture system or a system that does not use multiple cell types. In some embodiments, the systems or compositions disclosed herein comprise / contain one or more cells comprising any mutation. In some embodiments, at least 1, 100, 500, 1000, or more cell types comprise mutations relevant to a particular model of a human disease. Any of the disclosed systems may comprise cells having the mutations disclosed in Table B described hereinafter. In some embodiments, the cells of the present disclosure comprise or express an endogenous mutant protein or at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% mutant protein disclosed in Table B. Thus, these model systems may be useful for testing the efficacy or toxicity of a particular drug, biomolecule, or other therapeutic agent added to the system. The models may also be useful for understanding the underlying biology in the context of the form of action of environmental pollutants, pathogens, or endogenously expressed proteins, and such molecules on the nervous system, based on information such as axonal outgrowth, myelination, and response to substances involving demyelination, or morphological changes in the cells themselves.

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[0138] In some embodiments, at least one cell of the disclosed system comprises any one or more mutations at the locus identified in Table B. If Table B discloses an mRNA sequence, the one or more mutations of the cell may be present in the complementary endogenous DNA sequence disclosed in the GenBank sequence above. In some embodiments, the cell comprises one or more mutations in the sequence of Table B identified above, or at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the sequences disclosed in Table B, or if the sequence is an mRNA sequence, the cell comprises a mutation in one or more complementary DNA sequences of these sequences identified in Table B, or at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the sequences disclosed in Table B, or a mutation complementary to the sequence.

[0139] In some embodiments, the spheroids disclosed herein comprise 2, 3, 4, or 5, or more mutations in the genes identified in Table B. If a spheroid comprising a particular mutation identified in Table B is used within the system disclosed herein, the corresponding system may be used as an in vitro model of the corresponding medical condition identified above.

[0140] In some embodiments, any of the compositions, systems, or methods described in PCT / US2015 / 050061 may also be used in the embodiments of the present disclosure.

[0141] In some embodiments, the method is a method of manufacturing a system, culture plate, or apparatus for culturing cells, comprising: obtaining stem cells such as induced pluripotent stem cells; exposing the cells to one or more cell growth factors; differentiating the stem cells into nerve cells; seeding the cells into a solid substrate having first and / or second cavities or wells; The method includes the above steps. In some embodiments, the first and / or second cavities are U-bottom wells, curved-bottom wells, or flat-bottom wells. In some embodiments, the method includes seeding about 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 90,000, 100,000, 125,000, 150,000, 175,000, 200,000, 225,000, or 250,000 cells. In some embodiments, the step of seeding the cells includes seeding one or more types of cells into a series of cavities or wells separated within a solid substrate and each containing a cell culture medium. In some embodiments, the step of seeding the cavities or wells includes seeding the cells in a pattern disposed within the solid substrate such that each well contains a spheroid of cells and each spheroid grows in suspension or hanging drop format. In some embodiments, the method of manufacturing a system, culture plate, or apparatus for culturing the cells includes culturing the cells without disturbing them for a time sufficient for the cells to spontaneously form one or more spheroids.

[0142] The present disclosure also relates to a method for testing the toxicity of a drug by exposing the drug to one or more spheroids on or within a cavity or within a solid substrate or on or within a cavity or well. In some embodiments, the method comprises exposing the drug to the one or more spheroids for a time sufficient for the drug to be absorbed by one or more cell types of the one or more spheroids, and then measuring the viability of the cells by recording, observing, or a combination of both, morphological changes.

[0143] The present disclosure also relates to a method for forming spheroids of cells derived from stem cells or cells derived from the nervous system of a subject. In some embodiments, the method for forming the spheroids comprises (i) differentiating the cells from stem cells into one or more cell types that are one or more of neurons, astrocytes, Schwann cells, or any other cell disclosed herein, and then (ii) mixing the one or more cells for a time sufficient to form spheroids and. In some embodiments, the method does not include a step of differentiating any cells after the spheroids are formed. In some embodiments, the method does not include exposing the spheroids or any cells to one or more DRGs.

[0144] The following examples are intended to be non-limiting examples of the methods of manufacture and use of the embodiments disclosed in this application. Publications, patents, or patent applications disclosed in the examples or the body of the specification are incorporated by reference in their entirety.

Example

[0145] Example 1: Human Motor-Nerve-On-A-Chip for Preclinical Neurotoxicity Testing The goal was to develop an organotypic microphysiological model that mimics peripheral nerve morphology and supports clinically similar physiological measurements.The Nerve-On-A-Chip design was fabricated using a microengineered hydrogel scaffold (Figure 1, A and B).

[0146] The goal of this design was to direct and restrict 3D axonal outgrowth and cell positioning to mimic nerve fiber tracts (Figure 2). Robust neuronal growth, fasciculation, and glial interactions facilitated morphological and physiological outputs as a high-content screening assay for neurotoxicity and pharmacological manipulations (Figure 3).

[0147] The results showed a structure similar to the anatomy of native peripheral nerves, allowing examination of nerve density, fiber type, and myelination, as well as axonal outgrowth, cell migration, and glial differentiation (Figures 4-7). Example 2: Rat spinal cord nano-shuttle spheroid protocol Day 1 (Figure 12): 1. Isolate six spinal cords from E15 rat pups, ensuring all dorsal root ganglia are present Remove. 2. Using spring-handled scissors, cut all six spinal cords into small chunks. 3. Using a 1000 μL pipette, transfer the pelleted spinal cord and media to a 1.5 mL microcentrifuge tube. Pellet the spinal cord chunks by centrifugation at 4.700 rcf for 2 minutes and 30 seconds. 5. Remove the supernatant from the microcentrifuge tube, being careful not to disturb the pellet. 6.1 mL of 0.25% trypsin-EDTA is added to the microcentrifuge tube and the pellet is broken up and suspended in the trypsin. 7.Incubate at 37°C for 15 minutes. 8. Quench the trypsin-EDTA by adding the trypsin + cell suspension to a 15 mL tube containing 1.5 mL of trypsin inhibitor solution. Centrifuge at 9,700 rcf for 5 minutes to pellet the spinal cord mass. Carefully remove the supernatant from the 15 mL conical tube without disturbing the pellet. Add 2 mL of spinal cord seeding medium to the 15 mL conical tube containing the pellet. Using a 100 μL pipette, triturate 15 times (set the pipette to 1 mL volume) to break up the mass. Add an additional 3 mL of spinal cord seeding medium to the dissociated spinal cord solution. Pass all 5 mL of the dissociated spinal cord solution through a 40 μm cell strainer and collect the filtered medium in a Petri dish. Add 300 μL of the dissociated spinal cord solution to each 12-well PLL coverslip. The medium should remain bubbly on the coverslip, and care should be taken not to overfill the well with liquid beyond the edge of the coverslip. To ensure complete coverage of the coverslip, gently tilt the 12-well plate so that the dissociated spinal cord solution spreads across the entire coverslip. Repeat tilting in additional directions as needed to achieve complete coverage. Incubate the seeded cells at 37 °C for 2 hours. After 2 hours, gently remove the seeding medium and replace it with 700 μL of N2:NG medium. Add the N2:NG medium to the side of the well to prevent the seeded cells from being removed from the substrate by the fluid flow. Day 2: To count, sacrifice the seeded cells in one well by adding 0.5 mL of 0.25% trypsin-EDTA to the well. Incubate at 37 °C for 4 minutes. Transfer the trypsin and cells aspirated from the substrate to a 1.5 mL microcentrifuge tube containing 0.5 mL of neurobasal medium to dilute the trypsin. Triturate 10 times to disrupt cell-cell adhesion and obtain a cell solution. Count the cells present in the well using trypan blue and a hemocytometer. 6. Add the nanoshuttle to the remaining wells at a rate of 1 μL per 60,000 cells. Disperse the nanoshuttle solution over the entire area of the coverslip. 7. Return this to the incubator and incubate further. Day 3: 1. Add 0.5 mL of 0.25% trypsin-EDTA to each well. 2. Incubate at 37 °C for 4 minutes. 3. Transfer the trypsin and cells aspirated from the substrate to a 15 mL conical tube containing an amount of N2:NG medium equal to the amount that dilutes the trypsin. 4. Centrifuge the above cell solution at 700 rcf for 5 minutes. 5. Remove the supernatant and replace it with 2 mL of N2:NG medium. 6. Disrupt the cell pellet by grinding 5 times using a 1 mL syringe and a 20-gauge needle. 7. Add an additional 5.2 mL of N2:NG medium to the above cell suspension (total 7.2 mL). 8. Take a 10 μL sample from this cell suspension and calculate the number of cells per mL using trypan blue and a hemocytometer. 9. Calculate the amount of cell suspension required for addition to each well of a 96-well plate. For example, 500,000 cells, 400,000 cells, or 300,000 cells per well 10. Place the non-adhesive 96-well plate securely on the magnetic drive device (Figure 11), and then add an appropriate amount of cell solution to each well of the non-adhesive 96-well plate. If necessary, add N2:NG medium to the wells so that the volume in each of the 96 wells is 150 μL. 11. Return the above 96-well plate to the incubator and allow spheroids to form without disruption for 2 days. Day 5: 1. Prepare the PEG construct. Refer to other protocols if a guide is needed. 2. Wash 3 times with 2% anti / anti washing solution. 3. Store in the washing solution at 37 °C overnight. Day 6: 1. Remove the 96-well plate from above the magnetic drive device. 2. Using a 1000 μL pipette (set to 100 μL), gently aspirate / dispense the liquid in each well of the 96-well plate to suspend the spheroids. 3. Using a 10 μL pipette (set to 10 μL), remove the spheroids from the 96-well plate. 4. Add the above spheroids to 2 mL of medium in a Petri dish on ice. 5. Transfer the above spheroids back to an empty Petri dish on ice. Use a 10 μL pipette set to 4 μL. This operation helps to remove any cell debris transferred from the 96-well plate. 6. Prepare a 1:20 dilution of Matrigel for encapsulating cells by mixing Matrigel with N2:NG medium while keeping all solutions on ice (below 10 °C) at all times to prevent gelation (taking into account the medium added by the transfer of the spheroids). Example: For a 200 μL of 1:20 Matrigel dilution containing 4 spheroids, it contains 10 μL of Matrigel, 178 μL of N2:NG medium, and 12 μL of medium transferred with the spheroids. Thus, in this step, add 10 μL of Matrigel to 178 μL of N2:NG medium. 7. Add the above mixed Matrigel and N2:NG medium to the spheroids in the Petri dish in the upper airspace. 8. Attach a slide glass coated with Rain-X to the above magnetic positioning device (Figure 13). 9. Place a Transwell insert containing the above PEG construct on the above slide glass. 10. Operate the above magnetic drive device to adjust the position of the magnet below the void where the spheroids are to be placed. 11. Using a pipette set to 10 μL, transfer the spheroids and the 1:20 Matrigel solution to the above void. Release the spheroids above the magnet (B in Figure 14). 12. Repeat steps 10 and 11 for all constructs in the Transwell insert. Gelatinize Matrigel at 13.37 °C for 30 minutes. 14. Add N2:NG medium under the insert. 15. Incubate as desired.

[0148] Example 3: Spheroids for Directional Neurite Outgrowth Round-bottom / U-bottom plate: For either one differentiated cell type or a combination of differentiated cell types, a 96-well, clear "U-shaped" round-bottom, untreated spheroid microplate (Corning REF: 4415) was used. Using a hemocytometer, re-suspend the cells and count them again. Accordingly, using a micropipettor, add to each well the density required for each spheroid from 5,000 to a maximum of 100,000 cells. Then centrifuge this spheroid microplate for 5 minutes at the centrifugation speed corresponding to the cell type with the suspension, and place it in an incubator at 37 °C for more than 24 hours until spheroids are formed.

[0149] Hanging drop plate: The Perfecta3D hanging drop plate of 3D biomatrix was used for spheroid production. A known amount of neurons and glial cells derived from differentiated induced pluripotent stem cells, about 5,000 - 100,000, were suspended in a small amount of medium. The ratio of differentiated cells was varied to enable spheroid formation and growth characteristics after spheroid formation. The cells were suspended in a volume of 40 ul and pipetted into the access holes at the top of the plate. Then the cells were left standing in a conventional 5% CO2 incubator for at least 24 hours for self-organization to form spheroids.

[0150] The following table shows various methods of spheroid production.

Table 1-2

Table 2-1

Table 2-2

[0151] Migration of spheroids into the Nerve-On-A-Chip construct To move the spheroids for placement in the three-dimensional construct, within a 6-well tissue culture-treated plate (Transwell, 24 mm diameter, 0.4 um pore size, REF: 3450 - transparent), for each well, the construct is dried by removing 500 ul of the total 1500 ul of PBS used, where the top of the membrane is partially dried for placing the spheroids. Then 8% Matrigel is added inside the three-dimensional structure and then placed in an incubator at 37 °C for 30 minutes.

[0152] Next, the above spheroids are removed using a p1000 pipettor and placed as droplets on a 35 mm tissue culture-treated dish (Cell Treat catalog number: 229635). Then, using sterilized Dumont #4 forceps (11 cm in length, standard 0.13 × 0.08 mm Dumostar 11294 - 00), the spheroids are placed into the "valve part" of the three-dimensional construct. Then 1500 ul of medium is placed under the membrane of the above 6-well plate and placed in an incubator at 37 °C.

[0153] Example 4: Three-dimensional muscle cell encapsulation part of the neuromuscular junction (NMJ) Undifferentiated primary human myoblasts are seeded on a non-coated tissue culture vessel at the density specified by the supplier, and growth medium containing serum is supplied on day 1, day 2, day 4, etc., to trigger cell division until a culture density of 60% is reached. When the 60% culture density is reached, the cells have been passaged up to passage 6 with trypsin. At passage 6 (P6) and when the 60% culture density is reached, the primary human myoblasts are removed from the culture vessel with trypsin, centrifuged, resuspended in the medium for counting, centrifuged again, and resuspended in DMEM / F12 to a concentration of 8 million cells / mL.

[0154] Prepare a solution of 5% GelMA, the added laminin, and 0.05% LAP solution containing n - vinylpyrrolidone, and mix it with the cell suspension so that the cells reach a concentration of 2 million cells / mL. Pipette the myoblast / GelMA / LAP solution into a specific chamber of a pre - made polyethylene glycol (PEG) construct that is impenetrable to cells, where in the said construct, the said chamber is separated from any chamber that houses motor neurons. Realize the polymerization of cell - loaded GelMA / LAP containing 2 million cells / mL by exposing the solution in the chamber to UV light.

[0155] Another alternative method requires directly resuspending the said cells at a concentration of 2 million cells / mL into the said GelMA / LAP solution. (The steps of suspension in the medium and the second centrifugation are omitted).

[0156] Differentiation of myoblasts in three dimensions is realized by medium replacement. On days 1 - 3, it is necessary to supply the same growth medium as above to the said construct. The medium replacement on day 4 changes the medium to a differentiation medium consisting of DMEM / F12 and horse serum.

[0157] By the method of high - density encapsulation, as a result of the medium, the construct differentiates over a maximum of three weeks, and paracrine - type signal transduction is realized.

[0158] Differentiation is confirmed by using a histological technique that includes fluorescently labeling muscle cells with antibodies against proteins that are only expressed by multinucleated myotubes containing anti - desmin, anti - alpha - heavy - chain myosin, and DAPI, or by checking whether more than two nuclei are contained in a single cell body.

[0159] Example 5: Examination of spheroids In this study, we describe an in vitro, microengineered, biomimetic, all-human peripheral nerve (Human-Nerve-on-a-Chip [HNoaC]) composed of induced pluripotent stem cell (iPSC)-derived neurons (hN) and primary human Schwann cells (hSC) that can provide data suitable for the assessment of integrated nerve conduction velocity (NCV) and histopathology. This all-human system is an important extension of the in vitro "Nerve-on-a-Chip" (NoaC) platform that we previously developed using embryonic rat dorsal root ganglion (DRG) neurons and rat SCs. To the best of our knowledge, this combination of hN and hSC has not been achieved in any other stem cell-based in vitro nervous system to date. This model mimicked robust axonal outgrowth (up to ~5 mm) and showed the first evidence of myelination of human iPSC-derived neurons by human Schwann cells and the first evidence of testing nerve conduction velocity in an all-human in vitro system such as an in vitro model. Thus, the innovative HNoaC model of the human peripheral nerve has the potential to accelerate the fields of human disease modeling, drug discovery, and toxicity screening. 5 To the best of our knowledge, this combination of hN and hSC has not been achieved in any other stem cell-based in vitro nervous system to date. This model mimicked robust axonal outgrowth (up to ~5 mm) and showed the first evidence of myelination of human iPSC-derived neurons by human Schwann cells and the first evidence of testing nerve conduction velocity in an all-human in vitro system such as an in vitro model. Thus, the innovative HNoaC model of the human peripheral nerve has the potential to accelerate the fields of human disease modeling, drug discovery, and toxicity screening.

[0160] Culturing of Schwann cells A T-75 culture flask (353136; Corning, Corning, NY) was prepared by coating it with a sterile-filtered solution of 0.1% poly-L-ornithine (PLO; Sigma-Aldrich, St. Louis, MO) in sterile water (Sigma-Aldrich, St. Louis, MO). The flask was then washed four times with sterile water. 7.5 mL of a 10 μg / mL laminin (Sigma-Aldrich, St. Louis, MO) solution in phosphate-buffered saline (PBS; Caisson Labs, Smithfield, UT) was added to the flask and kept at 4 °C overnight. The laminin solution was aspirated, 15 mL of culture medium was placed directly into this T-75 culture flask, and after equilibration in an incubator at 37 °C, cell seeding was performed. The human Schwann cell (hSC) medium was purchased from ScienCell (Carlsbad, CA). The human Schwann cell line (catalog number 1700; ScienCell) was received in a cryopreserved vial and, according to the report, was more than 5×10 5 cells / mL. This vial was taken out of cryopreservation and thawed in a 37 °C water bath. The contents of the vial were evenly distributed onto the PLO / laminin-coated T-75 flask. The culture was left standing at 37 °C in a 5% CO2 atmosphere for at least 16 hours to promote attachment and growth. The medium was changed every 24 hours. When a culture density of 80% was reached, the hSCs were passaged by using 3 mL of Accutase® (Sigma-Aldrich), and Accutase® was added to the flask at 37 °C for 3 minutes. When the cells were completely detached, 8 mL of hSC medium was placed into the flask. The 11 mL solution of detached hSCs was transferred to a 15 mL conical tube and centrifuged at 200×g (Eppendorf 5810R centrifuge, radius 18 cm, Eppendorf, Hamburg, Germany) at room temperature (RT, approximately 22 °C) for 5 minutes. The supernatant was aspirated, and the pellet was resuspended in 1 mL of hSC culture medium. The cells were counted using a conventional hemocytometer (Hausser Scientific, Horsham, PA).

[0161] Culture of Motor Neurons

[0162] The iCell® motor neuron (hN) medium was prepared using 100 mL of iCell® Neurons Base Medium (FUJIFILM Cellular Dynamics, Inc) supplemented with 2 mL of iCell® Neural Supplement A (FUJIFILM Cellular Dynamics, Inc, Madison, WI) and 1 mL of iCell® Nervous System Supplement (FUJIFILM Cellular Dynamics, Inc). To prepare for thawing the motor neurons, the hN medium was warmed to room temperature and 1 mL of the hN medium was added to a sterile 50 mL conical tube. One vial of iCell® human motor neurons (hN; FUJIFILM Cellular Dynamics, Inc) was thawed in a 37°C water bath for approximately 2 minutes and 30 seconds. The contents of this vial were transferred to a 50 mL conical tube containing 1 mL of hN medium by dripping with a swirling motion to thoroughly mix the cell solution and minimize osmotic shock to the thawed cells. The cell vial was then rinsed with 1 mL of hN medium and transferred to the 50 mL tube. Next, the volume of this solution was adjusted to 10 mL by slowly adding hN medium dropwise (2 - 3 drops / second) while swirling the 50 mL centrifuge tube. The cell solution was then transferred to a 15 mL conical tube and centrifuged at 200×g for 5 minutes at room temperature. The supernatant was aspirated, the tube was tapped with a finger, and then the cells were resuspended in 1 mL of hN medium by pipetting up and down 2 - 3 times. Next, a 10 μL sample of the cell solution was taken and the cells were counted using a hemocytometer.

[0163] Generation of spheroids

[0164] An untreated, transparent, "U"-bottomed, 96-well spheroid microplate (4515; Corning) was used for monoculture of both human neurons (hN) and human Schwann cells (hSC) as well as co-culture of hN / hSC. The concentration expressed as cells / μL-medium was calculated for both hSC and hN, and the volumes required to generate spheroids of the following sizes and compositions, namely, monoculture of hN - 100,000, 75,000, 50,000, or 25,000 cells; monoculture of hSC - 75,000, 50,000, or 25,000 cells; co-culture, either 75,000 hN and 75,000, 50,000, or 25,000 hSC, were made calculable. The calculated volume was added to the microwell plate, and the volume of each well was made 200 μL by adding medium warmed to 37°C. The spheroid microplate was then centrifuged at 200×g for 5 minutes and placed in an incubator at 37°C in a 5% CO2 atmosphere until spheroid formation was observed (usually about 48 hours). The hN medium was changed every other day by replacing half of it with 95 μL and replenishing it with 100 μL of fresh warmed (37°C) hN medium.

[0165] 3D double hydrogel nerve growth construct

[0166] On the membrane of a Transwell® insert (0.4 μm / PES; Corning), as previously reported 6A double hydrogel scaffold was fabricated using a microphotolithography technique similar to the method. Unless otherwise stated, all solutions were prepared using sterile filtered PBS. The outer cell-restricting (i.e., growth-resistant) photo-translinkable hydrogel was prepared using a solution of polyethylene glycol dimethacrylate 1000 (PEGDMA; Polysciences, Warrington, PA) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Sigma Aldrich). First, a 10% w / v PEGDMA solution and a 1.1 mM LAP solution were prepared and mixed in a 1:1 solution. The resulting solution was sterile filtered and placed on a Transwell® insert placed on a slide glass treated with Rain-X (ITW Global Brands, Glenview, IL) in a volume of 0.6 mL, while being placed under the lens of a digital micromirror device (DMD, PRO4500 Wintech Production Ready Optical Engine; Wintech Digital Systems Technology Corp, Carlsbad, CA) (Figure 1). The mask and polymerization parameters were selected using commercially available software (DLP Lightcrafter 4500 Control Software, Texas Instruments, Dallas, TX), and the photo-translinkable solution was irradiated with ultraviolet light at a wavelength of 385 nm for 28 to 32 seconds. After treatment, the excess PEGDMA / LAP solution was removed from the voids created by the insert and the photomask. The construct was then washed three times for 10 minutes each at the top and bottom of the insert using a 2% antibiotic / antifungal wash buffer (Thermo Fischer Scientific, Walton, MA). The wash buffer was removed from the insert and the internal keyhole-shaped channels. To create a cell-permeable scaffold, the voids were carefully filled with an 8% growth factor-reduced Matrigel® matrix (Corning) and polymerized in an incubator at 37°C.

[0167] Migration of spheroids into hydrogel constructs To induce myelin formation in three-dimensional constructs, two media were prepared using hN medium (described above). The pre-myelination medium was prepared using hN medium, 10% HyClone fetal bovine serum (FBS; LaCell LLC, New Orleans, LA), and 1% antibiotic-antifungal buffer. The myelination medium was prepared using hN medium, 10% FBS, 10 ng / mL of recombinant rat β-nerve growth factor (NGF; R&D Systems, Minneapolis, MN), and 50 μg / mL of L-ascorbic acid (Sigma-Aldrich). After spheroids were formed, they were transferred from the microplate using a pipette and placed as droplets of hN medium onto a 35-mm tissue culture-treated dish (Cell Treat, Pepperell, MA). Then, using sterile Dumont #5 fine-tip forceps (11295-10; Dumont, Montignez, Switzerland), the spheroids were placed into the "valve part" of the three-dimensional construct within Matrigel. Finally, 1.5 mL of the pre-myelination medium was placed under the Transwell® membrane of the 6-well plate described above, and the loaded hydrogel construct was placed in an incubator with a 5% CO2 atmosphere at 37 °C for culturing. The medium was changed by half every other day. After the above constructs were maintained in the pre-myelination medium for one week, they were changed to the myelination medium and maintained for three weeks.

[0168] Immunocytochemistry All wells of the 6-well culture plate were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA), pH 7.4, for 30 minutes at room temperature and then washed four times with PBS for 15 minutes each. Next, the fixed samples were placed in a 1× blocking solution containing PBS, 5% goat normal serum (Jackson ImmunoResearch, West Grove, PA), 0.2% Triton-X-100 (Sigma-Aldrich), and 0.4% bovine serum albumin (Sigma-Aldrich) for 1 hour at room temperature, followed by labeling overnight at 4°C in the blocking solution with the following primary antibodies: rabbit-α-sl00 (ab868, 1:400; Abcam, Cambridge, MA) or mouse-α-βIII tubulin (ab78078, 1:500; Abcam). In another trial, rabbit-α-myelin basic protein (MBP, ab133620, 1:500; Abcam) was also used under the same incubation conditions. The next day, the wells were washed four times with PBS for 8 minutes each at room temperature. The plate was then labeled with secondary antibodies, Alexa 488 goat anti-rabbit IgG (1:300, Abam) or Alexa 568 goat anti-mouse IgG (1:300, Abam), and DAPI (1:200, Sigma-Aldrich). The secondary antibodies and DAPI were dissolved in the I× blocker solution for 90 minutes at room temperature in the dark. The plate was washed five times with PBS for 8 minutes each at room temperature in the dark. Next, the plate was sealed with parafilm, covered with metal foil, maintained at 4°C, and then microscopic observation was performed using a Nikon Al confocal microscope (Nikon, Tokyo, Japan).

[0169] Embedding with plastic resin All materials used for embedding were purchased from Electron Microscopy Sciences unless otherwise noted, handled under draft, and used while wearing recommended personal protective equipment. The hydrogel constructs were removed from the cultures and the two sides of the membrane-through wells were washed three times with PBS at room temperature and then fixed. The hydrogel constructs were then immersed in a solution of 4% PFA / 0.5% glutaraldehyde for 30 minutes at room temperature. Secondary fixation and staining of cell lipids were achieved by post-fixation for 2 hours at room temperature in the dark using a PBS, pH 7.4 solution of 1% osmium tetroxide. The constructs were then washed three times with PBS for 15 minutes each and then counterstained for 30 minutes at room temperature in the dark using an aqueous solution of 2% uranyl acetate. Dehydration was performed by stepwise ethanol washes at room temperature starting with a 10-minute wash in 50% ethanol / PBS, followed by a 10-minute wash in 70% ethanol / PBS, and an overnight wash in 90% ethanol / PBS. The next day, the constructs were washed twice with 100% ethanol for 30 minutes each at room temperature. Under a dissecting microscope, the hydrogel constructs were individually dissected from the membrane-through wells without removing the PEGDMA using a scalpel. The constructs were placed in a flat embedding mold (EMS 70902, Electron Microscopy Sciences). After allowing time for the residual ethanol to evaporate from the fixed hydrogels, they were replaced with an infiltration medium consisting of a 1:1 mixture of Spurr resin (low viscosity embedding medium Spurr kit; Electron Microscopy Sciences) and propylene oxide. After allowing the infiltration medium to stand for 75 minutes, it was replaced with 100% Spurr resin, which was cured in an oven at 70 °C overnight and at room temperature for 48 hours, after which thin sections were prepared using an ultramicrotome. Section Preparation and Transmission Electron Microscopy (TEM) Section preparation and evaluation by TEM were performed at the Shared Instrumentation Facility (SIF) of Louisiana State University (Baton Rouge, LA). Ultra-thin sections were cut to a thickness of 80 - 100 nm at four locations within the HNoaC specimen, i.e., within the tissue bulb (wherein, in the said bulb, the said bubble intersects the said channel and the said proximal channel (i.e., near the bulb), as well as the distal channel). The sections were placed on Formvar carbon-coated copper grids, 200 mesh, and impregnated with metal by floating them on droplets of 2% uranyl acetate for 20 minutes at room temperature. These sections were then rinsed three times for 1 minute with droplets of deionized water. For visualization, a JEOL 1400 TEM (Peabody, MA) was used at an accelerating voltage of 120 kV at various magnifications.

[0170] Morphometric analysis Metrics obtained from TEM images of HNoaC cross-sections included axon diameter and G-ratio (i.e., the ratio of axon diameter to the diameter of the entire fiber [axon + myelin sheath]). Axon diameter and G-ratio were determined by two different independent blinded researchers measuring both unmyelinated axons and axons surrounded by three or more layers of dark myelin wrapping. G-ratio and axon diameter were measured using Fiji 7Measurements were made using scale, threshold, and measurement functions in []. The G-ratio metric was calculated by randomly sampling 10 images to find axons with three or more myelin laminae, while unmyelinated fibers were measured by randomly sampling images of 10 axons from the distal channel. Axon diameter was measured by using a threshold function to find the total area of the axon. Then, assuming the axon was circular, the diameter was calculated from the area. The calculation of the G-ratio was based on a simple linear prediction of the diameter of the inner axon, while the outer diameter of the entire fiber (consisting of the axon and the darkly stained myelin lamellae surrounding it) was calculated by taking the average of the minimum and maximum diameters of a given nerve fiber. This averaging method for obtaining the outer diameter was necessary because the proximity of the myelin layers was not consistent throughout the entire circumference of the myelin sheath. The G-ratio was calculated by taking the inner diameter relative to the average outer diameter. When measuring the outer extent of the myelin sheath, large nucleated bodies of Schwann cells were excluded.

[0171] Electrophysiology After 1 month in co-culture, the Transwell® insert with the reconstituted nerve was placed on the stage for electrophysiological examination. Two tubes (one for supply and the other for aspiration) were placed along the edge of the Transwell® insert, and oxygenated artificial cerebrospinal fluid (ACSF) 5It was perfused into the tissue sample. To record the compound action potential (CAP), a glass capillary micropipette electrode (1 - 4 MΩ) was inserted into the valve of the channel near the clustered cell bodies, and the axons extending in the channel were stimulated using a concentric bipolar platinum - iridium electrode placed 1 - 3 mm distal from the above - mentioned valve. The platinum recording electrode was placed in an ACSF - filled glass micropipette and connected to an amplifier set with a gain of 100 times and high - pass filtering at 0.1 Hz to low - pass filtering at 3 kHz. The height and width of the stimulation pulse were maintained at 10 volts and 200 μs, respectively. The sample was stimulated at a maximum repetition rate of 1 Hz, and at least 50 stimulations were applied per sample. The CAP waveform was visualized using an analog - to - digital converter (PowerLab; AD Instmments, Colorado Springs, CO) and further saved using LabChart software (AD Instruments). After recording the CAP, snapshots of the stimulating and recording electrodes were taken using a stereomicroscope and a camera, and the distance between the above - mentioned electrodes was measured for calculating the nerve conduction velocity (NCV). The latency was determined by subtracting the position of the stimulation artifact from the CAP peak position. The NCV of the myelinated hMN / hSC co - cultures and the unmyelinated hMN monocultures was evaluated by dividing the distance between the stimulating and recording electrodes by the latency.

[0172] Statistical analysis One - way analysis of variance (ANOVA) with Tukey's post - hoc test was performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) to evaluate the differences in size between different types of spheroids. For the electrophysiological analysis, the mean value and standard deviation were calculated, and an independent two - sample t - test was performed (GraphPad Software). A p - value ≤ 0.05 was used to designate that there was a significant difference between the mean values.

[0173] Results Schwann cells improved the assembly of neurons into spheroids The inventors prepared spheroids with various cell densities in order to create spheroids that would fit appropriately within the dimensions of a Nerve-on-a-chip (NoaC) system (i.e., having a diameter of less than 1,000 μM and maintaining a large number of cells). The inventors also compared the sizes of the various spheroids to understand the interaction between hN and hSC. After placing the desired number of cells in a low-adhesion round-bottom plate, the formation of spheroids was monitored daily. In monoculture of hSC, spheroids were formed within about 2 days and were found to always have a shape with sharp edges (a-c in FIG. 43). In contrast, in monoculture of hN, self-organization into spheroids did not occur within 2 days, and instead many smaller spherical structures were formed (g-i in FIG. 43). In co-culture, hSC promoted the incorporation of hN into spheroids (about 2 days) when compared to spheroids formed from hN alone (about 3-9 days, FIG. 44). The edges of the co-culture spheroids (d-f in FIG. 43) were less clearly defined compared to spheroids containing only hSC, presumably due to the heterogeneous nature of the co-culture spheroids. Interestingly, the size of the co-culture spheroid composed of 75,000 hN and 75,000 hSC (1025 ± 52 μm) was found to be very similar to the size of the spheroid of 75,000 hSC alone (967 ± 51 μm), suggesting that the co-culture spheroid is more densely packed and thus confirming that the two cell types have an affinity for each other.

[0174] By measuring the diameters of various spheroid types (Figs. 43 and 44), the inventors determined that having 75,000 neurons is the optimal number of hNs to form the hNoaC system. This is because when the inventors produced co-culture spheroids using 25,000, 50,000, and 75,000 hSCs, it was found that the sizes of the spheroids were approximately 833 ± 108, 948 ± 39, and 1025 ± 52 μm, respectively. In the culture of neurons alone, it became clear that as the number of cells increased, the size of the spheroids increased as expected. It was revealed that for all four hN conditions (Fig. 44), the size of the spheroids increased continuously and significantly. From this, it became clear that the packing density did not substantially change across the four spheroids (25K, 50K, 75K, and 100K), and that the total number of cells contributed more to the size of the spheroids than the interactions between the various cell types within the spheroids.

[0175] Co-culture spheroids showed robust neurite outgrowth in the NoaC system The outer part of the double hydrogel system is composed of growth-resistant 10% PEGDMA, while the inner part of the channel is filled with fully concentrated (8 - 12 mg / mL) Matrigel as a growth-promoting substrate. After gel formation, the spheroids were gently transferred onto the valve part of the channel and grown in a medium containing 10% FBS, but it did not contain NGF to promote the growth and migration of hSCs while delaying neurite outgrowth from hNs. After one week, the above incubation solution was replaced with a medium supplemented with NGF and L-ascorbic acid to promote neurite outgrowth and myelination by hSCs in contact with the growing axons.

[0176] The three-dimensional characteristics of the reconstructed in vitro nerves were clarified by confocal imaging, revealing that both cell bodies and axons were present throughout the depth of the channels (Figure 45). The neurites extended at an average rate of approximately 1 mm per week. Since a basal medium containing ascorbic acid but not FBS did not support hSC migration and myelination (data not shown), the addition of FBS was an important factor in optimizing myelination. Immunostaining with S100 after 4 weeks revealed that hSC cells migrated approximately 1-1.5 mm outside the spheroid and grew along the extended axons (A-C in Figure 45). Meanwhile, the axons reached the distal end (approximately 5 mm) of the Matrigel-filled channels. Interestingly, for many co-culture samples, the spheroid appeared to affect axonal outgrowth, such that the axons appeared to turn back after growing a certain distance, presumably due to a chemotactic effect caused by growth factors released from hSCs in the spheroid. As the number of hSCs in the spheroid increased, the effect on the axons became more pronounced.

[0177] Myelination of In Vitro Human Nerves and Structure of Nerve Fibers Finally, embedding in plastic resin and section preparation were performed together with immunostaining and confocal microscopy, and the level and quality of myelination in the above system were evaluated by TEM. Evidence of effective myelination in the above system included, but was not limited to, non-compact myelin (Figure 47A), compact myelin (Figure 47B), and myelin in the process of compaction (Figure 47C). For axons where evidence of myelination was seen, the G ratio of myelinated nerve fibers was 0.57 ± 0.16. The axon diameters of myelinated and unmyelinated axons were 0.55 ± 0.33 and 0.40 ± 0.15 μm, respectively. Also, evidence of lamellar myelination without axons (Figure 47D), the presence of lamellar bodies in the cytoplasm (Figure 47E), and naked (unmyelinated) axons (Figure 47F) were seen. The appearance of cytoplasmic lamellar bodies consisting of relatively regularly spaced membranous whorls was interpreted as representing autophagosome production consistent with the recycling of aging organelles. The distribution of lamellar bodies was sparse, and apoptotic nuclei were not observed, indicating that the affected cells were not involved in programmed cell death.

[0178] In vitro human nerves exhibit effective composition-dependent electrical conductivity To determine whether the nerve conduction velocity (NCV) of induced pluripotent stem cell (iPSC)-derived human neurons (hN) can be measured in the presence and absence of human Schwann cells (hSC), a technique similar to brain slice electrophysiology was used. Axons within the channel were stimulated and compound action potentials (CAPs) from the cell bodies were recorded (A in Fig. 46). The axons were stimulated at a location approximately 1 to 3 mm away from the cell bodies, and the impulse travel distance between the stimulating electrode and the recording electrode was calculated. To determine the difference between the fastest signal and the peak signal, two types of NCV, namely onset NCV and peak NCV, were evaluated (B' and B'' in Fig. 46). Surprisingly, it was found that the onset and peak NCV were slower when using hN / hSC co-culture samples compared to hN monoculture samples. The onset NCV of 75K hN monoculture and 75K / 25K hN / hSC co-culture was measured to be 0.28 ± 0.07 and 0.20 ± 0.02 m / s, respectively, while the peak NCV was found to be 0.18 ± 0.04 and 0.13 ± 0.02 m / s, respectively (C in Fig. 46). For samples with a large number of SCs (hN / SC co-cultures at 75K / 75K and 75K / 50K), it was difficult to measure the onset and peak NCV. Qualitative tests of these samples revealed that the density of neurite outgrowth was slightly lower in co-culture samples, suggesting that NCV may decrease.

[0179] In this study, the inventors present the first biomimetic, fully human in vitro model of the peripheral nerve, assembled as a Nerve-on-a-Chip (NoaC) platform. This microengineered dual hydrogel system holds the nerve cell bodies in defined locations (i.e., “ganglia”) and confines dense three-dimensional axonal outgrowths within narrow channels that extend linearly outward from the clustered cell bodies (i.e., “nerves”). This system supports the functional (e.g., electrophysiological testing) and structural (e.g., qualitative and quantitative microscopic analysis) evaluation items that are the current “gold standard” required for the assessment of neuropathological diseases associated with peripheral nerve disorders, which represent growing medical concerns. Innovative aspects of this study include the reproducible production of neuron / Schwann cell co-culture spheroids, robust viability in vitro (≈4 weeks) and extensive neurite outgrowth (≈5 mm), effective myelination of human iPSC-derived neurons (hN) by primary human Schwann cells (hSC), and the ability to measure nerve conduction velocity (NCV) in an in vitro environment suitable for human disease modeling, drug discovery, and toxicity screening. Challenges in the fabrication of in vitro nerve systems In vitro myelination using primary hSC has been long challenging, partly due to the complex issues associated with the extraction of hSC from adult nerves 8、9 , contamination by fibroblasts 8~10 , and transformation of SCs into a proliferative / non-myelinating phenotype in vitro 11、12 Regarding the myelination of rat dorsal root ganglion (DRG) sensory neurons by embryonic, neonatal, and adult rodent SCs, co-culture conditions are well established 13~15 . However, myelination cannot be reproduced using human SCs cultured with rat DRG neurons under similar co-culture conditions 11When primary human SCs are purified strictly or human stem cells or human fibroblasts are differentiated into SC-like cells, it results in a restricted level of myelination of rat sensory neurons. However, the degree of the above-mentioned restriction seen in mixed-species cultures is significantly smaller compared to the degree of the above-mentioned restriction that occurs using embryonic rat SCs. 11、16 , which is presumably due to species differences or the density of SCs compared to the number of axons. Recently, Clark and his co-researchers 17 succeeded in demonstrating myelination of human stem cell-derived sensory neurons by rat stem cells. Nevertheless, an in vitro system showing myelination of human iPSC-derived neurons by human Schwann cells remains elusive.

[0180] In the past few years, many studies have focused on the generation of glioblastoma organoids, creating brain-like tissues in vitro. 18~22 . Interestingly, all of these strategies have focused on differentiating aggregates of neural progenitor cells into more defined neural structures. In contrast, the inventors reverse-engineered this process by combining two differentiated cell types and evaluating the potential for interaction and self-organization between them. To mimic the growth of embryonic dorsal root ganglia (DRGs) in vitro, neuron / Schwann cell spheroids were generated using ultra-low attachment 96-well plates, and the crosstalk between axons and SCs, which is important for the differentiation of SCs into a myelinating phenotype. 23、24The co-cultured spheroids promoted the formation of axons and SCs in close proximity to each other in the 3D spheroids, thus increasing the chances of cross-communication and successful myelination. Following the addition of the antioxidant ascorbic acid, we observed the first evidence of in vitro myelination of stem cell-derived human neurons by primary human Schwann cells. Although the rate of self-organization and spheroid formation differed for both hNs and hSCs individually, when combined, hSCs improved the quality and rate of spheroid self-organization compared to the neuron-only condition. Based on the spheroid diameter, the co-cultured spheroids were found to be more compact compared to either hNs or hSCs spheroids, indicating enhanced interactions between these two cell types.

[0181] Schwann cell migration from spheroids Schwann cell migration is a key event during peripheral nerve regeneration after development and injury. 25 The cues that direct the fate of neural crest cells to Schwann cell precursors and ultimately to Schwann cells are largely unknown. However, it has been known for decades that both precursor cells and Schwann cells depend on outgrowing axons for differentiation, proliferation, and functional maturation. 26 Here, for the first time, we were able to observe this migration into a human tissue of origin in vitro by generating mini-ganglia composed of hNs and hSCs. The axons extended outwards in concert with the migrating hSCs, synchronizing with the extending axons in the process. It was interesting to observe that the hSCs only migrated to the outermost ~1 mm of the spheroids, compared to a total axonal extension of ~5 mm. This is in contrast to typical 2D co-culture experiments, where we typically add more than 100,000 Schwann cells in a smaller 2D area. 17、27This may be due to the fact that, compared to , fewer hSCs compared to neurons were added during these experiments. This modest outgrowth of hSCs compared to axonal outgrowth may also be the result of the fact that the period before myelination is only one week and NGF was added to the medium after the first week of culture. It has been shown that NGF also enhances neuron-Schwann cell interactions and myelination 28 , from which it can be inferred that NGF may be a factor that reduces the migration of SCs. Based on the migration of human SCs outside the above spheroids, this HNoaC model can also be used to study the potential ability of SCs to migrate in the presence of therapeutic molecules, and thus can create candidates for therapeutic drugs for patients with peripheral nerve injury.

[0182] hSCs are known to behave differently in vitro compared to rat Schwann cells in terms of their responsiveness to mitogens and growth factors, and the fact that hSCs are unable to reproduce myelination. 29 . The three-dimensional spheroid model of human nerves developed by the present inventors exhibited the general characteristics of neural stem cells observed in nerves obtained by autopsy or biopsy procedures. Axons had a complete complement of organelles including cytoskeletal filaments and mitochondria and were often (but not always) associated with a myelin sheath characterized by closely apposed myelin membranes. The apposition of the myelin layers varied between nerve fibers and in some cases, lamellar myelin was formed in the absence of axons, and both of these findings are rarely seen in differentiated nerves harvested in vivo, indicating that some differences in the differentiated state actually occur in culture (as expected). Nevertheless, a sufficient number of myelinated axons were observed in the mini-nerve portion of the mixed culture system, making it a suitable alternative to a mixed somatic nerve (i.e., a somatic nerve containing densely myelinated axons, sparsely myelinated axons, and unmyelinated axons).

[0183] Evaluation of nerve conduction velocity (NCV) Various neuropathies are known to exhibit various types of neurophysiological characteristics30 Therefore, nerves generated by in vitro microengineering should be able to reveal electrophysiological changes as a means for conducting exploratory and mechanistic toxicological studies. In this study, which was the first to examine nerve conduction using human iPSC-derived neurons, differences in nerve conduction velocity (NCV) between myelinated and unmyelinated human axons were confirmed, demonstrating that this system is sufficiently sensitive to evaluate nerve function. Surprisingly, in myelinated hN / hSC co-culture samples, the NCV was found to be slower compared to samples of unmyelinated hN monocultures alone. Qualitative investigation of this culture revealed that the number of hSCs in the spheroid may have decreased the outgrowth and axon density in the channels of HNoaC, which is likely to lead to a decrease in NCV. Also, in co-cultures with high SC (50K and 75K) densities, many axons appeared to be folded, so the optimal length between the point where a stimulus that could affect NCV calculation was applied and the point where recording was done could not be measured. Furthermore, the presence of non-neuronal cell bodies in the co-culture spheroid decreased the probability of recording from appropriately stimulated cell bodies. Importantly, the NCV from hN was found to be considerably lower compared to NCV values obtained in human patients. 31~33 This is not particularly surprising considering the in vitro system composed of iPSC-derived neurons with a lower maturity compared to the mature myelinated axons of nerves evaluated in vivo at room temperature.

[0184] The simple design of this all-human NoaC system opens new avenues in bridging research of technologies. This platform can be used not only to screen drug candidates based on clinically relevant electrophysiological and histopathological metrics, but also to study the basic mechanisms underlying nerve diseases, including but not limited to toxic diseases, demyelinating diseases, and other neurodegenerative diseases. Regarding a specific treatment or therapy, our conceptually identical rat NoaC 6By comparing the data obtained from human NoaC, it will help to bridge the gap between non-clinical trials and the ability of the present invention to predict responses and potential safety risks in humans.

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Cultures of human Schwann cells isolated from fetal nerves. Brain Research 440, 261-266, doi:https: / / doi.org / 10.1016 / 0006-8993(88)90994-8 (1988). 11 Morrissey, T. K., Kleitman, N. & Bunge, R. P. Human Schwann cells in vitro. II. Myelination of sensory axons following extensive purification and heregulin-induced expansion. J Neurobiol 28, 190-201, doi:10.1002 / neu.480280206 (1995). 12 Porter, S., Glaser, L. & Bunge, R. P. Release of autocrine growth factor by primary and immortalized Schwann cells. Proceedings of the National Academy of Sciences 84, 7768 (1987). 13 Podratz, J. L., Rodriguez, E. H. & Windebank, A. J. Antioxidants are necessary for myelination of dorsal root ganglion neurons, in vitro. Glia 45, 54-58, doi:doi:10.1002 / glia.10302 (2004). 14 Windebank, A. J., Wood, P., Bunge, R. P. & Dyck, P. J. Myelination determines the caliber of dorsal root ganglion neurons in culture. J Neurosci 5, 1563-1569 (1985). 15 Paivalainen, S. et al. Myelination in mouse dorsal root ganglion / Schwann cell cocultures. Molecular and Cellular Neuroscience 37, 568-578, doi:https: / / doi.org / 10.1016 / j.mcn.2007.12.005 (2008). 16 Lehmann, H. C. et al. Human Schwann cells retain essential phenotype characteristics after immortalization. Stem Cells Dev 21, 423-431, doi:10.1089 / scd.2010.0513 (2012). 17 Clark, A. J. et al. Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination. Brain 140, 898-913, doi:10.1093 / brain / awx012 (2017). 18 Koito, H. & Li, J. Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies. Journal of Visualized Experiments : JoVE, 1304, doi:10.3791 / 1304 (2009). 19 Reynolds, B. A. & Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science (New York, N.Y.) 255, 1707-1710 (1992). 20 Pasca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods 12, 671-678, doi:10.1038 / nmeth.3415 (2015). 21 Bae, B. I. & Walsh, C. A. Neuroscience. What are mini-brains? Science (New York, N.Y.) 342, 200-201, doi:10.1126 / science.1245812 (2013). 22 Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373-379, doi:10.1038 / nature12517 (2013). 23 Sulaiman, O. A. R. & Gordon, T. Effects of short- and long-term Schwann cell denervation on peripheral nerve regeneration, myelination, and size. Glia 32, 234-246, doi:doi:10.1002 / 1098-1136(200012)32:3<234::AID-GLIA40>3.0.CO;2-3 (2000). 24 Salzer, J. L. Schwann Cell Myelination. Cold Spring Harbor Perspectives in Biology 7, doi:10.1101 / cshperspect.a020529 (2015). 25 Anton, E. S., Hadjiargyrou, M., Patterson, P. H. & Matthew, W. D. CD9 plays a role in Schwann cell migration in vitro. The Journal of Neuroscience 15, 584 (1995). 26 Bhattacharyya, A., Brackenbury, R. & Ratner, N. Axons arrest the migration of Schwann cell precursors. Development (Cambridge, England) 120, 1411-1420 (1994). 27 Zanazzi, G. et al. Glial Growth Factor / Neuregulin Inhibits Schwann Cell Myelination and Induces Demyelination. The Journal of Cell Biology 152, 1289 (2001). 28 Chan, J. R. et al. NGF Controls Axonal Receptivity to Myelination by Schwann Cells or Oligodendrocytes. Neuron 43, 183-191, doi:https: / / doi.org / 10.1016 / j.neuron.2004.06.024 (2004). 29 Monje, P. V., Sant, D. & Wang, G. Phenotypic and Functional Characteristics of Human Schwann Cells as Revealed by Cell-Based Assays and RNA-SEQ. Molecular Neurobiology 55, 6637-6660, doi:10.1007 / s12035-017-0837-3 (2018). 30 Fuller, G. How to get the most out of nerve conduction studies and electromyography. Journal of Neurology, Neurosurgery &Psychiatry 76, ii41 (2005). 31 Buchthal, F. & Rosenfalck, A. Evoked action potentials and conduction velocity in human sensory nerves. Brain Research 3, v-122, doi:https: / / doi.org / 10.1016 / 0006-8993(66)90056-4 (1966). 32 Palve, S. S. & Palve, S. B. Impact of Aging on Nerve Conduction Velocities and Late Responses in Healthy Individuals. Journal of Neurosciences in Rural Practice 9, 112-116, doi:10.4103 / jnrp.jnrp_323_17 (2018). 33 Mallik, A. & Weir, A. I. Nerve conduction studies: essentials and pitfalls in practice. Journal of Neurology, Neurosurgery &Psychiatry 76, ii23 (2005).

[0186] Example 6: Sensory Synapse Model

[0187] Co-culture of rat dorsal root ganglion (DRG) neurons and cells of the dorsal horn (DH) of the rat spinal cord has been reported previously (Ohshiro et al., 2007; Vikman et al., 2001). When co-cultured, the above DRG neurons form synapses on the dorsal horn cells. The development of this three-dimensional form of the synapse model from rat DRG to DH will be the first step towards the development of a human spinal cord DH afferent sensory synapse model.

[0188] The important point of this experiment is that DRG neurons extend axons through GelMA and form synapses on DH neurons. Past experiments in the laboratory have shown that the growth of spinal spheroids can be controlled by the rigidity of the gel and does not grow well in GelMa. The inventors aim to create a unidirectional neural circuit in which DH axons do not extend into GelMA but extend through the entire Matrigel through which compound action potentials (CAPs) can be recorded.

[0189] The above-mentioned dorsal horn and DRG were isolated and dissociated from the spinal cord of embryonic day 15 rats. The cells were then cultured individually in spheroid cultures in 96-well U-bottom plates. Two days after seeding, spheroids were formed and then placed in a double hydrogel construct to allow for three-dimensional neuron growth (A in Figure 48). DRG was placed in GelMA in the keyhole at the bottom of the construct, while the DH spheroid was placed in Matrigel in the central keyhole. When this culture was stained with β3-tubulin, it was confirmed that axons were extending from both spheroids in the culture at 28 DIV (B in Figure 48). The stimulating electrode was placed on the DRG axon, and CAP recordings were taken from the DH spheroid. This distance was measured to be 3.1 mm between the electrodes for this culture (C in Figure 48). An example of the CAP recording curve showed the response in the DH spheroid when the DRG axon was stimulated (D in Figure 48). This suggests that DRG synapses with and activates the DH neuron, which is a DH neuron and from which CAP responses are recorded. In further experiments, focus will be placed on confirming the formation of these synapses and converting this model into a human form.

[0190] Ohshiro H, Ogawa S, and Shinjo K. Visualizing sensory transmission between dorsal root ganglion and dorsal horn neurons in co-culture with calcium imaging. J Neurosci Methods, 2007, 165: 49-54.

[0191] Vikman K, Backstrom E, Kristensson K, and Hill R. A two-compartment in vitro model for studies of modulation of nociceptive transmission. J Neurosci Methods 2001;105: 175-184.

Claims

1. A composition comprising spheroids of cells containing nerve cells, wherein the spheroids are obtained by co-culturing nerve cells and Schwann cells at a cell ratio of 1:1 to 3:1, the spheroids have a diameter of about 800 μm to 1,000 μm, the spheroids contain 12,500 cells ± 5% or more, and the nerve cells are derived from stem cells selected from embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells.

2. The composition according to claim 1, wherein the spheroid further comprises one or more cells selected from glial cells, induced pluripotent stem cells, T cells, B cells, macrophages, and combinations thereof.

3. The composition according to claim 1 or 2, wherein the spheroid further comprises one or more astrocytes, and the ratio of astrocytes to nerve cells is 1:2 to 1:

4.

4. The spheroid is (a) one or more astrocytes at a ratio of 4 nerve cells per astrocyte; or (b) nerve cells and one or more glial cells at a ratio of 4 nerve cells per glial cell, comprising the composition according to any one of claims 1 to 3.

5. The composition according to any one of claims 1 to 4, further comprising a medium containing ascorbic acid at a concentration in the range of 0.001 wt / vol% to 0.01 wt / vol%.

6. The composition according to any one of claims 1 to 5, further comprising a solid substrate, wherein the solid substrate comprises an internal chamber, and the internal chamber comprises the following: (i) a first cavity in which the spheroid is disposed; (ii) a second cavity in which a second spheroid is disposed; and, (iii) at least one channel connecting the first cavity and the second cavity in fluid communication comprising wherein the channel comprises one or more bundled axons connecting the first spheroid and the second spheroid, and the one or more axons have a thickness of 10 microns to 1000 microns. composition.

7. The composition according to claim 6, wherein the second spheroid comprises spinal cord neurons or peripheral neurons.

8. (i) a cell culture container comprising a hydrogel, and (ii) one or more spheroids containing nerve cells, wherein the spheroids are obtained by co-culturing nerve cells and Schwann cells at a cell ratio of 1:1 to 3:1, the spheroids have a diameter of about 800 μm to 1,000 μm, and the spheroids contain 12,500 cells ± 5% or more, and the nerve cells are derived from stem cells selected from embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells, a spheroid, and (iii) an amplifier comprising a current generator, (iv) a voltmeter and / or an ammeter, (v) at least a first stimulating electrode and at least a first recording electrode A system comprising: The amplifier, the voltmeter and / or ammeter, and the electrodes are electrically connected to each other via a circuit through which current is supplied from the amplifier to the at least one stimulating electrode, received at the recording electrode, and supplied to the voltmeter and / or ammeter, and the stimulating electrode is arranged at or near one or more cell bodies of the nerve cells so that an electric field is established throughout the cell culture vessel, and the recording electrode is arranged at a predetermined distance distal to the cell body. The system.

9. The system according to claim 8, wherein the culture vessel comprises 96, 192, 384 or more internal chambers comprising Schwann cells and / or oligodendrocytes that are sufficiently close to the nerve cells such that one or more isolated Schwann cells or one or more oligodendrocytes accumulate myelin in response to axonal outgrowth from the nerve cells.

10. The system according to claim 8 or 9, further comprising a solid substrate on which the hydrogel matrix is crosslinked, the solid substrate comprising at least one plastic surface having pores with a diameter of 1 micron ± 5% to 5 microns ± 5%. (a) The solid substrate has a continuous outer surface and an inner surface, and such solid substrate includes at least one cylindrical or substantially cylindrical portion and at least one hollow interior, and at an end of the hollow interior, the hollow interior defined by at least one portion of the inner surface. The inner surface has one or more pores with a diameter of 0.1 micron ± 5% to 1.0 micron ± 5%. The hollow interior of the solid substrate is accessible through at least one opening from a point outside the solid substrate; the hollow interior portion includes a first portion proximate to the opening and at least a second portion distal to the opening; the one or more nerve cells are disposed in or proximate to the first portion of the hollow interior and are in physical contact with the hydrogel matrix; 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 nerve cells into the second interior portion of the hollow interior, and / or (b) At least a part of the solid substrate, at least a part of the inner surface of the solid substrate, is cylindrical or substantially cylindrical so as to define a cylindrical or substantially cylindrical hollow interior chamber in which the spheroid is disposed. System.

11. The hydrogel is (a) at least a polymer impenetrable to a first cell and a polymer penetrable to a first cell; (b) at least a polymer impenetrable to a first cell containing 15% or less of PEG, and a polymer penetrable to a first cell containing 0.05% to 1.00% of one or a combination of self-assembling peptides selected from RAD 16-I, RAD 16-II, EAK 16-I, EAK 16-II, and dEAK 16. (c) A first region and a second region, wherein the first region is a cylinder or a rectangular parallelepiped, and the axis in the longitudinal direction thereof is oriented in a direction penetrating the upper surface and the bottom surface of the cell culture vessel, and is formed in the shape of the cylinder or the rectangular parallelepiped, each of the cylinder or the rectangular parallelepiped having a space defined by the inner surface thereof, and being accessible through the space and one or more openings penetrating the upper surface of the cell culture vessel; the second region being adjacent to the first region and in fluid communication with the first region, and having a space formed in the shape of the inner wall of the second region having an opening in its side; the first region and the second region; and / or (d) At least 1% polyethylene glycol (PEG) The system according to any one of claims 8 to 10, comprising

12. The hydrogel comprises a series of two or more cavities that are in fluid communication with each other by a series of channels, at least one cavity comprising a spheroid, and at least a second cavity comprising a second spheroid, a suspension of cells, or a DRG, wherein the spheroid and the second spheroid, the suspension of cells, or the DRG are connected by three-dimensional axons. The system according to any one of claims 8 to 11.

13. (a) The height of the three-dimensional axon is at least 10 microns ± 5% at its lowest point, or at least three layers of a cell monolayer; and / or (b) The cavity is a well having a U-shaped or circular well disposed on a horizontal or substantially horizontal surface of the solid substrate, and each channel comprises one or more axons connected to one or more spheroids. The system according to claim 12.

14. (a) (i) One or more types of nerve cells, and / or (ii) One or more types of Schwann cells or oligodendrocytes A first spheroid containing, and (i) One or more types of peripheral neurons A second spheroid containing, and Each spheroid is disposed in the cavity; or (b) First, second, and third cavities, each cavity being configured to hold a spheroid and at least 50 microliters of a cell culture medium, the cavities being aligned such that the first cavity is proximal to the second cavity and distal to the third cavity, and optionally (i)Further comprising at least a fourth cavity, wherein the cavities are arranged in a pattern such that each cavity defines a corner of a square; or (ii)the cavities are aligned in a row such that an axon originating from the first spheroid in the first cavity extends to the second cavity, and an axon from the spheroid in the second cavity extends to the axon in the third cavity. The system according to claim 13.

15. A method for manufacturing a three-dimensional culture of one or more spheroids in a culture vessel comprising a solid substrate, comprising: (a)contacting one or more types of nerve cells with the solid substrate, the solid substrate comprising at least one outer surface, at least one inner surface, and at least one inner chamber defined by the at least one inner surface and accessible from a point outside the solid substrate through at least one opening; (b)placing one or more spheroids containing nerve cells, wherein the spheroids are obtained by co-culturing nerve cells and Schwann cells at a cell ratio of 1:1 to 3:1, the spheroids have a diameter of about 800 μm to 1,000 μm, and the spheroids contain 12,500 ± 5% or more cells; (c)applying a cell culture medium into the culture vessel in an amount sufficient to cover the at least one spheroid; comprising wherein at least a part of the inner surface comprises a polymer impenetrable to the first cells and a polymer penetrable to the first cells.

16. The spheroid is (a)in each case a suspension of nerve cells selected from one or a combination of motor neurons, sensory neurons, sympathetic neurons, parasympathetic neurons, cortical neurons, spinal cord neurons, peripheral neurons, each derived from stem cells; and / or (b)further comprises oligodendrocytes. The method according to claim 15.

17. Further comprising the following steps: (a)step (d) of growing neurites and / or axons on the spheroid for 12 hours to 1 year after step (c); and / or (b) (i)a step of isolating one or more types of nerve cells from a sample before step (a), and / or (ii) When the one or more spheroids include dorsal root ganglia (DRGs), isolating the DRGs from one or more mammals before step (b), and / or (iii) When the one or more spheroids include oligodendrocytes, isolating one or more Schwann cells and / or one or more oligodendrocytes; and / or (c) When introducing current into the stimulating electrode, arranging at least one stimulating electrode adjacent to or in the cell bodies of the one or more nerve cells so that the recording electrode can receive signals corresponding to one or more electrophysiological metrics measurable at the recording electrode, and arranging at least one recording electrode adjacent to or in the axons at the point most distal from the cell bodies. The method further includes wherein the one or more electrophysiological metrics are one or a combination of conduction velocity, action potential, amplitude of the wave accompanying the passage of an electrical impulse along the membrane of the one or more nerve cells, width of the electrical impulse along the membrane of the one or more nerve cells, latency of the electrical impulse along the membrane of the one or more nerve cells, and envelope of the electrical impulse along the membrane of the one or more nerve cells. The method according to claim 15 or 16.

18. (a) Culturing one or more spheroids in the composition according to any one of claims 1 to 7; (b) Exposing the one or more spheroids to at least one drug; (c) Measuring and / or observing one or more morphometric changes and / or one or more electrophysiological metrics of the one or more spheroids; and (d) Correlating one or more morphometric changes and / or one or more electrophysiological metrics of the one or more spheroids with the toxicity of the drug, such that when the morphometric changes and / or electrophysiological metrics indicate a decrease in cell viability, the drug is characterized as toxic, and when the morphometric changes and / or electrophysiological metrics indicate no change or an increase in cell viability, the drug is characterized as non-toxic and / or having a neuroprotective effect. A method for evaluating the toxicity and / or neuroprotective effect of a drug.

19. A method for determining myelination or demyelination of one or more axons of one or more spheroids, culturing one or more spheroids in the composition of any one of claims 1 to 4, in the presence or absence of a drug, under conditions and for a time sufficient to grow at least one axon; detecting the amount of myelination in one or more axons derived from the one or more spheroids; comprising; detecting comprises; (i) measuring and / or observing one or more morphometric changes and / or one or more electrophysiological metrics of the one or more spheroids, in the presence or absence of a drug; (ii) correlating one or more morphometric changes and / or one or more electrophysiological metrics of the one or more spheroids, in the presence or absence of a drug, with a quantitative or qualitative change in myelination of the spheroids; the method as described above. **Claim 20** quantifying the number or density of one or more spheroids and / or axons grown from spheroids; culturing one or more spheroids in the composition of any one of claims 1 to 4; calculating the number of cells within the spheroids and / or the number or density of axons grown from the spheroids in the composition, after culturing the spheroids for a period sufficient to grow the one or more axons or cells within the spheroids; comprising; step (b) comprising contacting the one or more spheroids with one or more drugs; step (c) comprising, after culturing the one or more spheroids, detecting an internal and / or external record of such one or more spheroids and correlating the record with a measured value of a similar record corresponding to a known or control number of cells, or step (c) comprises; (i) further measuring intracellular and / or extracellular records and / or morphometric changes before and after the step of contacting the one or more spheroids with one or more drugs; (ii) correlating the difference in the records and / or morphometric changes before contacting the one or more spheroids with the one or more drugs with the records and / or morphometric changes after contacting the one or more spheroids with the one or more drugs, with a change in the number of cells and / or the number or density of axons; A method for detecting and / or quantifying nerve cell growth and / or axonal degeneration, comprising **Claim 21** A method for measuring or quantifying the neuromodulatory effect of a drug, comprising: a) culturing one or more spheroids in the composition according to any one of claims 1 to 4 in the presence and absence of the drug; b) applying a potential across the one or more spheroids in the presence and absence of the drug; c) measuring one or more electrophysiological metrics from the one or more spheroids in the presence and absence of the drug; d) correlating the difference in the one or more electrophysiological metrics by the one or more spheroids with the neuromodulatory effect of the drug, such that a change in the electrophysiological metric in the presence of the drug as compared to the electrophysiological metric measured in the absence of the drug indicates a neuromodulatory effect, and no change in the electrophysiological metric in the presence of the drug as compared to the electrophysiological metric measured in the absence of the drug indicates that the drug has no neuromodulatory effect; or comprising: a) culturing one or more spheroids in the composition according to any one of claims 1 to 4 in the presence and absence of the drug; b) measuring and / or observing one or more morphometric changes in the one or more spheroids in the presence and absence of the drug; c) correlating the one or more morphometric changes with the neuromodulatory effect of the drug, such that a change in the morphological metric in the presence of the drug as compared to the morphological metric measured and / or observed in the absence of the drug indicates a neuromodulatory effect, and no change in the morphological metric in the presence of the drug as compared to the morphological metric measured and / or observed in the absence of the drug indicates that the drug has no neuromodulatory effect; The method as described above.

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