Cell systems using spheroids, as well as methods for manufacturing and using them.

Abstract

To provide cell systems using spheroids, as well as methods for manufacturing and using them. [Solution] This disclosure broadly relates to a cell culture system, and more specifically to a three-dimensional cell culture system of nerve cells that promotes both structural and functional properties that mimic the structural and functional properties of in vivo peripheral fibers, including cellular myelin formation. This disclosure provides a method, apparatus, and system for an in vitro spatially controlled three-dimensional model, enabling intracellular and extracellular electrophysiological measurements and recordings, using a dual hydrogel construct and a spheroid containing nerve cells. The three-dimensional hydrogel construct allows for the incorporation of cell types, geometric fabrication, and electrical manipulation, providing a system capable of culturing, disturbing, and testing biomimetic nerve extension with physiologically valid results.

Description

[Technical Field]

[0001] Disclosure of research supported by the federal government This invention was made with government support under NIH STTR Grant No. R42-TR001270. The U.S. Government has certain rights to this invention.

[0002] Cross-reference of related applications This application is an international application designated in the United States, filed under Section 120 of the United States Patent Act, claiming priority to U.S. Provisional Application No. 62 / 594,525, filed on 4 December 2017, which is incorporated herein by reference in its entirety.

[0003] This disclosure relates, broadly, to a cell culture system, and more specifically, to a three-dimensional cell culture system for spheroids that promotes both structural and functional properties that mimic the structural and functional properties of in vivo nerve fibers, including cellular myelin formation and the propagation of complex action potentials. [Background technology]

[0004] For peripheral nerve tissue, where long-distance bioelectrical conduction is one of the most relevant physiological outcomes, reproducing the physiological and functional aspects using benchtop equipment is particularly difficult. Therefore, three-dimensional tissue models of peripheral nerves lag behind those of epithelial, metabolic, and tumor tissues, where soluble analytes function as appropriate metrics. Recently, multi-electrode array technology has enabled the use of electrophysiological techniques for screening environmental toxins, as well as for disease modeling and therapeutic testing. While this technique is groundbreaking for peripheral nervous system (PNS) and central nervous system (CNS) studies, the dissociative nature of these cultures prevents the reproduction of important metrics for population-level environments and peripheral tissues. Instead, clinical methods for investigating peripheral neuropathy and neuroprotection include nerve conduction studies using composite action potential (CAP) measurements and nerve fiber density (NFD) using morphometric analysis of skin biopsies. [Overview of the Initiative] [Means for solving the problem]

[0005] This 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 provide only one or the other. Organ-type tissue samples can provide both native tissues and three-dimensional constructs, but these models are not suitable for very high-speed analysis.

[0006] This disclosure relates to a composition comprising a cell spheroid comprising one or a combination of cells and / or tissues selected from nerve cells, ganglia, stem cells, and immune cells. In some embodiments, the spheroid comprises 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, induced pluripotent stem cell-derived 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, astrocytic cells, oligodendrocytes, Schwann cells, microglia, 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 includes one or more glutamatergic neurons. In some embodiments, the spheroid includes one or more dopaminergic neurons. In some embodiments, the spheroid includes one or more serotonergic neurons. In some embodiments, the spheroid includes one or more trigeminal ganglion cells. In some embodiments, the spheroid includes one or more astrocytes. In some embodiments, the spheroid includes one or more oligodendrocytes. In some embodiments, the spheroid includes one or more Schwann cells. In some embodiments, the spheroid includes one or more microglia cells. In some embodiments, the spheroid includes one or more ependymal cells. In some embodiments, the spheroid includes one or more radial glial cells. In some embodiments, the spheroid includes one or more satellite cells. In some embodiments, the spheroid includes one or more intestinal glial cells. In some embodiments, the spheroid includes one or more pituitary cells.

[0007] In some embodiments, the spheroid comprises one or more immune cells selected from T cells, B cells, macrophages, and astrocytes. In some embodiments, the spheroid comprises one or more 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 may include each of the above cell types individually or in combination with one another.

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

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

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

[0011] In some embodiments, the spheroid contains approximately 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 approximately 75,000 or more cells. In some embodiments, the spheroid contains approximately 65,000 or more cells. In some embodiments, the spheroid contains approximately 60,000 or more cells. In some embodiments, the spheroid contains approximately 100,000 or more cells. In some embodiments, the spheroid contains approximately 125,000 or more cells. In some embodiments, the spheroid contains approximately 150,000 or more cells. In some embodiments, the spheroid contains approximately 175,000 or more cells. In some embodiments, the spheroid contains approximately 200,000 or more cells. In some embodiments, the spheroid contains approximately 225,000 or more cells. In some embodiments, the spheroid contains approximately 250,000 or more cells. In some embodiments, the spheroid contains approximately 12,500 or more cells. In some embodiments, the spheroid contains approximately 12,500 to approximately 250,000 cells. In some embodiments, the spheroid contains approximately 12,500 to approximately 100,000 cells. In some embodiments, the spheroid contains approximately 12,500 to approximately 75,000 cells.

[0012] In some embodiments, the spheroid further comprises one or more magnetic particles. In some embodiments, the magnetic particles have one or more hollow interiors. In some embodiments, the magnetic particles comprise one or more polymer layers on which the cells form spheroids.

[0013] This disclosure also, (i) A cell culture vessel equipped with 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 ammeter, (v) at least a first stimulating electrode and at least a first recording electrode A system equipped with, 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, the current is received by the recording electrode, and supplied to the voltmeter and / or ammeter. The system also relates to the system in which the stimulating electrode is positioned near or adjacent to one or more cell bodies of the nerve cells and / or isolated tissue explants, such that an electric field is established throughout the entire cell culture vessel, and the recording electrode is positioned at a predetermined distal distance from the cell bodies. 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, said 96, 192, 384 or more internal chambers contain the Schwann cells and / or oligodendrocytes located sufficiently close to said tissue explants and / or nerve cells such that one or more types of isolated Schwann cells or one or more types of oligodendrocytes accumulate myelin in response to axon outgrowth from said one or more types of isolated tissue explants and / or said one or more types of nerve cells.

[0015] In some embodiments, the system further comprises a solid substrate, on which the hydrogel matrix is cross-linked, and the solid substrate comprises 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, and the solid substrate comprises at least one portion that is cylindrical or substantially cylindrical, and at least one hollow interior, said hollow interior being defined by at least one portion of the inner surface at an end of said hollow interior, and the inner surface comprises 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 outside the solid substrate, the hollow interior portion comprises a first portion adjacent to the opening, and at least a second portion distal from the opening, said one or more types of nerve cells and / or said one or more types of tissue explants are disposed in or adjacent to said first portion of the hollow interior, and are in physical contact with the hydrogel matrix, said second portion of the at least one hollow interior is in fluid communication with said first portion, such that axons can grow from said one or more types of nerve cells and / or said one or more types of tissue explants into said second interior portion of the hollow interior.

[0016] In some embodiments, the system or composition does not comprise a sponge or does not contain a sponge, respectively. In some embodiments, the hydrogel comprises at least a first cell-impermeable polymer and a first cell-permeable polymer. In some embodiments, the at least one cell-impermeable polymer comprises about 15% or less PEG, and the at least one cell-permeable polymer comprises about 0.05% to about 1.00% of one or a combination of self-assembling peptides selected from the group consisting of RAD 16-I, RAD 16-II, EAK 16-I, EAK 16-II, and dEAK 16. In some embodiments, the composition does not comprise polyethylene glycol (PEG). In some embodiments, the hydrogel comprises a first region and a second region, the first region is shaped as a cylinder or a cuboid, the longitudinal axis of which is oriented in a direction penetrating the top surface and the bottom surface of the cell culture vessel, each of said cylinder or cuboid comprises a space defined by the inner surface of said cylinder or cuboid, said space being accessible through one or more openings penetrating through the top surface of said space and said cell culture vessel; The second region comprises a space adjacent to the first region and in fluid communication with the first region, the space being formed in the shape of the inner wall of the second region having an opening at a side thereof. In some embodiments, the composition comprises at least 1% polyethylene glycol (PEG).

[0017] In some embodiments, the system further comprises a cell culture medium comprising nerve growth factor (NGF) at a concentration of about 5 to about 20 picograms per milliliter and / or ascorbic acid at a concentration ranging from about 0.001 w / v% to about 0.01 w / v%.

[0018] In some embodiments, the system comprises one or more spheroids containing 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, astrocytic cells, oligodendrocytes, Schwann cells, microglia, ependymal cells, radial glial cells, satellite cells, enteric glial cells, and pituitary cells. In some embodiments, the system further comprises one or more 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 mammals.

[0019] In some embodiments, the spheroids are cultured for approximately 3, 30, 90, or 365 days or longer.

[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 internal chamber in which the spheroids are arranged. In some embodiments, the hydrogel comprises a series of two or more cavities fluidly communicating 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, a cell suspension, or DRG being connected by three-dimensional axons. In some embodiments, the cavities are wells having U-shaped or circular wells arranged on a horizontal or substantially horizontal plane of the solid substrate, with each channel comprising one or more axons connected to one or more spheroids.

[0021] In some embodiments, the one or more spheroids contain one or more nerve cells having axonal growth with a width of about 100 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 layers of a single cell.

[0022] In some embodiments, the system described above is (i) one or more types of nerve cells, and / or (ii) One or more Schwann cells or oligodendrocytes The first spheroid, which includes, (i) One or more types of peripheral neurons The second spheroid, which includes The system comprises a first, second, and third cavity, each configured to hold a spheroid and at least 50 microliters of cell culture medium, with the cavities aligned such that the first cavity is located proximal to the second cavity and distal to the third cavity. In some embodiments, the system comprises at least a fourth cavity, with the cavities arranged in a pattern such that each cavity defines a square corner. In some embodiments, the cavities are aligned in a line 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.

[0023] This disclosure also relates to a method for producing a three-dimensional culture of one or more spheroids in a culture vessel. In some embodiments, the above method is (a) Bringing one or more nerve cells into contact with the solid substrate, which comprises 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 outside the solid substrate through at least one opening, (b) Placing one or more spheroids containing nerve cells in at least one of the internal chambers, (c) Apply the cell medium to the culture vessel in an amount sufficient to cover at least one spheroid. Includes, At least a portion of the inner surface comprises a polymer into which the first cells cannot penetrate and a polymer into which the first cells can penetrate. In some embodiments, step (b) includes arranging 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 neurons, and peripheral neurons, which are 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 neurons, and peripheral neurons, which are 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) allowing neurites and / or axons to grow on the spheroid for about 12 hours to about 1 year after step (c). In some embodiments, the method further comprises isolating one or more types of nerve cells from a sample before step (a), and / or, if the one or more spheroids include dorsal root ganglia (DRGs), isolating DRGs from one or more mammals before step (b), and / or, if the one or more spheroids include Schwann cells or oligodendrocytes, isolating one or more types of Schwann cells and / or one or more oligodendrocytes.

[0026] In some embodiments, the method is configured such that when a current is introduced to the stimulating electrode, the recording electrode receives a signal corresponding to one or more electrophysiological metrics that can be measured at the recording electrode. At least one stimulating electrode is placed near or adjacent to the cell body of one or more of the above-mentioned nerve cells or tissue explants, At least one recording electrode is positioned on or near the axon at the point furthest from the cell body. It further includes, The above one or more electrophysiological metrics are one or a combination of the following: electrical 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] This disclosure also, (a) culturing one or more spheroids in any of the compositions disclosed herein, (b) Exposure to at least one of the above spheroids with 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 mentioned above, (d) The toxicity of the drug is correlated with one or more morphometric changes and / or electrophysiological metrics of one or more spheroids, and if the morphometric changes and / or electrophysiological metrics indicate a decrease in cell viability, the drug is characterized as toxic; if the morphometric changes and / or electrophysiological metrics indicate no change or an increase in cell viability, the drug is characterized as nontoxic and / or neuroprotective. This also relates to methods for evaluating the toxicity and / or neuroprotective effects of drugs, including those mentioned above.

[0028] This disclosure also relates to a method for determining whether myelin formation or demyelination has occurred in one or more axons of one or more spheroids, (a) Culturing one or more spheroids in any of the compositions disclosed herein for a time and under conditions sufficient to grow at least one axon, in the presence or absence of the drug, (b) To detect the amount of myelin formation in one or more axons derived from one or more spheroids, Includes, Detecting is optional. (i) The step of measuring and / or observing one or more morphometric changes and / or electrophysiological metrics of one or more spheroids in the presence or absence of the drug, (ii) A step of 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 the drug with quantitative or qualitative changes in myelin formation of the spheroids. This also relates to the above methods, including those mentioned above.

[0029] This disclosure also, (a) Quantify 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 for a period of time sufficient to grow one or more axons or cells within the spheroid, calculate the number of cells in the spheroid and / or the number or density of axons grown from the spheroid in the composition. The present invention also relates to methods for detecting and / or quantifying neuronal cell growth and / or axonal degeneration, including, in some embodiments. In some embodiments, step (b) optionally includes contacting one or more spheroids with one or more drugs. In some embodiments, step (c) optionally includes, after culturing one or more spheroids, detecting internal and / or external records of such one or more spheroids and correlating the records with measurements of similar records corresponding to a known or control number of cells. In some embodiments, step (c) optionally includes, (i) A further step of measuring intracellular and / or extracellular recordings and / or morphometric changes before and after the above step of bringing one or more spheroids into contact with one or more drugs, (ii) A step of correlating the difference between the recorded and / or morphometric changes after contacting one or more spheroids with one or more drugs and the recorded and / or morphometric changes before contacting one or more spheroids with one or more drugs with changes in cell number and / or axon number or density. Includes.

[0030] This disclosure also relates to a method for measuring or quantifying the neuromodulatory effects of a drug, (a) Culturing one or more spheroids in any of the compositions disclosed herein in the presence and absence of the above-mentioned drug, (b) Applying an electrical potential to the entire spheroid, both in the presence and absence of the above-mentioned drug, (c) Measuring one or more electrophysiological metrics from one or more spheroids in the presence and absence of the above-mentioned drug, (d) Correlating the above differences in one or more electrophysiological metrics caused by one or more spheroids with the neuromodulatory effect of the drug, the results showing that changes in the electrophysiological metrics in the presence of the drug compared to the electrophysiological metrics measured in the absence of the drug indicate a neuromodulatory effect, and that no change in the electrophysiological metrics in the presence of the drug compared to the electrophysiological metrics measured in the absence of the drug indicates that the drug does not exert a neuromodulatory effect. This also relates to the above methods, including those mentioned above.

[0031] This disclosure also relates to a method for measuring or quantifying the neuromodulatory effects of a drug, (a) Culturing one or more spheroids in any of the compositions disclosed herein in the presence and absence of the above-mentioned drug, (b) Measuring and / or observing one or more morphometric changes of the one or more spheroids in the presence and absence of the above-mentioned drug, (c) Correlating one or more of the above morphometric changes with the neuromodulatory effect of the above drug, the results showing that changes in morphological metrics in the presence of the above drug compared to the morphological metrics measured and / or observed in the absence of the above drug indicate a neuromodulatory effect, and that no change in morphological metrics in the presence of the above drug compared to the morphological metrics measured and / or observed in the absence of the above drug indicates that the above drug does not exert a neuromodulatory effect. This also relates to the above methods, including those mentioned above. In certain embodiments, for example, the following items are provided: (Item 1) A composition comprising a cell spheroid containing one or a combination of cells and / or tissues selected from nerve cells, ganglia, stem cells, and immune cells. (Item 2) The composition according to item 1, wherein the spheroid comprises tissue selected from the dorsal root ganglion and the trigeminal ganglion. (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, astrocytic cells, oligodendrocytes, Schwann cells, microglia, 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 nerve cells are derived from stem cells 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 approximately 200 microns to approximately 700 microns. (Item 8) The composition according to any one of items 1 to 7, wherein 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 four nerve cells per Schwann cell. (Item 9) The composition according to any one of items 1 to 8, wherein the spheroid comprises one or more types of nerve cells and one or more types of astrocytes, in a ratio of approximately 4 nerve cells per astrocyte. (Item 10) The composition according to any one of items 1 to 9, wherein the spheroid comprises one or more types of nerve cells and one or more types of astrocytes in a ratio of approximately one nerve cell per astrocyte. (Item 11) The composition according to any one of items 1 to 10, wherein the spheroid comprises one or more types of nerve cells and one or more types of Schwann cells, in a ratio of approximately 10 nerve cells per 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 in a ratio equal to about 4 nerve cells per glial cell. (Item 13) The composition according to any one of items 1 to 12, wherein one or more of the aforementioned 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 approximately 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 comprises one or more magnetic particles. (Item 19) (i) A cell culture vessel equipped with 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 ammeter, (v) at least a first stimulating electrode and at least a first recording electrode A system equipped with, 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, the current is received by the recording electrode, and supplied to the voltmeter and / or ammeter. The stimulating electrode is positioned near or adjacent to one or more cell bodies of the nerve cells and / or isolated tissue explants so that an electric field is established throughout the entire cell culture vessel, and the recording electrode is positioned at a predetermined distal distance from the cell bodies. The aforementioned system. (Item 20) The system according to item 19, wherein the spheroid is a spheroid described in 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 each contain the Schwann cells and / or oligodendrocytes in close proximity to the explant and / or nerve cells, such that the Schwann cells and / or oligodendrocytes of one or more isolated Schwann cells or one or more oligodendrocytes accumulate myelin for axonal extension from the explant and / or the one or more 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, wherein 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, the solid substrate comprising at least one cylindrical or substantially cylindrical portion and at least one hollow interior, the interior of the hollow defined at the end of the interior of the hollow by at least one portion of the inner surface, the inner surface comprising one or more pores with a diameter of about 0.1 microns to about 1.0 microns, The hollow interior of the solid substrate is accessible from the outside of the solid substrate through at least one opening. The hollow internal portion comprises a first portion adjacent 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 positioned in or near the first portion inside the hollow and are in physical contact with the hydrogel matrix. The second portion inside the at least one hollow is in fluid communication with the first portion so that axons can grow from the one or more nerve cells and / or the one or more tissue explants into the second internal portion inside the hollow. The system described in 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 into which the first cell cannot penetrate and a polymer into which the first cell can penetrate. (Item 27) The system according to item 26, wherein the PEG content of 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 comprises about 0.05% to about 1.00% of one or a combination of self-assembling peptides selected from RAD 16-I, RAD 16-II, EAK 16-I, EAK16-II, and dEAK 16. (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 formed in the shape of a cylinder or rectangular parallelepiped, wherein its longitudinal axis is oriented in a direction that penetrates the top and bottom surfaces of the cell culture vessel, and each of the cylinder or rectangular parallelepiped comprises a space defined by the inner surface of the cylinder or rectangular parallelepiped, which is accessible by one or more openings that penetrate the space and the top surface of the cell culture vessel; The second region is adjacent to the first region and has fluid communication with the first region, and comprises a space formed in the shape of the inner wall of the second region, with an opening on its side. A system described in any of items 19-28. (Item 30) The system according to any one of items 19 to 29, further comprising a cell medium containing nerve growth factor (NGF) at a concentration of approximately 5 to approximately 20 picograms per milliliter and / or ascorbic acid at a concentration ranging from approximately 0.001 wt / vol% to approximately 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, astrocytic cells, oligodendrocytes, Schwann cells, microglia, ependymal cells, radial glial cells, satellite cells, enteric glial cells, and pituitary cells. (Item 32) A system according to any one of items 19 to 31, further comprising one or more stem cells, pluripotent cells, myoblasts, and osteoblasts. (Item 33) The system according to any one of items 19 to 32, wherein one or more types of 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 comprises at least 1% polyethylene glycol (PEG). (Item 35) The system according to any one of items 19-34, wherein the spheroids are cultured for approximately 3, 30, 90, or 365 days or longer. (Item 36) The system according to any one of items 19 to 35, wherein at least a portion 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 located. (Item 37) The system according to any one of items 19 to 36, wherein the one or more spheroids contain one or more types of nerve cells having axonal growth with a width of approximately 100 microns to approximately 500 microns and a length of approximately 0.11 to approximately 10,000 microns. (Item 38) The system according to any one of items 19 to 37, wherein the hydrogel comprises a series of two or more cavities that are fluidly connected to 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, a cell suspension, or DRG are connected by a three-dimensional axon. (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 layers of a single cell. (Item 40) The system according to item 38, wherein the cavity is a well having a U-shaped or circular well located 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 types of nerve cells, and / or (ii) One or more Schwann cells or oligodendrocytes The first spheroid, which includes, (i) One or more types of peripheral neurons The second spheroid, which includes Equipped with, The system according to item 40, wherein each spheroid is located in the cavity. (Item 42) The first, second, and third cavities are provided, each configured to hold a spheroid and at least 50 microliters of cell culture medium. The cavities are aligned such that the first cavity is located proximal to the second cavity and distal to the third cavity. The system described in item 40. (Item 43) The system described in 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 equipped with a solid substrate, (a) Bringing one or more nerve cells into contact with the solid substrate, which comprises 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 outside the solid substrate through at least one opening; (b) Placing one or more spheroids containing nerve cells in the at least one internal chamber, (c) Applying the cell medium to the culture vessel in an amount sufficient to cover at least one spheroid. Includes, The method wherein at least a portion of the inner surface comprises a polymer into which the first cell cannot penetrate and a polymer into which the first cell can penetrate. (Item 46) The method according to item 45, wherein step (b) involves positioning 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 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 neurons, and peripheral neurons, which are optionally 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 the step of growing neurites and / or axons in the spheroid for about 12 hours to about 1 year after step (c). (Item 50) (i) a step of isolating one or more types of nerve cells from the sample prior to step (a), and / or (ii) If one or more spheroids include a dorsal root ganglion (DRG), a step of isolating the DRG from one or more mammalian species prior to step (b), and / or (iii) If the one or more spheroids contain Schwann cells or oligodendrocytes, the step of isolating one or more Schwann cells and / or one or more oligodendrocytes. The method described in any of items 45-49, further including the method described in any of items 45-49. (Item 51) When current is introduced to the stimulating electrode, the recording electrode is configured to receive signals corresponding to one or more electrophysiological metrics that can be measured at the recording electrode. At least one stimulating electrode is positioned near or adjacent to the cell body of one or more nerve cells or tissue explants, At least one recording electrode is positioned on or near the axon at the point furthest from the cell body. It further includes, The one or more electrophysiological metrics are one or a combination of the following: electrical 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 described in any of items 45-50. (Item 52) (a) Culturing one or more spheroids in any of the compositions described in items 1 to 18, (b) Exposing 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. A method for evaluating the toxicity and / or neuroprotective effects of drugs, including [specific example]. (Item 53) A method for determining whether myelin formation or demyelination has occurred in one or more axons of one or more spheroids, (a) Culturing one or more spheroids in any of the compositions described in items 1 to 18 for a time and under conditions sufficient to grow at least one axon, in the presence or absence of the drug, (b) To detect the amount of myelin formation in one or more axons derived from one or more spheroids. Includes, Detecting is optional. (i) measuring and / or observing one or more morphometric changes and / or electrophysiological metrics of the one or more spheroids in the presence or absence of the drug, (ii) A step of 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 the drug with quantitative or qualitative changes in myelin formation of the spheroids. The method comprising the above. (Item 54) (a) Quantify 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 described in item 1 to 18, (c) After culturing the spheroid for a period of time sufficient to grow one or more axons or cells within the spheroid, calculate the number of cells in the spheroid and / or the number or density of axons grown from the spheroid in the composition. Includes, Step (b) is optional and includes bringing one or more spheroids into contact with one or more drugs. Step (c) is optional and may include, after culturing one or more spheroids, detecting internal and / or external records of such one or more spheroids and correlating such records with measurements of similar records corresponding to a known or control number of cells, Step (c) is optional. (i) A further step of measuring intracellular and / or extracellular recordings and / or morphometric changes before and after the step of bringing one or more spheroids into contact with one or more drugs, (ii) A step of correlating the difference between the recorded and / or morphometric changes after contacting one or more spheroids with one or more drugs and the recorded and / or morphometric changes before contacting one or more spheroids with one or more drugs with a change in cell number and / or axon number or density. A method for detecting and / or quantifying neuronal cell growth and / or axonal degeneration, including the above. (Item 55) A method for measuring or quantifying the neuromodulatory effects of a drug, (a) Culturing one or more spheroids in any of the compositions described in items 1 to 18 in the presence and absence of the drug, (b) Applying an electrical potential to the entire one or more spheroids in the presence and absence of the drug, (c) Measuring one or more electrophysiological metrics from one or more spheroids in the presence and absence of the drug, (d) Correlating the difference in one or more electrophysiological metrics due to one or more spheroids with the neuromodulatory effect of the drug, the results showing that a change in the electrophysiological metrics in the presence of the drug compared to the electrophysiological metrics measured in the absence of the drug indicates a neuromodulatory effect, and no change in the electrophysiological metrics in the presence of the drug compared to the electrophysiological metrics measured in the absence of the drug indicates that the drug does not exert a neuromodulatory effect. Includes or, (a) Culturing one or more spheroids in any of the compositions described in items 1 to 18 in the presence and absence of the drug, (b) Measuring and / or observing one or more morphometric changes of the one or more spheroids in the presence and absence of the drug, (c) Correlating one or more morphometric changes with the neuromodulatory effect of the drug, the results showing that changes in morphological metrics in the presence of the drug compared to morphological metrics measured and / or observed in the absence of the drug indicate a neuromodulatory effect, and that no change in morphological metrics in the presence of the drug compared to morphological metrics measured and / or observed in the absence of the drug indicates that the drug does not exert a neuromodulatory effect. The method comprising the above. (Item 56) A method for manufacturing the system described in any of items 19 to 37, (a) Culturing nerve cells in cell culture medium for a period of time sufficient for the cells to form spheroids, (b) Placing the spheroid within the hydrogel, (c) Exposing the spheroids to the cell culture medium for a period sufficient to allow the growth of nerve processes or axons. The method comprising the above. (Item 57) The method according to item 56, comprising step (a) mixing the nerve cells with one or more magnetic particles. (Item 58) The method according to item 57, wherein step (b) includes using magnetic force to place the spheroid into the cavity of the hydrogel. (Item 59) The method according to item 56, wherein step (b) includes placing the hydrogel into the cavity using ultrasonic force, mechanical force or fluid force. (Item 60) The method according to item 56, wherein the spheroid is a spheroid disclosed in any of the compositions described in items 1 to 18. (Item 61) The method of item 52, further comprising correlating one or more morphometric changes and / or electrophysiological metrics of one or more spheroids with the toxicity of the drug, wherein if the morphometric changes and / or electrophysiological metrics indicate a decrease in cell viability, the drug is characterized as toxic, and if 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 neuroprotective. [Brief explanation of the drawing]

[0032] [Figure 1] Figures A and B show three-dimensional hydrogel scaffoldings fabricated for Nerve-on-a-chip design. [Figure 2]This figure shows typical spheroid and axonal growth within the hydrogel described above. The hydrogel construct can induce and restrict three-dimensional axonal growth and cell arrangement to mimic nerve fiber pathways. [Figure 3] This is a list of morphological and physiological measurements that can be obtained in the ganglia of the dorsal root ganglia, in the proximal nerve pathway, at the midpoint of the nerve pathway, and in the distal nerve pathway. [Figure 4] This is a stack of confocal images of unmyelinated nerve fiber tracts proximal to the dorsal root ganglia, midpoint, and distal to the ganglia, stained with β-III tubulin to show neurites, DAPI to show nuclei, and S100 to show Schwann cells. [Figure 5] This is a confocal depth map that displays 3D neurite density. [Figure 6] Figures A-C show transmission electron microscopy (TEM) images of cross-sections of nerve cultures. A shows dense, parallel, bundled unmyelinated nerve processes within a channel approximately 1.875 mm from the ganglion. B shows a focal point centered on a Schwann cell (SC)-covered axon (Ax) approximately 1 mm from the ganglion. C shows the myelin sheath around individual nerve fibers in a 25-day culture. [Figure 7] Images A and B are three-dimensional confocal images. Image A shows the immunohistochemistry of MBP protein. Image B shows the immunohistochemistry of MAG. The thickness of both cultures was 190 μm, confirming the three-dimensional myelin formation capability of this in vitro system. [Figure 8] This figure shows neurite outgrowth from spheroids formed from induced pluripotent stem cells (induced pluripotent stem cells). The neurites extend on a two-dimensional surface from spheroids of human motor neurons co-cultured with astrocytes (left) and Schwann cells (right). [Figure 9] This figure shows spheroids of motor neurons and astrocytes grown in the three-dimensional hydrogel system described above. These spheroids exhibited robust three-dimensional neurite outgrowth (approximately 5 mm). [Figure 10]This diagram shows nerve processes extending in three dimensions from human motor neurons / Schwann cell spheroids (top) and human sensory neurons (bottom). [Figure 11] This diagram shows a 96-well spheroid printing apparatus. A 96-well plate is placed on the apparatus, with one magnet positioned substantially in the center of each well. The magnetized cells are then attracted to the magnet, causing them to aggregate and form spheroids. [Figure 12] This figure shows a protocol for producing rat spinal cord spheroids. Spheroids can be formed by adding magnetic nanoparticles to cultured cells and culturing the cells in a non-adhesive plate on a magnet. (Not shown: Spheroids can also be formed by rotating cells in a non-adhesive round-bottom plate in the presence of magnetic nanoparticles.) Even with the addition of a hydrogel stretching matrix, one or more magnetic cell spheroids can be held in place by the magnet. [Figure 13] This diagram shows an apparatus for positioning magnetic spheroids in the voids of a hydrogel. A magnet is located in the center of the outer part of this diagram. The dark gray area is movable in the y-direction, while the inner / top section houses a microscope slide, adjusting the insert's position and being movable in the x-direction. The single magnet in the center allows for control over the placement of structures requiring a single magnet, regardless of the structure's shape. Other apparatus designs include those with multiple magnets for positioning spheroids in connected wells. [Figure 14] Figures A and B show the arrangement of spheroids in a hydrogel construct. Neurite outflow from rat embryonic spinal cord spheroids, as shown by β-III tubulin staining, is consistent with the hydrogel pattern of the outer structure. Figure A shows an improper spheroid arrangement without the use of a magnet. Figure B shows an appropriate arrangement using the apparatus shown in Figure 13. [Figure 15]This bar graph shows the reproducibility of spheroid formation. Consistency was obtained between batches when the same spheroid formation method was used. The average diameter of spheroids from 27,000 cells was 0.47 ± 0.03 mm, and the roundness in the x and y directions was 0.72 ± 0.15. [Figure 16] Figures A and B show typical phase images and growth potential of cell spheroids. These illustrate examples of reproducible, well-formed spheroid formation from primary embryonic rat spinal cord cells, exhibiting consistent size and shape. [Figure 17] This figure shows neurite extension from spheroids in Matrigel diluted at a 1:20 ratio. The neurites remain constrained to elongation channels within a three-dimensional environment. The neurites extended to a thickness of approximately 100–150 μm. [Figure 18] This figure shows a defined circuit created using a dorsal root ganglion (DRG) explant combined with Schwann cell spheroids in a gelatin-methacrylate hydrogel. This configuration enables unidirectional DRG neurite extension. [Figure 19] This figure shows a defined circuit arranged in a rectangular shape, prepared using multiple Schwann cell spheroids in Matrigel diluted at a 1:20 ratio. [Figure 20] This is a schematic diagram of a method for manufacturing a neuromicrophysiological system using magnetic spheroids. Using digital projection lithography, a hydrogel "mold" containing an extensible hydrogel can be cured. [Figure 21] This figure shows three-dimensional spheroid seeding, where spheroids are placed within a micropatterned hydrogel using a nanoshuttle. Spheroids can be formed by adding magnetic nanoparticles to cultured cells and culturing them in a non-adhesive plate on a magnet. Even with the addition of a hydrogel stretching matrix, one or more magnetic cell spheroids can be held in place using a magnet. [Figure 22] This bar graph shows that the number of seeded cells affects the diameter of the spheroid. [Figure 23] Figures A and B show the growth rate and three-dimensional structure of spheroids. A: Calcein staining shows high cell growth rate in spheroids formed from primary rat embryonic spinal cord tissue. B: Cross-sectional view of β-III tubulin-stained spheroids in a hydrogel construct obtained by fluorescence sheet microscopy shows the three-dimensional structure of the spheroids. [Figure 24] This figure shows a spheroid incorporating different cell types. This spheroid contains neurons (β-III), oligodendrocyte lineage cells (Olig2), astrocytes (GFAP), as well as microglia and macrophages (CDIIb), as indicated by antibody staining. [Figure 25] This figure shows the formation of a three-dimensional neural network using spheroids and micropatterned hydrogels. Spheroids formed from rat embryonic DRG cells (left) extend neurites, indicated by calcein staining, towards spinal cord spheroids (right) through methacrylate-esterified gelatin, but the reverse does not occur. [Figure 26] This figure shows the arrangement of multiple spinal cord spheroids in fixed positions and the neural network according to the geometry of their external shape. [Figure 27] These are confocal images (top 10x, bottom 20x) of co-cultured iPSC-derived motor neurons extending on human muscle cells and myotubes. The muscle cells were differentiated in growth medium for 3 days, then motor neurons were added, and the medium was replaced with motor neuron medium. [Figure 28] This is a confocal image (10x magnification) of a two-dimensional culture of mature myotubes that form a three-dimensional sheath expressing heavy chain myosin. The myotubes were cultured for a total of three weeks: three days in growth medium, seven days in differentiation medium, and the remainder in growth medium. [Figure 29] This is a 10x phase-contrast image of 3D muscle cells encapsulated in 5% GelMA after 3 weeks of culture. [Figure 30] This is a 10x maximum intensity projected Z-stack of desmin-expressing human skeletal muscle tubes after 3 weeks of culture at 255 μM. [Figure 31]This is a 10x maximum intensity projected Z-stack of heavy chain myosin-expressing human skeletal muscle tubes at 96 μM after 3 weeks of GelMA culture. [Figure 32] This figure shows the generation of three-dimensional spheroids in induced neurons with different seeding densities using the hanging-drop method. [Figure 33] This figure shows the generation of three-dimensional spheroids in U-shaped bottom wells in induced neurons at different seeding densities. [Figure 34] This figure shows the generation of three-dimensional spheroids in motor neurons with different seeding densities using the hanging-drop method. [Figure 35] This figure shows the generation of three-dimensional spheroids in U-shaped bottom wells in motor neurons at different seeding densities. [Figure 36] This figure shows the generation of three-dimensional spheroids in motor neurons at different seeding densities using the hanging-drop method over 24 or 48 hours. [Figure 37] This figure shows the generation of three-dimensional spheroids in U-shaped bottom wells in motor neurons at different seeding densities over 24 or 48 hours. [Figure 38] This figure shows the growth of hanging drop spheroids seeded with 25,000 iPSC-derived motor neurons and 25,000 astrocytes after 4 days. [Figure 39] This figure shows the growth of a U-shaped base wellspheroid seeded with 25,000 iPSC-derived motor neurons and 25,000 astrocytes after 4 days. [Figure 40] This figure shows the growth of hanging drop spheroids seeded with 40,000 iPSC-derived motor neurons and 10,000 astrocytes after 4 days. [Figure 41] This figure shows various combinations of neurons and glial cells over a 24-hour period. [Figure 42] This figure shows spheroids of oligodendrocyte progenitor cells (4x magnification (top) and 10x magnification (bottom)). [Figure 43-1]Figures A-J show the formation of spheroids composed of human neurons (hN) and / or human Schwann cells (hSC) in vitro after 2 days. Spheroid formation did not occur in 2 days under the condition of hN alone, while spheroid formation was promoted in the co-culture system with hSC present. hSC spheroids were produced at three different cell densities: 25,000 (a), 50,000 (b), and 75,000 (c). Co-cultured spheroids were produced at a constant hN density (75,000), but with varying hSC densities: 25,000 (d), 50,000 (e), and 75,000 (f). In parallel, hN spheroids were produced at three different densities: 50,000 (g), 75,000 (h), and 100,000 (i). (j) Comparing the diameters of different spheroids, it was found that co-cultured spheroids were more compact than single-cultured spheroids, indicating that the affinity of hSCs to hN results in more densely packed cell clusters. K: multiple of thousands. N=4, and the error bars represent the standard error of the mean (SEM). Scale bar: 100 μm. ****: p-value ≤ 0.0001, ***: p-value ≤ 0.001, **: p-value ≤ 0.01, *: p-value ≤ 0.05. [Figure 43-2] Same as above. [Figure 44] This figure shows the formation of spheroids composed solely of human neurons (hN). Qualitative examinations conducted more than two days after the start of hN-only cultures consistently showed a significant increase in spheroid size as the total number of cells increased (25K, 50K, 75K, 100K). The graph comparing the diameters of individual spheroids supported the qualitative examination results. K: multiple of thousands. N=4, and the error bars represent the standard error of the mean (SEM). Scale bar: 100 μm. ****: p-value ≤ 0.0001, **: p-value ≤ 0.01. [Figure 45]Figures A and B show Schwann cells migrating from spheroids and extending along axons. (A) Image showing human Schwann cells (hSCs) stained with the hSC marker S100 (light gray) migrating outside the spheroid over 4 weeks, along with axonal extension stained with βIII tubulin (gray). Nuclei are labeled with DAPI (dark gray). Scale bar: 1000 μm. (B) High-magnification image of the inset of image A. Scale bar: 25 μm. [Figure 46] Figures A-C show records of axons extending from spheroids during culture. Figures B' and B'' are graphs of the collected records. These graphs show the time course of nerve conduction velocity (NCV) of two spheroids, measured in meters per second. Figure C is a bar graph showing NCV at two time points, namely the start and peak of current initiation, representing the same NCV experiment as above. [Figure 47] Figures A-F show various stages of myelin formation observed in human neurons reconstructed in vitro. (A) Non-compact myelin. (B) Compact myelin. (C) Myelin in the process of compactification. (D) Myelin formation without axons. (E) Lamellar bodies in the cytoplasm. (F) Naked (unmyelinated) axons. [Figure 48] Figures A-D show the characterization of dorsal horn spheroids. (A) Micrograph showing a typical culture from DRG to DH synapse at 14DIV (14 days in vitro). (B) Fluorescence image showing β3-tubulin staining of synaptic culture at 28DIV. (C) Micrograph showing synaptic culture on an electrophysiology rig. The blue arrow indicates the distance between the stimulating electrode and the recording electrode, which was 3.1 mm. This image shows the stimulating electrode at the location of the DRG axon and the recording electrode at the location of the DH spheroid. (D) Electrical activity in response to a 20V stimulation of the DRG axon was recorded in the DH spheroid. The unit of the X axis is milliseconds and the unit of the Y axis is millivolts. [Modes for carrying out the invention]

[0033] Throughout this specification and the claims, various terms relating to the methods and other aspects of the disclosure are used. Unless otherwise indicated, such terms shall have their ordinary meanings in the art. Other clearly defined terms shall be construed in accordance with the definitions made herein.

[0034] In this specification and the appended claims, unless otherwise explicitly indicated, the singular forms "a," "an," and "the" encompass multiple referents.

[0035] In this specification, the term “greater than 2” is defined as any integer greater than the number 2, such as 3, 4, or 5.

[0036] In this specification, the term “about” means, when referring to measurable values ​​such as quantity, duration, etc., to include variations 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, provided that such variations are reasonable for carrying out the disclosed method.

[0037] In this specification and the claims, the phrase “and / or” should be understood to mean “either or both” of the elements joined by the phrase, that is, elements that exist in some cases as a combination and in other cases separately. Other elements other than those explicitly identified by the “and / or” clause may be present at their discretion, whether related to those explicitly identified elements or not, unless expressly contrary thereto. Thus, as a non-restrictive 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 in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in an enumeration, “or” or “and / or” should be interpreted as inclusive, that is, including not only at least one of the enumeration of numbers or elements, but also multiple of them, and optionally, any further items not enumerated. Terms that are explicitly contrary to the above, such as “only one of” or “solely one of” or, in the claims, “consisting of,” mean that only one of the elements of the enumeration of numbers or elements is included. In general, in this specification, the term “or” should be interpreted only as indicating an exclusive choice (i.e., “one or the other, but not both”) when preceded by the exclusive terms “either,” “one of,” “only one of,” or “solely one of.” When “essentially consists of” is used in the claims, it should have the usual meaning as used in the field of patent law.

[0039] In this specification, 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 any additional unlisted elements or steps of method.

[0040] In this specification, the phrase "integers X to Y" means any integer including the endpoints. That is, when a range is disclosed, each integer within that range including the endpoints is disclosed. For example, the phrase "integers X to Y" discloses 1, 2, 3, 4, or 5, as well as the range 1 to 5.

[0041] In this specification, the term “plural” is defined as any quantity greater than one or any number greater than one.

[0042] In this specification, “substantially equivalent” may mean, for example, that a given measured metric lies within a range known to correlate with an abnormal or normal range. For example, if the control sample is from a patient with the disease, substantially equivalent means that it lies within the abnormal range. If the control sample is from a patient known not to have the disease under test, substantially equivalent means that the given metric lies within the normal range.

[0043] This disclosure broadly relates to a system capable of housing and culturing one or more spheroids in a three-dimensional culture. In some embodiments, the system uses a solid substrate, such as a plastic or similar polymer, having pores, on which a hydrogel of any shape or size can be placed. In some embodiments, the hydrogel of the system serves as a support for the cells of the 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 hydrogel forming a cavity having the first region, namely a first region, which is similar to a well, having a flat or curved bottom and a diameter across the longitudinal surface of the sold support, and also having an opening on the upper surface of the region that allows external access to the system, and an opening on at least one side of the first region that is in fluid communication with the second 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 side defining the height of the channel. In some embodiments, the width of the channel is approximately 10 to approximately 750 microns. In some embodiments, the length of the channel is approximately 100 to approximately 10,000 microns. After growing spheroids from any one or combination of the cells specified in this disclosure, these spheroids may be placed in the first region containing cell culture medium. After placement, neurites may spontaneously elongate or elongation may be stimulated by exposure to one or more growth-stimulating molecules. Elongation of neurites and / or axons may originate in the first region of the system, pass through the at least one opening on the side of the hydrogel, enter the second region, and occur in the second region. After the neurites or axons have elongated to a desired length, the cell culture may be exposed to a drug to determine the effect of the drug on the elongation, morphology, or action potential of the axons or neurites.

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

[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. Other examples of non-nerve cells include organ-derived cells such as kidney cells, hepatocytes, and pancreatic cells. Examples of non-nerve cells 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 in the spheroid are primary human cells. In some embodiments, the cells are taken 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, canine cells, or bovine cells. In some embodiments, any of the disclosed systems may comprise a spheroid of nerve cells mixed with or unmixed with non-nerve cells.

[0046] The methods of the present disclosure include methods for culturing spheroids as disclosed herein, and methods for measuring the toxicity or biological effects of toxins, drugs, therapeutic agents, biomolecules, or contaminants, wherein such molecules, drugs, or therapeutic agents are exposed in the system to a culture of spheroids and axons or neurites extending from such spheroids. In some embodiments, the methods include methods for inducing unidirectional extension of axons and / or neurites in a culture from a first spheroid to a second spheroid. In some embodiments, any of the systems of the present disclosure includes agents that stimulate, accelerate, decelerate, or stop the extension of neurites and / or axons in a culture. In some embodiments, any of the methods of the present disclosure includes stimulating directional extension of axons in a culture. In some embodiments, agents are used to either attract guidance for axon and / or neurite extension or to repel the extension of axons and / or neurons. In some embodiments, an attractive guidance protein selected from netrin, neurotrophin, adherent extracellular matrix proteins, and cell adhesion receptors (such as cadherins, Ig-CAM, or integrins) is added to the system, and peptides that mimic the presumed binding sites of these proteins may also be used. In some embodiments, proteins that repel axonal and / or neurite extension are components of the system. Examples of repulsive proteins include ephrin (sometimes), semaphorin (mostly), slit, and chondroitin sulfate proteoglycans.

[0047] Methods for manufacturing spheroids with or without magnetic particles or magnetic beads are also disclosed. When magnetic particles are components of the spheroid, one or more spheroids can be positioned in a disclosed cavity formed by a hydrogel wall using any apparatus equipped with magnets. The disclosure broadly relates to an apparatus comprising a movable frame, the movable frame being movable in any lateral direction parallel to the horizontal plane on which the apparatus operates. The frame is attached to one or more magnets with a magnetic force sufficient to attract spheroids containing magnetic particles. In some embodiments, the frame, being movable in the x and y directions of the longitudinal plane of the apparatus, is mechanically attached to magnets by adhesive, polymer, or fasteners so that moving the frame moves the magnets, which have a magnetic force sufficient to move the spheroid in any direction when the spheroid is in the magnetic field of the magnets. In some embodiments, the apparatus comprises a first frame and a second frame, at least one of the first or second frame being movable laterally parallel to the longitudinal plane of the apparatus and being horizontal or substantially horizontal on the apparatus.

[0048] The term “bioreactor” means a container or partial container in which cells are cultured, optionally in a suspension. In some embodiments, the bioreactor means a container or partial container in which cells are cultured, and the cells may be present in a suspension or in contact with, or growing on or in, another non-liquid substrate, which includes, but is not limited to, a solid growth support material. In some embodiments, the solid growth support material or solid substrate includes 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 vessels in which nerve cells may be cultured, either in or in cell growth medium.

[0049] In this specification, the term “culture vessel” may be any vessel suitable for growing, culturing, cultivating, proliferating, propagating, or otherwise similarly manipulating cells. The culture vessel may also be referred to herein as “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 medium 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 does not contain bases or any other structures. In some embodiments, the culture vessel is designed to house a hydrogel or hydrogel matrix and various culture media. In some embodiments, the culture vessel consists of or is essentially made of a hydrogel or hydrogel matrix. In some embodiments, the only plastic components of the culture vessel are the components of the culture vessel that make up the side walls and / or bottom of the culture vessel, which isolate the volume of the cell growth well or zone from external points. 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 one or more nerve cells are seeded on the hydrogel and glial cells.

[0050] The term "electrical stimulation" refers to the process by which the cells are exposed to either alternating current (AC) or direct current (DC). The current may be introduced into the solid substrate or applied via the cell culture medium or other appropriate components of the cell culture system. In some embodiments, the electrical stimulation is applied to the apparatus or system by placing one or more electrodes at different locations within the apparatus or system to generate a potential throughout the cell culture vessel. The electrodes are operably connected by one or more wires to one or more amplifiers, voltmeters, ammeters, and / or electrochemical systems (such as batteries or generators). Such devices and wires form a circuit through which an electric current is generated, thereby generating a potential throughout the tissue culture system.

[0051] In this specification, the term “hydrogel” may mean, for example, any water-insoluble, cross-linked, three-dimensional network of polymer chains having voids between polymer chains that are filled with water or can be filled with water. In this specification, the term “hydrogel matrix” means, for example, any three-dimensional hydrogel construct, system, apparatus, or similar structure. Hydrogels and hydrogel matrices are known in the art, for example, in U.S. Patents 5,700,289 and 6,129,761, and Curley and Various types are described in Moore, 2011; Curley et al., 2011; Irons et al., 2008; and Tibbitt and Anseth, 2009 (each of which is incorporated by reference in whole). 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 with a wavelength greater than approximately 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 nerve pathways. In some embodiments, the hydrogel or hydrogel matrix contains poly(ethylene glycol) dimethacrylate (PEG). In some embodiments, the hydrogel or hydrogel matrix contains PuraMatrix. In some embodiments, the hydrogel or hydrogel matrix contains 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 includes explants derived from the nervous system of animals (such as mammals), and an auxiliary population of cells derived from the nervous system, which have been isolated and cultured to concentrate the population of such cells in the culture. In some embodiments, the hydrogel or hydrogel matrix includes explants such as retinal tissue explants, DRGs, or spinal cord tissue explants, as well as isolated and cultured populations of Schwann cells, oligodendrocytes, and / or microglia 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 accessible wells. In a multilayered tissue culture vessel, some embodiments may provide any number of such wells or independently accessible locations in the cell culture vessel such that the hydrogel matrix in one well or location is different from or identical to the hydrogel matrix in another well or location in the cell culture vessel.

[0052] In this specification, the term “immune cells” may be any cells 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 cells of the subject, or improving the symptoms of a disease caused by a pathogen. In some embodiments, immune cells include one or more B cells; T cells; antigen-presenting cells such as astrocytes, 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 immunomodulatory molecules. In some embodiments, the immunomodulatory molecules are, 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, IL-18 variants, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase Selected from ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon-responsive genes, 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.Immunomodulatory proteins are exemplified in U.S. Patent No. 8,008,265.

[0053] The term "immunomodulatory" refers to a substance that modulates the immune system. Such substances can be readily identified using standard assays that exhibit various aspects of the immune response, such as cytokine secretion, antibody production, NK cell activation, and T cell proliferation. 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. See al. (1997a) Eur. J. Immunol. 27:2340-44, WO98 / 55495, and WO00 / 61151. Thus, these and other methods can be used to identify, test, and / or confirm immunostimulatory substances such as immunostimulatory nucleotides and immunostimulatory isolated nucleic acids.

[0054] In some embodiments, the two or more hydrogels may contain different amounts of PEG and / or Puramatrix. In some embodiments, the densities of the two or more hydrogels may vary. In some embodiments, the two or more hydrogels may have varying degrees of penetrability, allowing cells to extend within the hydrogels. In some embodiments, the flexibility of the two or more hydrogels may vary. In some embodiments, the bioreactors, cell culture devices, or compositions disclosed herein include a hydrogel comprising two layers of polymer, i.e., a polymer into which cells can penetrate and a polymer into which cells cannot penetrate. In some embodiments, the layer into which cells can penetrate is laminated over at least one region of the upper surface of the layer into which cells cannot penetrate.

[0055] The term "polymer into which cells can penetrate" refers to a hydrophilic polymer having identical or mixed monomer units at a concentration and / or density sufficient to create spaces when crosslinked 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 extend during culture.

[0056] The term "cellularly impenetrable polymer" refers to a hydrophilic polymer having identical or mixed monomer units at a concentration and / or density sufficient to prevent the formation of spaces or compartments when crosslinking in a solid or semi-solid state on a solid substrate. In other words, a cellularly impenetrable 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” means any portion of a polypeptide or nucleic acid sequence that relates to the respective full-length polypeptide or nucleic acid and is long and structurally long enough to have at least similar or substantially similar biological effects to the full-length polypeptide or nucleic acid on which the fragment is based. In some embodiments, the functional fragment is a portion of a full-length or wild-type nucleic acid sequence encoding any one of the nucleic acid sequences disclosed herein, wherein the portion encodes a polypeptide of a certain length and / or structure that is shorter than the full length but still encodes a domain that is biologically functional compared to the full-length or wild-type protein. In some embodiments, the functional fragment may have reduced biological activity, substantially equivalent biological activity, or 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 with respect 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 with respect to the wild-type human sequence from which the sequence is derived.

[0058] Those skilled in the art will understand that the polymers into which cells cannot penetrate and the polymers into which cells can penetrate may contain the same or substantially the same polymer, but differences in concentration or density after crosslinking will result in a hydrogel matrix having some parts that promote the elongation of cells or parts 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 ​​about 100 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 150 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 200 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 250 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 300 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 350 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 400 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 450 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 500 μm to approximately 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 550 μm to approximately 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 600 μm to approximately 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 650 μm to approximately 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 700 μm to approximately 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 750 μm to approximately 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 650 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 550 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 450 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 300 μm to approximately 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 400 μm to approximately 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 ​​approximately 10 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 150 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 200 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 250 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 300 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 350 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 400 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 450 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 500 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 550 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 600 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 650 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 700 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 750 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 800 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 850 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 900 μm to approximately 3000 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 950 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 1000 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 1500 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 2000 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 2500 μm to approximately 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 2500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 2000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 1500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 1000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 950 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 900 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 850 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 650 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 600 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 550 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 450 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 100 μm to approximately 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 300 μm to approximately 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​approximately 400 μm to approximately 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: 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: 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, titin, 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: polylysine, polyglutamate, or polyglycine.

[0065] In some embodiments, the above hydrogel is used in Khoshakhlagh et al., “Photoreactive interpenetrating network of hyaluronic acid and Puramatrix as a selectively tunable scaffold for neurite growth” Acta Biomaterialia, January This includes one or a combination of polymers selected from the hydrogels published in 21, 2015.

[0066] Any hydrogel suitable for cell growth can be formed by placing one or a combination of the polymers disclosed herein under sufficient time, concentration, and conditions to produce two crosslinked polymers of distinct densities, i.e., one crosslinked polymer into which one type of cell can penetrate and one crosslinked polymer into which one type of cell cannot penetrate. The polymers may be polypeptides, such as synthetic polymers, polysaccharides, natural proteins, or glycoproteins, and / or selected from the following. Synthetic polymers Polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), polyacrylamide, silicones, combinations thereof, and derivatives thereof, etc. Polysaccharides (which may be synthetic or derived from natural sources) Hyaluronic acid, heparan sulfate, heparin, dextran, agarose, chitosan, alginates, combinations thereof, and their derivatives, etc. Natural proteins or glycoproteins Collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, combinations thereof, and derivatives thereof, etc. Polypeptides (synthetic or natural sources) Polylysine, and all of the RAD and EAK peptides already listed, etc.

[0067] In this specification, 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 one another. In some embodiments, the term three-dimensional means, in the context of the systems of this disclosure, that the thickness or height of the neurites and / or axons is about 10 to about 1000 microns. In some embodiments, the term three-dimensional means, in the context of the systems of this disclosure, that the thickness or height of the neurites and / or axons is about 10 to about 100 microns.

[0068] The term “isolated neuron” means a nerve cell that is an organism or culture from which the nerve cell was originally grown, and which has been removed or detached from the organism or culture. 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, including a tissue sample or a suspension with non-nerve cells. In some embodiments, the nerve cell is isolated at the stage when it is removed from the animal from which the nerve cell originates, 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 comprises at least one or more cells derived from one species or combination of species selected from sheep cells, goat cells, horse cells, bovine cells, human cells, monkey cells, mouse cells, rat cells, rabbit cells, dog cells, cat cells, pig cells, or other non-human mammals. In some embodiments, the isolated neuron is a human cell. In some embodiments, the isolated neuron is a stem cell that has been pretreated to have a differentiated phenotype similar to or substantially similar to that of human nerve cells. In some embodiments, the isolated neurons are human cells. In some embodiments, the isolated neurons are stem cells that have been pretreated to have a differentiated phenotype similar to or substantially similar to that of non-human nerve cells. In some embodiments, the stem cells are selected from mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, hematopoietic stem cells, epidermal stem cells, stem cells isolated from mammalian umbilical cord, or endodermal stem cells.

[0069] The term “neurodegenerative disease” is used throughout this specification to describe diseases resulting from damage to the central and / or peripheral nervous systems. Exemplary neurodegenerative diseases that may be studied using the disclosed models, systems, or apparatus include, for example, Parkinson’s disease; Huntington’s disease; amyotrophic lateral sclerosis (Lou Gehrig’s disease); Alzheimer’s disease; lysosomal storage disorders (e.g., “white matter disease” or glial / demyelinating disease as described by Folkerth, J. Neuropath. Exp. Neuro., 58, 9, Sep., 1999); Tay-Sachs disease (β-hexosaminidase deficiency); other genetic disorders; multiple sclerosis; brain injury or trauma resulting from ischemia, accident, environmental injury, etc.; spinal cord injury; ataxia; and alcoholism. Furthermore, the present invention may be used to test the potency, toxicity, or neurodegenerative effects of drugs on nerve cells in culture for the study of the treatment of neurodegenerative diseases. The term neurodegenerative diseases include, in particular, neurodevelopmental disorders such as autism and related neurological disorders (such as schizophrenia).

[0070] In this specification, the term “neuron” means a cell comprising, for example, at least one or a combination of dendrites, axons, and cell bodies, or any cell or group of cells isolated from nervous system tissue. In some embodiments, a neuron is any cell that includes or is capable of forming axons. In some embodiments, the neuron is a Schwann cell, glial cell, glial cell, cortical neuron, embryonic cell isolated from or derived from nervous tissue, or embryonic cell differentiated into a neuronal phenotype or a phenotype substantially similar to that of a neuron, pluripotent stem cell (iPS) differentiated into a neuronal phenotype, or mesenchymal stem cell derived from or differentiated into a neuronal phenotype. In some embodiments, the neuron is a neuron derived from dorsal root ganglion (DRG) tissue, retinal tissue, spinal cord tissue, or brain tissue from an adult, adolescent, infant, or fetal subject. In some embodiments, the neuron is any one or more cells isolated from the nervous tissue of the subject. In some embodiments, the neuron is a mammalian cell. In some embodiments, the cells are human cells and / or rat cells. In some embodiments, the cells are non-human mammalian cells or derived from cells isolated from non-human mammals. The nerve cells may include neurons isolated from multiple species when isolated or cleaved from the original animal from which the cells originate. In some embodiments, the spheroids do not contain DRG tissue.

[0071] In some embodiments, nerve cells are one or more of the following: 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, glial cells are one or more of the following: astrocytes, oligodendrocytes, Schwann cells, microglia, ependymal cells, radial glial cells, satellite cells, enteric glial cells, and pituitary cells. In some embodiments, immune cells are one or more of the following: macrophages, T cells, B cells, leukocytes, lymphocytes, monocytes, mast cells, neutrophils, natural killer cells, and basophils. In some embodiments, the stem cells are one or more of the following: hematopoietic stem cells, neural stem cells, embryonic stem cells, adipose-derived stem cells, bone marrow-derived stem cells, induced pluripotent stem cells, astrocytic cell-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-derived 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 spheroid may also include other cell types such as keratinocytes or endothelial cells.

[0072] In this specification, the term “neuronal cell culture medium” or simply “culture medium” may mean any nutrient suitable for supporting the growth, culture, cultivating, proliferating, propagating, or other forms of manipulation of nerve 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 in a concentration ranging from about 0.001 wt / vol% to about 0.01 wt / vol%. In some embodiments, the medium comprises ascorbic acid in a concentration ranging from about 0.001 wt / vol% to about 0.008 wt / vol%. In some embodiments, the medium comprises ascorbic acid in a concentration ranging from about 0.001 wt / vol% to about 0.006 wt / vol%. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.001% by weight / volume to about 0.004% by weight / volume. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.002% by weight / volume to about 0.01% by weight / volume. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.003% by weight / volume to about 0.01% by weight / volume. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.004% by weight / volume to about 0.01% by weight / volume. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.006% by weight / volume to about 0.01% by weight / volume. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.008% by weight / volume to about 0.01% by weight / volume. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.002% by weight / volume to about 0.006% by weight / volume. In some embodiments, the culture medium contains ascorbic acid at a concentration ranging from about 0.003% by weight / volume to about 0.005% by weight / volume.

[0073] In some embodiments, the hydrogel, hydrogel matrix, and / or neuronal cell culture medium may contain 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, It contains one or more of the following: IL-6, IL-10, IL-12, IL-17, IL-18, insulin, laminin, lipoxin, mac-1-saporin, methionine sulfoximine, minocycline, neuregulin-1, neuroprotectin, neuruturin, NGF, nitric oxide, NT-3, NT-4, percephin, platelet lysate, PMX53, poly-D-lysine (PLL), poly-L-lysine (PLL), propentophilin, resolvin, S100 calcium-binding protein B, selenium, substance P, TNF-α, type I-V collagen, and zymosan.

[0074] In this specification, the term "optogenetics" refers to a biological technique that involves the use of light to control cells in living tissues, typically neurons, that have been genetically modified to express photosensitive ion channels. Optogenetics is a neuromodulation method used in neuroscience that combines optical and genetic techniques to control and monitor the activity of individual neurons in living tissues (even in freely moving animals) and to accurately measure the effects of these genetic manipulations in real time. The main reagents used in optogenetics are photosensitive proteins. Optogenetic actuators such as channelrhodopsin, halorhodopsin, and archelodopsin can be used to achieve spatially precise control of neurons, while optogenetic sensors for calcium (aequorin, chameleon, GCaMP), chloride (chromeleon), or membrane voltage (mermaid) can be used to create temporally accurate records. In some embodiments, neurons modified 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), polylactic acid-coglycolic 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 polymers. In some embodiments, the plastic is a mixture of 3, 4, 5, 6, 7, 8 or more polymers.

[0076] In this specification, the term "seeding" means, for example, transferring a certain amount of cells to a new culture vessel. The above amount may be specified and may be based on the volume or number of cells. The above cells may be part of a suspension.

[0077] In this specification, the term “sequence identity” means a specified percentage of residues that are identical across a specified region in the context of two or more nucleic acid or polypeptide sequences. This term is synonymous with “sequence homology” or sequences that are “homologous” to another sequence. The above percentage can be calculated by optimally aligning the two sequences, comparing the two sequences across a specified region, determining the number of positions where identical 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 two sequences have different lengths, or if the sequence comparison results in one or more attached ends and the specified comparison region contains only a single sequence, 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 verification can be performed manually or by using computer sequencing algorithms such as BLAST or BLAST2.0.

[0078] In this specification, the term “solid substrate” means any material that is a solid support that is free from or substantially free from cytotoxins. In some embodiments, the solid substrate includes one or a combination of silica, plastics, and metals. In some embodiments, the solid substrate has pores of a size and shape sufficient to allow proteins, nutrients, and gases to diffuse or be inactively 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. Those skilled 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 cells growing on the solid substrate in a particular microenvironment. For example, those skilled in the art can determine whether any cultured cells in the system or device are viable under conditions where the solid substrate has pores of various sizes. In some embodiments, the solid substrate comprises a base having a predetermined shape that defines the shapes of the outer and inner surfaces. In some embodiments, the base comprises one or a combination of silica, plastic, ceramic, or metal, and the base is cylindrical or substantially cylindrical in shape such that a polymer that is impenetrable to the first cell and a polymer that is penetrable to the first cell cover the inner surface of the base, defining a cylindrical or substantially cylindrical internal chamber, with an opening located at one end of the cylindrical shape. In some embodiments, the base comprises one or more pores of sufficient size and shape to allow proteins, nutrients, and oxygen to diffuse through the solid substrate in the presence of cell culture medium.In some embodiments, the solid substrate comprises a plastic base with a pore diameter of 1 micron or less, and comprises at least one hydrogel matrix layer, wherein the hydrogel matrix comprises at least a polymer that is impenetrable to at least a first cell and a polymer that is penetrable to at least a first cell, the base has a predetermined shape such that the polymer that is impenetrable to at least a first cell and the polymer that is penetrable to at least a first cell are physically adhered to or chemically bonded around it, and the solid substrate comprises at least one compartment that is 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 located at one end of the solid substrate. In some embodiments, if the solid substrate comprises a hollow interior defined by at least one inner surface, the cells in a suspension or tissue explant may be seeded by positioning the cells in 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. The interior of the solid substrate can contain the cells within the at least one compartment or hollow space defined by the shape of the inner surface solid substrate, thereby promoting directional growth of the cells outward through the opening. In the case of nerve cells, the degree and shape of containment of the at least one compartment promotes axonal elongation from cell bodies located within the at least one compartment and in 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 multiple bifurcated shape of the interior of the hollow space of the solid is configured to allow axons to elongate in a multi-branched pattern, or the shape makes it possible for axons to elongate in a multi-branched pattern. If electrodes are positioned 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 this device or system.In some embodiments, the electrode is operably connected to a voltmeter, ammeter, and / or device, which is capable of generating an electric current on a wire of a certain length that is physically connected to the voltmeter, ammeter, and / or device.

[0079] This disclosure relates to appropriately filled hydrogels comprising a mixture of both a polymer into which cells can penetrate and a polymer into which cells cannot penetrate. In some embodiments, the hydrogel contains about 10% to about 20% PEG and has an overall modulus of elasticity of about 0.1 to about 200 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 0.5 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 10 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 50 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 75 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 90 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 100 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 125 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 150 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 175 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 200 Pa. In some embodiments, the modulus of elasticity of the hydrogel is about 230 Pa or less.

[0080] Spheroid In this specification, “spheroid” or “cellular spheroid” may mean, for example, a collection of any cells having a three-dimensional shape corresponding to an ellipse or circle or a convex or concave arc rotating around one of its principal axes (long axis or minor axis), and examples include three-dimensional oval, oblate and oblong, spherical, lenticular, 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 The range may be any range within the above range, but not limited to, such as approximately 1,000 μm, approximately 900 μm to approximately 1,000 μm, approximately 750 μm to approximately 1,500 μm, approximately 1,000 μm to approximately 5,000 μm, approximately 1,000 μm to approximately 10,000 μm, approximately 2,000 to approximately 50,000 μm, approximately 25,000 μm to approximately 40,000 μm, or approximately 3,000 μm to approximately 15,000 μm. 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, multiple spheroids are generated, and the width, length, thickness, and / or diameter of each of the multiple spheroids may vary by less than about 20%, for example, about 15%, 10%, or less than 5%. In some embodiments, the width, length, thickness, and / or diameter of each of the multiple spheroids may be within any of the above ranges.

[0082] The cells in the spheroid may have a specific orientation. In some embodiments, the spheroid may have an inner core and an outer surface. In some embodiments, the 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 inner core contains magnetic nanoparticles.

[0083] The stiffness of the above spheroids, measured, for example, by elastic modulus (Pascals, Pa), may vary. In certain embodiments, the elastic modulus of the spheroids is in the range of about 100 Pa to about 10,000 Pa, for example, about 100 Pa to about 12,000 Pa, or about 100 Pa to about 4,800 Pa. In some embodiments, the elastic modulus of the spheroids may be about 1,200 Pa. As another example, the elastic modulus of the spheroids 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 apparatus of the present disclosure comprises one or more wells, each well containing / containing one or more different spheroids, a first, second, third, fourth, or fifth spheroid, or a group of more spheroids. In one embodiment, the first spheroid has an elastic modulus of about 100 Pa to about 300 Pa, and the second spheroid has an elastic modulus of 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 inner core may be composed of one, two, three, or more different cell types. In some embodiments, the cells of the 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 nerve cells and non-nerve cells. In some embodiments, the spheroid comprises nerve cells and astrocytes in a nerve-to-astrocyte ratio of about 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the spheroid comprises nerve cells and non-nerve cells in a ratio of about 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the spheroid comprises nerve cells and non-nerve cells in a ratio of about 1:5:1:4, 1:3, or 1:2. Any combination of the cell types disclosed herein may be used in the spheroids of this disclosure in the ratios specified above.

[0086] Depending on the particular embodiment, groups of cells may be arranged according to any and appropriate shape, geometry, and / or pattern. In some embodiments, the cells are arranged on a sphere over the entire surface area of ​​beads or nanoparticles having a solid or hollow core. For example, groups of independent cells may be deposited as spheroids, and the spheroids may be arranged in a three-dimensional grid or other any and appropriate three-dimensional pattern. All of the independent spheroids may contain substantially the same number of cells and be substantially the same size, or different spheroids may have different numbers of cells and different sizes. In some embodiments, multiple spheroids may be arranged in shapes such as L-shapes or T-shapes, radially from a single or multiple points, in a single line or parallel line sequence, as spheroids, tubes, cylinders, torus, hierarchically branched vascular networks, high aspect ratio objects, thin closed shells, organoids, or other complex shapes that can correspond to the geometry of tissues, blood vessels, or other biological structures.

[0087] In the methods of the present disclosure, any and appropriate physiological response of the spheroid may be determined, evaluated, measured, and / or identified. In some embodiments, in the methods of the present disclosure, one, two, three, four, or more types of physiological responses 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 methods may include measuring a morphological change of the spheroid, which may include evaluating at least one morphological parameter before contacting the spheroid with a drug such as a chemical and / or biological compound, evaluating the at least one morphological parameter after contacting the spheroid with the drug, and calculating the difference between the at least one morphological parameter before and after contacting the spheroid with the drug to obtain a 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 the drug. The morphology of the spheroid described above may be measured using any method known to those skilled in the art, including, but not limited to, the 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, which may include evaluating a first volume before contacting the spheroid with the drug, evaluating a second volume after contacting the spheroid with the drug, and calculating the difference between the first and second volumes to obtain the change in the volume of the spheroid. In some embodiments, the physiological response of the spheroid may be a contraction or expansion of the spheroid in response to contact with the drug.

[0089] The above-mentioned agents may be any and appropriate compounds, such as organic compounds, small molecule compounds (e.g., small organic compounds), proteins, antibodies, oligonucleotides (e.g., DNA and / or RNA), gene therapy vehicles (e.g., viral vectors), and any combination thereof. In the method of the present invention, one or more agents (e.g., one, two, three, four, five, or more types) may be used. For example, the method of the present invention may involve contacting the spheroid of the present invention with two or more different agents. In some embodiments, the method of the present invention may indirectly modify 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. Culturing can be carried out using methods known to those skilled in the art. In some embodiments, cells and / or spheroids may be cultured for any desired period of time, such as several hours, several days, several weeks, or several months, but not limited to these. In some embodiments, 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 for 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] Suitable cell culture media for the method of the present invention are known in the art and include, but are not limited to, BEGM® Bronchial Epithelial Growth Medium, Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Modified Eagle Medium High Glucose (DMEM-H), McCoy 5A Modified Medium, RPMI, Ham Medium, Medium 199, and mTeSR. The above cell culture media may be supplemented with further components, but are not limited to, vitamins, minerals, salts, growth factors, carbohydrates, proteins, serum, amino acids, adhesion molecules, cytokines, growth factors, hormones, antibiotics, therapeutic agents, and buffers. The above cell culture components and / or conditions may be selected to enhance and / or stimulate the characteristics and / or properties of specific cells, and / or may be modified during the method of the present invention. Examples of seeding and cell culture methods are found in U.S. Patents 5,266,480, 5,770,417, 6,537,567, and 6,962,814, as well as Oberpenning et al. “De novo reconstitution of a functional mammalian urinary bladder by tissue engineering” Nature Biotechnology 17:149-155. These are described in (1999) (these are incorporated herein by reference in their entirety). In some embodiments, the cell culture medium is progressively modified to promote myelin formation of axons in the culture. The pre-myelin formation medium and the myelin formation medium comprise the following components: [Table 1-1]

[0092] In some embodiments, the solid substrate, cell culture apparatus, or nanoparticles comprises / includes spheroids containing one or more cell types disclosed herein. In this application, any of the particles may contain one, two, three, four, five, six, seven, eight, or more types of cell types, or any combination thereof.

[0093] The terms “nanoparticle” or “nanoshurt” (as these terms are sometimes used synonymously) refer to particles containing at least one region. Magnetic particles in the range of approximately 0.7 to 1.5 microns are described in patent documents, for example, U.S. Patents 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. Patent Application No. 20110250146 (each of which is invoked by reference in whole). 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 this disclosure include / encompassing cells in contact with a magnetically responsive element or spheroids containing a magnetically responsive element. In some embodiments, compositions and / or systems of this disclosure include / encompassing cells in contact with one or more magnetic nanoparticles or spheroids containing one or more magnetic nanoparticles. Herein, “magnetically responsive element” may be any element or molecule that responds to a magnetic field. One or more of the above nanoparticles must contain or be magnetically responsive elements. In some embodiments, the nanoparticles may be taken up by or adsorbed by any of the cells described herein. In some embodiments, the position, shape, pattern or movement of the cell or spheroid can be manipulated using a magnetic field.

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

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

[0096] In some embodiments, the nanoparticles comprise 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) oxides. In some embodiments, the nanoparticles comprise one or more of gold, iron oxide, and polylysine.

[0097] In some embodiments of this disclosure, coated magnetic particles are provided, comprising a nanoparticle core of a magnetic material and a base coating material on the magnetic core in an amount sufficient to prevent nonspecific binding of biomacromolecules to the magnetic core. These magnetic particles are characterized by extremely low nonspecific binding and highly efficient target capture, which are essential for achieving the concentration levels required to effectively isolate very rare cells, such as neurons or other cell types disclosed herein. In alternative embodiments, namely, i. A nanoparticle core of magnetic material, ii. A base coating material that forms a discontinuous coating on the magnetic core, wherein, when accessible, the base coating material provides at least one discontinuous region that contributes to the nonspecific binding of the base coat particles to the biopolymer, iii. Coated magnetic particles are provided, comprising a further coating material that prevents access to discontinuous regions by biomolecules. The magnetic core material of the particles described above may contain at least one transition metal oxide, and a preferred base coating material contains a protein. Suitable proteins for coating 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 component 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 components of the specific binding pair bind to the base coating material via a bifunctional linking compound. Examples of bifunctional linking compounds include succinimidyl-propiono-dithiopyridine (SPDP) and sulfosuccinimidyl-4-[maleimidomethyl]cyclohexane-1-carboxylate (SMCC). However, various other such heterodifunctional 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 is in the range of about 90-150 nm. The particles may be synthesized so that they are more monodisperse, for example, in the range of about 90-120 nm or about 120-150 nm. The particles of the present invention are generally suspended in a biologically compatible medium.

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

[0100] The nanoparticles may be positively or negatively charged. 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 alloys and / or oxides (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] This disclosure also, (i) Hydrogel matrix and (ii) One or more spheroids, (iii) Generator and, (iv) A voltmeter and / or ammeter, (v) at least a first stimulating electrode and at least a first recording electrode A system equipped with, The generator, the voltmeter and / or 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, the current is received by the recording electrode, and supplied to the voltmeter and / or ammeter. The system also relates to the system in which the stimulating electrode is positioned near or adjacent to one or more nerve cell bodies, and the recording electrode is positioned at a predetermined distal distance from the cell bodies, so that an electrical potential is established throughout 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 molded into a predetermined shape or form for seeding cells of a specific size suitable for axonal elongation. In some embodiments, the solid substrate or at least one base portion is shaped to have at least one branched internal tubular structure, the diameter of which decreases as the position of the tube becomes more distal to the position where the tissue explant or nerve cells are seeded (optionally). For example, the disclosure envisions a focal point at one end of a semi-cylindrical or cylindrical portion of the solid substrate, accessible to a point outside the solid substrate by an opening or hole in the outer surface. Cells (one or more of any of the disclosed cells or combinations thereof) can be positioned or seeded at the focal point using the opening or hole. While the cells grow over several days during culture, they are exposed for a sufficient period of time to the above to a medium containing any of the components disclosed herein at a concentration sufficient for axonal elongation from the nerve cells. If myelin formation is desired for the cells described above, or if myelin formation is desired for research purposes, glial cells may be introduced and seeded through the same pore before adding nerve cells or explants. As the axon extends within the semi-cylindrical or tubular structure, axonal extension can occur increasingly distal to the focal point. Using openings or access points in the solid substrate at points increasingly distal to the focal point (or seeding point), axonal extension in the axonal state can be addressed or observed. This disclosure envisions the structure of the solid substrate taking any form to facilitate axonal extension. In some embodiments, the internal chamber or compartment housing the axonal extension has a semi-circular or substantially cylindrical diameter. In some embodiments, the solid substrate branches into two or more internal compartments at points distal to the focal point.In some embodiments, this branching may resemble a keyhole shape or tree, with two, three, four, five, six, seven, or eight or more tubular or substantially cylindrical internal chambers that are in fluid communication with one another, resulting in axonal elongation originating from a point where one or more cell bodies are seeded and extending longitudinally along the internal chambers to one or more branches. In some embodiments, one or more electrodes may be placed in or near one or more openings so that recording can be taken over one or more locations along the length of the axon. This can also be used to examine one or more locations along the length of the axon.

[0104] This disclosure relates to a system for accurately measuring recordings between a ganglion of an artificial central nervous system and a ganglion of a peripheral nervous system, the system comprising at least a first spheroid and a second spheroid, the first spheroid comprising a dorsal root ganglion or a nerve cell of central nervous system origin or a mammalian embryonic cell, and the second spheroid comprising at least one nerve cell of peripheral nervous system origin or a primary mammalian stem cell. This disclosure relates to the manufacture of any of the systems disclosed herein, comprising arranging at least a first or second spheroid comprising 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 an axon running between a central nervous system ganglion or group of cells and a peripheral nervous system ganglion or group of cells, in some embodiments, a first spheroid is located in or near a first well or channel, and a second spheroid is located in a second well or channel, at a distance sufficient to allow axonal extension between the two spheroids after exposure to cell culture medium. The disclosure relates to measuring a recording between a central nervous system ganglion or group of cells and a peripheral nervous system ganglion or group of cells, and the method includes placing electrodes on or near the first spheroid, placing electrodes on or near the second spheroid, and stimulating the system with an electric current using an amplifier or generator equipped with a generator. In some embodiments, the method further includes measuring an electrophysiological response.

[0105] In this specification, the term “recording” means, for example, measuring the response of one or more types of nerve cells. Such responses may be electrophysiological responses, such as patch-clamp electrophysiological recordings or field potential recordings.

[0106] This disclosure provides methods and apparatus for obtaining physiological measurements of microscale organ-type models of in vitro nerve tissue that mimic clinical nerve conduction and NFD studies. Results obtained using these methods and apparatus can better predict clinical outcomes and enable a more cost-effective approach to selecting promising lead compounds with a higher probability of success in later stages of development. This disclosure includes the fabrication and use of a three-dimensional microengineering system that uniquely enables the extension of high-density, highly parallel nerve fiber pathways. Due to the localized nature of these nerve fiber pathways, 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 possesses novel capabilities similar to clinical histopathology and nerve conduction studies for evaluating tissue morphometry and population electrophysiology.

[0107] This disclosure also provides a method for measuring axon myelin formation induced 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 natural tissues, axons of varying diameters and degrees of myelin formation transmit and return sensory information to and from the central nervous system at varying rates. Schwann cells assist in sensory relay by myelinating axons and insulating them for faster conduction. Similarly, the three-dimensional elongation induced by this in vitro construct includes densely parallel, oriented axons of varying diameters spanning distances up to 3 mm. The presence of Schwann cells and sheathing were observed in confocal and TEM imaging.

[0108] While neuronal morphology is a useful indicator of phenotypic maturity, a clearer indication of a healthy neuron is its ability to conduct action potentials. Cell death alone is not a complete measure of neuronal health, as many pathological changes may occur before cell death appears. 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 endpoints can be measured. Similarly, CAP measurement indicates the overall health of myelin and provides further insight into the toxicity and neuroprotective mechanisms of various drugs or target compounds, while information gathered from imaging can determine quantitative metrics regarding the degree of myelin formation.

[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 pollutant. In some embodiments, the at least one agent comprises one or a combination of small chemical compounds selected from chemotherapeutic agents, analgesics, cardiovascular modifiers, cholesterol, neuroprotective agents, neuromodulators, immunomodulators, anti-inflammatory agents, and antibacterial agents.

[0110] In some embodiments, the above at least one drug is actinomycin, alitretinoin, all-trans retinoic acid, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epotilon, erlotinib, etoposide, fluorouracil, gefitinib, gemcitabine, hydroxyurea, idarubicin, imatinib, iri This includes one or a combination of chemotherapeutic agents selected from nitekane, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosourea, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tafluposide, temozolomide (oral dacarbazine), teniposide, thioguanine (formerly thioguanine), topotecan, tretinoin, barrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, bismodegib, and vorinostat.

[0111] In some embodiments, the at least one of the above-mentioned agents includes one or a combination of analgesics selected from paracetoamol, nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, opioids, flupirin, tricyclic antidepressants, carbamacepine, gabapentin, and pregabalin.

[0112] In some embodiments, the at least one of the above-mentioned agents includes one or a combination of cardiovascular modulators selected from nepicastat, cholesterol, niacin, scutellaria, prenylamine, dehydroepiandrosterone, monatepir, esketamine, niglidipine, asenapine, atomoxetine, flunarizine, milnacipran, mexiletine, amphetamine, thiopental sodium, flavonoids, bretillium, oxazepam, and honokiol.

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

[0114] In some embodiments, the at least one of the above-mentioned agents includes one or a combination of immunomodulators selected from clenoliximab, etoticumab, rigerizumab, simtuzumab, baterizumab, pulsatuzumab, imagatuzumab, tregalizumab, patechlizumab, namulumab, perakizumab, fararimomab, patrizumab, atinumab, ubrituximab, futuximab, and durigotumab.

[0115] In some embodiments, the above-mentioned 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 includes one or a combination of antimicrobial agents selected from antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, heat, radiation, and ozone.

[0117] This disclosure further discloses a method for measuring both intracellular and extracellular recordings of biomimetic neural tissue on a three-dimensional culture platform. Previously, electrophysiological experiments were performed using either dissociated surface seeding cultures or organ-type section specimens, each method having its own inherent limitations. Studies in dissociated cell cultures are typically limited to single-cell recordings due to the absence of organized multicellular neurite structures present in organ-type specimens. Organ-type specimens possess complete neural circuits, allowing for both intracellular and extracellular studies. However, acute brain sections exhibit complex, simultaneous variables with no means of controlling individual factors, and therefore inherently limit processing speed.

[0118] Intracellular recording in in vitro 3D cultures has been demonstrated to date. However, neuronal growth has not been limited to anatomically valid structures that support spatially examining extracellular populations. More biomimetic 3D neuronal cultures are needed to enable the testing of population-level electrophysiological behavior. This disclosure supports synchronous population-level events in extracellular field recordings derived from whole-cell patch-clamp techniques and limited neurite outgrowth in 3D geometry. Prior to this disclosure, the measurement of these endpoints, which are directly similar to clinical nerve conduction studies, had not been demonstrated in purely cellular in vitro studies.

[0119] Using the methods and apparatus disclosed herein, and employing field recordings, the complex extracellular changes in potential resulting from signal conduction in all recruited fibers are measured. A collectively induced response (CAP) is an electrically stimulated collective response. Electrically induced collective spikes are inherently stepwise and involve a combined effect of action potentials in slow and fast fibers. The spike is a response consisting solely of action potentials, characterized by a rapid rise and short duration, a single, concentrated event, or rapid signal conduction in the absence of synaptic input. The three-dimensional neural constructs disclosed herein also support CAPs stimulated from greater distances along nerve pathways or channels, demonstrating the ability of neural cultures to rapidly transport signals from distant stimuli, such as afferent peripheral nerves. The three-dimensional neural cultures of this disclosure support proximal and distal stimulation techniques useful for measuring conduction properties.

[0120] This disclosure may be used in conjunction with one or more growth factors that induce the recruitment of multiple fiber types, as is common in nerve fiber tracts. In particular, nerve growth factor (NGF) preferentially recruits small-diameter fibers, 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 elongation of larger-diameter proprioceptive fibers. Electrophysiological studies may be incorporated into growth-influencing factors such as bioactive molecules and pharmacological agents to allow for the systematic manipulation of conditions for investigating the mechanisms.

[0121] Three-dimensional nerve cultures produced using this disclosure can be used as a platform to investigate the underlying mechanisms of myelin-damaging diseases and peripheral neuropathy by examining the effects of known myelin-damaging substances, culture conditions that induce neuropathy, and toxic neuropathogenic compounds on these nerve cultures. This disclosure makes it possible to use conduction velocity as a functional measure of myelin and nerve fiber integrity under toxic and therapeutic conditions, thereby facilitating research on drug safety and efficacy. By incorporating gene mutations and drugs into nerve cultures produced using the techniques disclosed herein, it becomes possible to reproduce disease phenomena in a controlled form, which may lead to a better understanding of neurodegeneration and potential therapies.

[0122] This disclosure provides apparatus, methods, and systems for generating, maintaining, and physiologically investigating nerve cells and neural networks constructed by microengineering, designed to mimic the anatomical structure of natural nerve tissue. In some embodiments, the apparatus and systems comprise one or more cultured or isolated Schwann cells and / or one or more cultured or isolated oligodendrocytes in contact with one or more nerve cells in a cell culture vessel comprising a solid substrate, the substrate comprising at least one outer surface, at least one inner surface, and at least one internal chamber, the shape of the internal chamber being at least partially defined by the at least one inner surface and accessible from a point outside the solid substrate through at least one opening in the outer surface, the cell bodies of the one or more nerve cells being located at one end of the internal chamber, and the axons being able to extend within the internal chamber along at least one length of the internal chamber such that the position of the axon tip extends distally from the cell body. In some embodiments, the inner surface of the solid substrate is cylindrical or substantially cylindrical, and as a result, the cell bodies of the nerve cells are located close to an opening at one end of the cylindrical or substantially cylindrical inner surface, and the axons of the nerve cells include a certain 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 to the cell body.In some embodiments, the inner surface of the solid substrate is cylindrical or substantially cylindrical, and as a result, the cell bodies of the nerve cells are positioned close to an opening at one end of the cylindrical or substantially cylindrical inner surface, and the axons of the nerve cells comprise a certain 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 to the cell body, and if the cell culture vessel contains multiple types of nerve cells, the multiple axons extend from the multiple cell bodies (somata) (or cell bodies (soma)) such that the multiple axons define a bundle of axons that can extend distally from the cell body along the length of the inner surface. In some embodiments, the nerve cells extend on and within the penetrable polymer. In some embodiments, one or more electrodes are positioned at or close to the tip of at least one axon, and one or more electrodes are positioned on or close to the cell body, so that an electrical potential is established over the entire length of one or more types of nerve cells.

[0123] Another object of this disclosure is to provide medium-to-fast processing assays for 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 a clinical disease. In some embodiments, the agent is any drug or agent used to treat a human disease, such that toxicity, efficacy, or neuromodulation can be compared between the novel agent, which is a proposed mammalian therapeutic agent, and an existing therapeutic agent from a human disease. In some embodiments, the novel agent for the treatment of a human disease is a therapeutic agent for a neurodegenerative disease and is compared to an existing therapeutic agent for a neurodegenerative disease. In the case of multiple sclerosis as a non-limiting example, the effects of novel drugs (modified cells, antibodies, or small compounds) are compared and contrasted with the same effects of existing treatments for multiple sclerosis, such as Copaxone, Rebif, other interferon therapies, Tysabri, dimethyl fumarate, fingolimod, teriflunomide, mitoxantrone, prednisone, tizanidine, and baclofen.

[0124] Another object of this disclosure is the use of proprietary techniques such as the assembly of nerve bundles by two-dimensional and three-dimensional microengineering, in conjunction with electrophysiological stimulation and recording of neuronal populations.

[0125] Another objective of this disclosure is to provide a novel method for evaluating neurophysiology in vitro, using composite action potentials (CAPs) as a clinically similar metric to obtain results that are more sensitive to human physiology and predictive than those obtained with current methods.

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

[0127] Another purpose of this disclosure is to provide methods for replicating, manipulating, modifying, and evaluating the underlying mechanisms of myelin-damaging diseases and peripheral neuropathy.

[0128] Another object of this disclosure is to enable medium-to-rapid processing assays for neuromodulation in human nerve cells for screening the pharmacological and / or toxicological activities of chemical and biological agents.

[0129] Another object of this disclosure is the use of proprietary assemblies of techniques such as nerve bundles by two-dimensional and three-dimensional microengineering, in conjunction with optical and electrochemical stimulation and recording of human neuronal cell populations.

[0130] Another objective of this disclosure is to quantify evoked postsynaptic potentials in biomimetic, designed thalamocortical circuits. Our observations of retrogradely generated collective spikes in neural pathways suggest that they enable collective-level physiology, such as the conduction of compound action potentials and postsynaptic potentials.

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

[0132] Another object of this disclosure is to investigate whether selective 5-HT reuptake inhibitors (SSRIs) and second-generation antipsychotics alter the developmental maturation of such circuits by using microengineered circuits in trials of such drugs.

[0133] In one embodiment, a combination of polyethylene glycol dimethacrylate and Puramatrix hydrogel is micropatterned using projection photolithography with a digital micromirror device (DMD), as shown in Figure 1. This method allows for rapid and direct micropatterning of one or more hydrogels onto conventional cell culture materials. Since the photomask never comes into contact with the gel material, multiple hydrogels can be rapidly cured in succession, enabling the production of dozens of gel constructs in one hour without automation. This technique allows for the restraint of neurite elongation within a biomimetic elongation-promoting gel using polyethylene glycol (PEG), a mechanically robust cell elongation-inhibiting gel. In some embodiments, this elongation-promoting gel may be Puramatrix, agarose, or methacrylated dextran. When embryonic dorsal root ganglion (DRG) explants elongate in this restrained three-dimensional environment, axons elongate densely and bundled from the ganglion, as shown in Figures 5 and 6. Most axons appear as small-diameter, non-myelinating fibers that elongate to nearly 1 mm in length within 2–4 weeks. The structure of this culture model, with its dense, highly parallel, three-dimensional nerve fiber pathways extending from the ganglia, broadly resembles that of peripheral nerves. Its morphology can be evaluated using neuromorphometry, enabling clinically relevant assessments not available with conventional cell assays.

[0134] In some embodiments, the culture model described above provides the ability to record electrically evoked collective field potentials arising from composite action potentials (CAPs). Exemplary recording curves exhibit a characteristic, uniform, rapid, short-latency collective spike response, which maintains consistency at high-frequency (100 Hz) stimuli, as shown in Figure 8B. As shown in Figures 8E and 8F, CAPs are reversibly eliminated by tetrodotoxin (TTX), demonstrating that drugs can be applied and exert their effects. The delay to rise with distal pathway stimulation is measurably increased, as shown in Figures 8C and 8D. The response is insensitive to neurotransmitter blockers, indicating that the evoked response is primarily CAP, rather than synaptic potentials, as shown in Figure 10. Embryonic DRG cultures have been effectively used as models of peripheral neurobiology for decades. While conventional DRG cultures are highly useful as model systems, they have been found to be unpredictable in predicting clinical toxicity when evaluated using conventional cell viability assays. While single-cell patch-clamp recordings can be performed in DRG cultures, there are no reports on CAP recordings due to the lack of tissue structure. In preferred embodiments, this disclosure provides the ability to evaluate histomorphometry and population electrophysiology, as well as clinical histopathology and nerve conduction studies.

[0135] In some embodiments, the disclosure uses human nerve cells to grow nerve tissue in a three-dimensional environment, including a CAP, where the nerve cell bodies are bundled together and positioned separately from the axonal fiber pathway, mimicking native nerve structures and enabling the measurement of morphometric and electrophysiological data. In some embodiments, the disclosure uses nerve and glial cells derived from primary human tissue. In other embodiments, the nerve and glial cells may be derived from human stem cells, including induced pluripotent stem cells.

[0136] In another embodiment, the Disclosure uses conduction velocity as a functional measure of the state of nerve tissue under toxic and therapeutic conditions. Information regarding the degree of myelination, myelin integrity, axonal transport, mRNA transcription, and neuronal damage can be determined from electrophysiological analysis. Combined with morphometric analysis of nerve density, myelin formation rate, and nerve fiber type, the mechanism of action of the compound in question can be determined. In some embodiments, the apparatus, methods, and systems disclosed herein incorporate gene mutations and drugs to reproduce disease phenomena in controlled forms, leading to a better understanding of neurodegeneration and possible therapeutic methods.

[0137] This disclosure relates to systems comprising any of the disclosed compositions, and to methods of using these systems to obtain physiologically relevant ambient data than data collected using two-dimensional tissue culture systems or systems that do not use multiple cell types. In some embodiments, the systems or compositions disclosed herein comprise / contain one or more types of cells containing any mutation. In some embodiments, at least one, 100, 500, 1000, or more types of cells contain mutations associated with specific models of human diseases. Any of the disclosed systems may comprise cells having the mutations disclosed in Table B, described later. In some embodiments, the cells of this disclosure contain or express at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the endogenous mutant proteins disclosed in Table B. Thus, these model systems may be useful for testing the efficacy or toxicity of specific drugs, biomolecules, or other therapeutic agents added to the system. The above models may also be useful in understanding the fundamental biology in the context of environmental pollutants, pathogens, or endogenously expressed proteins, and the morphological effects of such molecules on the nervous system, based on information such as responses to substances involving axon elongation, myelin formation, and demyelination, or morphological changes in the cells themselves. [Table 3-1] Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 Table 3-27 Table 3-28 Table 3-29 Table 3-30 Table 3-31 Table 3-32 Table 3-33 Table 3-34 Table 3-35 Table 3-36 Table 3-37 Table 3-38 Table 3-39 Table 3-40 Table 3-41 Table 3-42 Table 3-43 Table 3-44 Table 3-45 Table 3-46 Table 3-47 Table 3-48 Table 3-49 Table 3-50 Table 3-51 Table 3-52 Table 3-53 Table 3-54 Table 3-55 Table 3-56 Table 3-57 Table 3-58 Table 3-59 Table 3-60 Table 3-61 Table 3-62 Table 3-63 Table 3-64 Table 3-65 Table 3-66 Table 3-67 Table 3-68 Table 3-69 Table 3-70 Table 3-71 Table 3-72 Table 3-73 Table 3-74 Table 3-75 Table 3-76 Table 3-77 Table 3-78 Table 3-79 Table 3-80 Table 3-81 Table 3-82 Table 3-83 Table 3-84 Table 3-85 Table 3-86 Table 3-87 Table 3-88 Table 3-89 Table 3-90 Table 3-91 Table 3-92 Table 3-93 Table 3-94 Table 3-95 Table 3-96 Table 3-97 Table 3-98 Table 3-99 Table 3-100 Table 3-101 Table 3-102 Table 3-103 [Table 3-104]

[0138] In some embodiments, at least one cell type of the disclosed system contains any one or more mutations at the loci identified in Table B. If Table B discloses mRNA sequences, the one or more mutations in the cell may be present in the complementary endogenous DNA sequences disclosed in the above GenBank sequences. In some embodiments, the cells include one or more mutations in sequences that have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences disclosed in Table B, or, if the sequences are mRNA sequences, the cells include one or more mutations in complementary DNA sequences of these sequences identified in Table B, or sequences that have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the sequences disclosed in Table B, or mutations that are complementary to the sequences.

[0139] In some embodiments, the spheroids disclosed herein contain two, three, four, five, or more mutations in the genes identified in Table B. When a spheroid containing the specific mutations identified in Table B is used in a system disclosed herein, the corresponding system may be used as an in vitro model of the corresponding disease identified above.

[0140] In some embodiments, any composition, system, or method described in PCT / US2015 / 050061 may be used in embodiments of the present disclosure.

[0141] In some embodiments, the method is a method for manufacturing a system, culture plate, or apparatus for culturing cells, Obtaining stem cells such as induced pluripotent stem cells, Exposing the above cells to one or more cell growth factors, Differentiating the above stem cells into nerve cells, The above cells are seeded in a solid substrate having a first and / or second cavity or well. The above method includes the following: In some embodiments, the first and / or second cavity is a U-shaped bottom well, a curved bottom well, or a flat bottom well. In some embodiments, the above 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 f cells into a series of cavities or wells separated within a solid substrate, each containing a cell culture medium. In some embodiments, the step of seeding the cavities or wells includes seeding the cells into a pattern arranged within the solid substrate such that each well contains a cell spheroid, and each spheroid grows in a suspension or hanging drop form. In some embodiments, a method for manufacturing a system, culture plate, or apparatus for culturing the cells includes culturing the cells without disturbing them for a sufficient time for the cells to spontaneously form one or more spheroids.

[0142] This 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 well in a solid substrate. In some embodiments, the method further includes exposing the one or more spheroids with the drug for a time sufficient to allow the drug to be absorbed by one or more cells of the one or more spheroids, and then measuring the viability of the cells by recording, observing, or a combination of morphological changes.

[0143] This disclosure also relates to a method for forming spheroids from stem cells or cells derived from the nervous system of a subject. In some embodiments, the method for forming the spheroids is as follows: (i) Differentiating cells from stem cells into one or more cell types, which are nerve cells, astrocytes, Schwann cells, or any other cells disclosed herein, and then (ii) Mixing one or more of the above-mentioned cells for a sufficient amount of time to form a spheroid. This includes. In some embodiments, the method does not include the step of differentiating any cells after the spheroid has been formed. In some embodiments, the method does not include exposing the spheroid 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 herein. Publications, patents, or patent applications disclosed in the examples or the text of the specification are incorporated by reference in their entirety. [Examples]

[0145] Example 1: Human motor nerve on a chip for preclinical neurotoxicity testing. The objective was to develop an organ-type microphysiological model that mimics the morphology of peripheral nerves and supports clinically similar physiological measurements. The Nerve-On-A-Chip was designed using a microengineered hydrogel scaffold (Figures 1A and B).

[0146] The goal of this design was to mimic nerve fiber pathways by directing and restricting three-dimensional axonal elongation and cell arrangement (Figure 2). Robust nerve growth, bundle formation, and glial interactions facilitated morphological and physiological outputs, making it a high-content screening assay for neurotoxicity and pharmacological manipulation (Figure 3).

[0147] The results showed a structure similar to the anatomy of natural peripheral nerves. This allowed for the examination of nerve density, fiber type, and myelin formation, as well as the study of axonal elongation, cell migration, and glial differentiation (Figures 4-7). Example 2: Nano-shuttle spheroid protocol in rat spinal cord Day 1 (Figure 12): 1. Six spinal cords were isolated from E15 rat offspring, ensuring all dorsal root ganglia were identified. Remove. 2. Using spring-handle scissors, cut all six spinal cords into small pieces. 3. Using a 1000 μL pipette, transfer the spinal cord and culture medium, which have been divided into small pieces, to a 1.5 mL microcentrifuge tube. 4. The spinal cord mass is pelletized by centrifuging at 4.700 rcf for 2 minutes and 30 seconds. 5. Carefully remove the supernatant from the microcentrifuge tube, taking care not to break up the pellet. Add 6.1 mL of 0.25% trypsin-EDTA to the above microcentrifuge tube, crush the pellet, and suspend it in the trypsin. Incubate at 7.37°C for 15 minutes. 8. Quench trypsin-EDTA by adding 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 pelletize the spinal cord mass. 10. Carefully remove the supernatant from the 15 mL conical tube, taking care not to break up the pellet. 11. Add 2 mL of spinal cord seeding medium to the 15 mL conical tube containing the pellet. Using a 12.100 μL pipette, crush the lumps 15 times (setting the pipette to a 1 mL volume) to break them up. 13. Add an additional 3 mL of spinal cord seeding medium to the dissociated spinal cord solution. 14. Pass the entire 5 mL of the dissected spinal cord solution through a 40 μm cell strainer, and collect the filtered culture medium in a Petri dish. 15. Add 300 μL of dissociated spinal cord solution to each 12-well PLL coverslip. This medium should remain foamy on the coverslip and should not overflow the edges of the coverslip to fill the wells. To ensure complete coverage of the coverslip, gently tilt the 12-well plate to spread the dissociated spinal cord solution across the entire coverslip. Repeat tilting in other directions as needed to ensure complete coverage. 16. Incubate the seeded cells at 37°C for 2 hours. After 17.2 hours, gently remove the seeding medium and replace it with 700 μL of N2:NG medium. Add N2:NG medium next to the well to prevent the seeded cells from being removed from the substrate by the fluid flow. Day 2: 1. Count the number of seeded cells in one well by adding 0.5 mL of 0.25% trypsin-EDTA to the well. 2. Incubate at 2.37°C for 4 minutes. 3. Transfer the trypsin and cells aspirated from the substrate to a 1.5 mL microcentrifuge tube containing 0.5 mL of basal neuronal culture medium, and dilute the trypsin. 4. Grind the cells 10 times to break down intercellular adhesion and obtain a cell solution. 5. Use trypan blue and a hemocytometer to count the cells present in the wells. 6. Add nanoshuttle to the remaining wells at a rate of 1 μL per 60,000 cells. Disperse the nanoshuttle solution across the entire surface 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 2.37°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 used to dilute 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. The cell pellet is crushed by grinding it five times using a 6.1 mL syringe and a 20 gauge needle. 7. Add an additional 5.2 mL of N2:NG medium to the 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. Calculate the amount of cell suspension needed to be added to each of the 9.96 wells. For example, 500,000 cells, 400,000 cells, or 300,000 cells per well. 10. Securely place the non-adhesive 96-well plate on the magnetic drive device (Figure 11), and then add an appropriate amount of cell solution to each of the 96 non-adhesive wells. If necessary, add N2:NG medium to each of the 96 wells so that the total volume of each well is 150 μL. 11. Return the 96-well plate to the incubator and allow the spheroids to form without disturbing them for two days. Day 5: 1. Prepare the PEG construct. Refer to other protocols if you need guidance. Wash three times with a 2.2% anti / anti cleaning solution. Store in the washing solution overnight at 3.37°C. Day 6: 1. Remove the 96-well plate from the top of the magnetic drive unit. 2. Using a 1000 μL pipette (set to 100 μL), gently draw up and dispense the liquid from each of the 96 wells to suspend the spheroids. Using a 3.10 μL pipette (set to 10 μL), remove the spheroids from the 96 wells. 4. Add the above spheroid to 2 mL of culture medium in a Petri dish on ice. 5. Transfer the spheroids again to an empty petri dish on ice. Use a 10 μL pipette set to 4 μL. This step helps to remove any cell fragments that may have moved from the 96 wells. 6. Prepare a 1:20 dilution of Matrigel for cell mounting by mixing Matrigel with N2:NG medium, ensuring that all solutions are kept on ice (below 10°C) to prevent gelation (the amount of medium added due to spheroid migration must be taken into consideration). For example, a 200 μL 1:20 Matrigel dilution containing 4 spheroids would include 10 μL of Matrigel, 178 μL of N2:NG medium, and 12 μL of medium transferred with the spheroids. Therefore, in this step, 10 μL of Matrigel is added to 178 μL of N2:NG medium. 7. Add the above-mentioned mixed Matrigel and N2:NG medium to the spheroid in an empty Petri dish. 8. Attach the slide glass coated with Rain-X to the magnetic positioning device described above (Figure 13). 9. Place the Transwell insert containing the PEG structure on the above-mentioned microscope slide. 10. Operate the magnetic drive device described above to adjust the position of the magnet below the gap where you want to place the spheroid. Using a pipette set to 11.10 μL, transfer the spheroid and 1:20 Matrigel solution into the aforementioned cavity. Release the spheroid over a magnet (Figure 14B). 12. Repeat steps 10 and 11 for all constructs during the Transwell insert. Allow the Matrigel to gel at 13.37°C for 30 minutes. 14. Add N2:NG medium below the above insert. 15. Cultivate as desired.

[0148] Example 3: Spheroid for directional extension of neurites Round-bottom / U-bottom plates: For either one differentiated cell type or a combination of differentiated cell types, a 96-well, transparent, "U-shaped" round-bottom, untreated spheroid microplate (Corning REF: 4415) was used. The resuspended cells were recounted using a hemocytometer. Accordingly, the required density for each spheroid, ranging from 5,000 to a maximum of 100,000 cells, was added to each well using a micropipette. The spheroid microplate was then centrifuged in suspension at a centrifuge rate corresponding to the cell type for 5 minutes and incubated in a 37°C incubator for at least 24 hours until spheroids formed.

[0149] Hanging Drop Plate: The Perfecta3D Hanging Drop Plate from 3D biomatrix was used for spheroid preparation. Approximately 5,000 to 100,000 differentiated induced pluripotent stem cell-derived neurons and glial cells, in known quantities, were suspended in a small amount of culture medium. The ratio of differentiated cells was varied to enable spheroid formation and subsequent growth characteristics. The cells were suspended in a volume of 40 μl and transferred by pipette to the access port at the top of the plate. The cells were then incubated in a conventional 5% CO2 incubator for at least 24 hours to allow self-organization and spheroid formation.

[0150] The following table shows various methods for producing spheroids. [Table 1-2] [Table 2-1] [Table 2-2]

[0151] Moving Spheroids to Nerve-On-A-Chip Constructs To move the spheroids to position them in a 3D construct, the constructs are dried in a 6-well tissue culture processing plate (Transwell, 24 mm diameter, 0.4 μm pore size, REF: 3450 - transparent) by removing 500 μl of the 1500 μl of PBS used from each well, with the top of the membrane partially dried for placement of the spheroids. Then, 8% Matrigel is added to the interior of the 3D structure, and it is then incubated in a 37°C incubator for 30 minutes.

[0152] Next, the spheroids were removed using a p1000 pipette and placed as droplets on a 35 mm tissue culture dish (Cell Treat catalog number: 229635). Then, using sterile Dumont No. 4 forceps (11 cm long, standard 0.13 × 0.08 mm Dumostar 11294-00), the spheroids were placed in the "valve portion" of the three-dimensional construct. Next, 1500 μl of culture medium was placed under the membrane of the 6-well plate and placed in a 37°C incubator.

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

[0154] A solution of 5% GelMA, added laminin, and 0.05% LAP solution containing n-vinylpyrrolidone is prepared and mixed with the cell suspension to a concentration of 2 million cells / mL. The myoblast / GelMA / LAP solution is pipettered into a specific chamber of a pre-prepared, cell-impenetrate polyethylene glycol (PEG) construct, where this chamber is separated from any chambers containing motor neurons. Polymerization of the cell-loaded GelMA / LAP containing 2 million cells / mL is achieved by exposing the solution in the chamber to UV light.

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

[0156] Myoblast differentiation in three dimensions is achieved by changing the culture medium. On days 1-3, the same growth medium as described above must be supplied to the construct. On day 4, the culture medium is changed to a differentiation medium consisting of DMEM / F12 and horse serum.

[0157] The high-density encapsulation method allows the constructs to differentiate over up to three weeks as a result of the culture medium, enabling paracrine signaling.

[0158] Differentiation is confirmed by histological methods, including fluorescent labeling of muscle cells with antibodies against proteins expressed only by multinucleated myotubes, including anti-desmin and anti-alpha heavy chain myosin and DAPI, or by confirming whether a single cell body contains two or more nuclei.

[0159] Example 5: Investigation of spheroids In this study, we describe an in vitro, microengineered, biomimetic, whole-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), which can provide suitable data for integrated nerve conduction velocity (NCV) and histopathological evaluation. This whole-human system is based on the in vitro "Nerve-on-a-Chip" (NoaC) platform previously developed by the inventors using embryonic rat dorsal root ganglion (DRG) neurons and rat SCs. 5 This represents a significant extension of the previous model. 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. This model mimics robust axonal elongation (approximately 5 mm) and provides the first evidence of myelin formation of human Schwann cells in human iPSC-derived neurons, as well as the first evidence of nerve conduction velocity testing in a whole-human in vitro system such as this in vitro model. Therefore, the above-described innovative HNoaC model of human peripheral nerves has the potential to accelerate the fields of human disease modeling, drug discovery, and toxicity screening.

[0160] Culture of Schwann cells A T-75 culture flask (353136; Corning, Corning, NY) was prepared by covering it with a sterile filtered 0.1% poly-L-ornithine (PLO; Sigma-Aldrich, St. Louis, MO) solution in sterile water (Sigma-Aldrich, St. Louis, MO). The flask was then washed four times with sterile water. 7.5 mL of 10 μg / mL laminin (Sigma-Aldrich, St. Louis, MO) phosphate-buffered saline (PBS; Caisson Labs, Smithfield, UT) solution was added to the flask and kept at 4°C overnight. Laminin solution was aspirated, and 15 mL of culture medium was directly added to this T-75 culture flask. After equilibration in a 37°C incubator, cell seeding was performed. Human Schwann cell (hSC) medium was purchased from ScienCell (Carlsbad, CA). The human Schwann cell line (catalog number 1700; ScienCell) was received in refrigerated vials, and according to reports, 5 × 10⁶ 5 The cell density was greater than 50 / mL. This vial was removed from cryopreservation and thawed in a 37°C water bath. The contents of the vial were uniformly distributed onto a T-75 flask coated with the above PLO / laminin. The culture was allowed to stand in a 5% CO2 atmosphere at 37°C for at least 16 hours to promote adhesion and growth. The medium was changed every 24 hours. When the culture density reached 80%, the hSCs were subcultured using 3 mL of Accutase® (Sigma-Aldrich), and Accutase® was added to the flask at 37°C for 3 minutes. Once the cells were completely detached, 8 mL of hSC medium was added to the flask. 11 mL of the detached hSC solution 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. Conventional hemocytometer (Hausser Scientific, Horsham, Cells were counted using PA.

[0161] Culture of motor neurons

[0162] iCell® Motor Neuron (hN) Medium contains 2 mL of iCell® Neural Supplement A (FUJIFILM Cellular). Dynamics, Inc. Madison, WI) and 1 mL of iCell® Nervous System Supplement (FUJIFILM The preparation was done using 100 mL of iCell® Neurons Base Medium (FUJIFILM Cellular Dynamics, Inc) supplemented with Cellular Dynamics, Inc. To prepare for thawing the motor neurons, the hN medium was warmed to room temperature, and 1 mL of 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 dropping with a swirling motion to completely mix the cell solution and minimize osmotic shock to the thawed cells. Next, this cell vial was rinsed with 1 mL of hN medium and transferred to a 50 mL tube. Then, the volume of this solution was increased to 10 mL by slowly adding hN medium dropwise (2-3 drops / second) while swirling in this 50 mL centrifuge tube. Next, the cell solution was transferred to a 15 mL conical tube and centrifuged at 200 × g at room temperature for 5 minutes. The supernatant was aspirated, the tube was flicked with a finger, and then the cells were resuspended in 1 mL of hN medium by pipetting up and down 2-3 times. A 10 μL sample of the cell solution was then taken, and the cells were counted using a hemocytometer.

[0163] Spheroid fabrication

[0164] Untreated, clear, U-shaped, round-bottomed, 96-well spheroid microplates (4515; Corning) were used for monoculture of both human neurons (hN) and human Schwann cells (hSC), as well as for co-culture of hN / hSC. The concentrations, expressed as cells / μL-medium, were calculated for both hSC and hN to allow for the calculation of the volume required to produce spheroids of the following sizes and compositions: hN monoculture - 100,000, 75,000, 50,000, or 25,000 cells; hSC monoculture - 75,000, 50,000, or 25,000 cells; and co-culture - 75,000 hN and 75,000, 50,000, or 25,000 hSC spheroids. The calculated volume was added to the microwell plate, and the volume of each well was adjusted to 200 μL by adding medium warmed to 37°C. Next, the spheroid microplate was 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 replaced every other day, with half the volume (95 μL) being replaced with 100 μL of fresh, warmed (37°C) hN medium.

[0165] 3D dual-hydrogel nerve growth construct

[0166] On the membrane (0.4 μm / PES; Corning) of the Transwell® insert, previously reported 6Double hydrogel scaffolds were prepared using a microphotolithography technique similar to the method described above. Unless otherwise noted, 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 ratio. The obtained solution was sterile filtered and added to a Transwell® insert placed on a Rain-X (ITW Global Brands, Glenview, IL) treated slide glass in a volume of 0.6 mL, while positioned 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 set using commercially available software (DLP Lightcrafter). Selective PEGDMA / LAP solution was applied using 4500 Control Software (Texas Instruments, Dallas, TX), and irradiation with a photo-translinkable solution was performed using ultraviolet light at a wavelength of 385 nm for 28–32 seconds. After treatment, excess PEGDMA / LAP solution was removed from the voids created by the insert and photomask. The construct was then washed three times with 2% antibiotic / antifungal wash buffer (Thermo Fischer Scientific, Walton, MA) for 10 minutes each on the top and bottom of the insert. The wash buffer was removed from the insert and the keyhole-shaped channels inside. To create a cell-permeable scaffold, the voids were carefully filled with 8% growth factor-reduced Matrigel® matrix (Corning) and polymerized in a 37°C incubator.

[0167] Transfer of spheroids to hydrogel constructs To induce myelin formation in three-dimensional constructs, two culture 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, and 10 ng / mL recombinant rat β-neuronal growth factor (NGF; R&D Systems, The preparation was done using Minneapolis (MN) and 50 μg / mL L-ascorbic acid (Sigma-Aldrich). After spheroid formation, the spheroids were transferred from the microplate using a pipette and placed as droplets of hN medium on a 35 mm tissue culture dish (Cell Treat, Pepperell, MA). Next, using sterile Dumont No. 5 fine-tipped tweezers (11295-10; Dumont, Montignez, Switzerland), the spheroids were placed in the "valve portion" of the three-dimensional construct within the Matrigel. Finally, 1.5 mL of pre-myelination medium was placed under the Transwell® membrane of the 6-well plate, and the hydrogel construct was placed in an incubator at 37°C in a 5% CO2 atmosphere for incubation. Half of the medium was replaced every other day. After the construct was kept in the pre-myelination medium for one week, it was replaced with myelination medium and kept for three weeks.

[0168] immunocytochemistry All wells of the above 6-well culture plate were immobilized at room temperature for 30 minutes using 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA), pH 7.4, and then washed four times with PBS for 15 minutes each time. Next, the immobilized samples were placed at room temperature for 1 hour in a 1× blocking solution containing PBS, 5% normal goat serum (Jackson ImmunoResearch, West Grove, PA), 0.2% Triton-X-100 (Sigma-Aldrich), and 0.4% bovine serum albumin (Sigma-Aldrich), and then labeled with the following primary antibodies, namely rabbit-α-sl00 (ab868, 1:400; Abcam, Cambridge, MA) or mouse-α-βIII tubulin (ab78078, 1:500; Abcam), and left overnight in the blocking solution at 4°C. In another trial, rabbit-α-myelin basic protein (MBP, ab133620, 1:500; Abcam) was also used under the same incubation conditions. The following day, the wells were washed four times with PBS at room temperature for 8 minutes each time. These plates were then labeled with a secondary antibody, 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 antibody and DAPI were dissolved in I× blocker solution at room temperature in the dark for 90 minutes. These plates were then washed five times with PBS at room temperature in the dark for 8 minutes each time. Next, these plates were sealed with Parafilm, covered with metal foil, maintained at 4°C, and then observed under 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 a fume hood, and used with the recommended personal protective equipment. The hydrogel construct was removed from the culture, washed three times on both sides of the transmembrane well with PBS at room temperature, and then fixed. The hydrogel construct was then immersed in a 4% PFA / 0.5% glutaraldehyde solution at room temperature for 30 minutes. Secondary fixation and staining of cellular lipids were achieved by post-fixation with a 1% osmium tetroxide solution in PBS, pH 7.4, at room temperature and in the dark for 2 hours. The construct was then washed three times with PBS for 15 minutes each, and counterstained with a 2% uranyl acetate aqueous solution at room temperature and in the dark for 30 minutes. Dehydration was performed by a stepwise ethanol wash at room temperature, starting with a 10-minute wash in 50% ethanol / PBS, followed by a 10-minute wash in 70% ethanol / PBS, and then an overnight wash in 90% ethanol / PBS. The following day, the construct was washed twice with 100% ethanol at room temperature for 30 minutes. Under a dissecting microscope, the hydrogel construct was individually dissected from the transmembrane well using a scalpel without removing PEGDMA. The construct was placed in a planar embedding mold (EMS 70902, Electron Microscopy Sciences). After allowing time for the residual ethanol to evaporate from the immobilized hydrogel, it was 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 the infiltration medium was allowed to stand for 75 minutes, it was replaced with 100% Spurr resin and cured overnight in a 70°C oven and at room temperature for 48 hours, after which thin sections were prepared using an ultramicrotome. Section preparation and transmission electron microscopy (TEM) Sectioning and TEM evaluation were performed at the Shared Instrument Facility (SIF) of Louisiana State University (Baton Rouge, LA). Ultrathin sections 80–100 nm thick were cut from four locations within the HNoaC specimen, i.e., within the tissue valve (where the valve intersects with the channel, the proximal channel (i.e., near the valve), and the distal channel). The sections were placed on a Formvar carbon-coated copper grid, 200 mesh, and impregnated with metal by suspending them on droplets of 2% uranyl acetate at room temperature for 20 minutes. These sections were then rinsed three times with deionized water droplets for 1 minute each. For visualization, a JEOL 1400 TEM (Peabody, MA) was used at various magnifications with an accelerating voltage of 120 kV.

[0170] Organizational morphological measurement and analysis Metrics obtained from TEM images of HNoaC 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. The G ratio and axon diameter were determined by Fiji 7Measurements were performed using scales, thresholds, and measurement functions. The G-ratio metric was calculated by randomly sampling 10 images to identify 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 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 G-ratio calculation was based on a simple linear prediction of the internal axon diameter, while the overall outer diameter of the fiber (consisting of the axon and the surrounding dark-stained myelin laminae) was calculated by adopting the average of the minimum and maximum diameters of a given nerve fiber. This averaging method was necessary to obtain the outer diameter because the proximity of the myelin layers is not consistent across the entire circumference of the myelin sheath. The G-ratio was calculated by adopting the internal diameter relative to the average outer diameter. When measuring the extent of the outer myelin sheath, large nucleated Schwann cells were excluded.

[0171] Electrophysiology One month after co-culture, the Transwell® insert containing the reconstituted nerves was placed on an electrophysiological testing stage. Two tubes (one for supply, the other for aspiration) were positioned along the edge of the Transwell® insert, and oxygenated artificial cerebrospinal fluid (ACSF) was supplied. 5The tissue sample was perfused with the solution. To record the composite action potential (CAP), a glass capillary micropipette electrode (1-4 MΩ) was inserted into the valve of a channel near the clustered cell bodies, and the axon extending through the channel was stimulated using a concentric bipolar platinum-iridium electrode positioned 1-3 mm distal to the valve. The platinum recording electrode was placed in an ACSF-filled glass micropipette and connected to an amplifier set to 100x gain and 0.1 Hz high-pass filtering to 3 kHz low-pass filtering. 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, with at least 50 stimulations applied per sample. The CAP waveform was visualized using an analog-to-digital converter (PowerLab; AD Instruments, Colorado Springs, CO) and then saved using LabChart software (AD Instruments). After recording the CAP, snapshots of the stimulating and recording electrodes were taken using a stereomicroscope and camera, and the distance between the electrodes was measured for the calculation of nerve conduction velocity (NCV). Latency was determined by subtracting the location of the stimulation artifact from the CAP peak location. The NCV of myelinated hMN / hSC co-cultures and unmyelinated hMN single cultures was evaluated by dividing the distance between the stimulating and recording electrodes by the latency.

[0172] Statistical processing GraphPad Prism software (GraphPad Software, Using GraphPad Software, we performed a one-way analysis of variance (ANOVA) using the Tukey post-hoc test to evaluate the size differences between different types of spheroids. For electrophysiological analysis, we calculated the mean and standard deviation and performed independent two-group t-tests. A p-value of ≤0.05 was used to specify a statistically significant difference between means.

[0173] result Schwann cells improved the assembly of neurons into spheroids. The inventors created spheroids with various cell densities to produce spheroids that fit appropriately within the dimensions of the Nerve-on-a-chip (NoaC) system (i.e., with a diameter of less than 1,000 μM and maintaining a large number of cells). The inventors also compared the sizes of various spheroids to understand the interaction between hN and hSC. After placing the desired number of cells in low-adhesion round-bottom plates, spheroid formation was monitored daily. In hSC monoculture, spheroids formed within approximately 2 days and were found to always have a shape with sharp edges (Figure 43 a-c). In contrast, hN monoculture did not self-organize into spheroids within 2 days, but instead formed many smaller spherical structures (Figure 43 g-i). In co-culture, hSC promoted the uptake of hN into spheroids (approximately 2 days) compared to spheroids formed from hN alone (approximately 3-9 days, Figure 44). The edges of the co-cultured spheroids (d-f in Figure 43) were not as clearly defined compared to spheroids containing only hSCs, likely due to the heterogeneous nature of the co-cultured spheroids. Interestingly, the size of the co-cultured spheroids composed of 75,000 hN and 75,000 hSC (1025 ± 52 μm) was found to be very similar to the size of 75,000 hSC alone (967 ± 51 μm). This suggests that the co-cultured spheroids are more densely packed, and therefore confirms that the two cell types have affinity for each other.

[0174] The inventors determined that having 75,000 neurons is the optimal number of hNs to constitute an hNoaC system by measuring the diameter of various spheroid types (Figures 43 and 44). This is because, when the inventors prepared co-cultured spheroids using 25,000, 50,000, and 75,000 hSCs, the spheroid sizes were found to be approximately 833±108, 948±39, and 1025±52 μm, respectively. In cultures of neurons alone, it was found that the spheroid size increased as expected with increasing cell numbers. Under all four hN conditions (Figure 44), the spheroid size increased continuously and significantly, indicating that the packing density remained substantially unchanged across all four spheroids (25K, 50K, 75K, and 100K), and that the total number of cells contributed more significantly to the spheroid size than the interactions between various cell types within the spheroid.

[0175] Co-cultured spheroids showed robust neurite outgrowth in the NoaC system. The outer portion of the dual hydrogel system is composed of growth-resistant 10% PEGDMA, while the inner portion of the channel is filled with fully concentrated (8-12 mg / mL) Matrigel as a growth-promoting substrate. After gel formation, spheroids were gently transferred onto the valve portion of the channel and grown in a medium containing 10% FBS, but without NGF to promote the proliferation and migration of hSCs while delaying neurite extension from hN. After one week, the incubation solution was replaced with a medium supplemented with NGF and L-ascorbic acid to promote neurite extension and myelin formation by hSCs in contact with the elongating axons.

[0176] Confocal imaging revealed the three-dimensional properties of the reconstructed in vitro neurons, showing that both the cell body and axon were present throughout the entire channel depth (Figure 45). Neurites elongated approximately 1 mm on average per week. Since the basic medium containing ascorbic acid but without FBS did not support hSC migration and myelin formation (data not shown), the addition of FBS was a crucial factor in optimizing myelin formation. Immunostaining with S100 after 4 weeks revealed that hSC cells migrated approximately 1–1.5 mm outside the spheroid and grew along the elongating axon (Figure 45A–C), while the axon reached the very end of the Matrigel-filled channel (approximately 5 mm). Interestingly, for many co-culture samples, the spheroid appeared to influence axon elongation, resulting in the axon folding back after elongating a certain distance, likely due to chemotaxis caused by growth factors released from hSCs in the spheroid. As the number of hSCs in the spheroid increased, the effect on the axon became more pronounced.

[0177] In vitro myelin formation and nerve fiber structure of human nerves Finally, along with immunohistochemical staining and confocal microscopy observation, embedding in plastic resin and thin section preparation were performed, and the level and quality of myelin formation in the system were evaluated by TEM. Evidence of effective myelin formation in the system included, but was not limited to, non-compact myelin (Figure 47A), compact myelin (Figure 47B), and myelin in the process of compactification (Figure 47C). For axons showing evidence of myelin formation, 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. Evidence of lamellar myelin formation without axons (Figure 47D), the presence of lamellar bodies in the cytoplasm (Figure 47E), and naked (unmyelinated) axons (Figure 47F) was also observed. The appearance of lamellar bodies in the cytoplasm, consisting of relatively regularly spaced helical membranes (membranen whorls), was interpreted as representing autophagosome production, consistent with the recirculation of senescent organelles. The distribution of lamellar bodies was sparse, and no apoptotic nuclei were observed, indicating that the affected cells were not involved in programmed cell death.

[0178] In vitro human neurons exhibit effective composition-dependent electrical conductivity. To determine whether the nerve conduction velocity (NCV) of iPSC-derived human neurons (hN) could be measured with and without human Schwann cells (hSCs), techniques similar to brain section electrophysiology were used. Axons within channels were stimulated, and compound action potentials (CAPs) from the cell bodies were recorded (Figure 46A). Axons were stimulated approximately 1–3 mm away from the cell bodies, and the distance the impulse traveled between the stimulating electrode and the recording electrode was calculated. Two types of NCV, namely the onset NCV and the peak NCV, were evaluated to determine the difference between the fastest signal and the peak signal (Figure 46B' and B''). Surprisingly, the onset and peak NCVs were found to be slower in hN / hSC co-culture samples compared to hN-only culture samples. The initial NCVs for 75K hN monoculture and 75K / 25K hN / hSC co-cultures were measured at 0.28±0.07 and 0.20±0.02 m / s, respectively, while the peak NCVs were found to be 0.18±0.04 and 0.13±0.02 m / s, respectively (Figure 46C). For samples with a large number of SCs (75K / 75K and 75K / 50K hN / SC co-cultures), it was difficult to measure the initial and peak NCVs. Qualitative testing of these samples revealed that the density of neurite outgrowth was slightly lower in the co-culture samples, which may explain the decrease in NCV.

[0179] In the present study, the present inventors disclose the first biomimetic fully human in vitro model of peripheral nerves assembled as a Nerve-on-a-Chip (NoaC) platform. This microengineered dual hydrogel system maintains the neuronal cell bodies at defined locations (i.e., "ganglia") and restricts dense three-dimensional axonal outgrowth within narrow channels that extend linearly outward from the clustered cell bodies (i.e., "nerves"). The system supports functional (e.g., electrophysiological examination) and structural (e.g., qualitative and quantitative microscopic analysis) endpoints, which are the current "gold standard" required for the assessment of neuropathological conditions associated with peripheral neuropathy representing a growing medical concern. Innovative aspects of this study include the reproducible fabrication of neuron / Schwann cell co-culture spheroids, robust in vitro viability (~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 neural systems In vitro myelination using primary hSCs involves the extraction of hSCs from adult nerves 8、9 , contamination by fibroblasts 8~10 , and transformation of SCs to a proliferative / non-myelinating phenotype in vitro 11、12 It has long been a problem, in part due to the complex issues associated with the above. Co-culture conditions have been well established for myelination of rat dorsal root ganglion (DRG) sensory neurons by embryonic, neonatal, and adult rodent SCs 13~15 . However, under similar co-culture conditions, myelination cannot be reproduced using human SCs cultured with rat DRG neurons 11Strict purification of primary human SCs or differentiation of human stem cells or human fibroblasts into SC-like cells results in a limitation of myelin formation in rat sensory neurons; however, the degree of this limitation observed in mixed-species cultures is significantly smaller than the degree of this limitation observed when using embryonic rat SCs. 11、16 This is likely due to differences in species or the density of SCs compared to the number of axons. Recently, Clark and his collaborators 17 This study successfully demonstrated myelin formation in human stem cell-derived sensory neurons using rat stem cells. However, an in vitro system demonstrating myelin formation in human iPSC-derived neurons using human Schwann cells remains elusive.

[0180] For the past few years, much research has focused on creating glial cell organoids, generating brain-like tissue in vitro. 18~22 Interestingly, all these strategies focused on differentiating aggregates of neural progenitor cells into more defined neural structures. In contrast, we reverse-engineered this process by combining two differentiated cell types and evaluating their potential for interaction and self-organization. To mimic the growth of embryonic dorsal root ganglia (DRGs) in vitro, we generated neuron / Schwann cell spheroids using ultra-low adhesion 96-well plates and studied the crosstalk between axons and SCs, which is crucial for the differentiation of SCs into the myelin-forming phenotype. 23、24This promoted cross-communication and enhanced the potential for good myelin formation by bringing axons and SCs into close proximity within the three-dimensional spheroid. Following the addition of the antioxidant ascorbic acid, we observed the first evidence of in vitro myelin formation of stem cell-derived human neurons by primary human Schwann cells. While the self-assembly rates and spheroid formation rates differed individually in both hN and hSC, when combined, hSCs improved quality and increased the rate of spheroid self-assembly compared to the neuron-only condition. Based on spheroid diameter, co-cultured spheroids were found to be more compact than either hN or hSC spheroids, indicating enhanced interaction between these two cell types.

[0181] Migration of Schwann cells from spheroids Schwann cell migration is an important phenomenon during development and peripheral nerve regeneration after injury. 25 Little is known about the clues that direct the fate of neural crest cells towards Schwann cell precursors and ultimately to Schwann cells. However, it has been known for decades that both progenitor cells and Schwann cells depend on elongating axons for differentiation, proliferation, and functional maturation. 26 In this specification, we are for the first time able to observe the migration of human-origin cells into tissue in vitro by constructing this mini-ganglion composed of hN and hSC. The axons extended outward in conjunction with the migrating hSCs, synchronized with the elongating axons in this process. Interestingly, we observed that the hSCs migrated only about 1 mm outside the spheroid, compared to a total axonal elongation of about 5 mm. This is in contrast to typical two-dimensional co-culture experiments that usually involve adding more than 100,000 Schwann cells into a smaller two-dimensional region. 17、27This may be due to the smaller number of hSCs added during these experiments compared to neurons. This modest hSC elongation compared to axonal elongation may also be a result of the short pre-myelin formation period of only one week and the addition of NGF to the culture medium after the first week of culture. It has been shown that NGF also enhances neuron-Schwann cell interactions and myelin formation. 28 Therefore, NGF may be a factor that reduces SC migration. Based on the migration of human SCs outside of spheroids described above, this HNoaC model can also be used to study the potential ability of SC migration in the presence of therapeutic molecules, and thus to generate candidate therapeutics for patients with peripheral nerve injury.

[0182] hSCs are known to behave differently in vitro from rat Schwann cells in terms of their responsiveness to mitogenic factors and growth factors, as well as their inability to replicate myelin formation. 29 The inventors' three-dimensional spheroid model of human nerves exhibited the general characteristics of nerve trunks observed in nerves obtained by autopsy or biopsy. Axons were associated with a myelin sheath, often (but not always) characterized by a closely spaced myelin membrane, and possessing complete complement of organelles including cytoskeletal filaments and mitochondria. The apposition of the myelin layer differed among nerve fibers, and in some cases, laminar myelin formed in the absence of axons. Both of these findings are rarely seen in differentiated nerves obtained in vivo, indicating that some differences in differentiation actually occur in culture (as expected). Nevertheless, a sufficient number of myelinated axons were observed in the mini-nerve portions of the mixed culture system, which would be a suitable substitute for mixed somatic nerves (i.e., somatic nerves including densely myelinated axons, sparsely myelinated axons, and unmyelinated axons).

[0183] Evaluation of nerve conduction velocity (NCV) Various neurological disorders are known to exhibit a variety of neurophysiological characteristics.30 Therefore, in vitro microengineered neurons should be able to reveal electrophysiological changes, serving as a means for conducting exploratory and mechanistic toxicological studies. In this study, the first to examine nerve conduction using human iPSC-derived neurons, we were able to confirm differences in nerve conduction velocity (NCV) between myelinated and unmyelinated human axons, demonstrating that this system is sufficiently sensitive to assess nerve function. Surprisingly, myelinated hN / hSC co-culture samples showed slower NCV compared to unmyelinated hN-only single-culture samples. Qualitative analysis of these cultures revealed that the number of hSCs in the spheroids may have reduced elongation and axon density in the HNoaC channels, which likely contributed to the decrease in NCV. Furthermore, in co-cultures with high SC densities (50K and 75K), many axons appeared folded, making it impossible to measure the optimal length between the stimulated point and the recorded point, which could affect NCV calculations. Furthermore, the presence of non-neuronal cell bodies in co-cultured spheroids reduces the probability of recording from properly stimulated cell bodies. Importantly, NCVs from hNs were 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, which consists of iPSC-derived neurons with lower maturity compared to mature myelinated axons of nerves evaluated in vivo at room temperature.

[0184] The simple design of this whole-human NoaC system opens new avenues in translational research. This platform can be used not only to screen drug candidates based on clinically relevant electrophysiological and histopathological metrics, but also to study the underlying mechanisms causing neurological diseases, including, but not limited to, toxic, demyelinating, and other neurodegenerative diseases. In relation to specific treatments or therapies, our conceptually identical rat NoaC system... 6By comparing this data with data obtained from human NoaC, it will be helpful to bridge the gap between non-clinical trials and the present invention's ability to predict human responses and potential safety risks.

[0185] References 1 Huh, D., Hamilton, GA & Ingber, D. E. From 3D cell culture to organs-on-chips. Trends in Cell Biology 21, 745-754, doi:https: / / doi.org / 10.1016 / j.tcb.2011.09.005 (2011). 2 Huh, D., Torisawa, Y.-s., Hamilton, GA, Kim, HJ & Ingber, DE Microengineered physiological biomimicry: Organs-on-Chips. Lab on a chip 12, 2156-2164, doi:10.1039 / C2LC40089H (2012). 3 Pankevich, Diana E., Altevogt, Bruce M., Dunlop, J., Gage, Fred H. & Hyman, Steve E. Improving and Accelerating Drug Development for Nervous System Disorders. Neuron 84, 546-553, doi:https: / / doi.org / 10.1016 / j.neuron.2014.10.007 (2014). 4 Bespalov, A. et al. Failed trials for central nervous system disorders do not necessarily invalidate preclinical models and drug targets. Nature Reviews Drug Discovery 15, 516, doi:10.1038 / nrd.2016.88 https: / / www.nature.com / articles / nrd.2016.88#supplementary-information (2016). 5 Huval, R. M. et al. Microengineered peripheral nerve-on-a-chip for preclinical physiological testing. Lab on a chip 15, 2221-2232, doi:10.1039 / c4lc01513d (2015). 6 Parastoo, K., Ashwin, S., Lauren, A. P., Daniel, W. S. & Michael, J. M. Methods for fabrication and evaluation of a 3D microengineered model of myelinated peripheral nerve. Journal of Neural Engineering 15, 064001 (2018). 7 Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nature methods 9, 676-682, doi:10.1038 / nmeth.2019 (2012). 8 Morrissey, T. K., Kleitman, N. & Bunge, R. P. Isolation and functional characterization of Schwann cells derived from adult peripheral nerve. J Neurosci 11, 2433-2442 (1991). 9 Scarpini, E., Kreider, B. Q., Lisak, R. P. & Pleasure, D. E. Establishment of Schwann cell cultures from adult rat peripheral nerves. Experimental Neurology 102, 167-176, doi:https: / / doi.org / 10.1016 / 0014-4886(88)90090-8 (1988). 10 Scarpini, E. et al. 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, AI Nerve conduction studies: essentials and pitfalls in practice. Journal of Neurology, Neurosurgery &Psychiatry 76, ii23 (2005).

[0186] Example 6: Sensory Synapse Model

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

[0188] A key aspect of this experiment is that DRG neurons extend their axons through GelMA and form synapses on DH neurons. Previous laboratory experiments have shown that spinal cord spheroid growth can be controlled by the stiffness of the gel and does not grow well in GelMA. Using this characteristic, the inventors aim to create a unidirectional neural circuit in which DH axons do not extend in GelMA but extend throughout the entire Matrigel, which can record compound action potentials (CAPs).

[0189] The dorsal horn and DRG were isolated and dissociated from the spinal cord of 15-day-old embryonic rats. The cells were then individually cultured in spheroid cultures in 96-well U-bottom plates. Two days after seeding, spheroids were formed and then placed in a bi-hydrogel construct to allow for three-dimensional neuronal growth (Figure 48A). The DRG was placed in GelMA in the keyhole at the bottom of the construct, while the DH spheroid was placed in Matrigel in the keyhole in the center. Staining of the culture with β3-tubulin confirmed axonal extension from both spheroids in the culture at 28DIV (Figure 48B). Stimulatory electrodes were placed on the DRG axons, and CAP recordings were taken on the DH spheroid. The distance between the electrodes in this culture was measured at 3.1 mm (Figure 48C). An example of a CAP recording curve showed the response in a DH spheroid when a DRG axon was stimulated (Figure 48, D). This suggests that the DRG is a DH neuron that synapses with and activates the DH neuron that records the CAP response from that neuron. Further experiments will focus on confirming the formation of these synapses and translating 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

[Claim 1] The composition or method described in the specification.

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