Nerve bundles and methods for manufacturing nerve bundles

Abstract

[Problem] To provide a method for producing a nerve fascicle, the method comprising efficiently elongating an axon of a nerve cell. [Solution] Nerve cells are cultured in the presence of feeder cells including at least one kind of cell selected from among angiogenic cells, perivascular cells, and oligodendrocytes.

Description

[Technical Field]

[0001] This invention relates to nerve bundles and methods for producing nerve bundles. [Background technology]

[0002] In recent years, methods for nerve transplantation have been developed to restore the function of nerves damaged by neurodegenerative diseases or physical causes. A widely used method for nerve transplantation involves injecting neural stem cells into the affected area. However, this method has a problem: the injected neural stem cells tend to migrate easily within the body and have difficulty engrafting, making it difficult to efficiently repair damaged areas of nerve tissue such as the brain, spinal cord, and complex peripheral nerves.

[0003] To solve this problem, a method has been developed in which bundles of nerve cells with extended axons (nerve bundles) are attached to the remaining nerves at the site of injury and then transplanted. As a method for obtaining nerve bundles for transplantation, a method has been reported in which nerve cells are cultured and the axons of the nerve cells are extended to create nerve bundles (Patent Documents 1 and 2). However, these methods cannot efficiently extend and thicken the axons of nerve cells, and there is a problem in that it is difficult to obtain nerve bundles with axons of sufficient length and thickness (diameter) for transplantation.

[0004] Furthermore, in cases of central nervous system injury, particularly spinal cord injury, it is necessary to restore nerve function within a short period after the injury from the perspective of maintaining vitality. Therefore, it is necessary to create nerve bundles with axons of sufficient length and diameter for transplantation in a short period of time.

[0005] Under these circumstances, there is a need for a method to efficiently obtain nerve bundles with axons of sufficient length and diameter for transplantation. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2017 / 187696 [Patent Document 2] Japanese Patent Publication No. 2014-136128

[0007] The inventors have discovered that by culturing nervous system cells in the presence of specific feeder cells and glial cells, the axons of nervous system cells can be efficiently extended. Furthermore, the inventors have also discovered that the axons can be hypertrophied in conjunction with their extension. According to this invention, nerve bundles with axons of sufficient length and diameter for transplantation can be efficiently produced. This invention is based on these findings.

[0008] The present invention encompasses the following inventions. [1] A method for producing nerve bundles, comprising culturing a population of nerve cells in the presence of at least one type of feeder cell selected from vascular constituent cells, perivascular cells and oligodendrocytes, and glial cells, and extending the axons of the nerve cells. [2] The method according to [1], wherein the feeder cells include at least one cell selected from the group consisting of pericytes, vascular endothelial cells, fibroblasts, smooth muscle cells and oligodendrocytes. [3] The method according to [1] or [2], wherein the feeder cells include cells that secrete at least one growth factor selected from the group consisting of VEGF, NGF, BDNF, FGF-2, NGFB, and EGF. [4] The method according to any one of [1] to [3], wherein the glial cells include oligodendrocytes. [5] The method according to any one of [1] to [4], wherein the nerve bundle has a myelin sheath containing oligodendrocytes. [6](a) A step of preparing a substrate having at least one recess and a groove connected to the recess, the groove being covered with feeder cells, (b) Adding the nerve cell population and glial cells to the recess, (c) A step of culturing the cell population containing the nerve cells and the glial cells to extend the axons of the nerve cells along the grooves. A method of any of [1] to [5], including the above. [7](a) A step of preparing a substrate having two recesses and a groove connecting the two recesses, the groove being covered with the feeder cells, (b) Adding the nerve cell population and glial cells to the recess, (c) A step of culturing the cell population containing the nerve cells and the glial cells to extend the axons of the nerve cells along the grooves. A method of any of [1] to [6], including the above. [8] The method according to [6] or [7], wherein in step (a), the groove is covered with fibroblasts before the groove is covered with the feeder cells. [9] The method according to any one of [1] to [8], wherein the cell population containing the nerve cells further comprises endothelial cells.

[10] The method according to [9], wherein the endothelial cells included in the cell population containing nerve cells are vascular endothelial cells.

[11] The method according to

[10] , wherein the vascular endothelial cells are vascular endothelial cells from at least one tissue selected from the group consisting of dental pulp, gingiva, subcutaneous tissue, intracavitary arteries, intracavitary veins, and umbilical cord.

[12] The method according to any one of [9] to

[11] , further comprising forming tubes derived from endothelial cells contained in the nerve cell population in step (c).

[13] The method according to any one of [9] to

[12] , wherein the nerve cells and endothelial cells are derived from the same individual.

[14] The method according to any one of [9] to

[13] , wherein the cell population containing the nervous system cells further comprises a biocompatible material, and the nervous system cells and endothelial cells are each present in layers on the surface of separate biocompatible materials.

[15] The method according to

[14] , wherein the biocompatible material comprises collagen. The method according to

[14] or

[15] , wherein the biocompatible material contains collagen beads. The method according to any one of [6] to

[16] , wherein the length of the groove portion is 3 mm or more. A nerve bundle produced by the method according to any one of [1] to

[17] . A method for producing a graft material, comprising covering a nerve bundle produced by the method according to any one of [1] to

[17] with a sheet of a biocompatible material. The method according to

[19] , wherein the sheet contains fibroblasts. The method according to

[19] or

[20] , wherein the graft material is a graft material for nerve regeneration. A graft material produced by the method according to any one of

[19] to

[21] .

[23] A method for extending axons of nervous system cells, comprising culturing the nervous system cells in the presence of feeder cells containing at least one kind of cells selected from vascular constituent cells, perivascular cells and oligodendrocytes, and glial cells.

[24] The method according to

[23] , wherein the feeder cells contain at least one kind of cells selected from the group consisting of pericytes, endothelial cells, fibroblasts, smooth muscle cells and oligodendrocytes.

[25] The method according to

[23] or

[24] , wherein the glial cells contain oligodendrocytes.

[26] A nerve bundle having axon-extended nervous system cells and endothelial cell tubes existing along the axons, the nerve bundle containing at least one kind of cells of HNK-1 glycan-expressing cells and p75NTR-expressing cells, and the axons having a myelin sheath containing oligodendrocytes.

[27] The nerve bundle according to

[26] , containing HNK-1 glycan-expressing cells and p75NTR-expressing cells.

[28] The nerve bundle according to

[26] and

[27] , containing at least one kind of cells selected from the group consisting of NS200-expressing cells, peripherin-expressing cells, myelin basic protein-expressing cells, S100-expressing cells, MPZ-expressing cells, paranodin-expressing cells, CD31-expressing cells and PDGFRβ-expressing cells.

[29] A nerve bundle according to any one of

[26] to

[28] , comprising NS200-expressing cells, perineurium-expressing cells, myelin basic protein-expressing cells, S100-expressing cells, MPZ-expressing cells, paranodin-expressing cells, CD31-expressing cells and PDGFRβ-expressing cells.

[30] The nerve bundle according to any one of

[26] to

[29] , having a cell layer containing at least one cell of fibroblasts and pericytes that covers at least a part of the axon along the axon of the nervous system cells, and the tube of the endothelial cells being present inside the cell layer.

[31] The nerve bundle according to any one of

[26] to

[30] , wherein the tube of the endothelial cells further contains pericytes.

[32] A transplantation material comprising the nerve bundle according to any one of

[26] to

[31] .

[33] The transplantation material according to

[32] , which is a transplantation material for nerve regeneration.

[34] An apparatus for manufacturing a nerve bundle having at least one first recess and at least one first groove having at least first and second ends, wherein the first groove has at least one second groove having at least first and second ends and extending parallel to the first groove, wherein the first recess is connected to the first ends of the first and second grooves respectively, the apparatus.

[35] The apparatus according to

[34] , wherein at least one kind of feeder cell selected from vascular constituent cells, perivascular cells and oligodendrocytes, and glial cells are arranged in at least one of the first and second grooves.

[36] The apparatus according to

[34] and

[35] , wherein the second ends of the first and second grooves are connected to a second recess.

[37] The first recess is a recess having an opening with a diameter of 3 to 10 mm and a bottom curved horizontally or concavely, and a depth of 4 to 8 mm, and the first groove is a groove having a bottom curved horizontally or concavely, a width of the trough part of 4 to 6 mm, a length from the first end to the second end of 2 to 3 cm, and a depth of 4 to 6 mm, according to any one of

[34] to

[36] .

[38] The apparatus according to any one of

[34] to

[37] , wherein the second groove portion has a horizontal or concave curved bottom, and the width of the rim is 2 to 2.5 mm and the depth is 1.5 to 2 mm.

[39] The apparatus according to any one of

[36] to

[38] , wherein the second recess is a recess 3 to 6 mm deep having an opening 2 to 5 mm in diameter and a horizontal or concave curved bottom.

[40] The apparatus according to any one of

[34] to

[39] , wherein the first groove portion has three of the second groove portions.

[41] The apparatus according to any one of

[34] to

[40] , wherein the apparatus comprises polydimethylsiloxane.

[0009] According to the present invention, the axons of nerve cells can be efficiently extended. Furthermore, according to the present invention, the axons of nerve cells can be efficiently hypertrophied. According to the present invention, nerve bundles having axons of sufficient length and diameter for transplantation can be efficiently produced. Moreover, according to the present invention, nerve bundles having nerve cells and endothelial cell tubes present along the axons of said nerve cells can be obtained. [Brief explanation of the drawing]

[0010] [Figure 1] Figures 1A and 1B are phase-contrast micrographs of two-dimensional reticular neural cells obtained by culturing, differentiating, and growing dental pulp-derived neural cells in petri dishes coated with ornithine and / or lysine. The arrows in Figure 1A indicate small cells that have developed and attached to the cell bodies of neural cells. The arrows in Figure 1B indicate small cells that have developed and attached to the axons of neural cells. [Figure 2] Figure 2A is an immunostained image of a spinal nerve bundle prepared using small cells that have developed and attached to the cell bodies of nervous system cells, stained using an anti-Olig2 antibody. Figure 2B is an immunostained image of a spinal nerve bundle prepared using small cells that have developed and attached to the cell bodies of nervous system cells, stained using an anti-S100 antibody. [Figure 3]Figure 3A is an immunostained image of a peripheral nerve bundle prepared using small cells that have developed and attached to the axonal portion of a nervous system cell, stained using an anti-Olig2 antibody. Figure 3B is an immunostained image of a peripheral nerve bundle prepared using small cells that have developed and attached to the axonal portion of a nervous system cell, stained using an anti-S100 antibody. [Figure 4] Figure 4 is a schematic diagram of a device for creating nerve bundles. Figure 4A is a top view of the device. Figure 4B is a cross-sectional view (cloverleaf shape) of the groove section of the device. Figure 4C is a schematic diagram of the entire device and the groove section when various cells are added to the device. The enlarged schematic diagram of a part of the groove section in Figure 4C (one lobe of the clover) shows the case when fibroblasts and various feeder cells are added in sequence. [Figure 5] Figure 5 is a graph showing the relationship between the number of culture days and the length of nerve fibers (axons) when various cells are used as feeder cells. [Figure 6] Figure 6A is a phase-contrast micrograph showing the shape of a nerve bundle when feeder cells (pericytes) are present. Figure 6B is a phase-contrast micrograph showing the shape of a nerve bundle when feeder cells (pericytes) are absent. [Figure 7] Figure 7 shows macroscopic and immunostained images of nerve bundles (vascularly endogenous nerve bundles for the spinal cord) prepared by the method of Example 7. Figure 7A is a macroscopic image. Figure 7B is an immunostained image using anti-MBP antibody and anti-NF200. Figure 7C is an immunostained image using anti-NF200 and anti-vWF antibody. [Figure 8] Figure 8 shows immunofluorescence staining images of cross-sections of nerve bundles stained with NF200 and myelin basic proteins. [Figure 9] Figure 9 shows immunofluorescence stained cross-sections of nerve bundles stained for NF200 and von Willebrand factor. [Figure 10] Figure 10A is a photograph of a rat with only the T9 vertebral arch removed. Figure 10B is a photograph of the resected area after the T9 vertebral arch and thoracic spinal cord have been removed from the rat. [Figure 11]Figure 11A is a photograph of the target site in the rat spinal cord (thoracic spinal cord) immediately before the implantation of nerve bundle graft material. Figure 11B is a photograph of the implantation site immediately after the implantation of nerve bundle graft material in the rat spinal cord (thoracic spinal cord). [Figure 12] Figure 12 is a graph showing the recovery of motor function (BBB score) after transplantation (resection) in rats with the thoracic spinal cord removed and rats that underwent nerve bundle grafting after the thoracic spinal cord was removed. [Figure 13] Figure 13A shows a hematoxylin-eosin (HE) stained longitudinal section of the resection site 6 weeks after thoracic spinal resection in rats that did not receive nerve bundle graft material after thoracic spinal resection. Figure 13B shows an HE stained longitudinal section of the graft site 6 weeks after nerve bundle graft material transplantation in rats that received nerve bundle graft material immediately after thoracic spinal resection. [Figure 14] Figure 14A shows a hematoxylin-eosin (HE) stained longitudinal section of the resection and transplantation site of a rat that received nerve bundle graft material immediately after thoracic spinal resection, 6 weeks after transplantation. Figure 14B is a magnified HE stained image of the junction between the nerve bundle graft material and the nerve portion of the resected thoracic spinal cord of the rat (host) in the stained image of Figure 14A (indicated by the single arrow in Figure 14A). [Figure 15] Figures 15A-C are immunofluorescent images of longitudinal sections of nerve bundles stained with immunofluorescence for S100, p75NTR, and DAPI, respectively. Figure 15D is a merged image of Figures 15A-C. [Figure 16] Figures 16A-D are immunofluorescent images of longitudinal sections of nerve bundles stained with immunofluorescence for HNK-1 glycans, p75NTR, MPZ, and DAPI, respectively. Figure 16E is a merged image of Figures 16A-D. [Figure 17] Figure 17 shows a fluorescent immunostained cross-section of a nerve bundle stained for NF200 and myelin basic protein. [Figure 18]Figure 18A is a transverse immunofluorescence image of a nerve bundle stained for NF200 and S100. Figure 18B is a transverse immunofluorescence image of a nerve bundle stained for NF200 and peripherin. [Figure 19] Figure 19A is a fluorescent immunostained cross-section of a nerve bundle stained with NF200 and periaxine. Figures 19B and C are fluorescent immunostained longitudinal sections of the same nerve bundle stained with NF200 and periaxine. [Figure 20] Figures 20A and 20B are immunofluorescent images of cross-sections of nerve bundles that have been immunofluorescently stained for CD31 and PDGFRβ, respectively. [Figure 21] Figure 21A is a fluorescent immunostained image of a cross-section of a nerve bundle stained with fluorescent immunostaining for CD31 and PDGFRβ. Figure 21B is a magnified fluorescent immunostained image of a portion of Figure 21A. [Figure 22] Figure 22 is a magnified immunofluorescence image of a portion of a cross-section of a nerve bundle that has been immunofluoresced for CD31 and PDGFRβ. [Figure 23] Figure 23A is a photograph of the transplant site 12 weeks after transplantation in rats that received autologous nerve transplants. Figure 23B is a photograph of the transplant site 12 weeks after transplantation in rats that received nerve bundle transplant material. Figure 23C is a photograph of the transplant site 12 weeks after transplantation in rats that received artificial nerve transplants. Figure 23D is a photograph of the transplant site 12 weeks after sciatic nerve transection in rats. [Figure 24] Figure 24A shows the formula for calculating the sciatic nerve function index. Figure 24B is a graph showing the sciatic nerve function index after transplantation (transection) for each transplanted rat and sciatic nerve-transected rat. [Figure 25] Figure 25 is a graph showing the wet weight of the gastrocnemius muscle at the distal end of the transplant site in each transplanted rat and the sciatic nerve-transected rat. [Figure 26] Figures 26A-D are immunofluorescent images of cross-sections of nerve bundles stained with STEM121, p75NTR, MPZ, and DAPI, respectively. Figure 26E is a merged image of Figures 26A-D. Detailed description of the invention

[0011] (Method of manufacturing nerve bundles) According to one aspect of the present invention, a method for producing a nerve bundle (hereinafter also referred to as "the production method of the present invention") is provided. According to the production method of the present invention, a nerve bundle having axons of sufficient length for transplantation can be produced.

[0012] A method for producing nerve bundles includes culturing a population of nerve cells in the presence of feeder cells, which include at least one type of cell selected from vascular constituent cells, perivascular cells, and oligodendrocytes, and glial cells (glial cells), thereby extending the axons of the nerve cells.

[0013] In this specification, "cell population containing nervous system cells" means a cell population containing nervous system cells as described below. The cell population containing nervous system cells may also contain cells other than nervous system cells.

[0014] The nervous system cells contained in the nervous system cell population include not only cells that constitute the nervous system, but also cells that can differentiate into cells that constitute the nervous system. Specifically, any of the following can be used as nervous system cells: neural stem cells, cells that can differentiate into nerve cells or glial cells, cells in the process of differentiating into nerve cells or glial cells (e.g., immature nerve cells, immature glial cells, etc.), or differentiated mature nerve cells (e.g., mature nerve cells, mature glial cells, etc.). These nervous system cells may be commercially available, isolated and prepared from living organisms, or differentiated and induced from pluripotent stem cells such as ES cells or iPS cells. Furthermore, the nervous system cells may be cells derived from the target individual for transplantation (autologous cells) or cells derived from an individual other than the target individual for transplantation (allogeneic cells). In a preferred embodiment, the nervous system cell population contains neural stem cells, cells that can differentiate into nerve cells, cells in the process of differentiating into nerve cells, and / or mature nerve cells. The nervous system cells included in the nervous system cell population are not particularly limited as long as they do not exhibit antigenicity in the target of the nerve bundle transplantation of the present invention, and can be used regardless of their origin. Examples of nerve cell origins include those from the same individual as the nerve bundle recipient, from a different individual, and from HLA (Human Leukocyte Antigen) homozygous donors. Additionally, nerve cell origins can include cells in which immune rejection has been suppressed by modifying the human leukocyte antigen (HLA) gene using genome editing technology, such as universal donor cells.

[0015] The cell population containing nervous system cells may take any form as long as the effects of the present invention are achieved, for example, in the form of a cell population containing nervous system cells suspended in a culture medium (αMEM, Dulbecco's modified Eagle medium (DMEM), Dulbecco's modified Eagle medium / Ham F-12 mixed medium (DMEM / F12), Ham 10, Ham 12, RPMI1640 medium, various nerve cell culture media, etc.), or in the form of suspended cells.

[0016] The amount of nerve cells in a cell population containing nerve cells is not particularly limited as long as the effects of the present invention are achieved, but for example, the number of nerve cells may be 10 3 ~10 10 pieces, preferably 10 4 ~10 9 10 comfortable 5 ~10 8 It is an individual.

[0017] Cells other than nerve cells included in the nerve cell-containing cell population include, for example, endothelial cells and red blood cells. Preferably, endothelial cells capable of forming blood vessels are used, and specifically, vascular endothelial cells are used. The cells other than nerve cells included in the nerve cell-containing cell population are not particularly limited as long as they do not show antigenicity in the target of nerve bundle transplantation of the present invention, and can be used regardless of their origin. Examples of the origin of cells other than nerve cells include the same individual as the target of nerve bundle transplantation, a different individual from the target of nerve bundle transplantation, and HLA (Human Leukocyte Antigen) homozygous donors. As for the origin of nerve cells, examples include cells in which immune rejection has been suppressed by modifying the human leukocyte antigen (HLA) gene using genome editing technology, such as universal donor cells.

[0018] If a population of cells containing nerve cells also contains endothelial cells, culturing the population of nerve cells in the presence of feeder cells causes the endothelial cells to proliferate and differentiate, forming tubes derived from the endothelial cells along the extended axon. As a result, the resulting nerve bundle has not only a bundle of nerve cells (nerve fibers) with an extended axon, but also tubes (blood vessels) of endothelial cells that extend in the same direction as the nerve fiber (axon) and are attached to the nerve fiber (axon).

[0019] As the endothelial cells contained in the cell population containing nervous system cells, any of endothelial stem cells, cells in the process of differentiating into endothelial cells, and differentiated mature endothelial cells can be used. In addition, the origin of the endothelial cells contained in the cell population containing nervous system cells is not particularly limited, but preferably endothelial cells derived from blood vessels, more preferably endothelial cells derived from the blood vessels of dental pulp, gingiva, subcutaneous tissue, intra-cavity artery, intra-cavity vein or umbilical cord, and even more preferably endothelial cells derived from the blood vessels of dental pulp are used. These endothelial cells may be commercially available, or may be differentiated and induced from pluripotent stem cells such as ES cells and iPS cells. Further, the endothelial cells may be autologous cells derived from the individual to be transplanted, or may be allogeneic cells derived from an individual other than the transplant recipient.

[0020] The content of endothelial cells in the cell population containing nervous system cells is not particularly limited as long as the effects of the present invention are achieved. For example, the number of endothelial cells is 2×10 3 ~3×10 10 cells, preferably 2×10 4 ~3×10 9 cells, more preferably 2×10 5 ~3×10 8 cells. In a preferred embodiment, the number of endothelial cells in the cell population containing nerve cells is larger than the number of nervous system cells.

[0021] [[ID=,20]]The cell population containing nervous system cells may contain a biocompatible material in addition to the cells described above. The biocompatible material can be used without particular limitation as long as the cells contained in the cell population containing nervous system cells can adhere thereto and are metabolized and consumed by the cells during the process of cell growth and proliferation. Examples of such biocompatible materials include collagen, laminin, fibronectin, gelatin, Matrigel (registered trademark), and the like.

[0022] The shape of the biocompatible material is not particularly limited as long as the above-described effects are achieved. Examples thereof include beads (spherical, substantially spherical) shape, rod shape, film shape, and the like.

[0023] The size of the biocompatible material, for example, when it is bead-shaped, is 50 to 400 μm in diameter, preferably 100 to 300 μm, and more preferably 100 to 200 μm.

[0024] Bead-shaped biocompatible materials can be prepared using known methods, for example, by dropping the biocompatible material into an oil phase to form droplets within the oil phase. The oils constituting the oil phase are not particularly limited as long as droplets of biocompatible material are formed, and examples include edible oils such as corn oil, rapeseed oil, and sesame oil, as well as mineral oils derived from petroleum, natural gas, coal, etc. The diameter of the beads (droplets) can be adjusted as appropriate by changing the dropping rate.

[0025] It is preferable that the nerve cells included in the cell population are arranged in layers on the surface of the biocompatible material. Specifically, by arranging the nerve cells in layers on the surface of the biocompatible material, after culturing, the individual nerve cells with extended axons form a single bundle, which has the advantage of easily producing large (thick) nerve bundles.

[0026] Known methods can be used to arrange nerve cells in layers on the surface of a biocompatible material. For example, one method involves mixing the biocompatible material with a population of nerve cells and then culturing the mixture in a container such as a non-adhesive petri dish by swirling.

[0027] The amount of nerve cell population per unit mass (g) of biocompatible material is, for example, 200 to 1000 nerve cells, preferably 300 to 800, and more preferably 400 to 600. The swirling conditions for swirling culture are, for example, 30 to 60 rpm, preferably 35 to 55 rpm, and more preferably 40 to 50 rpm.

[0028] When a cell population containing nerve cells includes endothelial cells, it is preferable that the endothelial cells in the nerve cell population be arranged in layers on the surface of the biocompatible material. Specifically, by arranging endothelial cells in layers on the surface of the biocompatible material, multiple biocompatible materials having layers of endothelial cells derived from the nerve cell population come into contact and fuse during the culture process. As a result, a tube of endothelial cells can be formed, with the surface composed of endothelial cells and the interior containing biocompatible material. The endothelial cells constituting the tube surface then take in, metabolize, and consume the biocompatible material inside the tube during their growth and proliferation, thereby forming a hollow tube of endothelial cells. The formed tube of endothelial cells can act as a vessel (blood vessel) supplying oxygen, nutrients, etc., to the nerve cells of the nerve bundle being produced, which has the advantage of preventing the nerve bundle from dying. The method for arranging endothelial cells in layers on the surface of the biocompatible material is the same method as the method for arranging nerve cells in layers on the surface of the biocompatible material described above.

[0029] To achieve the advantages of having nerve cells and endothelial cells arranged in layers on the surface of a biocompatible material, it is preferable to arrange the nerve cells and endothelial cells in layers on the surface of separate biocompatible materials. Specifically, it is preferable to arrange nerve cells in layers on the surface of one biocompatible material and endothelial cells in layers on the surface of another biocompatible material.

[0030] Biocompatible materials may be pre-mixed with substances that promote the growth and proliferation of nerve cells, such as nerve growth factor (NGF), fibroblast growth factor β (β-FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and trophic factors. By including nerve growth factors in the biocompatible material, the growth of nerve cells present on the surface of the biocompatible material can be promoted more efficiently.

[0031] The proportion of nerve cell growth factor mixed into the biocompatible material can be appropriately set depending on the size of the nerve bundle being produced, for example, 1 to 1000 ng / mg, 5 to 750 ng / mg, or 10 to 500 ng / mg relative to the total mass of the biocompatible material.

[0032] Nerve cell growth factor may be used alone, but it may also be mixed with ganglioside 3 (GD3), a type of sugar chain. The amount of GD3 mixed with the biocompatible material is not particularly limited, but for example, it may be 10-100 ng / mg, 10-75 ng / mg, or 10-50 ng / mg. When the biocompatible material and nerve cell growth factor are added separately to the recess, axonal elongation can be further promoted by placing the nerve cell growth factor (or nerve cell growth factor and GD3 if GD3 is used) in the recess and then introducing the nerve cells.

[0033] If the cell population containing nerve cells includes endothelial cells, the biocompatible material may be pre-mixed with substances that promote the growth and proliferation of endothelial cells, such as vascular endothelial growth factor (VEGF). By including endothelial growth factors in the biocompatible material, the growth of endothelial cells present on the surface of the biocompatible material can be promoted more efficiently.

[0034] The proportion of endothelial cell growth factor to be mixed with the biocompatible material can be appropriately set depending on the size of the nerve bundle to be produced, for example, 10-300 ng / mg, 20-200 ng / mg, 30-100 ng / mg, etc., relative to the total mass of the biocompatible material.

[0035] When biocompatible materials and endothelial cells are added separately to a recess, it is preferable to add the biocompatible material and endothelial cells to the recess in such a way that the endothelial cells adhere to the surface of the biocompatible material. By adding the biocompatible material and endothelial cells in this way, the formation of blood vessels with a lumen can be further promoted.

[0036] If the cell population containing nerve cells includes endothelial cells, the biocompatible material may be pre-mixed with red blood cells. By including red blood cells in the biocompatible material, it is possible to have red blood cells present inside the hollow endothelial cell tubes formed during the culture process of the nerve cell population. As a result, it is possible to create endothelial cell tubes with complete hollowness. Then, through the created hollow endothelial cell tubes, oxygen is supplied to the nerve cells in the created nerve bundle by red blood cells and culture medium, thereby preventing the nerve bundle from dying.

[0037] The amount of red blood cells mixed with the biocompatible material can be appropriately set depending on the size of the nerve bundle to be produced, for example, it is 2 to 20% by mass, preferably 3 to 10% by mass, and more preferably 4 to 7% by mass relative to the amount of biocompatible material.

[0038] In this specification, "feeder cell" refers to a cell that produces and secretes substances that induce or maintain the survival, proliferation, and differentiation of nerve cells and optionally endothelial cells, and promotes the elongation and hypertrophy of nerve cells. The substances produced and secreted by feeder cells are not particularly limited as long as they have the functions described above, but examples include nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF-β), trophic factors, growth hormone-like substances, IGF-1, etc.

[0039] Feeder cells include cells that produce and secrete the substances described above, and specifically include at least one type of cell selected from vascular constituent cells that make up blood vessels, perivascular cells that surround blood vessels, and oligodendrocytes.

[0040] Oligodendrocytes are a type of glial cell in the central nervous system, also known as oligodendrocytes. While feeder cells may also contain oligodendrocytes if the glial cells described later include them, feeder cells preferably do not.

[0041] The vascular constituent cells are not particularly limited as long as the effects of the present invention are achieved, and examples include pericytes (e.g., vascular pericytes) and endothelial cells (e.g., vascular endothelial cells). Examples of perivascular cells include fibroblasts (e.g., perivascular fibroblasts) and smooth muscle cells (e.g., vascular smooth muscle cells).

[0042] In this specification, "glial cells" refers to all cells that constitute the nervous system but are not nerve cells, and that assist in the survival, development, and function of nerve cells. Examples include oligodendrocytes, astrocytes, Schwann cells, microglia, and ependymal cells. In a preferred embodiment of the present invention, glial cells include oligodendrocytes.

[0043] According to a preferred embodiment of the present invention, glial cells used in the present invention are prepared by the following method. First, the dental pulp obtained from extracted teeth is treated with enzymes (trypsin, collagenase, dispase, etc.) to isolate the cells that make up the dental pulp. Next, the isolated cells are seeded in a hydrophilic petri dish and cultured for 1-2 days at 37°C and a CO2 concentration of 4.7-5% in a neuronal induction medium (for example, a differentiation induction medium described in Takahashi et al. (Human Cell, Vol.30, Issue 2, pp60-71) with the optional addition of EGF and / or FGF) to obtain small spherical cells. Next, the small spherical cells are seeded in a cell-non-adsorbent petri dish and cultured in a swirling culture medium (for example, B27 Plus Neuronal Culture System) for about 1 day at 37°C and a CO2 concentration of 4.7-5%. The swirling speed for swirling culture is preferably 30-100 rpm, more preferably 40-80 rpm, and even more preferably 50-60 rpm. Cell aggregates (spheroids) are formed. Next, the formed cell aggregate is treated with trypsin / EDTA, collagenase, or dispase to separate the cells constituting the aggregate. Then, the separated cells are seeded in a hydrophilic petri dish to obtain nervous system cells. Next, the obtained nervous system cells are cultured in a petri dish or flask coated with ornithine and / or lysine to differentiate and grow into a two-dimensional reticular structure. Small cells develop and attach to the cell body and axon portions of the differentiated and grown nervous system cells, respectively. These small cells are all glial cells, and the small cells that develop and attach to the cell body portion of the nervous system cells differentiate into oligodendrocytes in particular, while the small cells that develop and attach to the axon portion differentiate into Schwann cells. The fact that glial cells, especially oligodendrocytes and Schwann cells, can be efficiently prepared by this method is a surprising and previously unknown finding. Furthermore, it can be easily confirmed that the small cells that develop and attach to the cell body portion of nerve cells are oligodendrocytes or Schwann cells using conventionally known methods such as immunohistochemistry.

[0044] According to one embodiment, nerve bundles produced by the method of the present invention may have myelin sheaths containing oligodendrocytes, a type of glial cell, in the axonal portion of at least some, preferably all, of the nervous system cells constituting the nerve bundle. Therefore, nerve bundles produced by the method of the present invention can be used as nerve bundles in the central nervous system, particularly the brain and spinal cord. Specifically, using nerve bundles produced by the method of the present invention can restore motor function in the limbs or both lower limbs in patients who have completely or partially lost motor function in the limbs or both lower limbs due to severe spinal cord injury.

[0045] The method for producing nerve bundles is not particularly limited in that it includes a step of culturing a population of cells containing nerve cells in the presence of feeder cells, which include at least one type of cell selected from vascular constituent cells, perivascular cells, and oligodendrocytes, and glial cells, to extend the axons of nerve cells, for example, as described in steps (a) to (e) below.

[0046] Process (a) In this process, a substrate is prepared having at least one recess and at least one groove connected to the recess, which is covered with feeder cells. In a preferred embodiment, the substrate has at least one recess and a plurality of grooves connected to the recess and arranged radially, each groove having another recess (tip recess) at the tip opposite to the recess. The number of grooves is not particularly limited, but can be, for example, 3 to 30, 5 to 20, 7 to 15, etc. By having a substrate with multiple grooves, multiple nerve bundles can be produced at once. In a preferred embodiment, endothelial cells are added to the recess of the substrate in advance before covering it with feeder cells, and the recess is then covered with endothelial cells. Specifically, about 10 endothelial cells are added to the recess. 6 After adding the cells and letting it stand for 6 hours, the depressions are covered with endothelial cells by washing with Hanks' solution.

[0047] The substrate material is not particularly limited as long as the effects of the present invention are achieved, but examples include dimethylpolysiloxane, polystyrene, polypropylene, and the like.

[0048] The shape and dimensions of the substrate can be set as appropriate, as long as the effects of the present invention are achieved. Preferably, the substrate has at least one plane, and can be a rectangular parallelepiped, cubic, cylindrical, or the like, with a rectangular parallelepiped being preferred.

[0049] The dimensions of the substrate are not particularly limited as long as it has at least one recess and groove. For example, if the shape is a rectangular parallelepiped, its dimensions are 5-20cm, 5-15cm, and 5-10cm in length and width, and 1-10cm, 1-5cm, and 1-3cm in height (depth).

[0050] The substrate has at least one recess for accommodating a population of cells containing nerve cells and glial cells. If the substrate has multiple recesses, the arrangement of the multiple recesses is not particularly limited as long as the effects of the present invention are achieved, but preferably the multiple recesses are located on the same plane of the substrate. For example, the substrate has two recesses, and the two recesses are located on the same plane of the substrate.

[0051] The shape and dimensions of the recess can be set as appropriate, as long as the effects of the present invention are achieved. Examples of recess shapes include cylindrical, rectangular, cuboidal, cubic, and hemispherical shapes.

[0052] If the shape of the recess is cylindrical, its dimensions are, for example, an opening diameter of 1 to 15 mm, preferably 1 to 10 mm, more preferably 1 to 7 mm, and a height (depth) of 2 to 8 mm, preferably 3 to 7 mm, more preferably 4 to 6 mm. If the substrate has multiple recesses, their shapes and / or dimensions may be the same or different from each other.

[0053] The substrate is provided with grooves that connect to recesses. The grooves may be coated with fibroblasts before being coated with feeder cells. If the grooves are coated with fibroblasts, the grooves are coated with feeder cells on top of the coated fibroblasts. The grooves may also be further coated with glial cells. If the grooves are coated with glial cells, the glial cells are preferably coated on top of the feeder cells. In a preferred embodiment of the present invention, the grooves are first coated with fibroblasts, then with endothelial cells as feeder cells, then with pericytes as feeder cells on top of the coated feeder cells, and then with glial cells on top of the coated feeder cells (see Figure 4C).

[0054] The arrangement of the grooves is not particularly limited as long as the effects of the present invention are achieved, but the grooves and recesses are on the same plane. Furthermore, if the substrate has multiple recesses, each recess is connected to a groove, and preferably the multiple recesses are connected via the grooves. In a preferred embodiment, the substrate has one central recess and multiple recesses radially surrounding the central recess, and grooves connecting the central recess and the radially surrounding recesses. The number of radially surrounding recesses is not particularly limited, but can be, for example, 3 to 30, 5 to 20, 7 to 15, etc. By having such a structure in the substrate, multiple nerve bundles or thick nerve bundles formed by bundling them together can be produced at once.

[0055] The shape (cross-sectional shape) and dimensions (length, depth) of the groove can be set as appropriate, as long as the effects of the present invention are achieved. Examples of groove shapes include clover-shaped, concave, V-shaped, U-shaped, and Ω-shaped grooves. In a preferred embodiment, the groove shape is clover-shaped. When the substrate has a clover-shaped groove, the diameter of the clover leaf portion in its cross-sectional shape can be, for example, 0.5 to 2 mm, 0.75 to 1.5 mm, or 1 to 1.2 mm.

[0056] The length of the groove (distance from the recess) is, for example, 3 to 50 mm, preferably 15 to 40 mm, and more preferably 20 to 30 mm. The depth of the groove is, for example, 1 to 10 mm, preferably 2 to 7 mm, and more preferably 3 to 5 mm. The width of the groove is, for example, 1 to 5 mm, preferably 1.5 to 4 mm, and more preferably 2 to 3 mm. If the substrate has multiple recesses, the length of the groove refers to the length (distance) between the recesses.

[0057] Process (b) In this step, a population of cells containing nerve cells and glial cells are added to the recess. In a preferred embodiment, the population of cells containing nerve cells and glial cells are each added to the recess in a layered manner on the surface of separate biocompatible materials, preferably beads such as collagen beads. If the population of cells containing nerve cells includes endothelial cells, the nerve cells and endothelial cells contained in the population of cells containing nerve cells are each added to the recess in a layered manner on the surface of separate biocompatible materials, preferably beads such as collagen beads. The amount of the population of cells containing nerve cells added to the recess is not particularly limited as long as the effects of the present invention are achieved, but for example, the number of nerve cells may be 10 3 ~10 10 pieces, preferably 10 4 ~10 9 10 comfortable 5 ~10 8 The number is [number]. Furthermore, if the cell population containing nerve cells also contains endothelial cells, the amount of endothelial cells added to the recess is, for example, 1:1 to 1:10, 1:2 to 1:7, or 1:3 to 1:5, as a ratio of the number of nerve cells to the number of endothelial cells (number of nerve cells: number of endothelial cells).

[0058] The amount of glial cells added to the recess is not particularly limited as long as the effects of the present invention are achieved, but for example, the number of glial cells is 10 4 ~10 6 pieces, preferably 5 × 10 4 ~5×10 5 pc, more comfortable 8×10 4 ~10 5The number of cells is [number]. Furthermore, the ratio of the number of glial cells to the number of nerve cells in a cell population containing nerve cells (number of glial cells: number of nerve cells) is, for example, 1:3 to 1:100, 1:5 to 1:50, and 1:10 to 1:30.

[0059] Process (c) In this process, culture medium is added to the recesses and grooves of the substrate, and the cell population containing nerve cells and glial cells added to the recesses are cultured, causing the axons of nerve cells to extend along the grooves of the substrate. In a preferred embodiment, the culture of the cell population containing nerve cells and glial cells is performed by perfusion culture. In parallel with the extension of the axons of nerve cells, glial cells (oligodendrocytes) attach to the axons of nerve cells, and myelin sheaths are formed on the oligodendrocytes.

[0060] The culture medium to be added is not particularly limited as long as it is a culture medium commonly used for culturing nerve cells. Examples include nerve cell culture medium, DMEM, RPMI1640 medium, EMEM, HAMM10, HAMM12 and other basic media, and DMEM / F12, which is an improved basic medium. Various amino acids such as L-glutamine and L-alanine, additives such as fetal bovine serum (FBS), and albumin may be added to these media. When FBS is added to the medium, its concentration is, for example, 5-20% by mass, 7-15% by mass, and 10-15% by mass relative to the medium. As for the method of adding the culture medium, the culture medium may be added to the recesses and grooves respectively, or the substrate may be immersed in a container that has been pre-filled with culture medium, and the culture medium may be supplied to the recesses and grooves respectively.

[0061] The culture conditions are not particularly limited as long as the cells and glial cells included in the cell population containing nervous system cells grow and proliferate, but examples include static culture at a temperature of 35-38°C, a CO2 concentration of 3-5%, and a culture time of 20-24 hours. Depending on the degree of axonal elongation of the nervous system cells, static culture and perfusion culture may be combined. For example, the conditions for perfusion culture can be a temperature of 35-38°C, a CO2 concentration of 3-5%, a perfusion rate of 1-10 ml / hour, preferably 2-5 ml / hour, and a culture time of 72 hours to 60 days.

[0062] (Nerve bundles produced by the manufacturing method of the present invention) According to one aspect of the present invention, a nerve bundle produced by the above method is provided. The nerve bundle produced by the manufacturing method of the present invention can be used as a transplant material, particularly as a transplant material for central nervous system regeneration, by appropriate processing as needed.

[0063] Multiple nerve bundles can be combined as needed to form a bundle of nerve bundles. A bundle of nerve bundles can be made by rolling (covering) multiple nerve bundles with a pre-prepared sheet of biocompatible material. The sheet of biocompatible material is not particularly limited as long as the effects of the present invention are achieved, and examples include a sheet of fibroblasts, a sheet of collagen fiber network, a sheet of elastic fiber network, and the like.

[0064] In one embodiment, multiple nerve bundles can be arranged on a pre-prepared sheet of fibroblasts, and these multiple nerve bundles can be rolled up (like a sushi roll) on the sheet of fibroblasts to form a bundle of nerve bundles. The thickness (diameter) of the bundle of nerve bundles can be arbitrarily changed by the number of nerve bundles arranged on the sheet of fibroblasts. When nerve bundles are rolled up on a sheet of fibroblasts in this way, they can be easily transplanted by suturing the ends of the sheet of fibroblasts to the damaged part (nerve stump) of the nerve to be transplanted.

[0065] In another embodiment, multiple nerve bundles are attached to the surface of a pre-prepared sheet of fibroblasts in an aligned and gapless manner. Two such sheets of fibroblasts are prepared, and the two sheets of biocompatible material are stacked so that the surfaces with the attached nerve bundles are in contact with each other and the orientation of the nerve bundles is aligned, thereby forming a bundle of nerve bundles. The thickness of the bundle of nerve bundles can be arbitrarily changed by the number of nerve bundles attached to the surface of the fibroblast sheet. In yet another embodiment, one sheet of fibroblasts with attached nerve bundles is prepared, and a tube is prepared that is filled with a cell mixture of neural cell beads, endothelial cell beads, and glial cell beads in a mass ratio of approximately 3:1:1. The prepared fibroblast sheet and tube are then arranged so that at least one end of the nerve bundles on the sheet surface is in contact with the end of the tube. The nerve bundles and tube are rolled together with the fibroblast sheet to form a tube, and then the cell mixture filled inside the tube is released into the tube to fill it. Next, a tubular sheet of fibroblasts filled with the cell mixture is cultured statically for 3 hours, followed by perfusion culture. This causes the cell mixture to differentiate and proliferate, further extending the nerve bundle. As a result, it becomes possible to obtain a longer nerve bundle. Glass tubes are one example of the tubes used. The diameter of the tube is not particularly limited and can be set appropriately according to the desired nerve bundle.

[0066] Nerve bundles can be used as graft material (nerve regeneration graft material) to replace damaged nerves. The target of grafting is not particularly limited; for example, they can be transplanted into the central nervous system, peripheral nervous system, etc., but are preferably transplanted into the central nervous system.

[0067] Nerve bundles can be prepared using cells derived from the recipient individual (autologous cells) or using cells derived from an individual other than the recipient individual (allogeneic cells). Therefore, by preparing the nerve bundles of the present invention using autologous cells, it becomes possible to transplant the nerve bundles (autotransplantation) with virtually no risk of rejection to the individual from which the cells used as material for the nerve bundles originate.

[0068] (Methods of axon extension in nervous system cells) According to one aspect of the present invention, a method for extending the axons of nerve cells (hereinafter also referred to as "the method of the present invention") is provided. The method of the present invention makes it possible to efficiently extend the axons of nerve cells. Furthermore, the method of the present invention also makes it possible to efficiently increase the diameter of nerve axons.

[0069] The method of the present invention includes the step of culturing a population of cells containing nervous system cells in the presence of feeder cells and glial cells, which include at least one type of cell selected from vascular constituent cells, perivascular cells, and oligodendrocytes, to extend the axons of the nervous system cells.

[0070] In the method of the present invention, the cell population containing nerve cells, feeder cells, and glial cells can be the same as those described in the method for producing nerve bundles described above.

[0071] In the method of the present invention, the specific conditions for the step of culturing a population of cells containing nerve cells in the presence of feeder cells and glial cells, which include at least one type of cell selected from vascular constituent cells, perivascular cells, and oligodendrocytes, in order to extend the axons of nerve cells, can be the same as those described in the method for producing nerve bundles described above.

[0072] (Nerve bundle) According to one aspect of the present invention, a nerve bundle is provided having axons extending from nerve cells and tubes of endothelial cells present along the axons, comprising at least one type of cell, HNK-1 glycan-expressing cells and p75NTR-expressing cells, wherein the axons have myelin sheaths containing oligodendrocytes (hereinafter also referred to as "the nerve bundle of the present invention").

[0073] The HNK-1 (Human Natural Killer-1) glycan, also known as CD57, was discovered as a glycan antigen expressed on human natural killer cells recognized by the monoclonal antibody HNK-1. The HNK-1 glycan is known to be expressed in the nervous systems of many vertebrates, including humans.

[0074] Furthermore, p75NTR (p75 Neurotrophin Receptor), also known as the low-affinity neurotrophic factor receptor, is a 75 kDa single-pass transmembrane receptor for neurotrophic factors belonging to the tumor necrosis factor (TNF) receptor superfamily.

[0075] Both HNK-1 glycans and p75NTR are known as neural crest stem cell markers that are mainly expressed in vertebrates (especially humans) during the early stages of development when neural crest cells separate from the dorsal neural tube. Specifically, while HNK-1 glycans and p75NTR are expressed in the immature nervous system of vertebrates during embryonic development, their expression is drastically reduced or absent in the nervous system of vertebrates after embryonic development. Therefore, in the mature nervous system of normal adult vertebrates, both HNK-1 glycans and p75NTR are expressed very weakly or not at all.

[0076] Generally, obtaining nerve bundles from the immature nervous system of an embryo in the early stages of development is extremely difficult, and practically impossible. Therefore, nerve bundles containing cells expressing HNK-1 glycans and / or p75NTR have not existed to date. Accordingly, the nerve bundles of the present invention are highly unique in that they substantially express HNK-1 glycans and / or p75NTR.

[0077] In a preferred embodiment, the nerve bundle of the present invention comprises HNK-1 glycan-expressing cells and p75NTR-expressing cells. The HNK-1 glycan-expressing cells and p75NTR-expressing cells may be the same cell or different cells. That is, both HNK-1 glycan and p75NTR may be expressed in one cell, or HNK-1 glycan and p75NTR may be expressed in different cells, respectively.

[0078] Regarding HNK-1 glycans and p75NTR, "HNK-1 glycan-expressing cells" and "p75NTR-expressing cells" refer to cells that substantially express HNK-1 glycans and p75NTR, respectively. Here, expressions such as "substantially expressed" and "substantially expressed" refer to a state in which gene transcripts or translation products are produced within the cell. Specifically, p75NTR binds to nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), etc., and regulates axonal elongation of nerve cells. In addition, HNK-1 glycans show increased expression during the developmental stages of nerves and are involved in neural circuit formation. Both p75NTR and HNK-1 glycans are markers of neural crest stem cells, and it is known that their expression is extremely weak or absent in the mature nervous system. Therefore, the nerve bundles of the present invention that express these markers are different from conventional nerve bundles and transplant materials obtained using normal peripheral nerves or nerve cells from a mature nervous system.

[0079] In this invention, the expression of HNK-1 glycans and p75NTR in cells can be confirmed by detecting the expression of each gene by immunostaining using antibodies against HNK-1 glycans and p75NTR, respectively.

[0080] In a preferred embodiment, the HNK-1 glycan-expressing cells and / or p75NTR-expressing cells are nervous system cells. That is, in a preferred embodiment, the nerve bundle of the present invention comprises nervous system cells expressing HNK-1 glycan and / or p75NTR.

[0081] In a preferred embodiment, the nerve bundle of the present invention includes at least one cell selected from the group consisting of cells expressing NS200 (NS200-expressing cells), cells expressing peripherin (peripherin-expressing cells), cells expressing myelin basic protein (myelin basic protein-expressing cells), cells expressing S100 (S100-expressing cells), cells expressing MPZ (MPZ-expressing cells), cells expressing periasin (periasin-expressing cells), cells expressing CD31 (CD31-expressing cells), and cells expressing PDGFRβ (PDGFR-expressing cells). The cells expressing each of the above genes may be the same cell or different cells. That is, the embodiment also includes cases where two or more of the above genes are expressed in a single cell. For example, an NS200-expressing cell may be a cell that expresses only NS200 among the above genes, or it may be a cell that expresses NS200 in addition to any of the above genes.

[0082] In a particularly preferred embodiment, the nerve bundle of the present invention includes all of the following: NS200-expressing cells, peripherin-expressing cells, myelin basic protein-expressing cells, S100-expressing cells, MPZ-expressing cells, periasin-expressing cells, CD31-expressing cells, and PDGFR-expressing cells.

[0083] The genes NF200, peripherin, myelin basic protein, S100, MPZ, and periaxin are all known as expression markers in the nervous system. Specifically, NF200 (Neurofilament 200) and peripherin are known as neuronal markers in myelinated nerves. S100 is also known as a marker for Schwann cells. Myelin basic protein, S100, MPZ (Myelin protein zero), and periaxin are known as markers for myelin sheaths.

[0084] On the other hand, both the CD31 and PDGFRβ genes are known as expression markers for the vascular system. Specifically, CD31 is known as a marker for vascular endothelial cells. PDGFRβ (Platelet-derived growth factor receptor β) is known as a marker for perivascular cells and fibroblasts.

[0085] The presence or absence of expression of each of the above genes can be confirmed using the same method as for confirming the presence or absence of expression of HNK-1 glycan and p75NTR as described above.

[0086] The nerve bundle of the present invention has endothelial cell tubules that are present along the axons of nerve cells. At least some of the endothelial cells constituting the tubules are preferably vascular endothelial cells, more preferably endothelial cells derived from dental pulp, gingiva, subcutaneous tissue, intracavitary arteries, intracavitary veins, or umbilical cord blood vessels, and even more preferably endothelial cells derived from dental pulp blood vessels. Furthermore, the endothelial cells may be autologous cells derived from the individual to which the nerve bundle is transplanted, or allogeneic cells derived from an individual other than the transplanted individual. The endothelial cell tubules have the advantage of being able to act as tubes (blood vessels) that supply oxygen, nutrients, etc. to the nerve cells of the nerve bundle, and can prevent the nerve bundle from dying.

[0087] In a preferred embodiment, the endothelial cell tubules described above further include pericytes. The inclusion of pericytes in the endothelial cell tubules reinforces them, specifically by the pericytes lining the endothelial cell tubules. If the nerve bundle of the present invention has endothelial cell tubules that function as blood vessels, the increased rigidity of the endothelial cell tubules allows blood to flow stably through them.

[0088] In a preferred embodiment, the nerve bundle of the present invention has a cell layer comprising at least one type of cell, fibroblasts and pericytes, which covers at least a portion, preferably all, of the axon along the axon of a nerve cell. In a particularly preferred embodiment, the nerve bundle of the present invention has endothelial cell tubules described above within the cell layer.

[0089] The nerve bundle of the present invention can be used as a replacement transplant material for damaged nerves, particularly as a nerve regeneration transplant material. The target of the nerve bundle transplant is not particularly limited; for example, it can be transplanted into the central nervous system, peripheral nervous system, etc., but it is preferably transplanted into the peripheral nervous system.

[0090] [Device for manufacturing nerve bundles] According to one aspect of the present invention, an apparatus for manufacturing nerve bundles (hereinafter also referred to as "the apparatus of the present invention") is provided. The apparatus of the present invention has at least one first recess and at least one first groove having at least first and second ends, wherein the first groove has at least one second groove having at least first and second ends and extending parallel to the first groove, and the first recess and the first ends of the first and second grooves are connected.

[0091] In one embodiment, the first recess is a recess with a depth of 4 to 8 mm, having an opening with a diameter of 3 to 10 mm and a horizontal or concave curved bottom. In a preferred embodiment, the first recess is a recess with a depth of 6 mm, having an opening with a diameter of 8 mm and a horizontal or concave curved bottom.

[0092] In one embodiment, the first groove has a horizontal or concave curved bottom, a rim width of 4-6 mm, a length of 2-3 cm from the first end to the second end, and a depth of 4-6 mm. In a preferred embodiment, the first groove has a horizontal or concave curved bottom, a rim width of 5 mm, a length of 2-3 cm from the first end to the second end, and a depth of 6 mm.

[0093] In one embodiment, the second groove has a horizontal or concave curved bottom, with a rim width of 2 to 2.5 mm and a depth of 1.5 to 2 mm. In a particularly preferred embodiment, the second groove has a horizontal or concave curved bottom, with a rim width of 2.5 mm and a depth of 2 mm.

[0094] The first groove may have one second groove or multiple second grooves. In a preferred embodiment, the first groove has three second grooves. In this case, the arrangement of the second grooves in the first groove is not particularly limited as long as the effects of the present invention are achieved, but the arrangement shown in Figure 4B is preferred.

[0095] The apparatus of the present invention may have a second recess in addition to the first recess. The second recess may be one or more. In a preferred embodiment, the second ends of the first and second grooves are connected to the second recess. If there are multiple second recesses, the second ends of different first and second grooves are each connected to the second recess.

[0096] In one embodiment, the second recess is a recess 3 to 6 mm deep with an opening 2 to 5 mm in diameter and a horizontal or concave curved bottom. In a preferred embodiment, the second recess is a recess 4 mm deep with an opening 3.5 mm in diameter and a horizontal or concave curved bottom.

[0097] The first and / or second grooves are preferably surface-treated to improve hydrophilicity. The hydrophilic treatment is not particularly limited as long as it does not cause cytotoxicity, but examples include plasma treatment.

[0098] The size of the apparatus of the present invention is not particularly limited as long as it can accommodate the above-described parts, but for example, it may be 4 to 10 cm in length, 4 to 10 cm in width, and 0.8 to 1.5 cm in thickness, with the above-described parts formed on a plane having the aforementioned length and width. In a preferred embodiment, the size of the apparatus of the present invention is 8 cm in length, 8 cm in width, and 1 cm in thickness.

[0099] The materials used in the apparatus of the present invention are not particularly limited as long as they do not cause cytotoxicity, but examples include polydimethylsiloxane.

[0100] In one embodiment, nerve cells, vascular endothelial cells, and oligodendrocytes are arranged in the first recess. Each of these cells can be the same as those described in the manufacturing method of the present invention described above.

[0101] In one embodiment, at least one of the first and second grooves contains at least one type of feeder cell selected from vascular constituent cells, perivascular cells, and oligodendrocytes, as well as glial cells. Each of these cells can be the same as those described in the manufacturing method of the present invention described above.

[0102] Using the apparatus of the present invention, nerve bundles can be produced according to the manufacturing method of the present invention described above. [Examples]

[0103] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0104] Example 1: Preparation of glial cells Glial cells used in this example were prepared according to the following procedure. Dental pulp obtained from wisdom teeth (M3) was treated with an enzyme (0.1% trypsin / 0.02% EDTA / PBS(-)) to isolate the cells constituting the dental pulp. Next, the isolated cells were seeded in a hydrophilic petri dish and cultured for 1-2 days at 37°C and 4.7% CO2 concentration in nerve induction medium (differentiation induction medium described in Takahashi et al., Human Cell, Vol.30, Issue 2, pp60-71) to differentiate into small spherical cells. Next, the small spherical cells were collected by pipetting, centrifuged (1500 rpm), seeded in a cell-non-adsorbent petri dish using nerve maintenance medium, and cultured in a swirling manner (60 rpm) at 37°C and 4.7% CO2 concentration for about 1 day to form cell aggregates (spheroids). Next, the formed cell aggregates were treated with the above trypsin / EDTA / PBS(-) to isolate the cells constituting the cell aggregates. Next, the isolated cells were seeded in a hydrophilic petri dish to obtain nervous system cells. These nervous system cells were then cultured in a nerve maintenance medium containing ornithine and lysine to differentiate and grow into a two-dimensional reticular structure. Phase-contrast microscope images of the two-dimensional reticular differentiated and grown nervous system cells are shown in Figures 1A and 1B. Next, small cells that had developed and attached to the cell bodies and axons of the reticular differentiated and grown nervous system cells were collected (the arrows in Figure 1A and Figure 1B indicate small cells that developed and attached to the cell bodies and axons of the nervous system cells, respectively). Immunostaining of each type of small cell using anti-S100 antibody and anti-Olig2 antibody confirmed that the small cells that developed and attached to the cell bodies of the nervous system cells were oligodendrocytes, a type of glial cell, and the small cells that developed and attached to the axons were Schwann cells, another type of glial cell. Specifically, as shown in Figures 2A and 2B, the small cells that developed and attached to the cell body were negative for S100, one of the Schwann cell markers, but positive for Olig2, one of the oligodendrocyte markers. On the other hand, as shown in Figures 3A and 3B, the small cells that developed and attached to the axon were positive for S100, one of the Schwann cell markers, but negative for Olig2, one of the oligodendrocyte markers.

[0105] Example 2: Preparation of a cell population containing nervous system cells The cell population containing nervous system cells used in this example was prepared by following the procedure below. Mesenchymal cells from the oral cavity, collected from human dental pulp, gingival epithelial tissue sublayer, and oral epithelial tissue sublayer, were seeded in cell-adhesion petri dishes (Falcon). They were cultured for 3 to 7 passages at 37°C and a CO2 concentration of 4.5-5.5% using Dulbecco's modified Eagle medium / Ham F12 (DMEM / F12). Cells were separated using trypsin-EDTA solution, and the separated cells were thinly seeded into new petri dishes. Large colonies with high proliferative capacity were obtained by conical cloning to obtain dental pulp-derived mesenchymal stem cells. The obtained stem cells were cultured at 37°C and a CO2 concentration of 4.5-5.5% in a culture medium prepared by adding 10 ng / ml of epidermal growth factor (EGF) and 10 ng / ml of fibroblast growth factor β (β-FGF) to the differentiation induction medium described in Takahashi et al. (Human Cell, Vol.30, Issue 2, pp60-71), and differentiation was induced to produce a cell population containing neural cells. Analysis by immunohistochemistry and RT-PCR confirmed that the differentiated cells included neural stem cells, immature neurons, immature glial cells, mature neurons, and mature glial cells. Furthermore, immunohistochemistry for tyrosine hydroxylase confirmed the presence of dopamine cells among the differentiated cells.

[0106] Example 3: Preparation of endothelial cells The endothelial cells used in this example were prepared according to the following procedure. Vascular network-containing tissue was collected from the oral cavity of a human, treated with digestive enzymes to isolate cells, and primary cultures were performed. Colonies of morphologically distinguishable endothelial cells were isolated by colonial cloning, and these were propagated to obtain endothelial cells.

[0107] Example 4: Preparation of neural cell beads The neural cell beads used in this example were prepared according to the following procedure. To atelocollagen solution (manufactured by Koken Co., Ltd.), a 10-fold concentrated Dulbecco's modified Eagle medium / Ham F-12 mixed medium (DMEM / F12) was added to the atelocollagen solution at a concentration of 10% by mass, and the mixture was stirred and mixed to obtain an atelocollagen mixture. The obtained atelocollagen mixture was dropped into corn oil to produce biocompatible material (collagen beads) with a particle (bead) diameter of 200 μm. The obtained collagen beads and the nerve cell-containing cell population prepared in Example 2 were mixed in a mass ratio of 1:500. After mixing, the mixture was placed in a non-adhesive petri dish (manufactured by IWAKI Co., Ltd.) and cultured at a swirling speed of 40-60 rpm to allow the nerve cell-containing cell population to remain in layers on the surface of the collagen beads, thereby producing nerve cell beads.

[0108] Example 5: Preparation of endothelial cell beads The endothelial cell beads used in this example were prepared according to the following procedure. To atelocollagen solution (manufactured by Koken Co., Ltd.), RPMI1640 medium concentrated 8 to 10 times was added to the atelocollagen solution at a concentration of 10% by mass. Furthermore, vascular endothelial growth factor (VEGF) (manufactured by Sigma) was added at a concentration of 50 ng / ml, and then red blood cells were added at a concentration of 5% by mass. The mixture was then stirred and mixed to obtain an atelocollagen mixture. The obtained atelocollagen mixture was dropped into corn oil to produce biocompatible material (collagen beads) with a particle (bead) diameter of 100 μm. The obtained collagen beads and endothelial cells prepared in Example 3 were mixed at a mass ratio of 1:500. After mixing, the mixture was placed in a non-adhesive petri dish (manufactured by IWAKI Co., Ltd.) and cultured at a swirling speed of 40 to 60 rpm to retain endothelial cells in layers on the surface of the collagen beads, thereby producing endothelial cell beads.

[0109] Example 6: Fabrication of a nerve bundle preparation device The device for creating nerve bundles was fabricated according to the following procedure. A cylindrical recess with a diameter of 5 mm and a depth of 5 mm was formed in the center of the surface of a dimethylpolysiloxane substrate (8 cm long x 8 cm wide x 1 cm thick). Furthermore, twelve grooves (3 cm long, 4 mm deep, and 2.5 mm wide) were formed radially from the center of this recess. A cylindrical recess (tip recess, 3 mm in diameter, and 5 mm deep) was formed at the tip of each groove (the tip opposite to the side connecting to the recess). Cloverleaf-shaped grooves were formed in each groove so that the diameter of each lobule was approximately 1 mm, thereby creating a device for preparing nerve bundles. Schematic diagrams of the prepared device and the grooves are shown in Figures 4A and 4B, respectively.

[0110] Example 7: Preparation of nerve bundles Prior to creating nerve bundles, we investigated feeder cells suitable for extending the axons of nerve cells using the following procedure. Four of the devices prepared in Example 6 were prepared, and vascular constituent cells (pericytes, endothelial cells, vascular-derived smooth muscle cells), perivascular cells (perivascular fibroblasts), oligodendrocytes, and Schwann cells were seeded as feeder cells in the grooves of each device. Then, each device was placed in a culture vessel, and DMEM / F12 with 15% fetal bovine serum (FBS) added was added to the culture vessel. The culture vessels were placed in a CO2 incubator (temperature 37°C, CO2 concentration 4.7%) to culture the cells in the grooves of each device, forming a monolayer of each cell in the grooves. Next, the neural cell beads prepared in Example 4 and the endothelial cell beads prepared in Example 5 were mixed in a mass ratio of 3:1 and added to each of the two recesses of each device using a micropipette. DMEM / F12 medium was added to each recess, and each device was cultured in a CO2 incubator (temperature 37°C, CO2 concentration 4.7%) for 20 hours. Next, each device was placed in a chamber (6 cm long x 6 cm wide x 2 cm thick) and subjected to perfusion culture for 72 to 168 hours (flow rate 2 ml / min, temperature 37°C, CO2 concentration 4.7%) to allow the axons of nerve cells to extend onto each cell layer in the groove, and the relationship between the number of culture days and the length of nerve bundles was examined. The results are shown in Figure 5. Although not shown in Figure 5, when pericytes, endothelial cells, perivascular fibroblasts, and vascular-derived smooth muscle cells were used as feeder cells, the axon diameters at 30 days after the start of culture were approximately 100-150 μm, 50-80 μm, 20-30 μm, and 10-20 μm, respectively. On the other hand, when no feeder cells were used, the axons of nerve cells hardly elongated or hypertrophied, and at 30 days after the start of culture, their length was approximately 5-10 μm and their diameter was approximately 5-10 μm.

[0111] The results shown in Figure 5 demonstrate that when vascular constituent cells, perivascular cells, or oligodendrocytes are used as feeder cells, the axons of nervous system cells elongate efficiently. In particular, when vascular constituent cells such as pericytes or endothelial cells, or oligodendrocytes, are used as feeder cells, the axons of nervous system cells elongate very efficiently.

[0112] Furthermore, as a preliminary step, we investigated the effect of the presence or absence of feeder cells on nerve bundle elongation when nerve bundles were fabricated using the apparatus created in Example 6. Specifically, we observed nerve bundle elongation with and without perivascular fibroblasts (vascular pericytes) present in the grooves of the apparatus. Phase-contrast microscope images of nerve bundles with and without perivascular fibroblasts are shown in Figures 6A and 6B, respectively. From the image in Figure 6A, it was suggested that when perivascular fibroblasts are present, oligodendrocytes elongate linearly, resulting in linear nerve bundle elongation. On the other hand, from the image in Figure 6B, it was suggested that when perivascular fibroblasts are absent, oligodendrocytes elongate in a branching manner, resulting in branching nerve bundle elongation rather than linear elongation. Generally, when nerve bundles are used as transplant material, linear nerve bundles are preferred, suggesting that it is preferable to use feeder cells, especially perivascular fibroblasts, when fabricating nerve bundles.

[0113] Below, nerve bundles were created using perivascular cells as feeder cells, following the procedure outlined below. Fibroblasts were seeded in each groove of the apparatus prepared in Example 6 to form a single layer of fibroblasts. Next, human-derived pericytes were seeded on top of the fibroblasts to form single to multilayer pericytes. Then, the apparatus was placed in a culture vessel, and DMEM / F12 with 15% by mass of fetal bovine serum (FBS) was added to the culture vessel. The culture vessel was placed in a CO2 incubator (temperature 37°C, CO2 concentration 4.7%) to culture the fibroblasts and pericytes in each groove of the apparatus, forming a single layer of fibroblasts and a single layer of pericytes adhered to the fibroblast layer in each groove. Next, glial cells (oligodendrocytes) prepared in Example 1, neural cell beads prepared in Example 4, and endothelial cell beads prepared in Example 5 were mixed in a mass ratio of approximately 1:4:2 and added to each of the recesses of the apparatus using a micropipette. DMEM / F12 medium was added to each recess, and the device was placed in a CO2 incubator (temperature 37°C, CO2 concentration 4.7%) for 20 hours of incubation. Next, the device was placed in a chamber (6 cm long x 6 cm wide x 2 cm thick) and perfusion cultured for 30 days (flow rate 2 ml / min, temperature 37°C, CO2 concentration 4.7%), allowing the axons of the nervous system cells to extend onto the fibroblast layer and vascular pericyte layer of each groove, thereby obtaining bundles of nervous system cells (nerve fibers) with extended axons (nerve bundles). The obtained nerve bundles contained not only bundles of nervous system cells (nerve fibers) with extended axons, but also endothelial cell tubes (blood vessels) that extended in the same direction as the nerve fibers (axons) and attached to the nerve fibers (axons). The length of the nerve bundles was approximately 3 cm, and the diameter was approximately 1500-2200 μm. Furthermore, the fibroblasts constituting the fibroblast layer grew and proliferated, and sheets of fibroblasts were formed in each groove. Figure 7 shows micrographs (Figure 7A) and immunohistochemical images (Figures 7B and C) of the obtained nerve bundles, respectively. Figure 7B shows the myelin sheath of oligodendrocytes and the axons of nervous system cells, and Figure 7C shows the tubules (blood vessels) of endothelial cells and the axons of nervous system cells. These results confirm that the nerve bundles prepared by the method of this example have, in addition to nervous system cells with extended axons, tubules (blood vessels) of endothelial cells along the axons and myelin sheaths of oligodendrocytes along the axons.

[0114] Furthermore, the nerve bundles prepared as described above were subjected to immunofluorescence staining for NF200 and myelin basic protein to confirm the presence or absence of NF200 and myelin basic protein expression. Next, the nerve bundles were cut perpendicular to the direction of axonal extension of the nerve cells. Figure 8 shows a photograph of the cross-section (transverse plane) of the cut nerve bundle. From the photograph in Figure 8, the expression of NF200 and myelin basic protein in and around it was observed. Since NF200 is known as a neuronal marker for myelinated nerves and myelin basic protein is known as a marker for oligodendrocytes, the photograph in Figure 8 suggests that the nerve bundle contains myelin-containing nerve cells of oligodendrocytes.

[0115] Similarly, the nerve bundles were immunofluorescently stained for NF200 and von Willebrand factor to confirm the presence or absence of NF200 and von Willebrand factor expression. Next, the nerve bundles were cut perpendicular to the direction of axonal extension of the nerve cells. Figure 9 shows a photograph of the cross-section of the severed nerve bundle. From the photograph in Figure 9, circular expression of NF200 and von Willebrand factor around it was observed. Since NF200 is known as a neuronal marker for myelinated nerves and von Willebrand factor is known as a marker for vascular endothelial cells, the photograph in Figure 9 suggests that the nerve bundles contain nerve cells and vascular endothelial cell tubes along the axons of the nerve cells.

[0116] Next, the nerve bundles formed in each groove were covered by rolling them with a sheet of fibroblasts formed in the groove. Then, each nerve bundle was cut at the boundary between the groove and the recesses at both ends and removed from the apparatus. Six of the collected nerve bundles were arranged side by side without gaps on the surface of each of two large fibroblast sheets that had been prepared in a petri dish beforehand, and left to stand for 1 hour to allow each nerve bundle to adhere to the fibroblast sheet. The two fibroblast sheets with the six nerve bundles attached were placed on top of each other so that the surfaces with the attached nerve bundles were in contact and the orientation of the nerve bundles was aligned, and left to stand for 30 minutes. Next, the fibroblast sheets were cut along the long axis of the nerve bundles to be used as transplant material and used for transplantation as described later.

[0117] Example 8: Nerve bundle transplantation 1 (transplantation into rat spinal cord) The nerve bundle graft material prepared in Example 7 was transplanted into a spinal cord injury model rat according to the following method. Rats (Wistar rats, 8 weeks old, approximately 200g in weight, manufactured by Nippon SLC Co., Ltd.) were anesthetized by intraperitoneal administration of a three-component anesthetic mixture (medetomidine hydrochloride (0.15mg / 0.15ml / kg), midazolam (2mg / 0.4ml / kg), butorphanol tartrate (2.5mg / 0.5ml / kg), and physiological saline (1.45ml / kg)). The T9 vertebral arch and a portion of the thoracic spinal cord (one segment, 2mm) were resected. Photographs of the rats after the T9 vertebral arch and portion of the thoracic spinal cord are shown in Figure 10 (A: after T9 laminectomy, B: after T9 laminectomy and thoracic spinal cord resection). Nerve bundle graft material (approximately 5-8mm in length and 2-3mm in diameter) prepared in Example 7 was transplanted into the nerve portion of the resected thoracic spinal cord. Photographs of the transplant site before and after the nerve bundle graft material transplant are shown in Figure 11 (A: before transplantation, B: after transplantation). The white area in the center of the photograph in Figure 11B represents the transplanted nerve bundle material. Observations of the rats after nerve bundle transplantation showed that they functioned similarly to control rats that received autologous nerve transplants, and movement in both lower limbs recovered. Furthermore, no adverse effects such as shortened lifespan were observed.

[0118] The recovery of motor function in 10 rats with their thoracic spinal cords removed and 15 rats with nerve bundle grafts transplanted after thoracic spinal cord removal was evaluated using the Basso-Beattie-Bresnahan Locomotor score (BBB score), a commonly used indicator of motor function recovery in spinal cord injury rats. The evaluation results are shown in Figure 12. The weekly changes in the BBB score (weeks 1-6 post-transplant) in the group of rats with nerve bundle grafts were 0.5, 1.5, 2.0, 4.0, 4.7, and 5.4. On the other hand, the weekly changes in the BBB score in the group of rats with their thoracic spinal cords removed were 0.8, 1.3, 1.9, 2.1, 2.2, and 2.3. Statistical analysis revealed significant differences between the group of rats with nerve bundle grafts and the group of rats with their thoracic spinal cords removed at weeks 4, 5, and 6 post-transplant. Furthermore, in the group of rats that received nerve bundle graft material, recovery of lower limb motor function was observed from 4 weeks after transplantation. On the other hand, in the group of rats that received nerve bundle graft material, when the transplant site was re-excised 6 weeks after transplantation, lower limb motor function deteriorated and the BBB score decreased to 1. These results indicate that rats that received nerve bundle graft material after thoracic spinal cord resection showed significantly higher recovery of motor function compared to rats that remained without thoracic spinal cord resection.

[0119] Figure 13 shows hematoxylin-eosin (HE) stained longitudinal sections of the resection (transplant) site at 6 weeks after thoracic spinal cord resection (transplant) in rats that received nerve bundle graft material immediately after thoracic spinal cord resection and in rats that did not receive nerve bundle graft material. Figure 13A is the HE stained longitudinal section of the resection site at 6 weeks after thoracic spinal cord resection in rats that did not receive nerve bundle graft material after thoracic spinal cord resection. Figure 13B is the HE stained longitudinal section of the transplant site at 6 weeks after nerve bundle graft material transplantation in rats that received nerve bundle graft material immediately after thoracic spinal cord resection. The photograph in Figure 13B shows that the nerve bundle graft material and the resection site of the thoracic spinal cord become connected when nerve bundle graft material is transplanted to the thoracic spinal cord resection site.

[0120] Figure 14 shows HE staining images of a longitudinal section of the transplant site in rats that received nerve bundle graft material immediately after thoracic spinal cord resection (transplantation) 6 weeks after thoracic spinal cord resection (transplantation). Figure 14A is an HE staining image showing the entire transplant site. Figure 14B is a magnified HE staining image of the junction between the nerve bundle graft material and the nerve portion of the resected thoracic spinal cord of the rat (host) in the staining image of Figure 14A (the area indicated by the single arrow in Figure 14A). In particular, the photograph in Figure 14B shows that the transplanted nerve bundle graft material maintained the continuity of nerves with cells even 6 weeks after transplantation and was junctioned with the nerve portion of the resected thoracic spinal cord of the rat (host).

[0121] These results demonstrate that nerve bundle graft material transplanted to the resected area of ​​the thoracic spinal cord connects the resected portion of the thoracic spinal cord and functions in place of the nerve portion of the thoracic spinal cord.

[0122] Example 9: Immunofluorescence staining of nerve bundles 1 The nerve bundles prepared in Example 7 were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for p75NTR and S100 to confirm the presence or absence of p75NTR and S100 expression. Next, the nerve bundles were cut parallel to the direction of axonal extension of the nerve cells. Photographs of the cross-section (longitudinal section) of the cut nerve bundles are shown in Figure 15. Photographs in Figures 15A-C show the expression of S100, p75NTR, and DAPI, respectively. Photograph in Figure 15D is a merged image of Photographs 15A-C. From the photographs in Figure 15, the expression of p75NTR and S100 was observed in the nerve bundles along the direction of axonal extension of the nerve cells.

[0123] Example 10: Immunofluorescence staining of nerve bundles 2 The nerve bundles prepared in Example 7 were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for HNK-1 glycans, p75NTR, and MPZ to confirm the presence or absence of expression of HNK-1 glycans, p75NTR, and MPZ. Next, the nerve bundles were cut parallel to the direction of axonal extension of the nerve cells. Photographs of the cross-section (longitudinal section) of the cut nerve bundles are shown in Figure 16. Photographs in Figures 16A to D show the expression of HNK-1 glycans, p75NTR, MPZ, and DAPI, respectively. Figure 16E is a merged image of Photographs 16A to D. From Photographs 16A to C, expression of HNK-1, p75NTR, and MPZ was observed in the nerve bundles along the direction of axonal extension of the nerve cells. Furthermore, from Photograph 16E, it was confirmed that HNK-1, p75NTR, and MPZ were expressed at almost the same location. Since MPZ is known as a marker of myelin, particularly immature myelin, the photograph in Figure 16E suggests that the nerve bundle contains nerve cells with myelin, and that HNK-1 and p75NTR are expressed in the myelin.

[0124] Example 11: Immunofluorescence staining of nerve bundles 3 The nerve bundles prepared in Example 7 were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for NF200 and myelin basic protein to confirm the presence or absence of NF200 and myelin basic protein expression. Next, the nerve bundles were cut perpendicular to the direction of axonal extension of the nerve cells. A photograph of the cross-section (transverse plane) of the cut nerve bundle is shown in Figure 17. From the photograph in Figure 17, the expression of NF200 and myelin basic protein in and around it was observed. Since NF200 is known as a neuronal marker for myelinated nerves and myelin basic protein is known as a marker for Schwann cells, the photograph in Figure 17 suggests that the nerve bundles contain myelin-containing nerve cells of Schwann cells.

[0125] Example 12: Immunofluorescence staining of nerve bundles 4 The nerve bundles prepared in Example 7 were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for NF200, S100, and peripherin to confirm the presence or absence of NF200, S100, and peripherin expression. Next, the nerve bundles were cut perpendicular to the direction of axonal extension of the nerve cells. Photographs of the cross-section (transverse plane) of the cut nerve bundles are shown in Figure 18. Photograph 18A shows the expression of NF200 and S100. Photograph 18B shows the expression of S100 and peripherin. From the photograph in Figure 18A, the expression of NF200 and S100 around it was observed. Since NF200 is known as a neuronal marker for myelinated nerves and S100 is known as a marker for Schwann cells, the photograph in Figure 18A suggests that the nerve bundle contains nerve cells with myelin sheaths that are Schwann cells. Furthermore, from the photograph in Figure 18B, the expression of peripherin and S100 around it was observed. Since peripherins are primarily known as neuronal markers in the peripheral nervous system, Figure 18B also suggests that the nerve bundle contains myelinated nerve cells (Schwann cells).

[0126] Example 13: Immunofluorescence staining of nerve bundles 5 The nerve bundles prepared in Example 7 were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for NF200 and periaxine to confirm the presence or absence of NF200 and periaxine expression. Next, the nerve bundles were cut perpendicular and parallel to the direction of axonal extension of the nerve cells. Photographs of the cross-section (transverse plane) of the cut nerve bundles are shown in Figure 19A. Photographs of the longitudinal section (longitudinal plane) of the nerve bundles are shown in Figures 19B and C. From the photograph in Figure 19A, the expression of NF200 and periaxine around it was observed. Furthermore, from Figures 19B and C, the expression of NF200 and periaxine was observed along the direction of axonal extension of the nerve cells. Since NF200 is known as a neuronal marker for myelinated nerves and periaxine is known as a marker for Schwann cells, the photographs in Figures 19A-C suggest that the nerve bundles contain myelinated nerve cells of Schwann cells.

[0127] Example 14: Immunofluorescence staining of nerve bundles 6 The nerve bundles prepared in Example 7 were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for CD31 and PDGFRβ to confirm the presence or absence of CD31 and PDGFRβ expression. Next, the nerve bundles were cut perpendicular to the direction of axon extension of the nerve cells. Photographs of the cross-sections of the cut nerve bundles are shown in Figures 20A and 20B. From the photographs in Figures 20A and 20B, the expression of CD31 and PDGFRβ was observed around the nerve cells (axons) of the nerve bundles. Since CD31 is known as a marker for vascular endothelial cells and PDGFRβ is known as a marker for vascular pericytes and fibroblasts, the photographs in Figures 20A and 20B suggest that the nerve bundles contain a layer of pericytes or fibroblasts around the axons of the nerve cells, and that this layer contains vascular endothelial cell tubes.

[0128] Example 15: Immunofluorescence staining of nerve bundles 7 The nerve bundles prepared in Example 7 were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for CD31 and PDGFRβ to confirm the presence or absence of CD31 and PDGFRβ expression. Next, the nerve bundles were cut perpendicular to the direction of axonal extension of the nerve cells. Figure 21 shows a photograph of the cross-section of the cut nerve bundle. Figure 21A is a magnified photograph of a part of the cross-section of the nerve bundle, and Figure 21B is a magnified photograph of the part in Figure 21A where the expression of CD31 and PDGFRβ was particularly strongly observed. From the photographs in Figures 21A and 21B, it was confirmed that CD31 was expressed in a circular pattern, and furthermore, PDGFRβ was expressed surrounding the outside of the circle of CD31. Since CD31 is known as a marker for vascular endothelial cells and PDGFRβ is known as a marker for vascular pericytes and fibroblasts, the photographs in Figures 21A and 21B suggest that nerve bundles contain a layer of pericytes or fibroblasts around the axons of nerve cells, and that these layers contain vascular endothelial cell tubes, and that these vascular endothelial cell tubes are lined with vascular pericytes and / or fibroblasts.

[0129] Example 16: Immunofluorescence staining of nerve bundles 8 Figure 22 shows a magnified photograph of a section (cross-section) of another nerve bundle that was immunofluorescently stained and sectioned in the same manner as in Example 15, in which the expression of CD31 and PDGFRβ was particularly strongly observed. From the photograph in Figure 22, it was confirmed that CD31 was expressed in a circular pattern, and furthermore, PDGFRβ was expressed surrounding the outside of the circle of CD31. Therefore, the photograph in Figure 22 also suggests that the nerve bundle contains a layer of pericytes or fibroblasts around the axon of the nerve cell, and that there are vascular endothelial cell tubes present in this layer, and furthermore, that the vascular endothelial cell tubes are lined with vascular pericytes and / or fibroblasts.

[0130] Example 17: Nerve bundle transplantation 2 (transplantation to rat sciatic nerve) The nerve bundle graft material prepared in Example 7 was transplanted into the sciatic nerve of a rat according to the following method. A rat (nude rat F344 / NJcl-rnu / rnu, 15 weeks old, male, manufactured by CLEA Japan) was anesthetized by intraperitoneal administration of a three-component anesthetic (a mixture of medetomidine hydrochloride (0.15 mg / 0.15 ml / kg), midazolam (2 mg / 0.4 ml / kg), butorphanol tartrate (2.5 mg / 0.5 ml / kg), and physiological saline (1.45 ml / kg)), and a portion of the sciatic nerve was resected. Each of the two ends of the nerve bundle graft material (approximately 1 cm in length and 1 mm in diameter) prepared in Example 6 was sutured three times with surgical suture (10-0 nylon) to each of the two nerve stumps after sciatic nerve resection, and the nerve bundle graft material was transplanted to the resected sciatic nerve portion. Following a similar method, rats were prepared in which the excised nerve was transplanted with its distal and proximal ends reversed (autologous nerve transplant rats), and rats were prepared in which artificial nerves (Nervebridge®, manufactured by Toyobo Co., Ltd.) were transplanted (artificial nerve transplant rats). In addition, rats were prepared in which the sciatic nerve had been transected (sciatic nerve transected rats). Photographs of the transplantation and transecting sites of each rat 12 weeks after transplantation are shown in Figure 23 (A: autologous nerve transplant rat, B: nerve bundle transplant material transplant rat, C: artificial nerve transplant rat, D: sciatic nerve transected rat). The photographs in Figure 23 show that in the autologous nerve transplant rat, nerve bundle transplant material transplant rat, and artificial nerve transplant rat, the transplanted autologous nerve, nerve bundle transplant material, and artificial nerve were joined to the rat's sciatic nerve, respectively. Furthermore, it was shown that when nerve bundle transplant material was transplanted (Figure 23B), the nerve bundle was significantly covered by fibroblasts compared to when autologous nerve or artificial nerve was transplanted (Figures 23A and C, respectively).

[0131] Furthermore, six rats were prepared with autologous nerve transplants, three rats with nerve bundle transplant materials, artificial nerve transplants, and four rats with sciatic nerve transection, respectively. The changes in the sciatic functional index (SFI) over time after transplantation (or transection) were observed in each type of rat. The sciatic functional index is described in the literature "Bain et al., Plast Reconstr Surg 83: 129-139 (1989)". The sciatic functional index was calculated based on the formula shown in Figure 24A by applying ink to the soles of each rat's feet and having them walk, and then analyzing the resulting footprints. The changes in the sciatic functional index over time for each type of rat are shown in Figure 24B. From Figure 24B, it was shown that in autologous nerve transplant rats, nerve bundle transplant material rats, and artificial nerve transplant rats, the sciatic functional index increased 6 weeks after transplantation compared to immediately after transplantation. Furthermore, in rats with autologous nerve transplants and rats with nerve bundle transplant materials, the sciatic nerve function index was shown to increase even further at 12 weeks after transplantation compared to 6 weeks after transplantation.

[0132] Furthermore, for each rat from which the sciatic nerve function index was calculated, the gastrocnemius muscle at the distal end of the transplant site was excised 12 weeks after transplantation (or transection), and its weight was measured as a weight ratio to the wet weight of the gastrocnemius muscle in rats without sciatic nerve transection. The measurement results are shown in Figure 25. From Figure 25, it can be seen that in rats with autologous nerve transplantation, rats with nerve bundle transplantation material, and rats with artificial nerve transplantation, the weight of the gastrocnemius muscle was greater compared to rats with sciatic nerve transection, indicating that the autologous nerve, nerve bundle transplantation material, and artificial nerve each functioned as the sciatic nerve in the rat.

[0133] Example 18: Immunostaining of nerve bundles after grafting to the sciatic nerve In Example 17, the nerve bundle graft material transplanted into the sciatic nerve of rats was excised 12 weeks after transplantation. The excised nerve bundle graft material was fixed with 4% paraformaldehyde, and immunofluorescence staining was performed for STEM121, p75NTR, and MPZ to confirm the presence or absence of expression of STEM121, p75NTR, and MPZ. Next, the nerve bundle graft material was cut perpendicular to the direction of axonal extension of the nerve cells. Photographs of the cut surface (cross-section) of the cut nerve bundle graft material are shown in Figure 26. Photographs in Figures 26A to D show the expression of STEM121, p75NTR, DAPI, and MPZ, respectively. Figure 26E is a merged image of Figures 26A to D. From the photographs in Figures 26A, B, and D, it was observed that STEM121, p75NTR, and MPZ were expressed at approximately the same location. Since STEM121 represents nerve cells in nerve bundle graft material, it was suggested that p75NTR and MPZ are expressed in nerve bundles, particularly in nerve cells, within the nerve bundle graft material. [Industrial applicability]

[0134] According to the present invention, it is possible to efficiently elongate and enlarge the axons of nerve cells. As a result, nerve bundles having axons of sufficient length and diameter for transplantation can be efficiently produced, and nerve bundle transplantation material necessary for nerve transplantation can be efficiently provided. [Explanation of symbols]

[0135] 1. Nerve bundle preparation device 2 Dimethylpolysiloxane substrate 3 recesses 4. Recess (tip recess) 5 grooves

Claims

1. A method for producing nerve bundles, comprising culturing a cell population containing nerve cells in the presence of feeder cells including vascular constituent cells, perivascular cells, and oligodendrocytes, and glial cells, and extending the axons of nerve cells, The aforementioned cell population containing nerve cells includes neural stem cells, immature nerve cells, immature glial cells, mature nerve cells, and mature glial cells. The vascular constituent cells include at least one type of cell selected from the group consisting of vascular pericytes and vascular endothelial cells. The method wherein the perivascular cells include at least one type of cell selected from the group consisting of perivascular fibroblasts and vascular smooth muscle cells.

2. The method according to claim 1, wherein the feeder cells include at least one cell selected from the group consisting of pericytes, vascular endothelial cells, fibroblasts, smooth muscle cells, and oligodendrocytes.

3. The method according to claim 1 or 2, wherein the feeder cells include cells that secrete at least one growth factor selected from the group consisting of VEGF, NGF, BDNF, FGF-2, NGFB, and EGF.

4. The method according to any one of claims 1 to 3, wherein the glial cells include oligodendrocytes.

5. The method according to any one of claims 1 to 4, wherein the nerve bundle has a myelin sheath containing oligodendrocytes.

6. (a) A step of preparing a substrate having at least one recess and a groove connected to the recess, the groove being covered with feeder cells, (b) Adding the neuronal cell population and glial cells to the recess, (c) A step of culturing the cell population containing the nerve cells and the glial cells to extend the axons of the nerve cells along the grooves. The method according to any one of claims 1 to 5, including the method described in any one of claims 1 to 5.

7. (a) A step of preparing a substrate having two recesses and a groove connecting the two recesses, the groove being covered with the feeder cells, (b) Adding the neuronal cell population and glial cells to the recess, (c) A step of culturing the cell population containing the nerve cells and the glial cells to extend the axons of the nerve cells along the grooves. The method according to any one of claims 1 to 6, including the method described in any one of claims 1 to 6.

8. The method according to claim 6 or 7, wherein, in step (a), the groove is covered with fibroblasts before the groove is covered with the feeder cells.

9. The method according to any one of claims 1 to 8, wherein the nerve cell-containing cell population further comprises endothelial cells.

10. The method according to claim 9, wherein the endothelial cells included in the aforementioned cell population containing nerve cells are endothelial cells of vascular origin.

11. The method according to claim 10, wherein the vascular endothelial cells are vascular endothelial cells from at least one tissue selected from the group consisting of dental pulp, gingiva, subcutaneous tissue, intracavitary arteries, intracavitary veins, and umbilical cord.

12. The method according to any one of claims 9 to 11, further comprising in step (c) forming tubes derived from endothelial cells contained in the population of nerve cells.

13. The method according to any one of claims 9 to 12, wherein the nerve cells and endothelial cells originate from the same individual.

14. The method according to any one of claims 9 to 13, wherein the cell population containing the nerve cells further comprises a biocompatible material, and the nerve cells and endothelial cells are each present in layers on the surface of a separate biocompatible material.

15. The method according to claim 14, wherein the biocompatible material includes collagen.

16. The method according to claim 14 or 15, wherein the biocompatible material includes collagen beads.

17. The method according to any one of claims 6 to 16, wherein the length of the groove is 3 mm or more.

18. A nerve bundle manufactured by the method described in any one of claims 1 to 17.

19. A method for producing a transplant material, comprising covering a nerve bundle produced by the method described in any one of claims 1 to 17 with a sheet of biocompatible material.

20. The method according to claim 19, wherein the sheet contains fibroblasts.

21. The method according to claim 19 or 20, wherein the transplant material is a nerve regeneration transplant material.

22. A transplant material manufactured by the method described in any one of claims 19 to 21.

23. A method for extending the axons of nervous system cells, comprising culturing nervous system cells in the presence of feeder cells including vascular constituent cells, perivascular cells and oligodendrocytes, and glial cells, The aforementioned nervous system cells include neural stem cells, immature nerve cells, immature glial cells, mature nerve cells, and mature glial cells. The vascular constituent cells include at least one type of cell selected from the group consisting of vascular pericytes and vascular endothelial cells. The method wherein the perivascular cells include at least one type of cell selected from the group consisting of perivascular fibroblasts and vascular smooth muscle cells.

24. The method according to claim 23, wherein the feeder cells include at least one cell selected from the group consisting of pericytes, endothelial cells, fibroblasts, smooth muscle cells, and oligodendrocytes.

25. The method according to claim 23 or 24, wherein the glial cells include oligodendrocytes.

26. A nerve bundle having axons extending from nerve cells and tubes of endothelial cells along the axons, wherein the nerve bundle comprises at least one type of cell, HNK-1 glycan-expressing cells and p75NTR-expressing cells, and the axons have myelin sheaths containing oligodendrocytes.

27. The nerve bundle according to claim 26, comprising HNK-1 glycan-expressing cells and p75NTR-expressing cells.

28. The nerve bundle according to claims 26 and 27, comprising at least one cell selected from the group consisting of NF200-expressing cells, periferin-expressing cells, myelin basic protein-expressing cells, S100-expressing cells, MPZ-expressing cells, periaxin-expressing cells, CD31-expressing cells, and PDGFRβ-expressing cells.

29. A nerve bundle according to any one of claims 26 to 28, comprising NF200-expressing cells, periferin-expressing cells, myelin basic protein-expressing cells, S100-expressing cells, MPZ-expressing cells, periasin-expressing cells, CD31-expressing cells, and PDGFRβ-expressing cells.

30. The nerve bundle according to any one of claims 26 to 29, wherein the nerve bundle has a cell layer comprising at least one cell, a fibroblast, and a pericyte, which covers at least a portion of the axon along the axon of the nervous system cell, and the endothelial cell tubes are located inside the cell layer.

31. The nerve bundle according to any one of claims 26 to 30, wherein the tubes of endothelial cells further include pericytes.

32. A transplant material comprising a nerve bundle according to any one of claims 26 to 31.

33. The implant material according to claim 32, which is a implant material for nerve regeneration.

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