WO2021166984A1

WO2021166984A1 - Method for manufacturing nerve cell device including myelinated nerve cells

Info

Publication number: WO2021166984A1
Application number: PCT/JP2021/006037
Authority: WO
Inventors: 正樹須藤; 耕史木下; 純輝宇田
Original assignee: 株式会社幹細胞＆デバイス研究所
Priority date: 2020-02-20
Filing date: 2021-02-18
Publication date: 2021-08-26
Also published as: JPWO2021166984A1

Abstract

The present invention provides: a method for manufacturing a nerve cell device, which includes nerve cells having highly myelinated axons, by co-culturing Schwann cells and nerve cells on a fiber sheet; and a nerve cell device manufactured by said method.

Description

Methods for Manufacturing Nerve Cell Devices Containing Myelinated Nerve Cells

The present invention relates to a method for producing a nerve cell device containing a nerve cell having myelinated axons by co-culturing Schwann cells and a nerve cell on a fiber sheet, and a nerve cell device produced by the method. ..

The axons of nerve cells are covered with a myelin sheath in which the cell membranes of glial cells are spirally stacked in multiple layers. Schwann cells in the peripheral nerves and oligodendrocytes in the central nerves are involved in the formation of myelin sheaths. Since nerve axons are electrically insulated by this myelin sheath, electrical signals generated by firing of nerve cells (generation of action potentials) are transmitted very quickly along the axons by saltatory conduction. .. The myelin sheath also has a protective effect on nerve axons. Demyelination means that the myelin sheath is damaged or shed by some mechanism, or the myelin sheath is dysplasia and the maintenance / regeneration disorder of the myelin sheath is dysplastic. It is a condition that collapses. When demyelination occurs, the transmission of nerve stimuli is extremely slow, and smooth nerve function is not maintained, resulting in various nerve dysfunctions. Demyelinating diseases in which demyelinating is the basis of lesions include peripheral neuropathy such as neurosheath disease, Charcot-Marie-Tooth disease, Gillan Valley syndrome and diabetic peripheral neuropathy, as well as multiple sclerosis and neuromyelitis optica. Includes central neuropathy such as inflammation, acute disseminated neuromyelitis and Periceus-Merzbacher's disease.

In order to develop a therapeutic drug for demyelinating disease, an appropriate drug screening system that reproduces the pathophysiology of demyelinating disease is required. In general, in vivo drug screening using animals is complicated and time-consuming in the process of creating a disease model and evaluating a drug. Therefore, it is required to develop a simple in vitro drug screening system with high throughput using a disease model prepared from cultured cells. In order to construct such an in vitro demyelinating disease model, it is necessary to prepare cells involved in the formation of myelin sheath. For that purpose, for example, a method of inducing differentiation of embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) to obtain Schwann cell precursors and Schwann cells has been reported (Patent Document 1, Non-Patent). Documents 1 to 3). Further, it has been reported that myelin formation can be induced by co-culturing Schwann cells and nerve cells (Patent Document 1, Non-Patent Documents 2 to 4). However, when Schwann cells derived from human stem cells are used, long-term culture is required before myelination is observed, and the efficiency of myelination cannot be said to be high.

As a method of promoting the maturation of Schwann cells, a method of culturing Schwann cells on a fibrous cell scaffold has been reported. For example, when human or rodent Schwann cells are cultured on oriented electrospun poly-ε-caprolactone fibersheets or polylactic-glycolic acid copolymer (PLGA) fibersheets, the cells are along the fiber axis. It has been reported that the expression of myelin-related glycoprotein (MAG), which is an early myelination marker, and myelin protein P0, which is a cell adhesion molecule characteristic of peripheral myelin, is enhanced (Non-Patent Document 5 and Non-Patent Document 5). 6). Further, a method of culturing human Schwan cells together with carbon fibers suspended in a culture solution away from the culture well surface has been reported (Non-Patent Document 7). In this method, Schwann cells have been shown to wrap around carbon fiber and express myelin basic protein (MBP) and myelin-related glycoprotein (MAG). When Schwann cells are cultured by these methods, the cells express myelination markers, but since they are not co-cultured with nerve cells, axons covered with a myelin sheath cannot be reproduced.

WO2018 / 090002

In order to reproduce the pathophysiology of peripheral neuropathy due to demyelination in humans, a disease model constructed using human-derived neurons and Schwann cells is desirable. However, in the conventional culture method, it takes several months or more for human-derived nerve cells and Schwann cells to functionally mature, and cell detachment is likely to occur during culture, which are problems in practical use. There is. In addition, in order to observe the demyelination process in detail using a disease model, it is important to use a disease model in which the myelin sheath is sufficiently formed. An object of the present invention is to stably culture human-derived nerve cells and Schwann cells, to mature the cells at an early stage, and to obtain a culture method having high myelin formation efficiency and the method. To provide an in vitro disease model.

The present inventors have found that co-culturing human-derived Schwann cells and nerve cells on a fiber sheet promotes the maturation of Schwann cells and nerve cells and improves the efficiency of myelin formation. The invention was completed.

That is, the object of the present invention is achieved by the following invention.
[1]
The process of inducing the differentiation of Schwann progenitor cells into Schwann cells, and
The step of co-culturing the Schwann cells with nerve cells or nerve cell spheroids on a cell scaffold,
A method for manufacturing a nerve cell device, including.
[2]
The method according to [1], wherein the step of inducing differentiation is performed on the cell scaffold.
[3]
The method according to [1] or [2], wherein the cell scaffold is a fiber sheet made of a polymer material.
[4]
The method according to [3], wherein the fiber sheet has an oriented structure.
[5]
The method according to [3] or [4], wherein the fiber sheet is coated with an extracellular matrix protein selected from polylysine, polyornithine, laminin, fibronectin, Matrigel® and Geltrex®.
[6]
The method according to [3] or [4], wherein the fiber sheet is coated with polyethyleneimine.
[7]
The method according to any one of [1] to [6], wherein the Schwann cell and the nerve cell form a three-dimensional structure on the cell scaffold and / or in the cell scaffold.
[8]
The method according to any one of [1] to [7], wherein the Schwann cell is a Schwann cell derived from a pluripotent stem cell.
[9]
The method according to [8], wherein the Schwann cell derived from the pluripotent stem cell is a Schwann cell derived from a mammal.
[10]
The method according to any one of [1] to [9], wherein the nerve cell is a primary cultured cell or a nerve cell derived from a pluripotent stem cell.
[11]
The method according to [10], wherein the primary cultured cell or a nerve cell derived from a pluripotent stem cell is a nerve cell derived from a mammal.
[12]
The method according to any one of [1] to [11], wherein the nerve cell comprises a glutamatergic, cholinergic, γ-aminobutyric acid, dopaminergic or histaminergic nerve cell.
[13]
^{The co-culture is started by seeding the nerve cells at a density of 1 to 10 × 10 4} cells / 0.07 cm ² with respect to the cell scaffold, according to any one of [1] to [12]. the method of.
[14]
The nerve cell spheroid is a nerve cell spheroid ^{prepared from 0.5 to 5 × 10 4} nerve cells, and the co-culture seeds the nerve cell spheroid at a density of ^{1 to 10 cells / 0.07 cm 2.} The method according to any one of [1] to [12], which is started by the above-mentioned method.
[15]
The culture medium used for the co-culture is a culture medium containing 5 to 20% (v / v) of the culture supernatant of IFRS1 which is a rat Schwann cell line and 0.000001 to 0.001% (w / v) of lipids [1]. ] To [14].
[16]
A nerve cell device produced by the method according to any one of [1] to [15].
[17]
The nerve cell device according to [16], further comprising a frame that holds the periphery of the nerve cell device.
[18]
The nerve cell device according to [17], wherein the frame is 2 mm × 2 mm to 15 mm × 15 mm, respectively, and the frame is circular or polygonal.
[19]
A neuron device-mounted Petri dish having the neuron device according to any one of [16] to [18].
[20]
In a multi-well plate having a plurality of wells, a nerve cell device mounting plate comprising the nerve cell device according to any one of [16] to [18] in at least one of the wells included in the plate.

According to the method of the present invention, co-culturing human-derived neurons and Schwann cells on an oriented fiber sheet enables stable culture with less cell detachment during culture and promotes maturation of neurons. be able to. Further, according to the present invention, it is possible to prepare an in vitro disease model in which myelination is advanced in axons extending from nerve cells or nerve cell spheroids. By using the nerve cell device of the present invention, an in vitro drug screening system can be rapidly constructed, so that efficient drug evaluation becomes possible.

This is the result of measuring the number of SOX10-positive cells contained in human iPS cell-derived Schwann progenitor cells with a flow cytometer. Schwan progenitor cells fixed with 2% paraformaldehyde were reacted with FITC-labeled anti-SOX10 antibody (Sample1-Posi) or FITC-labeled anti-IgG antibody (Sample1-N), and the latter was used as a negative control. Approximately 1% of the FITC-positive region contained in the main cell population of the negative control sample was set, and the number of SOX10-positive cells was counted. It is an immunostaining photograph of the human iPS cell-derived Schwann cell which induced the differentiation on the cell scaffold. Human Schwann progenitor cells seeded on the cell scaffold were induced to differentiate into Schwann cells, and immunostaining with anti-SOX10 antibody and anti-S100B antibody was performed 16 days after the start of differentiation induction. It is a phase difference optical micrograph showing the result of co-culturing human iPS cell-derived motor neurons and Schwann cells. Rat immortalized Schwann cells or human iPS cell-derived Schwann cells were seeded into the motor nerves on the Petri dish and co-cultured. The day after seeding Schwann cells, the cells were observed with a retardation optical microscope. As a result, it was observed that Schwann cells adhered along the nerve cell axons. It is a photograph showing myelination by co-culture of rat immortalized Schwann cells and human iPS cell-derived motor neurons. The neuronal device obtained by co-culture was immunostained with anti-MBP antibody and anti-CASPR antibody. It is an immunostaining photograph which shows the result of co-culturing a human iPS cell-derived Schwann cell and a human iPS cell-derived motor neuron. Three weeks after the start of co-culture, immunostaining was performed with an anti-βIII-Tubulin antibody. As a result, it was shown that nerve cell axons were engrafted on the fiber sheet. It is a photograph which shows the result of culturing the human iPS cell-derived motor neuron spheroid on the fiber sheet which is a cell scaffold. The figure on the left is an optical micrograph with transmitted light. The figure on the right shows the results of immunostaining with anti-βIII-Tubulin antibody and nuclear staining with DAPI. From this figure, it is shown that axons extend along the fibers from the human iPS cell-derived motor nerve spheroids seeded on the fiber sheet.

[Cell scaffolding]
A cell scaffold is a substrate on which cells seeded on a cell scaffold can adhere and proliferate or grow. The cell scaffold preferably used in the present invention is composed of fibers made of a polymer material. The cell scaffold is preferably a fiber sheet having the shape of a sheet in which fibers are accumulated. The fiber sheet has an oriented structure. The oriented structure means that the fibers constituting the fiber sheet are arranged along one direction, and when the angle of the one direction (orientation axis) is 0 °, 80% or more of the fibers are preferably 95%. The structure is such that the above number of fibers are arranged along an angle within a range of ± 5 °, preferably within a range of ± 1 °.

The polymer material constituting the fiber may be any material that does not exhibit cytotoxicity when the cells are cultured in contact with the cells, depending on the purpose of use of the cells grown on the fiber sheet. , Biodegradable or non-biodegradable polymeric materials can be used. Examples of the biodegradable polymer material include a copolymer of polylactic acid and polyglycolic acid (PLGA), polyglycolic acid (PGA), polybutyric acid (PLA), polyvinyl alcohol (PVA), and polyethylene glycol (PEG). , Polyethylene vinyl acetate (PEVA), polyethylene oxide (PEO) and the like, but are not limited thereto. Examples of the non-biodegradable polymer material include polystyrene (PS), polysulfone (PSU), polytetrafluoroethylene (PTFE), polycarbonate (PC), polymethylmethacrylate (PMMA), polyvinyl chloride (PVC), and the like. Examples include, but are not limited to, polyethylene terephthalate (PET), polyamide (PA) and polymethylglutalimide (PMGI).

The fiber sheet can be produced, for example, from a solution containing a polymer material by an electrospinning method. The fiber sheet having an oriented structure is not particularly limited, but for example, a rotating drum is used, and a solution containing a polymer material is sprayed from a nozzle onto the rotating surface of the drum while rotating the drum. Then, the fiber sheet can be manufactured by winding the fiber formed on the rotating drum. As long as it is a PTFE sheet, for example, commercially available Poaflon (registered trademark, Sumitomo Electric Industries, Ltd.) can be used. The solution of the polymer material may be an organic solvent that dissolves the polymer material to be used at a concentration of 10 to 30% (w / v) at room temperature, for example, 1,1,1,3,3,3. -Hexafluoro-2-propanol (HFIP), N, N-dimethylformamide (DMF) and the like can be mentioned.

The average diameter of the orthogonal cross sections of the fibers constituting the fiber sheet is not particularly limited, but is, for example, 1 to 7 μm, preferably 2 to 6 μm, and more preferably 3 to 5 μm.

Of the fibers that make up the fiber sheet, the distance between the core wires of adjacent fibers is the pitch. If the pitch is too large, the cells may not be retained and may fall out when seeded. On the other hand, if the pitch is too small, the cells cannot extend into the fiber sheet, and it becomes difficult to form a three-dimensional structure. The pitch of the fiber sheet used in the present invention is 6 μm to 60 μm, preferably 6 μm to 50 μm, and more preferably 6 μm to 30 μm.

The porosity of the fibers constituting the fiber sheet may vary depending on the polymer material used. The porosity is not particularly limited, but is, for example, 10 to 60%, preferably 15 to 50%, more preferably 20 to 40%, and even more preferably 30% to 40%. Here, the porosity is the ratio of the area where fibers do not exist to a certain area of the fiber sheet plane in the fiber sheet which is one layer in the direction perpendicular to the fiber sheet plane.

The fiber sheet used in the present invention is composed of two or more fiber sheet layers (laminated or multilayer) even if it is composed of one fiber sheet layer (single layer) in the direction perpendicular to the fiber sheet plane. , For example, 2 layers, 3 layers, 4 layers, 5 layers, 6 layers, etc.). When laminating fiber sheets, the upper and lower fiber sheets are in contact with each other. The orientation axes of the upper and lower fiber sheets intersect at 5 ° to 25 °, preferably 10 ° to 20 °, and more preferably 13 ° to 17 °. The thickness of the fiber sheet (single layer) is, for example, 1 to 40 μm, preferably 5 to 35 μm, and more preferably 10 to 30 μm.

The fiber sheet used in the present invention is an extracellular matrix such as polylysine, polyornithine, laminin, fibronectin, Matrigel® or Geltrex® to promote adhesion, elongation and proliferation of seeded cells. It may be coated with protein or polyethyleneimine, which is a cationic water-soluble polymer. The coating can be performed by immersing the fiber sheet in a solution in which the extracellular matrix protein or polyethyleneimine is dissolved in physiological saline, phosphate buffered saline, cell culture solution or the like.

〔cell〕
Nerve cells (also called neurons) and glial cells that differentiate from each progenitor cell of the nervous system form functional nervous tissue through the interaction of nerve cells with glial cells. Schwann cells are the major glial cells in the peripheral nerves. Schwann cells support the metabolic activity of nerve cells and play a role as myelin-forming cells. Schwann cells are cells that differentiate from stem cells through Schwann progenitor cells and then immature Schwann cells.

The method of the present invention is a step of seeding and culturing Schwann precursor cells on a cell culture vessel such as Petridish or a multi-well plate or a cell scaffold in which fibers produced by using the polymer material are accumulated, and the above-mentioned. The step of inducing the differentiation of Schwann precursor cells into Schwann cells is included. Since Schwann progenitor cells have the ability to differentiate into mature Schwann cells, they can be differentiated into Schwann cells by adherent culture on a cell culture vessel or the cell scaffold. The Schwann progenitor cells are positive for the transcription factor SOX10, which is an undifferentiated Schwann cell marker, and OCT6, which is a mature Schwann cell marker, is positive for the subsequent induction of differentiation. Becomes positive. These markers can be used to identify Schwann progenitor cells, mature Schwann cells, or Schwann cell populations involved in myelination.

The Schwann progenitor cells are preferably cells induced to differentiate from pluripotent stem cells. As the pluripotent stem cells, for example, ES cells and iPS cells can be used, but iPS cells are more preferable. As the cells for reprogramming into iPS cells, cells derived from mammals such as mice or rats which are rodents, or monkeys or humans which are primates are preferable, and cells derived from humans are particularly preferable. Patient-derived cells can also be used. By using patient-derived cells, it becomes possible to manufacture a nerve cell device that reflects the disease state. Patient diseases include CMT subtypes such as Charcot-Marie-Tooth disease (CMT) or CMT type 1. Since Schwann progenitor cells can be cryopreserved, the cryopreserved Schwann cells can be thawed and used when producing the nerve cell device of the present invention.

Examples of the culture medium used in the step of inducing the differentiation of Schwann progenitor cells into Schwann cells include DMEM (Dulbecco's Modified Eagle Medium) and MEM / Ham F-12, and the induction of differentiation of Schwann progenitor cells into Schwann cells. Is not limited as long as is possible. It is preferable that serum and / or cell growth factor is added to the culture medium. Examples of serum include fetal bovine serum (FBS), neonatal bovine serum, bovine serum albumin, goat serum, rabbit serum, mouse serum, monkey serum, human serum, etc., and the induction of differentiation of Schwann precursor cells into Schwann cells is performed. It is not limited as long as it is possible. Examples of cell growth factors include PDGF (platelet-derived growth factor), EGF (Epidermal Growth Factor), and FGF (Fibroblast growth factor). It is not limited as long as it is possible to induce the differentiation of Schwan precursor cells into Schwan cells. In addition, other well-known substances or antibiotics, or combinations thereof, may be added to the culture medium in order to maintain the cultured cells and promote the induction of differentiation.

The method of the present invention includes a step of inducing differentiation of the Schwann progenitor cells into Schwann cells, then seeding nerve cells or nerve cell spheroids on the cell scaffold and co-culturing with the Schwann cells. Nerve cells are also called neurons and can be classified according to the difference in neurotransmitters produced by nerve cells. Neurotransmitters produced by nerve cells used in the method of the present invention include monoamines such as dopamine, noradrenaline, adrenaline, serotonin and histamine, non-peptide neurotransmitters such as acetylcholine, γ-aminobutyric acid and glutamate, and , Adrenocorticotropic hormone (ACTH), α-endorphin, β-endorphin, γ-endorphin and vasopressin and other peptide neurotransmitters. For example, neurons using glutamate, acetylcholine, γ-aminobutyric acid, dopamine and histamine as neurotransmitters are classified into glutamate-operated neurons, cholinergic neurons, γ-aminobutyric acid-operated neurons, dopaminergic neurons and histamine-actuated neurons, respectively. It is called a sex neuron.

Examples of nerve cells used in the present invention include primary cultured cells. Since primary cultured cells retain many cell functions that are inherent in the living body, they are important as a system for evaluating the effects of drugs and the like in the living body. As the primary cultured cells, neurons of a mammal such as a rodent mouse or rat, or a primate monkey or human central nervous system and peripheral nervous system can be used. When preparing and culturing these nerve cells, the animal dissection method, tissue collection method, nerve separation / isolation method, nerve cell culture medium, culture conditions, etc. are known depending on the type of cells to be cultured. You can choose from the methods. As commercially available primary cultured divine cell products, for example, rat brain nerve cells manufactured by Lonza (Switzerland) and human brain nerve cells manufactured by ScienCell Research Laboratories (USA) can be used.

Further, as the nerve cell used in the present invention, a nerve cell induced to differentiate from a pluripotent stem cell can be mentioned. As the pluripotent stem cells, for example, embryonic stem cells (ES cells) and iPS cells can be used, but iPS cells are preferable. As the cells for reprogramming into iPS cells, cells derived from mammals such as rodent mice or rats, or primates monkeys or humans can be used, but human-derived cells can be used. Is preferred, and patient-derived cells are particularly preferred. By using patient-derived cells, it becomes possible to manufacture a nerve cell device that reflects the disease state. Patient diseases include CMT subtypes such as Charcot-Marie-Tooth disease (CMT) or CMT type 1.

Pluripotent stem cells can obtain various types of nerve cells by inducing differentiation using a known method for inducing nerve differentiation. For example, nerve cells can be obtained by a differentiation induction method using a small molecule compound described in the literature (Honda M, et al. Biochem Biophys Res Communi. 2016; 469: 587-592). In addition, commercially available pluripotent stem cell-derived neural cell products, such as iCell neurons from Cellular Dynamics International (USA), various neural stem cells from Axol Bioscience (UK), and various neural cell precursors from BrainXell (USA). Cells and XCL-1 neurons from XCell Science (USA) can also be used. These commercially available nerve cells can be cultured using the attached culture medium.

In the method of the present invention, nerve cell spheroids can be used. A nerve cell spheroid is a three-dimensional structure (cell mass) formed by adhesion and aggregation of nerve cells. As a method for producing a nerve cell spheroid, for example, a certain number of nerve cells are added to each well of a microwell having a cell non-adhesive surface and cultured to adhere the nerve cells to each other in the microwell. The method of producing is exemplified. The number of nerve cells added to each well of the microwell is preferably ^{0.5 to 5 × 10 4.} In addition, a method of rotating a cell culture chamber containing nerve cells and a culture solution and bringing suspended nerve cells into contact with each other in the chamber is exemplified. However, the method is not limited to these methods as long as the number and size of the obtained neuronal spheroids are relatively uniform.

When iPS cells are used to obtain Schwann progenitor cells and neurons, peripheral blood-derived mononuclear cells are preferable as a source of cells for reprogramming into iPS cells because of their low invasiveness. Not limited to. Other preferable sources of collection include body tissues such as skin containing fibroblasts.

[Co-culture]
In the method of the present invention, a nerve cell or a nerve cell spheroid is further seeded on the cell scaffold on which the Schwann cell differentiated from the Schwann progenitor cell grows, and co-cultured with the Schwann cell. Alternatively, the nerve cells or nerve cell spheroids seeded on the cell scaffold are cultured for a certain period of time, axons are extended from the respective cells and spheroids, and then the Schwann cells are seeded and co-cultured. When seeding the nerve cells, it is preferable ^{to seed the nerve cells at a density of 1 to 10 × 10 4} cells / 0.07 cm ² with respect to the cell scaffold on which Schwann cells grow. When seeding the Schwann cells, it is preferable to seed the cells or the cell scaffold on which the nerve cell spheroids grow at a density of ^{0.5 to 3 × 10 4} cells / 0.07 cm ^2. These cell seeding densities may be seeded at a density lower than the seeding density or higher than the seeding density, depending on the growth situation of each cell. When seeding the nerve cell spheroid, it is preferable to seed the cell scaffold on which Schwann cells grow at a ^{density of 1 to 10 spheroids / 0.07 cm 2.} The number of spheroids added on the cell scaffold can be adjusted so that all of the added spheroids are in contact with the grown and extended Schwann cells on the cell scaffold.

The culture medium used in the step of co-culturing the Schwann cell and the nerve cell or the nerve cell spheroid is DMEM culture solution, DMEM / Ham F-12 equal volume mixed culture solution, 1 × Neurobasal (registered trademark, Gibco) and the like. By way of example, but not limited to, if neuronal axonal and myelin formation is possible. It is preferable that serum and / or cell growth factor is added to the culture medium. Examples of serum include fetal bovine serum (FBS), neonatal bovine serum, bovine serum albumin, goat serum, rabbit serum, mouse serum, monkey serum, human serum, etc., and axonal formation and myelin formation of nerve cells are possible. If so, there is no limit. Cell growth factors include NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), GDNF (glial cell-derived neurotrophic factor), and NT. Examples thereof include -3 (neurotrophin-3; neurotrophin-3), NRG-1 (neuregulin-1; neurotrophin-1), PDGF, EGF, FGF, etc. Unrestricted if possible. In addition, other well-known substances or antibiotics, or combinations thereof, may be added to the culture medium to promote axonal formation and myelination of nerve cells.

The culture solution (IFRS1 culture supernatant) after culturing IFRS1 which is a rat Schwann cell line may be added to the culture solution used in the co-culture step at a concentration of 1 to 20% (v / v). can. IFRS1 is an immortalized cell line established from the dorsal root ganglion and peripheral nerve tissue of mature Fisher344 rats. IFRS1 can be purchased from Cosmo Bio, and can be cultured using a culture medium for IFRS1 (Cosmo Bio) using DMEM as a basal medium. It is preferable that lipid is further added to the culture solution used in the co-culturing step. Examples of the lipid include, but are not limited to, linolenic acid, oleic acid, palmitic acid, cholesterol, phosphatidylcholine, etc., as long as they can form axons and myelin in nerve cells. The lipid may be added alone or as a lipid concentrate consisting of a mixture of the lipids. Examples of the lipid concentrate include, but are not limited to, a lipid concentrate (Gibco). The concentration of the lipid to be added is preferably 0.000001 to 0.001% (w / v), more preferably 0.000003 to 0.0003% (w / v).

By co-culturing the Schwann cell with a nerve cell or a nerve cell spheroid, the nerve cell in which the cell forms a three-dimensional structure on the fiber sheet which is a cell scaffold or laminated and / or in the fiber sheet. You get the device. Forming a three-dimensional structure means a state in which Schwann cells and nerve cells adhere along the fibers constituting the fiber sheet and grow on one or both sides of the fiber sheet and in the fiber sheet.

[Nerve cell device]
The nerve cell device produced by the method of the present invention has a neural network in which myelinated nerve cells form a three-dimensional structure on and / or in the fiber sheet. Such neuronal devices are preferably stored in contact with cell culture medium in order to keep the cells alive. In one embodiment of the invention, a Petri dish or plate comprising the neuronal device of the invention is provided.

The nerve cell device of the present invention can form a fiber sheet by fixing or holding the periphery of the fiber sheet constituting the device with a frame. In order to fix or hold the fiber sheet on the frame, for example, a commercially available biocompatible adhesive such as silicone one-component condensation type RVT rubber (Shin-Etsu Chemical Co., Ltd., Catalog No. KE-45) can be used, but cells can be used. The adhesive is not particularly limited as long as it does not affect the culture.

Examples of the material of the frame include polydimethylsiloxane (PDMS), polystyrene (PS), polycarbonate (PC), stainless steel, and the like, but the material is not limited to these as long as it does not affect cell culture. The thickness of the frame is not particularly limited, but is 0.1 to 4 mm, preferably 0.25 to 3 mm, and more preferably 0.5 to 2 mm. The shape of the frame can be changed depending on the purpose of use, but it is circular or polygonal, and it is preferable that the length and width are 2 mm × 2 mm to 15 mm × 15 mm, respectively.

The nerve cell device of the present invention is a petri dish for cell culture having a diameter of 35 mm, 60 mm, 100 mm, etc., or a mulch having a plurality of wells such as 6 wells, 12 wells, 24 wells, 48 wells, and 96 wells. It can be directly attached or placed in at least one of the wells contained in the well plate. The same applies to a nerve cell device having a fiber sheet in which the periphery of the fiber sheet is fixed or held by the frame.

The present invention will be described in more detail and concretely by showing examples below, but the examples should not be construed as limiting the scope of the present invention.

[Preparation of oriented fiber sheet]
PLGA (SIGMA P1941) was dissolved in HFIP (Wako Pure Chemical Industries, Ltd. 089-04233) at room temperature to prepare a 20% (w / v) solution. This solution was filled in a syringe (Norm-Ject Syringes 5 mL volume, Osaka Chemical) and then placed in a nanofiber electrospinning device NANON-03 (Mech Co., Ltd.) equipped with a 22 G flat-edged needle. Next, a PLGA fiber sheet was prepared on the drum collector under the conditions of a voltage of 20 kV, an injection flow rate of 1 mL / h, and a drum rotation speed of 750 rpm. When preparing a PS fiber sheet, PS (Fluka) was dissolved in DMF (Wako Pure Chemical Industries, Ltd.) at room temperature to prepare a 30% (w / v) solution. This solution was filled in a syringe (Norm-Ject Syringes 5 mL volume, Osaka Chemical) and then placed in a nanofiber electrospinning device NANON-03 (Mech Co., Ltd.) equipped with a needle with a flat cutting edge of 25 G. Next, a PS fiber sheet was prepared on the drum collector under the conditions of a voltage of 10 kV, an injection flow rate of 1.5 mL / h, and a drum rotation speed of 2000 rpm.

[Adhesion of frame to fiber sheet]
A polycarbonate frame (15 mm x 15 mm) or a stainless steel circular frame (outer diameter 6) is used for the produced fiber sheet using silicone one-component condensation type RVT rubber (Shin-Etsu Chemical, Catalog No. KE-45). mm, inner diameter 3 mm) was adhered.

[Induction of differentiation of Schwann progenitor cells into Schwann cells on a cell scaffold]
Schwann progenitor cells were prepared by inducing differentiation of human-derived iPS cells according to known conditions. In the obtained Schwann progenitor cells, SOX10, which is a Schwann cell-specific transcription factor, was expressed in 86% or more of the cells (Fig. 1). These Schwann progenitor cells were seeded on an oriented PS fiber sheet and cultured in a 5% CO ₂ environment at 37 ° C. for about 10 days using a differentiation-inducing culture medium to differentiate into Schwann cells. As the differentiation-inducing culture medium, low glucose DMEM (Sigma, D5546) was used as the basal culture medium, and 1% FBS and 2 mM L-glutamine were added to the culture medium. In the differentiation-inducing culture medium, 200 ng / mL NRG1 (Peprotec, 100-03) and 100 nM all-trans retinoic acid (Peprotec, 100-03) and 100 nM all transretinoic acid (200 ng / mL NRG1 (Peprotec, 100-03)) Sigma, R2625), 10 ng / mL PDGF-BB (Gibco, PHG0044) and 4 μM forskolin (Sigma, F6886) were added and 200 ng / mL NRG1 and 10 ng / mL PDGF were added on days 5-6. -BB was added, and after the 7th day, the cells were cultured using a culture solution containing 200 ng / mL NRG1. On days 10 and 13, half of the culture was replaced with fresh culture. On the 16th day, immunostaining of Schwann cell markers SOX10 and S100B was performed, and almost all cells were positive (Fig. 2).

[Co-culture of nerve cells with rat or human-derived Schwann cells]
On human-derived motor / sensory nerve cells cultured on a cultured Petri dish or plate, 0.5 to 5 × 10 ⁴ cells / 0.07 cm ^{2 of rat immortalized Schwan cells (IFRS1) or human Schwan cells induced to differentiate in Example 2} The cells were sown at the same density as above, and cultured at 37 ° C. in _{a 5% CO 2 environment using a co-cultivation culture solution.} The culture medium for co-culture uses Neurobasal (registered trademark, Thermo Fisher) as the basal culture solution, 2% B-27 (registered trademark, Thermo Fisher), 20 ng / mL NRG1, 10 ng / mL BDNF, 10 ng /. mL GDNF, 0.1 mM mercaptoethanol, 0.5 μM forskolin, 1% penicillin-streptomycin and 0.3% growth factor reduced Matrigel (Corning) were added. The day after seeding the rat or human Schwann cells, observation with a phase-difference optical microscope confirmed that each Schwann cell was engrafted along the nerve cell axon (Fig. 3).

[Co-culture of rat-derived Schwann cells and nerve cells on a cell scaffold]
^{Rat immortalized Schwann cells (IFRS1) were seeded at a density of 0.5 to 5 × 10 4} cells / 0.07 cm ² into human-derived motor neurons cultured on an oriented PS fiber sheet, and a co-culture medium was used. The cells were cultured at 37 ° C in a 5% CO _{2 environment.} The culture medium for co-culture uses Neurobasal (registered trademark, Thermo Fisher) as the basal culture solution, and is 2% B-27 (registered trademark, Thermo Fisher), 20 ng / mL NRG1, 10 ng / mL BDNF, 10 ng /. mL GDNF, 0.1 mM mercaptoethanol, 0.5 μM forskolin, 1% penicillin-streptomycin were added. To the co-culture solution, 50 μg / mL ascorbic acid is added to the co-culture solution after the 6th day, with the sowing date of nerve cells (co-culture start date) as the 0th day, and myelinated culture is performed. Used as a liquid. From the 6th day onward, half of the culture broth was replaced with fresh culture broth every 2 to 3 days. When immunostaining of the obtained nerve cell device was performed 4 to 6 weeks after the start date of co-culture, expression of myelin formation-related proteins MBP and CASPR was observed at multiple sites (Fig. 4). In addition, almost no cell detachment from the cell scaffold was observed. Therefore, it is shown that the nerve cell device produced by the method of the present invention is useful as an in vitro neural tissue model.

[Co-culture of human-derived Schwann cells and nerve cells on a cell scaffold]
Human-derived Schwann cells differentiated on an oriented PS fiber sheet, and primary cultured neurons derived from the rat spinal posterior ganglion, which are peripheral sensory neurons, or neurons derived from human motor / sensory neurons, 1 to 5 The cells were seeded at a density of × 10 ⁴ cells / 0.07 cm ² , and cultured at 37 ° C. in _{a 5% CO 2} environment using a co-cultivation culture medium. The culture medium for co-culture uses Neurobasal (registered trademark, Thermo Fisher) as the basal culture solution, and is 2% B-27 (registered trademark, Thermo Fisher), 20 ng / mL NRG1, 10 ng / mL BDNF, 10 ng /. mL GDNF, 0.1 mM mercaptoethanol, 0.5 μM forskolin, 1% penicillin-streptomycin, culture supernatant of 5-10% (v / v) rat Schwann cell line IFRS1 and 0.00001% (w / v) lipid concentrate The culture solution was prepared by adding (Gibco). 50 μg / mL ascorbic acid was added to the co-culture solution on the 0th day after the seeding date of nerve cells (co-culture start date) to promote myelin formation. It is shown that in the nerve cell device produced by the method of the present invention, aggregation and cell detachment of nerve cell axons did not occur even one month after the start of co-culture (Fig. 5). Therefore, it is shown that the nerve cell device produced by the method of the present invention is useful as an in vitro neural tissue model.

[Elongation of nerve axons of nerve cell spheroids on cell scaffolds]
Nerve cell spheroids were prepared by adding ^{2 × 10 4} motile / sensory nerve cells induced to differentiate from human iPS cells to each well of the microwell and culturing them. The nerve cell spheroids were seeded on a cell scaffold, which is a PS fiber sheet coated with polylysine and laminin, at a ^{density of 1 to 2 cells / 0.07 cm 2} , and a co-culture medium was used in a 5% CO ₂ environment. , 37 ° C. The culture medium for co-culture uses Neurobasal (registered trademark, Thermo Fisher) as the basal culture solution, and is 2% B-27 (registered trademark, Thermo Fisher), 20 ng / mL NRG1, 10 ng / mL BDNF, 10 ng /. mL GDNF, 0.1 mM mercaptoethanol, 0.5 μM forskolin, 1% penicillin-streptomycin, culture supernatant of 5-10% (v / v) rat Schwann cell line IFRS1 and 0.00001% (w / v) lipid concentrate The culture solution was prepared by adding (Gibco). One week after the start date of co-culture, immunostaining of nerve cells was performed. As a result, axon elongation from nerve cell spheroids was observed (Fig. 6). It was confirmed that this axon elongation was maintained for 3 weeks after seeding of nerve cell spheroids on the cell scaffold.

[Co-culture of nerve cell spheroids and Schwann cells on a cell scaffold]
Rat immortalized Schwann cells or human-derived Schwann cells were ^{seeded at a density of 0.5 to 5 × 10 4} cells / 0.07 cm ² in the nerve cell spheroids cultured on the cell scaffold, and the above-mentioned co-culture medium was used. The cells were cultured at 37 ° C in a 5% CO _{2 environment.} The seeding date (co-culture start date) of the nerve cell spheroid was set as the 0th day, and after the 6th day, 50 μg / mL ascorbic acid was added to the co-culture culture solution to promote myelin formation. As a result, it was confirmed that Schwann cells were engrafted along the nerve cell axons. Therefore, it is shown that the nerve cell device produced by the method of the present invention is useful as an in vitro neural tissue model.

Claims

The process of inducing the differentiation of Schwann progenitor cells into Schwann cells, and
The step of co-culturing the Schwann cells with nerve cells or nerve cell spheroids on a cell scaffold,
A method for manufacturing a nerve cell device, including.
The method according to claim 1, wherein the step of inducing differentiation is performed on the cell scaffold.
The method according to claim 1 or 2, wherein the cell scaffold is a fiber sheet made of a polymer material.
The method according to claim 3, wherein the fiber sheet has an oriented structure.
The method of claim 3 or 4, wherein the fiber sheet is coated with an extracellular matrix protein selected from polylysine, polyornithine, laminin, fibronectin, Matrigel® and Geltrex®.
The method according to claim 3 or 4, wherein the fiber sheet is coated with polyethyleneimine.
The method according to any one of claims 1 to 6, wherein the Schwann cells and the nerve cells form a three-dimensional structure on and / or in the cell scaffold.
The method according to any one of claims 1 to 7, wherein the Schwann cell is a Schwann cell derived from a pluripotent stem cell.
The method according to claim 8, wherein the Schwann cell derived from the pluripotent stem cell is a Schwann cell derived from a mammal.
The method according to any one of claims 1 to 9, wherein the nerve cell is a primary cultured cell or a nerve cell derived from a pluripotent stem cell.
The method according to claim 10, wherein the primary cultured cell or a nerve cell derived from a pluripotent stem cell is a nerve cell derived from a mammal.
The method according to any one of claims 1 to 11, wherein the nerve cell comprises a glutamatergic, cholinergic, γ-aminobutyric acid, dopaminergic or histaminergic nerve cell.
The co-culture is started by seeding the nerve cells at a density of 1 to 10 × 10 4 cells / 0.07 cm 2 with respect to the cell scaffold, according to any one of claims 1 to 12. the method of.
The nerve cell spheroid is a nerve cell spheroid prepared from 0.5 to 5 × 10 4 nerve cells, and the co-culture seeds the nerve cell spheroid at a density of 1 to 10 cells / 0.07 cm 2. The method according to any one of claims 1 to 12, which is started by the above method.
Claimed that the culture medium used for the co-culture is a culture medium containing 1 to 20% (v / v) of the culture supernatant of IFRS1 which is a rat Schwann cell line and 0.000001 to 0.0001% (w / v) of phospholipids. Item 2. The method according to any one of Items 1 to 14.
A nerve cell device produced by the method according to any one of claims 1 to 15.
The nerve cell device according to claim 16, further comprising a frame that holds the periphery of the nerve cell device.
The nerve cell device according to claim 17, wherein the vertically long × horizontally long frames are 2 mm × 2 mm to 15 mm × 15 mm, respectively, and the frame is circular or polygonal.
A neuron device-mounted Petri dish comprising the neuron device according to any one of claims 16-18.
A nerve cell device mounting plate comprising the nerve cell device according to any one of claims 16 to 18, in at least one of the wells included in the multi-well plate having a plurality of wells.