Nerve bundle and method for producing the nerve bundle

Abstract

To provide a method for producing a nerve fascicle that includes efficiently elongating the axon of a neural cell.SOLUTION: A neural cell is cultured in the presence of a feeder cell including at least one cell selected from vascular constituent cells and perivascular cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to nerve bundles and methods for making nerve bundles. [Background technology]

[0002] In recent years, methods of nerve transplantation have been developed to restore the function of nerves damaged by neurodegenerative diseases or physical causes. A widely used method of nerve transplantation is to inject neural stem cells into the affected area. However, this method has the problem that the injected neural stem cells tend to migrate in the body and are difficult to engraft, making it difficult to efficiently repair damaged areas of nerve tissues such as the brain, spinal cord, and complex peripheral nerves.

[0003] In order to solve this problem, a method has been developed in which a bundle of nerve cells with elongated axons (nerve bundle) is joined to the remaining nerve at the damaged site for transplantation. As a method for obtaining a nerve bundle for transplantation, a method has been reported in which nerve cells are cultured and the axons of the nerve cells are elongated to produce a nerve bundle (Patent Documents 1 and 2), but these methods cannot efficiently elongate and enlarge the axons of the nerve cells, and there is a problem in that it is difficult to obtain a nerve bundle having an axon of sufficient length and thickness (diameter) for transplantation.

[0004] Under these circumstances, there is a need for a method for efficiently obtaining nerve bundles having axons of sufficient length and diameter for transplantation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 187696 [Patent Document 2] JP 2014-136128 A

[0006] The present inventors have found that by culturing nervous system cells using specific cells as feeder cells, the axons of the nervous system cells can be efficiently elongated. The present inventors have also found that the axons of the nervous system cells can be enlarged as they elongate. According to the present invention, it is possible to efficiently prepare nerve bundles having axons of sufficient length and diameter for transplantation. The present invention is based on these findings.

[0007] The present invention includes the following inventions. [1] A method for producing a nerve bundle, comprising culturing a cell population containing nervous system cells in the presence of at least one type of feeder cell selected from vascular cells and perivascular cells, and extending the axons of the nervous system cells. [2] The method according to [1], wherein the feeder cells comprise at least one type of cell selected from the group consisting of pericytes, vascular endothelial cells, fibroblasts and smooth muscle cells. [3] The method according to [1] or [2], wherein the feeder cells comprise cells secreting 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 of [1] to [3], wherein the nerve bundle has a myelin sheath containing Schwann cells. [5] (a) preparing a substrate having at least one recess and a groove connected to the recess, the groove being coated with feeder cells; (b) adding the neural cell-containing cell population to the well; (c) culturing the neural cell-containing cell population to allow the axons of the neural cells to extend along the grooves; The method according to any one of [1] to [4], [6] (a) preparing a substrate having two recesses and a groove connecting the two recesses, the groove being coated with the feeder cells; (b) adding the neural cell-containing cell population to the well; (c) culturing the neural cell-containing cell population to allow the axons of the neural cells to extend along the grooves; The method according to any one of [1] to [5], [7] The method according to [5] or [6], wherein in the step (a), the grooves are coated with fibroblasts before being coated with the feeder cells. [8] The method according to any of [1] to [7], wherein the neural cell-containing cell population further comprises endothelial cells. [9] The method according to [8], wherein the endothelial cells contained in the neural cell-containing cell population are endothelial cells derived from blood vessels.

[10] The method according to [9], wherein the endothelial cells derived from blood vessels are endothelial cells derived from blood vessels of at least one tissue selected from the group consisting of dental pulp, gingiva, subcutaneous tissue, intracavitary artery, intracavitary vein and umbilical cord.

[11] The method according to any of [8] to

[10] , further comprising forming tubes derived from endothelial cells contained in the neural cell-containing cell population in the step (c).

[12] The method according to any of [8] to

[11] , wherein the nervous system cells and endothelial cells are derived from the same individual.

[13] The method according to any of [8] to

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

[14] The method according to

[13] , wherein the biocompatible material comprises collagen.

[15] The method according to

[13] or

[14] , wherein the biocompatible material comprises collagen beads.

[16] The method according to any one of [5] to

[15] , wherein the length of the groove is 3 mm or more.

[17] A nerve bundle produced by a method according to any one of the methods described in [1] to

[16] .

[18] A method for producing a transplant material, comprising covering a nerve bundle produced by the method according to any one of the methods described in [1] to

[16] with a sheet of a biocompatible material.

[19] The method according to

[18] , wherein the sheet comprises fibroblasts.

[20] The method according to

[18] or

[19] , wherein the transplant material is a transplant material for nerve regeneration.

[21] A transplant material produced by the method according to any one of

[18] to

[20] .

[22] A method for extending axons of nervous system cells, comprising culturing nervous system cells in the presence of feeder cells containing at least one type of cell selected from vascular cells and perivascular cells. [twenty three] The method according to

[22] , wherein the feeder cells comprise at least one type of cell selected from the group consisting of pericytes, endothelial cells, fibroblasts and smooth muscle cells.

[0008] According to the present invention, it is possible to efficiently extend the axons of nervous system cells. Also, according to the present invention, it is possible to efficiently thicken the axons of nervous system cells. According to the present invention, it is possible to efficiently prepare a nerve bundle having axons of sufficient length and diameter for transplantation. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of an apparatus for creating nerve bundles. [Diagram 2] FIG. 2 is a graph showing the relationship between the number of days of culture and the length of nerve fibers (axons) when various cells were used as feeder cells. [Diagram 3] Figure 3A is a stereomicroscope photograph of the prepared nerve bundle, and Figure 3B is a hematoxylin-eosin (HE) stained image (longitudinal section) of the prepared nerve bundle. [Figure 4] Figure 4 shows immunostained images of nerve bundles prepared by the method of the present embodiment. Figure 4A shows a photograph of immunostained nerve bundles using anti-vWF and anti-F200 antibodies. Figure 4B shows a photograph of immunostained nerve bundles using anti-vWF and anti-S100 antibodies. [Diagram 5] Figure 5A is a photograph of the graft site immediately after implantation of the nerve bundle graft material into the sciatic nerve of a rat, and Figure 5B is a photograph of the graft site 14 days after implantation of the nerve bundle graft material into the sciatic nerve of a rat. [Figure 6]Figure 6A is a photograph of a rat in which only the T9 vertebral arch was removed, and Figure 6B is a photograph of the resected portion after the T9 vertebral arch and thoracic spinal cord were removed. [Figure 7] Figure 7A is a photograph of the graft site immediately before and immediately after the nerve bundle graft material is implanted in the spinal cord (thoracic spinal cord) of a rat. Figure 7B is a photograph of the graft site immediately after the nerve graft material is implanted in the spinal cord (thoracic spinal cord) of a rat. Description of the Invention

[0010] (Method of manufacturing nerve bundle) According to one embodiment of the present invention, there is provided a method for producing a nerve bundle (hereinafter, also referred to as the "production method of the present invention"). According to the production method of the present invention, a nerve bundle having an axon of sufficient length for transplantation can be produced.

[0011] The method for producing a nerve bundle includes a step of culturing a cell population containing neural cells in the presence of feeder cells containing at least one type of cell selected from vascular cells and perivascular cells, and allowing the axons of the neural cells to extend.

[0012] As used herein, the term "nervous system cell-containing cell population" refers to a cell population containing nervous system cells, which will be described later. The nervous system cell-containing cell population may contain cells other than nervous system cells.

[0013] The nervous system cells contained in the nervous system cell-containing population include not only cells constituting the nervous system, but also cells that can differentiate into cells constituting the nervous system. Specifically, any of the following can be used as the 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.), and differentiated mature nerve cells (e.g., mature nerve cells, mature glial cells, etc.). These nervous system cells may be commercially available, may be isolated and prepared from a living body, or may be differentiated or induced from pluripotent stem cells such as ES cells and iPS cells. In addition, the nervous system cells may be cells derived from an individual to be transplanted (autologous cells) or cells derived from an individual other than the transplanted individual (allogeneic cells). In a preferred embodiment, the nervous system cell-containing 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 contained in the nervous system cell-containing cell population are not particularly limited as long as they do not show antigenicity in the transplanted subject of the nerve bundle of the present invention, and any origin can be used. Examples of the origin of the nervous system cells include the same individual as the subject of nerve bundle transplantation, an individual different from the subject of nerve bundle transplantation, an HLA (Human Leukocyte Antigen) homozygous donor, and universal donor cells.

[0014] The cell population containing nervous system cells may be in any form as long as the effects of the present invention are achieved, and may be, for example, in the form of a cell population containing nervous system cells suspended in a culture medium (such as αMEM, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium / Ham's F-12 mixed medium (DMEM / F12), Ham's 10, Ham's 12, RPMI1640 medium, various neuronal culture media, etc.), or in the form of floating cells.

[0015] The content of neural cells in the neural cell-containing cell population is not particularly limited as long as the effects of the present invention are achieved. For example, the number of neural cells is 10 3 ~10 10 pcs, preferably 10 4 ~10 9pcs, more preferably 10 5 ~10 8 There are 10 pieces.

[0016] According to one embodiment of the present invention, the nerve bundle produced by the method of the present invention has at least a part, preferably all, of the nervous cells constituting the nerve bundle having myelin sheaths containing Schwann cells, a type of glial cell, in their axonal portions, and therefore the nerve bundle produced by the method of the present invention can be used as a nerve bundle, particularly in the peripheral nervous system.

[0017] Examples of cells other than nervous system cells contained in the cell population containing nervous system cells include endothelial cells and red blood cells. As the endothelial cells, endothelial cells capable of forming blood vessels are preferably used, specifically vascular endothelial cells. The cells other than nervous system cells contained in the cell population containing nervous system cells are not particularly limited as long as they do not show antigenicity in the transplantation target of the nerve bundle of the present invention, and any origin can be used. Examples of the origin of the cells other than nervous system cells include the same individual as the transplantation target of the nerve bundle, an individual different from the transplantation target of the nerve bundle, an HLA (Human Leukocyte Antigen) homozygous donor, a universal donor cell, etc.

[0018] When the cell population containing neural cells contains endothelial cells, the endothelial cells proliferate and differentiate by culturing the cell population containing neural cells in the presence of feeder cells, and tubes derived from the endothelial cells are formed along the extended axons. As a result, the resulting nerve bundle contains, in addition to a bundle of neural cells (nerve fibers) with extended axons, tubes (blood vessels) of endothelial cells that extend in the same direction as the nerve fibers (axons) and attach to the nerve fibers (axons).

[0019] The endothelial cells contained in the cell population containing nervous system cells can be any of endothelial stem cells, cells in the process of differentiating into endothelial cells, and differentiated mature endothelial cells. 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 dental pulp, gingiva, subcutaneous tissue, intracoelastic artery, intracoelastic vein, or umbilical cord blood vessels, and even more preferably, endothelial cells derived from blood vessels in dental pulp. These endothelial cells may be commercially available, or may be differentiated or induced from pluripotent stem cells such as ES cells or iPS cells. The endothelial cells may be autologous cells derived from an individual to be transplanted, or allogeneic cells derived from an individual other than the individual to be transplanted.

[0020] The content of endothelial cells in the cell population containing neural 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 pcs, preferably 2 x 10 4 ~3×10 9 pcs, more preferably 2×10 5 ~3×10 8 In a preferred embodiment, the number of endothelial cells in the neural cell-containing population is greater than the number of neural cells.

[0021] The cell population containing neural cells may contain a biocompatible material in addition to the above-mentioned cells. Any biocompatible material can be used without particular limitation as long as the cells contained in the cell population containing neural cells can adhere to the biocompatible material and the biocompatible material can be metabolized and consumed by the cells during the growth and proliferation of the cells. Examples of such biocompatible materials include collagen, laminin, fibronectin, gelatin, Matrigel (registered trademark), etc.

[0022] The shape of the biocompatible material is not particularly limited as long as the above-mentioned effects are achieved, but examples include beads (spherical, approximately spherical), rods, and membranes.

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

[0024] The bead-shaped biocompatible material can be prepared appropriately using a known method, for example, by dropping the biocompatible material into an oil phase to form droplets in the oil phase. The oil constituting the oil phase is not particularly limited as long as droplets of the biocompatible material can be formed, and examples of the oil include edible oils such as corn oil, rapeseed oil, and sesame oil, and mineral oils derived from petroleum, natural gas, and coal. The diameter of the beads (droplets) can be adjusted appropriately by changing the amount of dropping.

[0025] The nervous cells contained in the cell population containing nervous cells are preferably present in a layer on the surface of a biocompatible material. Specifically, by having the nervous cells present in a layer on the surface of a biocompatible material, the nervous cells with their axons extended form a bundle after culture, which is advantageous in that a nerve bundle with a large diameter (thickness) can be easily produced.

[0026] Known methods can be used to cause nervous system cells to be present in a layer on the surface of a biocompatible material, and examples include a method in which a biocompatible material is mixed with a cell population containing nervous system cells, and the mixture is placed in a container such as a non-adhesive petri dish and cultured in a rotational manner.

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

[0028] When the cell population containing nervous system cells includes endothelial cells, the endothelial cells contained in the cell population containing nervous system cells are preferably present in a layer on the surface of a biocompatible material. Specifically, by having the endothelial cells present in a layer on the surface of a biocompatible material, during the culture process, a plurality of biocompatible materials having a layer of endothelial cells derived from the endothelial cells contained in the cell population containing nervous system cells are brought into contact and fused, and as a result, a tube of endothelial cells whose surface is composed of endothelial cells and whose interior contains a biocompatible material can be formed. Then, the endothelial cells constituting the surface of the tube take up, metabolize, and consume the biocompatible material inside the tube during the process of growth and proliferation, and as a result, a hollow tube of endothelial cells can be formed. The formed tube of endothelial cells can play the role of a tube (blood vessel) that supplies oxygen, nutrients, etc. to the nervous cells of the nerve bundle to be produced, and has the advantage of preventing necrosis of the nerve bundle. The method for having the endothelial cells present in a layer on the surface of a biocompatible material can be the same as the method for having the nervous cells present in a layer on the surface of a biocompatible material described above.

[0029] In order to achieve the advantage of having the nervous system cells and the endothelial cells present in layers on the surface of a biocompatible material, the nervous system cells and the endothelial cells are preferably present in layers on the surface of separate biocompatible materials. Specifically, it is preferable to have the nervous system cells present in a layer on the surface of a biocompatible material and the endothelial cells present in a layer on the surface of another biocompatible material.

[0030] The biocompatible material may be mixed in advance with a substance (neuron growth factor) that promotes the growth and proliferation of nervous system cells, such as nerve growth factor (NGF), fibroblast growth factor β (β-FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), trophic factor, etc. By including nerve growth factor in the biocompatible material, the growth of nervous system cells present on the surface of the biocompatible material can be promoted more efficiently.

[0031] The ratio of nerve cell growth factor to be mixed into the biocompatible material can be appropriately set depending on the size of the nerve bundle to be produced, and is, 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] The nerve cell growth factor may be used alone, or may be further mixed with ganglioside 3 (GD3), a type of sugar chain. The amount of GD3 to be mixed with the biocompatible material is not particularly limited, but may be, for example, 10 to 100 ng / mg, 10 to 75 ng / mg, or 10 to 50 ng / mg. When the biocompatible material and the nerve cell growth factor are added separately to the wells, the nerve cell growth factor (when GD3 is used, the nerve cell growth factor and GD3) are placed in the wells, and then the nervous system cells are placed in the wells, thereby further promoting the extension of the axon.

[0033] When the cell population containing neural cells contains endothelial cells, a substance that promotes the growth and proliferation of endothelial cells (endothelial cell growth factor), such as vascular endothelial growth factor (VEGF), may be mixed in advance with the biocompatible material. By including an endothelial cell growth factor in the biocompatible material, the growth of endothelial cells present on the surface of the biocompatible material can be promoted more efficiently.

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

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

[0036] When the cell population containing nervous system cells contains endothelial cells, red blood cells may be mixed in advance with the biocompatible material. By including red blood cells in the biocompatible material, red blood cells can be present inside the hollow endothelial cell tube formed in the process of culturing the cell population containing nervous system cells. As a result, a completely hollow endothelial cell tube can be produced. Then, oxygen is supplied to the nervous system cells by the red blood cells in the produced nerve bundle through the produced hollow endothelial cell tube, so that the nerve bundle can be prevented from necrosis.

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

[0038] As used herein, the term "feeder cells" refers to cells that produce and secrete substances that induce or maintain the survival, proliferation, and differentiation of nervous cells and, optionally, endothelial cells, and promote the elongation and hypertrophy of nervous cells. The substances produced and secreted by feeder cells are not particularly limited as long as they have the above-mentioned functions, and examples thereof 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] The feeder cells include cells that produce and secrete the substances described above, and specifically include at least one type of cell selected from vascular cells that form blood vessels and perivascular cells that exist around blood vessels.

[0040] The vascular cells are not particularly limited as long as the effects of the present invention are achieved, and examples thereof include pericytes (e.g., vascular pericytes), endothelial cells (e.g., vascular endothelial cells), etc. Furthermore, examples of perivascular cells include fibroblasts (e.g., perivascular fibroblasts), smooth muscle cells (e.g., vascular smooth muscle cells), etc.

[0041] The method for producing a nerve bundle is not particularly limited as long as it includes a step of culturing a cell population containing nervous system cells in the presence of feeder cells containing at least one type of cell selected from vascular cells and perivascular cells, and extending the axons of the nervous system cells. The step can be carried out, for example, as shown in the following steps (a) to (e).

[0042] Process (a) In this step, a substrate is prepared that includes at least one recess and a groove portion that is connected to the recess and is coated with feeder cells.

[0043] The material of the substrate is not particularly limited as long as the effects of the present invention are achieved, but examples thereof include dimethylpolysiloxane, polystyrene, polypropylene, and the like.

[0044] The shape and dimensions of the substrate can be appropriately set as long as the effects of the present invention are achieved. The substrate preferably has at least one flat surface, and is, for example, a rectangular parallelepiped, cubic, cylindrical, or other shape, preferably a rectangular parallelepiped.

[0045] The dimensions of the substrate are not particularly limited as long as it has at least one recess and groove. For example, when the substrate has a rectangular parallelepiped shape, the dimensions are length and width of 5 to 20 cm, 5 to 15 cm, and 5 to 10 cm, and height (depth) of 1 to 10 cm, 1 to 5 cm, and 1 to 3 cm.

[0046] The substrate has at least one recess for accommodating a cell population containing neural cells. When the substrate has a plurality of recesses, the arrangement of the plurality of recesses is not particularly limited as long as the effects of the present invention are achieved, but preferably the plurality of recesses are present on the same plane of the substrate. For example, the substrate has two recesses, and the two recesses are present on the same plane of the substrate.

[0047] The shape and dimensions of the recesses can be appropriately set as long as the effects of the present invention are achieved. Examples of the shape of the recesses include a cylindrical shape, a rectangular parallelepiped shape, a cubic shape, and a hemispherical shape.

[0048] When the recess has a cylindrical shape, 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. When the substrate has multiple recesses, their shapes and / or dimensions may be the same or different from each other.

[0049] The substrate has a groove connected to the recess, and the groove is covered with feeder cells. The groove may be covered with fibroblasts before being covered with feeder cells. When the groove is covered with fibroblasts, the groove is preferably first covered with fibroblasts, and then the covered fibroblasts are covered with feeder cells. The arrangement of the groove is not particularly limited as long as the effect of the present invention is achieved, but the groove and the recess are present on the same plane. In addition, when the substrate has multiple recesses, each recess is connected to a groove, and preferably the multiple recesses are connected via the groove. For example, the substrate has two recesses on the same plane, and the two recesses are connected via a groove present on the same plane as the two recesses.

[0050] The shape (cross-sectional shape) and dimensions (length, depth) of the grooves can be appropriately set as long as the effects of the present invention are achieved. Examples of the shape of the grooves include a concave shape, a V shape, a U shape, and an Ω shape.

[0051] 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, 50 to 300 μm, preferably 75 to 250 μm, and more preferably 100 to 200 μm. The width of the groove is, for example, 50 to 300 μm, preferably 75 to 250 μm, and more preferably 100 to 200 μm. When the substrate has a plurality of recesses, the length of the groove refers to the length (distance) between the recesses.

[0052] Process (b) In this step, a cell population containing nervous system cells is added to the wells. In a preferred embodiment, the cell population containing nervous system cells is added to the wells in a state in which it is present in a layer on the surface of a biocompatible material, preferably a bead such as collagen beads. When the cell population containing nervous system cells contains endothelial cells, the nerve cells and endothelial cells contained in the cell population containing nervous system cells are added to the wells in a state in which they are present in a layer on the surface of separate biocompatible materials, preferably a bead such as collagen beads. The amount of the cell population containing nervous system cells to be added to the wells is not particularly limited as long as the effects of the present invention are achieved, but for example, 10 3 ~10 10 pcs, preferably 10 4 ~10 9 pcs, more preferably 10 5 ~10 8 When the cell population containing neural cells contains endothelial cells, the amount of endothelial cells added to the wells is, for example, 1:1 to 1:10, 1:2 to 1:7, or 1:3 to 1:5, as the ratio of the number of neural cells to the number of endothelial cells (number of neural cells: number of endothelial cells).

[0053] Process (c) In this process, a culture medium is added to the recesses and grooves of the substrate, and the neural cell-containing cell population added to the recesses is cultured, causing the axons of the neural cells to extend along the grooves of the substrate.

[0054] The culture medium to be added is not particularly limited as long as it is a culture medium that is usually used for culturing neural cells, and examples thereof include basic media such as nerve cell culture medium, DMEM, RPMI1640 medium, EMEM, Ham 10, and Ham 12, and DMEM / F12 which is an improved basic medium. These media may contain various amino acids such as L-glutamine and L-alanine, additives such as fetal bovine serum (FBS), albumin, etc. When FBS is added to the medium, the concentration of FBS is, for example, 5 to 20 mass%, 7 to 15 mass%, or 10 to 15 mass% relative to the medium. In addition, as a method of adding the culture medium, the culture medium may be added to each of the recesses and grooves, or the substrate may be immersed in a container filled with the culture medium in advance, and the culture medium may be supplied to each of the recesses and grooves.

[0055] The culture conditions are not particularly limited as long as the cells contained in the cell population containing neural cells grow and proliferate, and examples thereof include static culture at a temperature of 35 to 38°C, a CO2 concentration of 3 to 5%, and a culture time of 20 to 24 hours. Note that static culture and perfusion culture may be combined depending on the degree of axonal elongation of the neural cells. The conditions for perfusion culture may be, for example, a temperature of 35 to 38°C, a CO2 concentration of 3 to 5%, a perfusion rate of 1 to 10 ml / hour, preferably 2 to 5 ml / hour, and a culture time of 72 hours to 60 days.

[0056] (nerve bundle) According to one embodiment of the present invention, there is provided a nerve bundle produced by the above method (hereinafter, also referred to as the "nerve bundle of the present invention"). According to the present invention, the axons of the nerve bundle can be elongated to a length sufficient for transplantation.

[0057] If necessary, a plurality of nerve bundles can be combined and used as a bundle of nerve bundles. A bundle of nerve bundles can be prepared by rolling (covering) a plurality of nerve bundles with a sheet of a biocompatible material prepared in advance. The sheet of a biocompatible material is not particularly limited as long as the effect of the present invention is exhibited, and examples thereof include a sheet of fibroblasts, a sheet of collagen fiber network, a sheet of elastic fiber network, etc.

[0058] According to one embodiment of the present invention, a bundle of nerve bundles can be prepared by arranging a plurality of nerve bundles on a previously prepared sheet of fibroblast cells, and rolling the plurality of nerve bundles in a roll (seaweed roll) shape with the fibroblast sheet. The thickness (diameter) of the bundle of nerve bundles can be changed as desired depending on the number of nerve bundles arranged on the fibroblast sheet. In this way, when the nerve bundle is rolled with the fibroblast sheet, it can be easily transplanted by suturing the end of the fibroblast sheet and the damaged part (nerve stump) of the nerve to be transplanted.

[0059] The nerve bundle can be used as a transplant material (a nerve regeneration transplant material) to replace a damaged nerve. The transplant target is not particularly limited, and can be, for example, transplanted into the central nervous system, peripheral nervous system, etc., but is preferably transplanted into the peripheral nervous system.

[0060] The nerve bundle can be produced using cells derived from the individual to be transplanted (autologous cells) or cells derived from an individual other than the individual to be transplanted (allogeneic cells). Therefore, by producing the nerve bundle of the present invention using autologous cells, it becomes possible to transplant the nerve bundle (autologous transplant) with almost no rejection reaction in the individual from which the cells that are the material for the nerve bundle are derived.

[0061] (Method for extending axons of nervous system cells) According to one embodiment of the present invention, a method for extending an axon of a nervous system cell (hereinafter, also referred to as "the method of the present invention") is provided. According to the method of the present invention, it is possible to efficiently extend an axon of a nervous system cell. Furthermore, according to the method of the present invention, it is also possible to efficiently increase the diameter of a nerve axon.

[0062] The method of the present invention comprises the step of culturing a cell population containing neural cells in the presence of feeder cells comprising at least one type of cell selected from vascular cells and perivascular cells, to extend the axons of the neural cells.

[0063] In the method of the present invention, the neural cell-containing cell population and feeder cells can be the same as those explained in the above-mentioned method for producing a nerve bundle.

[0064] In the method of the present invention, the specific conditions for the step of culturing a cell population containing nervous system cells in the presence of feeder cells containing at least one type of cell selected from vascular cells and perivascular cells and extending the axons of the nervous system cells can be the same as those described in the above-mentioned method for producing a nerve bundle. EXAMPLES

[0065] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

[0066] Example 1: Preparation of a cell population containing neural cells The neural cell-containing cell population used in this example was prepared according to the following procedure. Oral mesenchymal cells collected from human dental pulp, gingival epithelial tissue sublayer, and oral epithelial tissue sublayer were seeded onto cell adhesive dishes (IWAKI) and cultured for 3 to 7 passages at 37°C and CO2 concentration of 4.5 to 5.5% using Dulbecco's modified Eagle's medium / Ham's F12 (DMEM / F12). The cells were then detached using trypsin-EDTA solution, and the isolated cells were thinly seeded (in small numbers) onto new dishes. Large colonies with high proliferation potential were subjected to colonial cloning to obtain dental pulp-derived mesenchymal stem cells. The obtained stem cells were cultured in neural induction medium (culture induction medium described in Takahashi et al., Human Cell, Vol.30, Issue 2, pp60-71) supplemented with epidermal growth factor (EGF) at 10ng / ml and fibroblast growth factor β (β-FGF) at 10ng / ml at 37℃ and CO2 concentration of 4.5-5.5% to induce differentiation and produce a cell population containing neural cells. Analysis by immunostaining and RT-PCR confirmed that the cells after differentiation induction included neural stem cells, immature neurons, immature glial cells, mature neurons, and mature glial cells. In addition, immunostaining using tyrosine hydroxylase antibody confirmed that the cells after differentiation induction included dopamine cells.

[0067] Example 2: Preparation of endothelial cells The endothelial cells used in this example were prepared according to the following procedure. Vascular network-containing tissue collected from the human oral cavity was treated with digestive enzymes to separate cells, which were then cultured as primary cells. Morphologically distinguishable colonies of endothelial cells were isolated by colonial cloning, and these were then grown to obtain endothelial cells. Alternatively, primary culture cells were seeded on a Matrigel-coated petri dish, VEGF (10-50ng / ml) was added to the culture medium, and the cells were cultured to isolate structures that form lumens, and the cells that make up these structures were grown to obtain endothelial cells.

[0068] Example 3: Preparation of neural cell beads The neural cell beads used in this example were produced according to the following procedure. A 10-fold concentrated Dulbecco's modified Eagle medium / Ham's F-12 mixed medium (DMEM / F12) was added to an atelocollagen solution (manufactured by Koken Co., Ltd.) so that the amount was 10% by mass relative to the atelocollagen solution, and the mixture was stirred and mixed to obtain an atelocollagen mixture. The obtained atelocollagen mixture was dropped into corn oil so that the atelocollagen particle (bead) diameter was 200 μm to prepare a biocompatible material (collagen beads). The obtained collagen beads were mixed with the neural cell-containing cell population prepared in Example 1 at a mass ratio of 1:500, and after mixing, the mixture was placed in a non-adhesive petri dish (manufactured by IWAKI Co., Ltd.) and cultured at a rotation speed of 40 to 60 rpm, and the neural cell-containing cell population was held in a layer on the surface of the collagen beads to prepare neural cell beads.

[0069] Example 4: Preparation of endothelial cell beads The endothelial cell beads used in this example were made according to the following procedure. RPMI1640 medium concentrated 8 to 10 times was added to the atelocollagen solution (manufactured by Koken Co., Ltd.) so that the concentration was 10% by mass relative to the atelocollagen solution. Furthermore, vascular endothelial growth factor (VEGF) (manufactured by Sigma) was added to a concentration of 50 ng / ml, and red blood cells were added to a concentration of 5% by mass, followed by stirring and mixing to obtain an atelocollagen mixture. The obtained atelocollagen mixture was dropped into corn oil so that the atelocollagen particle (bead) diameter was 100 μm to prepare a biocompatible material (collagen beads). The obtained collagen beads were mixed with the endothelial cells prepared in Example 2 at a mass ratio of 1:500, and after mixing, the mixture was placed in a non-adhesive petri dish (manufactured by IWAKI Co., Ltd.) and cultured at a rotation speed of 40 to 60 rpm, and the endothelial cells were held in a layer on the surface of the collagen beads to prepare endothelial cell beads.

[0070] Example 5: Preparation of a nerve bundle preparation device The apparatus for nerve bundle creation was fabricated according to the following procedure. Two recesses, each 5 mm in diameter and 5 mm deep, were formed 3 cm apart on the surface of a dimethylpolysiloxane substrate (5 cm long x 5 cm wide x 1 cm thick). A linear groove, 150 μm wide and 150 μm deep, was then formed between the two recesses to connect them, creating a device for nerve bundle creation. A schematic diagram of the device is shown in Figure 1.

[0071] Example 6: Creation of a nerve bundle Prior to the preparation of the nerve bundle of the present invention, feeder cells suitable for extending axons of nervous system cells were examined by the following procedure. Four devices prepared in Example 5 were prepared, and vascular constituent cells (vascular pericytes, vascular endothelial cells, blood vessel-derived smooth muscle cells) or perivascular cells (perivascular fibroblasts) were seeded as feeder cells in the grooves of each device. Then, each device was placed in a culture vessel, and DMEM / F12 to which fetal bovine serum (FBS) was added to make 15% by mass 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 cells in the grooves of each device, and a monolayer of each cell was formed in the grooves. Next, the nervous system cell beads prepared in Example 3 and the endothelial cell beads prepared in Example 4 were mixed in a mass ratio of 3:1, and added to each of the two recesses of each device with a micropipette. DMEM / F12 medium was added to each recess, and each device was placed in a CO2 incubator (temperature 37°C, CO2 concentration 4.7%) to culture for 20 hours. Next, each device was placed in a chamber (length 6 cm x width 6 cm x thickness 2 cm) and cultured under reflux for 72 to 168 hours (flow rate 2 ml / min, temperature 37°C, CO2 concentration 4.7%), and the axons of the nervous system cells were extended onto each cell layer in the groove to examine the relationship between the number of days of culture and the length of the nerve bundle. The results are shown in Figure 2. Although not shown in Figure 2, the diameters of the axons 30 days after the start of culture when vascular pericytes, vascular endothelial cells, perivascular fibroblasts, and vascular-derived smooth muscle cells were used as feeder cells were about 100 to 150 μm, about 50 to 80 μm, about 20 to 30 μm, and about 10 to 20 μm, respectively. On the other hand, when feeder cells were not used, the axons of the nervous system cells hardly extended or enlarged, and the length was about 5 to 10 μm and the diameter was about 5 to 10 μm 30 days after the start of culture.

[0072] The results in Figure 2 show that when vascular cells or perivascular cells are used as feeder cells, the axons of nervous cells extend efficiently. In particular, when vascular pericytes or endothelial cells, which are vascular cells, are used as feeder cells, the axons of nervous cells extend very efficiently.

[0073] Hereinafter, nerve bundles were prepared using vascular pericytes as feeder cells according to the following procedure. Fibroblasts were seeded in the grooves of the device prepared in Example 5 to form a monolayer of fibroblasts. Then, human-derived vascular pericytes were seeded on the fibroblasts to form a monolayer to multilayer of vascular pericyte layers. Then, the device was placed in a culture vessel, and DMEM / F12 to which fetal bovine serum (FBS) was added to make the concentration 15% by mass 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 vascular pericytes in the grooves of the device, forming a monolayer of fibroblasts in the grooves and a monolayer of vascular pericytes attached to the fibroblast layer. Next, the neural cell beads prepared in Example 3 and the endothelial cell beads prepared in Example 4 were mixed in a mass ratio of 3:1 and added to each of the two recesses of the device with a micropipette. DMEM / F12 medium was added to each well, and the device was placed in a CO2 incubator (temperature 37°C, CO2 concentration 4.7%) and cultured for 20 hours. The device was then placed in a chamber (length 6 cm x width 6 cm x thickness 2 cm) and cultured under reflux for 30 days (flow rate 2 ml / min, temperature 37°C, CO2 concentration 4.7%), and the axons of the nervous cells were extended onto the fibroblast layer and vascular pericyte layer in the groove to obtain a bundle of nervous cells (nerve fibers) with extended axons (nerve bundle of the present invention). The obtained nerve bundle had, in addition to the bundle of nervous cells (nerve fibers) with extended axons, a tube (blood vessel) of endothelial cells that extended in the same direction as the nerve fibers (axons) and attached to the nerve fibers (axons). The length of the nerve bundle was about 3 cm, and the diameter was about 100 to 150 μm. The fibroblasts that constitute the fibroblast layer grew and proliferated, and a sheet of fibroblasts was formed in the groove. The micrograph and immunostained image of the obtained nerve bundle are shown in Figure 3 (A: stereomicroscope image, B: hematoxylin-eosin (HE) stained image (longitudinal section)). In Figures 3A and 3B, the endothelial cell tubes (blood vessels) run along the nerve bundle. The immunostained image of the obtained nerve bundle is shown in Figure 4. In Figures 4A and 4B, the white or gray linear parts running along the nerve bundle are endothelial cell tubes (blood vessels). That is, the nerve bundle prepared by the method of this example has endothelial cell tubes (blood vessels) running along the axons in addition to the nervous system cells with extended axons.

[0074] Next, the nerve bundle formed in the groove was rolled and covered with the fibroblast sheet formed in the groove. A plurality of nerve bundles covered with the fibroblast sheet were prepared, and a large fibroblast sheet that had been prepared in advance in a petri dish was rolled and covered with the plurality of nerve bundles to prepare a transplant material, which was then used for transplantation, as described below.

[0075] Example 7: Transplantation of nerve bundles (transplantation into the sciatic nerve of rats) The nerve bundle graft material prepared in Example 6 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 Japan CLEA) was anesthetized by intraperitoneal administration of a triple anesthetic (a mixture of medetomidine hydrochloride (0.15mg / 0.15ml / kg), midazolam (2mg / 0.4ml / kg), butorphanol tartrate (2.5mg / 0.5ml / kg) and saline (1.45ml / kg)) and a part of the sciatic nerve was excised. Each of the two ends of the nerve bundle graft material (approximately 1cm long and 1mm in diameter) prepared in Example 6 was sutured with three stitches using surgical sutures (10-0 nylon) to each of the two nerve stumps after sciatic nerve excision, and the nerve bundle graft material was transplanted into the excised sciatic nerve portion. On the 14th day after transplantation, the nerve bundle graft material and the rat's sciatic nerve were joined, and no necrosis of the nerve graft material was observed. Figure 5 shows photographs of the graft site immediately after and 14 days after the nerve bundle graft material was transplanted (A: immediately after grafting, B: 14 days after grafting). In addition, when the rats were observed following the transplantation of the nerve bundle graft material, their toes were able to open sufficiently and they were as active as the control rats that had received autologous nerve transplants. Furthermore, no effects such as a shortened lifespan were observed. These results indicate that the transplanted nerve bundle graft material functions in place of the resected sciatic nerve.

[0076] Example 8: Transplantation of nerve bundles (implantation into rat spinal cord) The nerve bundle graft material prepared in Example 6 was transplanted into the spinal cord of a rat according to the following method. Rats (Wistar, 6-8 weeks old, female, manufactured by Japan SLC) were anesthetized in the same manner as described in Example 7, and a portion of the T9 vertebral arch and thoracic spinal cord (2 mm, which is one spinal cord segment) was excised. Photographs of the rat after the excision of the T9 vertebral arch and a portion of the thoracic spinal cord are shown in FIG. 6 (A: after T9 vertebral arch excision, B: after T9 vertebral arch and thoracic spinal cord excision). The nerve bundle graft material (length about 5-8 mm, diameter about 2-3 mm) prepared in Example 6 was transplanted into the nerve portion of the excised thoracic spinal cord. Photographs of the transplant site before and after the transplantation of the nerve bundle graft material are shown in FIG. 7 (A: before transplantation, B: after transplantation). The white part in the center of the photograph in FIG. 7B is the transplanted nerve bundle graft material. When the rats were observed after the transplantation of the nerve bundle graft material, they were active in the same way as the control rats to which autologous nerves were transplanted, and the movement of the left and right lower limbs was restored. In addition, no effects such as shortening of lifespan were observed. These results demonstrated that the implanted nerve bundle graft material functioned in place of the resected nerve portion of the thoracic spinal cord. [Industrial Applicability]

[0077] According to the present invention, it is possible to efficiently extend and enlarge the axons of nervous system cells, which results in efficient production of nerve bundles having axons of sufficient length and diameter for transplantation, and efficient provision of nerve bundle graft materials required for nerve transplantation. [Explanation of symbols]

[0078] 1. Nerve bundle creation device 2 Dimethylpolysiloxane substrate 3. Recess 4 Groove

Claims

1. 1. A method for producing a nerve bundle, comprising: The method includes a step of culturing a neural cell-containing cell population containing neural cells and endothelial cells induced from dental pulp-derived mesenchymal stem cells in the presence of at least one feeder cell selected from the group consisting of vascular pericytes, vascular endothelial cells, vascular-derived smooth muscle cells, and perivascular fibroblasts, and extending the axons of the neural cells, the step comprising: (a) preparing a substrate comprising at least one recess and a groove connected to the recess, the groove being coated with the feeder cells; (b) adding the neural cell-containing cell population to the well; (c) culturing the neural cell-containing cell population to allow the axons of the neural cells to extend along the grooves; A method for producing a nerve bundle, comprising:

2. The method of claim 1, wherein the feeder cells comprise cells secreting at least one growth factor selected from the group consisting of VEGF, NGF, BDNF, FGF-2, NGFB and EGF.

3. The method of claim 1 or 2, wherein the nerve bundle has a myelin sheath containing Schwann cells.

4. (a) preparing a substrate having two recesses and a groove connecting the two recesses, the groove being coated with the feeder cells; (b) adding the neural cell-containing cell population to the well; (c) culturing the neural cell-containing cell population to allow the axons of the neural cells to extend along the groove. The method according to any one of claims 1 to 3, comprising:

5. The method according to any one of claims 1 to 4, wherein in the step (a), the grooves are coated with fibroblasts before coating the grooves with the feeder cells.

6. The method according to any one of claims 1 to 5, wherein the endothelial cells contained in the neural cell-containing cell population are endothelial cells derived from blood vessels.

7. The method according to claim 6, wherein the endothelial cells derived from blood vessels are endothelial cells derived from blood vessels of at least one tissue selected from the group consisting of dental pulp, gums, subcutaneous tissue, intracavitary arteries, intracavitary veins and umbilical cord.

8. The method according to any one of claims 1 to 7, further comprising forming tubes derived from endothelial cells contained in the neural cell-containing cell population in the step (c).

9. The method according to any one of claims 1 to 8, wherein the nervous system cells and endothelial cells are derived from the same individual.

10. The method according to any one of claims 1 to 9, wherein the neural cell-containing cell population further comprises a biocompatible material, and the neural cells and endothelial cells are each present in layers on the surface of a separate biocompatible material.

11. The method of claim 10 , wherein the biocompatible material comprises collagen.

12. The method of claim 10 or 11, wherein the biocompatible material comprises collagen beads.

13. The method according to any one of claims 1 to 12, wherein the length of the groove is 3 mm or more.

14. A nerve bundle produced by the method according to any one of claims 1 to 13.

15. A method for producing an implant comprising covering a nerve bundle produced by the method according to any one of claims 1 to 13 with a sheet of biocompatible material.

16. The method of claim 15 , wherein the sheet comprises fibroblasts.

17. The method according to claim 15 or 16, wherein the transplant material is a transplant material for nerve regeneration.

18. A transplant material produced by the method according to any one of claims 15 to 17.

19. 1. A method for extending an axon of a nervous system cell, comprising: The method includes a step of culturing a neural cell-containing cell population containing neural cells and endothelial cells induced from dental pulp-derived mesenchymal stem cells in the presence of feeder cells containing at least one type of cell selected from the group consisting of vascular pericytes, vascular endothelial cells, vascular-derived smooth muscle cells, and perivascular fibroblasts, (a) preparing a substrate comprising at least one recess and a groove connected to the recess, the groove being coated with the feeder cells; (b) adding the neural cell-containing cell population to the well; (c) culturing the neural cell-containing cell population to allow the axons of the neural cells to extend along the grooves; A method for extending an axon of a nervous system cell, comprising:

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