WO2020013269A1 - Method for functional maturation of neurons - Google Patents

Method for functional maturation of neurons Download PDF

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WO2020013269A1
WO2020013269A1 PCT/JP2019/027476 JP2019027476W WO2020013269A1 WO 2020013269 A1 WO2020013269 A1 WO 2020013269A1 JP 2019027476 W JP2019027476 W JP 2019027476W WO 2020013269 A1 WO2020013269 A1 WO 2020013269A1
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cells
cell
nerve
fiber sheet
oriented
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French (fr)
Japanese (ja)
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郁郎 鈴木
あおい 小田原
一博 饗庭
紀江 遠井
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学校法人東北工業大学
株式会社幹細胞＆デバイス研究所
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Publication of WO2020013269A1 publication Critical patent/WO2020013269A1/en

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to a method for promoting functional maturation of a neural network obtained by culturing neural cells.
  • neurotoxicity is one of the main causes of drug development discontinuation, along with cardiotoxicity and hepatotoxicity. Therefore, accurate neurotoxicity evaluation is necessary from the early stage of drug development, but neurotoxicity evaluation in non-clinical studies is mainly performed by in vivo evaluation systems such as observation of symptoms by animal experiments and evaluation of brain histopathology. A simpler system for evaluating neurotoxicity in vitro has not been established.
  • in-vivo evaluation systems such as behavioral analysis using experimental animals, brain tissue damage experiments and pathological tissue evaluation, and analysis using knockout animals can be mentioned.
  • these in vivo evaluation systems are technically complicated.
  • the in vitro test method can be easily performed, and a large number of compounds can be easily evaluated.
  • Efficacy evaluation and toxicity evaluation in vitro using human-derived neurons are usually performed using cells cultured in two dimensions. From the nerve cells, axons, which are long processes, and short dendrites, which are complexly branched, extend. These neurites are connected to other nerve cells to form a neural network, which is a neural circuit. Nerve activity is based on the electrical activity expressed by the interaction of a plurality of nerve cells in a neural network.
  • Non-Patent Document 1 The microelectrode array measurement method is a method in which nerve cells are dispersed and cultured in two dimensions on an electrode array chip, cultured until spontaneous electrical activity occurs and synaptic propagation is observed, and then the function of the nerve cells is evaluated. . Investigations for actually evaluating neurotoxicity by this technique have been reported (Non-Patent Documents 2 and 3).
  • a nanofiber composed of polyolefin, polyamide, polyurethane, polyester, fluoropolymer, polylactic acid, polyvinyl alcohol, or the like, or a cell scaffold composed of the nanofiber adsorbing a protein component
  • Effectively perform culture or tissue regeneration (Patent Document 1); supply of nutrients and oxygen to cultured cells and supply of cultured cells by three-dimensional cell culture using a cell scaffold having a hollow fiber membrane mesh and a nanofiber layer Highly efficient removal of metabolic wastes of a plant (Patent Document 2); pluripotent stem cells using a nanofiber containing gelatin, collagen or cellulose, or a cell scaffold composed of the crosslinked nanofiber Supply a large amount of and suppress cell death (Patent Document 3); Using a cell scaffolding material in which glycolic acid is used as a support and nanofibers made of polyglycolic acid, gelatin, etc.
  • Patent Document 4 a method for early maturation of a neural network obtained by culturing a nerve cell and expressing a function similar to a biological tissue in the extracellular potential measurement by MEA.
  • the spheroids When nerve cells dispersed and cultured on the MEA form spheroids, the spheroids are partially detached from the MEA, and the number of electrodes in contact with the cells decreases, so that it is impossible to measure nerve activity stably. Furthermore, when culturing nerve cells at a high cell density, oxygen and nutrients may not sufficiently reach the inside of the cell population, and the cells may die. In addition, extracellular potentials obtained from two-dimensionally cultured neurons are weak, and in order to analyze neural activity with high accuracy, it is necessary to improve the S / N ratio. The present invention aims to overcome these problems.
  • the present inventors have conducted intensive studies with the aim of overcoming the above-mentioned problems of the prior art, and as a result, by culturing nerve cells on a fiber sheet as a cell scaffold, functional maturation of a neural network has been achieved. We found a way to promote it and completed the present invention.
  • a method for functionally maturating a neural network comprising culturing nerve cells using a cell scaffold.
  • the cell scaffold is a fiber sheet formed of a polymer material.
  • the fiber sheet has an oriented structure, a non-oriented structure, or a mixed structure of oriented and non-oriented.
  • the method according to 2 or 3 wherein the fiber sheet is coated with an extracellular matrix protein selected from polylysine, polyornithine, laminin, fibronectin, Matrigel (registered trademark) and Geltrex (registered trademark).
  • the nerve cell has formed a three-dimensional structure on and / or within the cell scaffold.
  • the nerve cell is a primary cultured cell or a neural cell derived from a pluripotent stem cell.
  • the primary cultured cells or the pluripotent stem cell-derived nerve cells are mammalian-derived nerve cells.
  • the nerve cells include glutamatergic, dopaminergic, ⁇ -aminobutyratergic, monoaminergic, histaminergic or cholinergic neurons. .
  • a neural activity comprising contacting a nerve cell functionally matured using the method according to any one of 1 to 9 with a microelectrode array and measuring an extracellular potential of the nerve cell. Evaluation method.
  • the present invention by culturing nerve cells using a cell scaffold formed by a fiber sheet, cell adhesion is improved, and stable culturing including reduction of cell detachment during culturing becomes possible. As a result, the functional maturation of the formed neural network is promoted.
  • the use of the nerve cells functionally matured according to the present invention enables measurement of neural activities such as spike firing and synchronous burst firing without detaching the seeded nerve cells from the electrodes of the microelectrode array. The time before it becomes is shortened. Further, since the aggregation of nerve cells is suppressed, a high-frequency spike firing and a strong synchronous burst firing can be observed. Neurons functionally matured by the method of the present invention are useful for neurotoxicity evaluation, drug screening for neurological diseases, and the like.
  • the fibers constituting the fiber sheet serve as a support for the nerve cell, and cell aggregation is suppressed, so that cells are stacked and spheroids are formed even when the cells are in contact with each other. Instead, it is possible to perform three-dimensional culturing in a planar and densely stacked state.
  • Such high-density culture allows early observation of an increase in spike firing frequency of neurons and strong synchronous burst firing due to synaptic propagation. That is, the functional maturation of the nerve cell is realized early.
  • nerve cells can be cultured at a high density in a planar manner, the number of electrodes in contact with the cells on the MEA is large, and it is possible to stably acquire an electric signal. Furthermore, since a cell scaffold containing nerve cells can be placed on the MEA during the measurement, the nerve cells do not need to be cultured on the MEA in advance, and electrode contamination during the culture period can be avoided. Therefore, since the measurement can be performed while the electrode impedance is kept in a low impedance state, it is hardly affected by external noise and the S / N ratio is increased. In addition, since it is not necessary to culture neurons on the MEA in advance, the operating rate of the MEA probe is improved. Furthermore, since the culturing period of the nerve cells is shortened, the cost required for culturing the cells can be reduced.
  • FIG. 9 is a diagram showing the influence of cell scaffolding and cell seeding density on extracellular potential of iPS cell-derived nerve cells measured using a MEA probe in which 16 (4 ⁇ 4) microelectrodes are arranged. The upper histograms in each of FIGS.
  • a to C are diagrams showing the time change of the integrated value of the number of spikes measured by 16 microelectrodes (vertical axis: array-wide spike detection rate (AWSDR, number of spikes / Seconds)).
  • the lower part of each figure of A to C is a figure showing spikes measured with time for each electrode (vertical axis: channel number of electrode).
  • B Cells were seeded on an oriented PLGA fiber sheet at 8 ⁇ 10 5 cells / cm 2 and cultured for 4 weeks.
  • FIG. 4 is a diagram showing the influence of cell scaffolding and cell seeding density on spike firing frequency and drug responsiveness of nerve cells.
  • Human iPS cell-derived neurons were seeded on oriented PS fiber sheets at a density of 3 ⁇ 10 5 cells / cm 2 or 12 ⁇ 10 5 cells / cm 2 .
  • FIG. 9 is a diagram showing the results of measuring the extracellular potential of human iPS cell-derived nerve cells (XCL-1 neuron, XCell Science) using an MEA probe on which 16 (4 ⁇ 4) microelectrodes are arranged. is there. The upper histograms in each of FIGS.
  • a to C are diagrams showing the time change of the integrated value of the number of spikes measured by 16 microelectrodes (vertical axis: array-wide spike detection rate (AWSDR, number of spikes / Seconds)).
  • the lower part of each figure of A to C is a figure showing spikes measured with time for each electrode (vertical axis: channel number of electrode).
  • B Cells were seeded at 3 ⁇ 10 5 cells / cm 2 on an oriented PS fiber sheet and cultured for 4 weeks.
  • FIG. 9 is a diagram showing the results of measuring the extracellular potential of human iPS cell-derived cerebral cortical neurons (Axol Bioscience) using an MEA probe on which 16 (4 ⁇ 4) microelectrodes are arranged. The upper histograms in each of FIGS.
  • a to C are diagrams showing the time change of the integrated value of the number of spikes measured by 16 microelectrodes (vertical axis: array-wide spike detection rate (AWSDR, number of spikes / Seconds)).
  • the lower part of each figure of A to C is a figure showing spikes measured with time for each electrode (vertical axis: channel number of electrode).
  • B Cells were seeded on an oriented PS fiber sheet at 8 ⁇ 10 5 cells / cm 2 and cultured for 4 weeks.
  • the cell scaffold 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 can have an oriented structure, a non-oriented structure, or a mixed structure of oriented and non-oriented.
  • the oriented structure is a structure in which the fibers constituting the fiber sheet are arranged in one direction, and when the angle in one direction is 0 °, 80% or more of the fibers exist in a range of ⁇ 30 °.
  • the distance between the fibers (the distance between the cores of adjacent fibers) is not particularly limited, but is preferably 5 to 50 ⁇ m.
  • the non-oriented structure is a structure in which the directions of the fibers are randomly arranged.
  • a biodegradable or non-biodegradable polymer material is preferable.
  • PLGA polylactic acid polyglycolic acid
  • PS polystyrene
  • PSU polysulfone
  • PTFE polytetrafluoroethylene
  • the diameter of the cross section of the fiber constituting the fiber sheet is not particularly limited, it is, for example, 0.1 to 8 ⁇ m, preferably 0.5 to 7 ⁇ m, and more preferably 1 to 6 ⁇ m.
  • the thickness of the fiber sheet is, for example, 1 to 40 ⁇ m, preferably 5 to 35 ⁇ m, and more preferably 10 to 30 ⁇ m.
  • the porosity of the fibers constituting the fiber sheet can vary depending on the polymer material used.
  • the porosity is not particularly limited, but is, for example, 10 to 50%, preferably 15 to 45%, and more preferably 20 to 40%.
  • the porosity is a ratio of an area where no fiber exists to a fixed area of the fiber sheet plane.
  • the fiber sheet can be manufactured by, for example, an electrospinning method from a solution containing a polymer material.
  • a fiber sheet having an oriented structure although not particularly limited, for example, using a rotating drum, while rotating the drum, spray the solution containing the polymer material from the nozzle to the rotating surface of the drum By winding the fiber formed on the rotating drum, a fiber sheet can be manufactured.
  • a fiber sheet having a non-oriented structure a fiber sheet can be manufactured by spraying a solution containing a polymer material onto a flat plate.
  • a fiber sheet having a mixed structure of an oriented structure and a non-oriented structure for example, it can be produced by combining the above-described production methods for producing a fiber sheet having an oriented structure and a non-oriented structure. .
  • polytetrafluoroethylene (PTFE) sheet for example, commercially available Pouflon (registered trademark) of Sumitomo Electric Industries, Ltd. can be used.
  • the solution of the polymer material may be any organic solvent that dissolves the polymer material used at room temperature at 10 to 30% by weight.
  • organic solvent for example, 1,1,1,3,3,3-hexafluoro-2- And propanol (HFIP) and N, N-dimethylformamide (DMF).
  • the fiber sheet can be fixed or held around the frame.
  • the frame is fixed or held on the fiber sheet, it is not particularly limited as long as it does not affect the cell culture.
  • a commercially available biocompatible adhesive such as a silicone one-pack condensation type RVT rubber (Shin-Etsu Chemical, Catalog No. KE- 45) can be used to bond the frame and the fiber sheet.
  • the material of the frame is not particularly limited as long as it does not affect the cell culture.
  • PDMS polydimethylsiloxane
  • PS polycarbonate
  • stainless steel and the like are exemplified.
  • 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, and the length and width are preferably 2 mm ⁇ 2 mm to 15 mm ⁇ 15 mm, respectively, and are circular or polygonal.
  • a cell scaffold in which nerve cells are seeded for example, a fiber sheet, or the fiber sheet fixed or held around the fiber sheet by a frame, is attached to at least one of the wells included in a cell culture dish or a multi-well plate having a plurality of wells. It can be placed as it is.
  • a nerve cell means a neuron composed of a cell body, dendrites, and axons, and is also called a neuron. Nerve cells can be classified according to the difference in neurotransmitters produced by the nerve cells. Examples of neurotransmitters include monoamines such as dopamine, noradrenaline, adrenaline and serotonin, and non-peptides such as acetylcholine, ⁇ -aminobutyric acid, and glutamic acid. Sexual neurotransmitters and peptide neurotransmitters such as adrenocorticotropic hormone (ACTH), ⁇ -endorphin, ⁇ -endorphin, ⁇ -endorphin, vasopressin and the like. For example, neurons that transmit dopamine, acetylcholine and glutamate as transmitters are called dopaminergic neurons, cholinergic neurons and glutamate neurons, respectively.
  • ACTH adrenocorticotropic hormone
  • ⁇ -endorphin ⁇ -endorphin
  • Primary cultured cells can be used as nerve cells.
  • Primary cultured cells retain many of the inherent cell functions in vivo, and are therefore important as a system for evaluating the effects of drugs and the like in vivo.
  • neurons of the central nervous system and peripheral nervous system of mammals such as rodents of mice or rats, or monkeys or human primates can be used.
  • animal dissection methods, tissue collection methods, nerve isolation / isolation methods, nerve cell culture media, culture conditions, etc. depend on the type of cells to be cultured and the purpose of the cells. Can be selected from known methods.
  • As commercially available products of primary cultured myeloid cells for example, rat brain nerve cells from Lonza (Switzerland) and human brain nerve cells from ScienCell Research Laboratories (USA) can be used.
  • a neural cell derived from a pluripotent stem cell can be further used.
  • pluripotent stem cells include embryonic stem cells (ES cells) and iPS cells.
  • ES cells embryonic stem cells
  • iPS cells iPS cells.
  • Various types of neural cells can be obtained by inducing differentiation of pluripotent stem cells using a known neural differentiation inducing method.
  • nerve cells can be obtained by a differentiation induction method using a low molecular compound described in the literature (Honda et al. Biochemical and Biophysical Research Communications 469 (2016) 587-592).
  • neural cell products derived from pluripotent stem cells such as iCell neurons from Cellular Dynamics International (US), various neural stem cells from Axol Bioscience (UK), and various neural cells from BrainXell (US) Cells and XCL-1 neurons from XCell Science (USA) can also be used. These commercially available neurons can be cultured using the attached culture solution.
  • Neurocytes can be cultured with glial cells derived from mammalian brain or glial cells differentiated from mammalian iPS cells. Glial cells include astrocytes, oligodendrocytes, microglia and the like. Alternatively, a culture solution after culturing astrocytes (astrocyte culture supernatant) can be added to a culture solution for nerve cells at a final concentration of 5 to 30% for culturing.
  • a neural network is a type of neural network in which axons, which are elongated processes, and a plurality of intricately branched dendrites, are connected to other nerve cells via these neurites, and the resulting neural circuit is formed. It is.
  • a nerve cell presynaptic cell
  • the electrical signal transmitted through the axon is transmitted from the presynaptic cell to the next nerve cell (postsynaptic cell) at the synapse. It transmits electrical signals to dendrites via neurotransmitters.
  • the site where axons and dendrites connect is a synapse.
  • the functional maturation of a neural network refers to a state in which nerve cells forming a neural network are functionally matured.
  • increased spike firing of neurons and strong synchronous burst firing due to synaptic propagation of spikes are observed.
  • spike firing is a rapid change in the action potential of a nerve cell
  • synchronous burst firing is a phenomenon in which nerve cells forming a neural network fire at the same time due to synaptic propagation.
  • a neural network is derived from stem cell-derived neural cells, established neural cells, primary neural cells, stem cells and neural progenitor cells that differentiate into neural cells in the course of culture, and the like, and cells that constitute neural tissue such as glial cells.
  • One or more selected cells are formed by culturing not a single cell but a plurality of cells on a cell scaffold.
  • a nerve cell suspended in a culture solution is applied to a fiber sheet or a fiber sheet whose periphery is fixed or held by a frame at 1 ⁇ 10 4 to 4 ⁇ 10 6 cells / cm 2 , preferably 1 ⁇ 10 5 to 4 ⁇
  • the cells are seeded at a high density of 10 6 cells / cm 2 , more preferably 8 ⁇ 10 5 to 4 ⁇ 10 6 cells / cm 2 , and still more preferably 2 ⁇ 10 6 to 4 ⁇ 10 6 cells / cm 2.
  • Forming a three-dimensional structure refers to a state in which nerve cells have grown on one or both sides of the fiber sheet and inside the fiber sheet.
  • a nerve cell When a nerve cell is seeded on a fiber sheet having an oriented structure, the nerve cell adheres along a fiber constituting the fiber sheet and extends along the orientation direction of the fiber.
  • neurons When neurons are seeded on a fiber sheet and cultured, there is no cell aggregation or spheroid formation in the culture observed when seeding directly into a culture dish without using a fiber sheet, and there is no cell detachment and uniformity. The cells are maintained in a spread state.
  • Fiber sheets are used to promote adhesion and spreading of seeded nerve cells, extracellular matrix proteins such as polylysine, polyornithine, laminin, fibronectin, Matrigel® or Geltrex®, or cationic aqueous May be coated with polyethyleneimine which is a hydrophilic polymer. Coating can be performed by immersing the fiber sheet in a solution in which the above-mentioned extracellular matrix protein or polyethyleneimine is dissolved in physiological saline, phosphate buffered saline, cell culture solution, or the like.
  • extracellular matrix proteins such as polylysine, polyornithine, laminin, fibronectin, Matrigel® or Geltrex®, or cationic aqueous May be coated with polyethyleneimine which is a hydrophilic polymer. Coating can be performed by immersing the fiber sheet in a solution in which the above-mentioned extracellular matrix protein or polyethyleneimine is dissolved in physiological saline,
  • a cell scaffold seeded with nerve cells for example, a fiber sheet or a fiber sheet fixed or held around a fiber sheet with a frame, is brought into contact with a microelectrode array to form a nerve cell contained in the cell scaffold.
  • Extracellular potential can be measured in an environment of 5% CO 2 and 37 ° C.
  • the microelectrode array a large number of planar microelectrodes are arranged on a substrate, and electric signals from a plurality of cells can be simultaneously observed.
  • fluorescent calcium indicators such as calcium-sensitive dyes and calcium-sensitive fluorescent proteins and fluorescent potential indicators such as voltage-sensitive dyes and voltage-sensitive fluorescent proteins are used (Grienberger, C. and Konnerth, A., Neuron 73, 862-885, 2012; Antic, S. D., et al. J Neurophysiol 116: 135-152, 2016; Miller, E. W., Curr Opin Chem Biol. 33: 74- 80, 2016).
  • fluctuations in calcium and potential of nerve cells included in the nerve cell device of the present invention can also be measured by a cell imaging device.
  • a random fiber sheet was prepared under the conditions of kV and a feed rate of 1 ml / h, and in the case of PSU, under the conditions of a needle diameter of 27 G, a voltage of 15 kV and a feed rate of 1 ml / h.
  • (2) Preparation of Oriented Fiber Sheet Oriented PLGA fiber sheet is prepared by dissolving PLGA (SIGMA P1941) at room temperature with HFIP (Wako Pure Chemicals, 089-04233) to a concentration of 20% by weight.
  • Oriented PS fiber sheet is prepared by dissolving PS (Fluka) with DMF (N, N-dimethylformamide, Wako Pure Chemical) at room temperature so as to have a concentration of 30% by weight, and dissolving the solution with a syringe (Norm-Ject Syringes 5).
  • FIG. 1A and those on the MEA electrode chip are shown in FIG. 1B.
  • the MEA electrode chip On the MEA electrode chip, it was shown that the cells aggregated into spheroids and detached from the electrode. On the other hand, it was shown that the cells spread uniformly and grew on the oriented PLGA fiber sheet.
  • nerve cells were seeded directly on the MEA probe at 8 ⁇ 10 5 cells / cm 2 , cultured for 4 weeks in a 5% CO 2 , 37 ° C. incubator, and the nerve activity was measured similarly.
  • the results are shown in FIG.
  • cells seeded at a higher density on oriented PLGA fiber sheets have higher levels of activity when neural activity is not sufficiently observed on cells on MEA probes. Spikes were confirmed by frequency. Also, strong synchronization bursts were observed.
  • Example 2 [Effect of cell scaffolding and cell seeding density on spike firing frequency and drug responsiveness of nerve cells]
  • the oriented PS fiber sheet produced in Example 1 was coated with poly-D-lysine and laminin.
  • human iPS cell-derived neurons (XCL-1 neurons, XCell Science, USA) were cultured at 3 ⁇ 10 5 cells / cm 2 (low density seeding) or 12 ⁇ 10 5 cells / cell.
  • the cells were seeded at a density of cm 2 (high-density seeding), cultured in a 5% CO 2 , 37 ° C. incubator for 2, 4 and 6 weeks, and then the nerve activity was measured in the same manner as in Example 3.
  • the spike firing which is a neural activity, is performed between the cells directly seeded on the MEA probe and the low-density or high-density seeded cells on the oriented PS fiber sheet.
  • the frequencies were compared.
  • spikes were observed more frequently in cells seeded at high density on oriented PS fiber sheets than in cells seeded directly on MEA probes (conventional method) (FIG. 3A).
  • FIG. 3B spikes were observed more frequently in cells seeded at low density on the oriented PS fiber sheet as compared to cells by the conventional method
  • 4-AP is a drug that induces changes in the membrane potential of nerve cells by blocking potassium channels on the cell membrane. Due to the addition of 4-AP, cells seeded directly on the MEA probe and cells seeded at low density or high density on the oriented PS fiber sheet all responded to 4-AP, The number of spikes was increased compared to the case (FIG. 3C). However, compared to cells according to the conventional method (cells seeded directly on the MEA probe), the number of spikes is higher in cells seeded on the oriented PS fiber sheet. It was clear when the neurons were seeded in. These results indicated that the functional maturation of the neural network was promoted by culturing neurons using the oriented PS fiber sheet. Furthermore, it was shown that seeding neurons at higher density on oriented PS fiber sheets promotes functional maturation.
  • the oriented PS fiber sheet provided with the stainless steel circular frame prepared in Example 1 (3) was hydrophilized by plasma treatment using a desktop plasma treatment device (Strex), and then poly-D-lysine and laminin were used. Coating treatment.
  • human iPS cell-derived neurons XCL-1 neuron, XCell Science, USA
  • XCL-1 neuron XCell Science, USA
  • human iPS cell-derived cerebral cortical neurons (Axol Bioscience, UK) were seeded at a density of 8 ⁇ 10 5 cells / cm 2 or 24 ⁇ 10 5 cells / cm 2 and 5% CO 2 , 37 ° C. Cultured for 4 weeks in an incubator. The obtained cell sheet was placed on a probe of a microelectrode array (MEA) MED64-Presto (Alpha Med Scientific), and the cell was brought into contact with the electrode of the MEA probe to measure a nerve action potential.
  • MEA microelectrode array
  • neural cells were directly plated on the MEA probe at 3 ⁇ 10 5 cells / cm 2 (for XCell Science, XCL-1 neurons) or 8 ⁇ 10 5 cells / cm 2 (Axol Bioscience, human iPS cells (In case of derived cerebral cortical neurons) and cultured for 4 weeks.
  • FIG. 4 shows the results of measurement of extracellular potential in human iPS cell-derived neuronal XCL-1 neurons (XCell Science). 4A and 4B, even at the same cell seeding density, a stronger synchronous burst was observed in the cells cultured using the oriented PS fiber sheet than in the cells cultured on the MEA probe. In addition, cells were seeded at a high density on the oriented PS fiber sheet, and the cultured cells showed higher spike firing and higher frequency than when the cells were seeded at a low density and directly on the MEA probe. Strong synchronous burst firing was observed (FIGS. 4C and D).
  • FIG. 5 shows the measurement results of extracellular potentials in human iPS cell-derived cerebral cortical neurons (Axol Bioscience). In the cells seeded and cultured on the oriented PS fiber sheet, higher frequency spike firing and strong synchronous burst firing were clearly observed compared to the cells seeded and cultured directly on the MEA probe.

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Abstract

The present invention provides a method for promoting the functional maturation of a neural network by culturing neurons on a fiber sheet serving as a cell scaffold.

Description

神経細胞の機能的成熟化法Methods for functional maturation of nerve cells
　本発明は、神経細胞を培養することにより得られる神経ネットワークの機能的成熟化を促進する方法に関する。 (4) The present invention relates to a method for promoting functional maturation of a neural network obtained by culturing neural cells.
　医薬品開発における安全性評価において、神経毒性は、心毒性や肝毒性と並び、医薬品開発が中止される主な原因の一つである。そのため、医薬品開発の初期段階から的確な神経毒性評価が必要であるが、非臨床試験における神経毒性の評価は、動物実験による症状観察や脳の病理組織評価などのin vivo評価系が主体であり、より簡便なin vitroで神経毒性を評価する系は確立されていない。認知機能などの中枢神経系の高次機能の評価においては、実験動物を用いた行動解析、脳組織損傷実験および病理組織評価、またノックアウト動物を用いた解析などのin vivo評価系が挙げられる。しかしながら、これらのin vivo評価系は手技的に煩雑である。これに対して、in vitro試験法は、簡便に実施することができ、また多数の化合物の評価も容易に行うことができる。 に お い て In safety evaluation in drug development, neurotoxicity is one of the main causes of drug development discontinuation, along with cardiotoxicity and hepatotoxicity. Therefore, accurate neurotoxicity evaluation is necessary from the early stage of drug development, but neurotoxicity evaluation in non-clinical studies is mainly performed by in vivo evaluation systems such as observation of symptoms by animal experiments and evaluation of brain histopathology. A simpler system for evaluating neurotoxicity in vitro has not been established. In order to evaluate higher-order functions of the central nervous system such as cognitive functions, in-vivo evaluation systems such as behavioral analysis using experimental animals, brain tissue damage experiments and pathological tissue evaluation, and analysis using knockout animals can be mentioned. However, these in vivo evaluation systems are technically complicated. On the other hand, the in vitro test method can be easily performed, and a large number of compounds can be easily evaluated.
　ヒト由来神経細胞を用いたin vitroでの有効性評価や毒性評価は、通常２次元で培養した細胞を用いて行われている。神経細胞からは、長い突起である軸索と、複雑に分岐した短い樹状突起が伸びており、これらの神経突起は、別の神経細胞とつながり、神経回路である神経ネットワークを形成する。神経活動は、神経ネットワークにおいて複数の神経細胞が相互に作用することで発現する電気活動に基づくため、ヒト由来神経細胞の機能を指標とする有効性評価や毒性評価において、単離神経細胞または多能性幹細胞から誘導した神経細胞を、微小電極アレイ（microelectrode array、以下MEAと略す）上に分散培養させることにより神経回路網を再構成し、細胞外電位を測定することにより神経活動を観察する手法が開発されている（非特許文献１）。微小電極アレイ測定法は、電極アレイチップ上に神経細胞を２次元で分散培養し、自発的電気活動が生じてシナプス伝播が観察されるまで培養した後、神経細胞の機能評価を行う方法である。この手法により、実際に神経毒性を評価する検討が報告されている（非特許文献２および３）。 有効 Efficacy evaluation and toxicity evaluation in vitro using human-derived neurons are usually performed using cells cultured in two dimensions. From the nerve cells, axons, which are long processes, and short dendrites, which are complexly branched, extend. These neurites are connected to other nerve cells to form a neural network, which is a neural circuit. Nerve activity is based on the electrical activity expressed by the interaction of a plurality of nerve cells in a neural network. Therefore, in the evaluation of efficacy and toxicity using the function of human-derived nerve cells as an index, isolated nerve cells or multiple Nerve cells derived from competent stem cells are dispersed and cultured on a microelectrode array (MEA) to reconstruct the neural network, and observe the neural activity by measuring extracellular potential A method has been developed (Non-Patent Document 1). The microelectrode array measurement method is a method in which nerve cells are dispersed and cultured in two dimensions on an electrode array chip, cultured until spontaneous electrical activity occurs and synaptic propagation is observed, and then the function of the nerve cells is evaluated. . Investigations for actually evaluating neurotoxicity by this technique have been reported (Non-Patent Documents 2 and 3).
　神経細胞の培養は一般に困難とされているが、神経細胞が接着する足場を提供して、神経細胞の増殖を促進する様々な手段が報告されている。例えば、多孔性の３次元ハイドロゲルに、ポリカプロラクトンまたはゼラチンと混合したポリカプロラクトンから成るマイクロファイバーを整列して埋め込んだ足場に神経細胞を播種することにより、神経細胞の増殖が促進されることが報告されている（非特許文献４）。また、医療用材料などへの応用を目的として、細胞の足場として用いる足場材料に関する報告がいくつかある。例えば、ポリオレフィン、ポリアミド、ポリウレタン、ポリエステル、フッ素系高分子、ポリ乳酸、ポリビニルアルコールなどで構成されるナノファイバー、またはタンパク質成分を吸着させた該ナノファイバーにより構成される細胞足場材料を使用し、細胞培養または組織再生を有効に行う（特許文献１）；中空糸膜メッシュとナノファイバー層とを有する細胞足場材料を使用する３次元細胞培養により、培養細胞への栄養や酸素の供給および培養細胞からの代謝老廃物の除去を高い効率で行う（特許文献２）；ゼラチン、コラーゲンもしくはセルロースを含有するナノファイバー、または架橋された該ナノファイバーにより構成される細胞足場材料を使用し、多能性幹細胞の大量供給を行う、および細胞死を抑制する（特許文献３）；ポリグリコール酸を支持体として用い、その上にポリグリコール酸やゼラチンなどからなるナノファイバーを塗布した細胞足場材料を使用し、ヒト多能性幹細胞の増殖率を向上させる（特許文献４）などが報告されている。しかしながら、MEAによる細胞外電位測定において、神経細胞を培養することにより得られ、生体組織に類似した機能を発現する神経ネットワークを、早期に成熟化させる方法については報告されていない。 培養 Culture of nerve cells is generally considered to be difficult, but various means have been reported to provide a scaffold to which nerve cells adhere and to promote proliferation of nerve cells. For example, by seeding neurons on a scaffold in which microcapsules made of polycaprolactone or polycaprolactone mixed with gelatin are aligned and embedded in a porous three-dimensional hydrogel, the proliferation of nerve cells can be promoted. It has been reported (Non-Patent Document 4). In addition, there are some reports on scaffold materials used as cell scaffolds for the purpose of application to medical materials and the like. For example, using a nanofiber composed of polyolefin, polyamide, polyurethane, polyester, fluoropolymer, polylactic acid, polyvinyl alcohol, or the like, or a cell scaffold composed of the nanofiber adsorbing a protein component, Effectively perform culture or tissue regeneration (Patent Document 1); supply of nutrients and oxygen to cultured cells and supply of cultured cells by three-dimensional cell culture using a cell scaffold having a hollow fiber membrane mesh and a nanofiber layer Highly efficient removal of metabolic wastes of a plant (Patent Document 2); pluripotent stem cells using a nanofiber containing gelatin, collagen or cellulose, or a cell scaffold composed of the crosslinked nanofiber Supply a large amount of and suppress cell death (Patent Document 3); Using a cell scaffolding material in which glycolic acid is used as a support and nanofibers made of polyglycolic acid, gelatin, etc. are applied thereon to improve the proliferation rate of human pluripotent stem cells (Patent Document 4) Have been. However, there is no report on a method for early maturation of a neural network obtained by culturing a nerve cell and expressing a function similar to a biological tissue in the extracellular potential measurement by MEA.
特開2006-254722JP 2006-254722 特開2011-239756JP 2011-239756 特開2013-247943JP 2013-247943 WO2016/068266WO2016 / 068266
　ヒト特有の副作用などを検出するためには、ヒト由来の神経細胞を用いることが望ましいが、ヒト由来神経細胞は機能的に成熟する速度が遅いことが実用化する上での課題となっている。MEA上に直接播種した神経細胞は、生体組織に類似した機能を発現し、神経活動測定に使用できるまでには長期の培養が必要とされている。しかしながら、長期の培養により、細胞の剥離や凝集、また細菌などのコンタミネーションが生じ、神経活動を評価する上で悪影響が出る可能性がある。培養する際の細胞密度を高めると、神経ネットワーク機能の成熟化が促進されるが、その一方で神経細胞が凝集し、スフェロイドとなる傾向がある。MEA上で分散培養した神経細胞がスフェロイドを形成すると、該スフェロイドはMEAから部分的に剥離し、細胞と接触する電極数が減るため、安定的に神経活動を測定することができない。さらに、高い細胞密度で神経細胞を培養すると、酸素と栄養が細胞集団の内部まで十分に到達せず、細胞が死滅する場合がある。また、２次元培養した神経細胞から得られる細胞外電位は微弱であり、神経活動を高い精度で解析するためには、S/N比の向上が必要である。本発明は、これらの問題点を克服することを目的とする。 To detect side effects unique to humans, it is desirable to use human-derived nerve cells, but the slowness of functional maturation of human-derived nerve cells has been a problem in practical use. . Neurons directly seeded on MEA exhibit functions similar to those of living tissues, and long-term culture is required before they can be used for measuring neural activity. However, long-term culture may cause detachment and aggregation of cells and contamination of bacteria and the like, which may have an adverse effect on evaluating neural activity. Increasing the cell density during culture promotes the maturation of the neural network function, but on the other hand, the nerve cells tend to aggregate and become spheroids. When nerve cells dispersed and cultured on the MEA form spheroids, the spheroids are partially detached from the MEA, and the number of electrodes in contact with the cells decreases, so that it is impossible to measure nerve activity stably. Furthermore, when culturing nerve cells at a high cell density, oxygen and nutrients may not sufficiently reach the inside of the cell population, and the cells may die. In addition, extracellular potentials obtained from two-dimensionally cultured neurons are weak, and in order to analyze neural activity with high accuracy, it is necessary to improve the S / N ratio. The present invention aims to overcome these problems.
　本発明者らは、上記の従来技術の問題点を克服することを目的とし、鋭意検討した結果、細胞足場としてのファイバーシート上で神経細胞を培養することにより、神経ネットワークの機能的成熟化を促進する方法を見出し、本発明を完成するに至った。 The present inventors have conducted intensive studies with the aim of overcoming the above-mentioned problems of the prior art, and as a result, by culturing nerve cells on a fiber sheet as a cell scaffold, functional maturation of a neural network has been achieved. We found a way to promote it and completed the present invention.
　すなわち、本発明の目的は、以下の発明により達成される。
（１）細胞足場を用いて神経細胞を培養することを含む、神経ネットワークを機能的に成熟化させる方法。
（２）前記細胞足場が高分子材料で形成されたファイバーシートである、１に記載の方法。
（３）前記ファイバーシートが、配向性構造、非配向性構造または配向性と非配向性との混合構造を有する、２に記載の方法。
（４）前記ファイバーシートが、ポリリジン、ポリオルニチン、ラミニン、フィブロネクチン、マトリゲル（登録商標）およびゲルトレックス（登録商標）から選ばれる細胞外マトリックスタンパク質でコーティングされた、２または３に記載の方法。
（５）前記ファイバーシートが、ポリエチレンイミンでコーティングされた、２または３に記載の方法。
（６）前記神経細胞が、細胞足場上および／または細胞足場内で3次元構造を形成した、１～５のいずれかに記載の方法。
（７）前記神経細胞が、初代培養細胞または多能性幹細胞由来の神経細胞である、１～６のいずれかに記載の方法。
（８）前記初代培養細胞または前記多能性幹細胞由来の神経細胞が、哺乳類由来の神経細胞である、７に記載の方法。
（９）前記神経細胞が、グルタミン酸作動性、ドーパミン作動性、γ-アミノ酪酸作動性、モノアミン作動性、ヒスタミン作動性またはコリン作動性の神経細胞を含む、１～８のいずれかに記載の方法。
（１０）１～９のいずれかに記載の方法を使用して機能的に成熟化させた神経細胞を用いる、神経活動の評価方法。
（１１）１～９のいずれかに記載の方法を使用して機能的に成熟化させた神経細胞を微小電極アレイと接触させ、該神経細胞の細胞外電位を測定することを含む、神経活動の評価方法。 That is, the object of the present invention is achieved by the following inventions.
(1) A method for functionally maturating a neural network, comprising culturing nerve cells using a cell scaffold.
(2) The method according to 1, wherein the cell scaffold is a fiber sheet formed of a polymer material.
(3) The method according to 2, wherein the fiber sheet has an oriented structure, a non-oriented structure, or a mixed structure of oriented and non-oriented.
(4) The method according to 2 or 3, wherein the fiber sheet is coated with an extracellular matrix protein selected from polylysine, polyornithine, laminin, fibronectin, Matrigel (registered trademark) and Geltrex (registered trademark).
(5) The method according to (2) or (3), wherein the fiber sheet is coated with polyethyleneimine.
(6) The method according to any one of (1) to (5), wherein the nerve cell has formed a three-dimensional structure on and / or within the cell scaffold.
(7) The method according to any one of 1 to 6, wherein the nerve cell is a primary cultured cell or a neural cell derived from a pluripotent stem cell.
(8) The method according to 7, wherein the primary cultured cells or the pluripotent stem cell-derived nerve cells are mammalian-derived nerve cells.
(9) The method according to any one of (1) to (8), wherein the nerve cells include glutamatergic, dopaminergic, γ-aminobutyratergic, monoaminergic, histaminergic or cholinergic neurons. .
(10) A method for evaluating nerve activity, using a nerve cell functionally matured using the method according to any one of 1 to 9.
(11) A neural activity comprising contacting a nerve cell functionally matured using the method according to any one of 1 to 9 with a microelectrode array and measuring an extracellular potential of the nerve cell. Evaluation method.
　本発明によれば、ファイバーシートにより形成された細胞足場を用いて神経細胞を培養することにより、細胞接着性が向上し、培養中の細胞剥離の低減などを含む安定した培養が可能となる。その結果、形成される神経ネットワークの機能的成熟化が促進される。本発明により機能的に成熟化させた神経細胞を用いると、微小電極アレイの電極から、播種した神経細胞が脱離することがなく、スパイク発火および同期バースト発火などの神経活動の測定が可能になるまでの期間が短縮される。さらに、神経細胞の凝集が抑制されるため、高頻度のスパイク発火および強い同期バースト発火を観察することができる。本発明の方法により機能的に成熟化させた神経細胞は、神経毒性評価や神経疾患に対する薬物スクリーニングなどに有用である。 According to the present invention, by culturing nerve cells using a cell scaffold formed by a fiber sheet, cell adhesion is improved, and stable culturing including reduction of cell detachment during culturing becomes possible. As a result, the functional maturation of the formed neural network is promoted. The use of the nerve cells functionally matured according to the present invention enables measurement of neural activities such as spike firing and synchronous burst firing without detaching the seeded nerve cells from the electrodes of the microelectrode array. The time before it becomes is shortened. Further, since the aggregation of nerve cells is suppressed, a high-frequency spike firing and a strong synchronous burst firing can be observed. Neurons functionally matured by the method of the present invention are useful for neurotoxicity evaluation, drug screening for neurological diseases, and the like.
　本発明により神経細胞を培養すると、ファイバーシートを構成するファイバーが神経細胞の支持体となり、細胞凝集が抑制されるため、細胞が積層化し、細胞同士が接触した状態であってもスフェロイドが形成されず、平面状で高密度に積層された３次元培養が可能となる。かかる高密度培養により、神経細胞のスパイク発火頻度の上昇およびシナプス伝播による強い同期バースト発火を早期に観察することができる。すなわち、神経細胞の機能的成熟化が早期に実現される。また、神経細胞を高密度で平面状に培養できるため、MEA上で細胞と接触する電極数が多く、安定して電気信号を取得することが可能となる。さらに、測定時に神経細胞を含む細胞足場をMEA上に載せて測定できることから、あらかじめMEA上で神経細胞を培養する必要がなく、培養期間中の電極の汚染を避けることができる。そのため、電極インピーダンスを低インピーダンス状態にしたまま測定できるため、外来ノイズの影響を受けにくく、S/N比が高くなる。また、あらかじめMEA上で神経細胞を培養する必要がないため、MEAプローブの稼働率も向上する。さらに、神経細胞の培養期間が短縮されるので、細胞培養に要する経費の削減が可能である。 When a nerve cell is cultured according to the present invention, the fibers constituting the fiber sheet serve as a support for the nerve cell, and cell aggregation is suppressed, so that cells are stacked and spheroids are formed even when the cells are in contact with each other. Instead, it is possible to perform three-dimensional culturing in a planar and densely stacked state. Such high-density culture allows early observation of an increase in spike firing frequency of neurons and strong synchronous burst firing due to synaptic propagation. That is, the functional maturation of the nerve cell is realized early. In addition, since nerve cells can be cultured at a high density in a planar manner, the number of electrodes in contact with the cells on the MEA is large, and it is possible to stably acquire an electric signal. Furthermore, since a cell scaffold containing nerve cells can be placed on the MEA during the measurement, the nerve cells do not need to be cultured on the MEA in advance, and electrode contamination during the culture period can be avoided. Therefore, since the measurement can be performed while the electrode impedance is kept in a low impedance state, it is hardly affected by external noise and the S / N ratio is increased. In addition, since it is not necessary to culture neurons on the MEA in advance, the operating rate of the MEA probe is improved. Furthermore, since the culturing period of the nerve cells is shortened, the cost required for culturing the cells can be reduced.
ヒトiPS細胞由来神経細胞を、8×105細胞／cm2の密度で、配向性PLGAファイバーシート上（Ａ）またはMEAプローブ上（Ｂ）に播種し、3週間培養した後の細胞の状態を示す光学顕微鏡写真（倍率×4）である。Human iPS cell-derived neurons were seeded on an oriented PLGA fiber sheet (A) or MEA probe (B) at a density of 8 × 10 5 cells / cm 2 , and cultured for 3 weeks. It is an optical microscope photograph (magnification × 4) shown. 16個（4個×4個）の微小電極が配置されたMEAプローブを用いて測定するiPS細胞由来神経細胞の細胞外電位に対する細胞足場および細胞播種密度の影響を示す図である。Ａ～Ｃの各図の上段のヒストグラムは、16個の微小電極で測定されたスパイク数の積算値の時間変化を示す図である（縦軸：array-wide spike detection rate（AWSDR、スパイク数／秒））。Ａ～Ｃの各図の下段は、各電極で経時的に測定されたスパイクを示す図である（縦軸：電極のチャネル番号）。（Ａ）MEAプローブ上に、8×105細胞／cm2で細胞を直接播種し、4週間培養した。（Ｂ）配向性PLGAファイバーシート上に、8×105細胞／cm2で細胞を播種し、4週間培養した。（Ｃ）配向性PLGAファイバーシート上に、24×105細胞／cm2で細胞を播種し、4週間培養した。FIG. 9 is a diagram showing the influence of cell scaffolding and cell seeding density on extracellular potential of iPS cell-derived nerve cells measured using a MEA probe in which 16 (4 × 4) microelectrodes are arranged. The upper histograms in each of FIGS. A to C are diagrams showing the time change of the integrated value of the number of spikes measured by 16 microelectrodes (vertical axis: array-wide spike detection rate (AWSDR, number of spikes / Seconds)). The lower part of each figure of A to C is a figure showing spikes measured with time for each electrode (vertical axis: channel number of electrode). (A) Cells were directly seeded at 8 × 10 5 cells / cm 2 on the MEA probe and cultured for 4 weeks. (B) Cells were seeded on an oriented PLGA fiber sheet at 8 × 10 5 cells / cm 2 and cultured for 4 weeks. (C) Cells were seeded on an oriented PLGA fiber sheet at 24 × 10 5 cells / cm 2 and cultured for 4 weeks. 神経細胞のスパイク発火頻度および薬物応答性に対する細胞足場および細胞播種密度の影響を示す図である。ヒトiPS細胞由来神経細胞を、配向性PSファイバーシート上に、3×105細胞／cm2または12×105細胞／cm2の密度で播種した。MEAプローブ上には、3×105細胞／cm2で播種した。これらを2、4および6週間培養した。培養後、MEAにより自発スパイク数を5分間測定した。結果は、平均±標準誤差（MEAプローブn=4, ファイバーシートn=2）で示される。（Ａ）2週間培養後の自発スパイク数を示す図である。（Ｂ）4週間培養後の自発スパイク数を示す図である。（Ｃ）6週間培養後の自発スパイク数（「添加前」と表示）および4-アミノピリジン（100μM）添加後（「添加後」と表示）の自発スパイク数を示す図である。FIG. 4 is a diagram showing the influence of cell scaffolding and cell seeding density on spike firing frequency and drug responsiveness of nerve cells. Human iPS cell-derived neurons were seeded on oriented PS fiber sheets at a density of 3 × 10 5 cells / cm 2 or 12 × 10 5 cells / cm 2 . The cells were seeded at 3 × 10 5 cells / cm 2 on the MEA probe. These were cultured for 2, 4 and 6 weeks. After the culture, the spontaneous spike count was measured for 5 minutes by MEA. Results are shown as mean ± standard error (MEA probe n = 4, fiber sheet n = 2). (A) shows the number of spontaneous spikes after two weeks of culture. (B) shows the number of spontaneous spikes after 4 weeks of culture. (C) is a diagram showing the number of spontaneous spikes after culturing for 6 weeks (displayed as “before addition”) and the number of spontaneous spikes after addition of 4-aminopyridine (100 μM) (displayed as “after addition”). ヒトiPS細胞由来神経細胞（XCL-1ニューロン、XCell Science社）の細胞外電位を、16個（4個×4個）の微小電極が配置されたMEAプローブを用いて測定した結果を示す図である。Ａ～Ｃの各図の上段のヒストグラムは、16個の微小電極で測定されたスパイク数の積算値の時間変化を示す図である（縦軸：array-wide spike detection rate（AWSDR、スパイク数／秒））。Ａ～Ｃの各図の下段は、各電極で経時的に測定されたスパイクを示す図である（縦軸：電極のチャネル番号）。（Ａ）MEAプローブ上に、3×105細胞／cm2で細胞を直接播種し、4週間培養した。（Ｂ）配向性PSファイバーシート上に、3×105細胞／cm2で細胞を播種し、4週間培養した。（Ｃ）配向性PSファイバーシート上に、9×105細胞／cm2で細胞を播種し、4週間培養した。（Ｄ）10分間で測定された総スパイク数を表す図である。結果は、平均±標準誤差（MEAプローブn=12、ファイバーシートn=6）で示される。FIG. 9 is a diagram showing the results of measuring the extracellular potential of human iPS cell-derived nerve cells (XCL-1 neuron, XCell Science) using an MEA probe on which 16 (4 × 4) microelectrodes are arranged. is there. The upper histograms in each of FIGS. A to C are diagrams showing the time change of the integrated value of the number of spikes measured by 16 microelectrodes (vertical axis: array-wide spike detection rate (AWSDR, number of spikes / Seconds)). The lower part of each figure of A to C is a figure showing spikes measured with time for each electrode (vertical axis: channel number of electrode). (A) Cells were directly seeded at 3 × 10 5 cells / cm 2 on the MEA probe and cultured for 4 weeks. (B) Cells were seeded at 3 × 10 5 cells / cm 2 on an oriented PS fiber sheet and cultured for 4 weeks. (C) Cells were seeded at 9 × 10 5 cells / cm 2 on an oriented PS fiber sheet and cultured for 4 weeks. (D) It is a figure showing the total number of spikes measured in 10 minutes. The results are shown as the mean ± standard error (MEA probe n = 12, fiber sheet n = 6). ヒトiPS細胞由来大脳皮質ニューロン（Axol Bioscience社）の細胞外電位を、16個（4個×4個）の微小電極が配置されたMEAプローブを用いて測定した結果を示す図である。Ａ～Ｃの各図の上段のヒストグラムは、16個の微小電極で測定されたスパイク数の積算値の時間変化を示す図である（縦軸：array-wide spike detection rate（AWSDR、スパイク数／秒））。Ａ～Ｃの各図の下段は、各電極で経時的に測定されたスパイクを示す図である（縦軸：電極のチャネル番号）。（Ａ）MEAプローブ上に、8×105細胞／cm2で細胞を直接播種し、4週間培養した。（Ｂ）配向性PSファイバーシート上に、8×105細胞／cm2で細胞を播種し、4週間培養した。（Ｃ）配向性PSファイバーシート上に、24×105細胞／cm2で細胞を播種し、4週間培養した。（Ｄ）10分間で測定された総スパイク数を表す図である。結果は、平均±標準誤差（MEAプローブn=5、ファイバーシートn=6）で示される。FIG. 9 is a diagram showing the results of measuring the extracellular potential of human iPS cell-derived cerebral cortical neurons (Axol Bioscience) using an MEA probe on which 16 (4 × 4) microelectrodes are arranged. The upper histograms in each of FIGS. A to C are diagrams showing the time change of the integrated value of the number of spikes measured by 16 microelectrodes (vertical axis: array-wide spike detection rate (AWSDR, number of spikes / Seconds)). The lower part of each figure of A to C is a figure showing spikes measured with time for each electrode (vertical axis: channel number of electrode). (A) Cells were directly seeded at 8 × 10 5 cells / cm 2 on the MEA probe and cultured for 4 weeks. (B) Cells were seeded on an oriented PS fiber sheet at 8 × 10 5 cells / cm 2 and cultured for 4 weeks. (C) Cells were seeded at 24 × 10 5 cells / cm 2 on an oriented PS fiber sheet and cultured for 4 weeks. (D) It is a figure showing the total number of spikes measured in 10 minutes. The results are shown as the mean ± standard error (MEA probe n = 5, fiber sheet n = 6).
　本発明において使用される細胞足場は、高分子材料で生成されたファイバーで構成される。細胞足場は、好ましくは、ファイバーを集積したシートの形状を有するファイバーシートである。該ファイバーシートは配向性構造、非配向性構造または配向性と非配向性との混合構造を有することができる。配向性構造とは、ファイバーシートを構成するファイバーが一方向に配置され、該一方向の角度を0°とした場合、80％以上のファイバーが±30°の範囲内に存在する構造である。配向性構造において、ファイバー間の距離（隣接するファイバーの芯線間の距離）は特に限定されないが、5～50 μmであるのが好ましい。非配向性構造とは、ファイバーの方向がランダムに配置された構造である。ファイバーを構成する高分子材料としては、生分解性または非生分解性の高分子材料が好ましく、例えば、PLGA (ポリ乳酸ポリグリコール酸)、ポリスチレン（PS）、ポリスルホン（PSU）およびポリテトラフルオロエチレン（PTFE）が挙げられるが、これらに限定されない。ファイバーシートを構成するファイバーの直交断面の直径は、特に限定されないが、例えば0.1～8 μｍであり、好ましくは0.5～7 μｍであり、より好ましくは1～6 μｍである。ファイバーシートの厚さは、例えば1～40 μｍであり、好ましくは5～35 μｍであり、より好ましくは10～30 μｍである。ファイバーシートを構成するファイバーの空隙率は、用いる高分子材料によって変動し得る。該空隙率は特に限定されないが、例えば10～50％であり、好ましくは15～45％であり、より好ましくは20～40％である。ここで空隙率とは、ファイバーシート平面の一定面積に対する、ファイバーが存在していない面積の比率のことである。 細胞 The cell scaffold 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 can have an oriented structure, a non-oriented structure, or a mixed structure of oriented and non-oriented. The oriented structure is a structure in which the fibers constituting the fiber sheet are arranged in one direction, and when the angle in one direction is 0 °, 80% or more of the fibers exist in a range of ± 30 °. In the oriented structure, the distance between the fibers (the distance between the cores of adjacent fibers) is not particularly limited, but is preferably 5 to 50 μm. The non-oriented structure is a structure in which the directions of the fibers are randomly arranged. As the polymer material constituting the fiber, a biodegradable or non-biodegradable polymer material is preferable. For example, PLGA (polylactic acid polyglycolic acid), polystyrene (PS), polysulfone (PSU) and polytetrafluoroethylene (PTFE), but is not limited thereto. Although the diameter of the cross section of the fiber constituting the fiber sheet is not particularly limited, it is, for example, 0.1 to 8 μm, preferably 0.5 to 7 μm, and more preferably 1 to 6 μm. The thickness of the fiber sheet is, for example, 1 to 40 μm, preferably 5 to 35 μm, and more preferably 10 to 30 μm. The porosity of the fibers constituting the fiber sheet can vary depending on the polymer material used. The porosity is not particularly limited, but is, for example, 10 to 50%, preferably 15 to 45%, and more preferably 20 to 40%. Here, the porosity is a ratio of an area where no fiber exists to a fixed area of the fiber sheet plane.
　ファイバーシートは、例えば、高分子材料を含む溶液からエレクトロスピニング法によって製造することができる。配向性構造を有するファイバーシートを製造する場合は、特に限定されないが、例えば回転ドラムを用い、該ドラムを回転させながら、ノズルから該ドラムの回転面に対して高分子材料を含む溶液を噴霧し、回転ドラム上で形成されたファイバーを巻き取ることにより、ファイバーシートを製造することができる。非配向性構造を有するファイバーシートを製造する場合は、高分子材料を含む溶液を平坦なプレート上に噴霧することにより、ファイバーシートを製造することができる。配向性構造と非配向性構造との混合構造を有するファイバーシートを製造する場合は、例えば、配向性構造および非配向性構造のファイバーシートを製造する上記の製造方法を組み合わせて製造することができる。 The fiber sheet can be manufactured by, for example, an electrospinning method from a solution containing a polymer material. When producing a fiber sheet having an oriented structure, although not particularly limited, for example, using a rotating drum, while rotating the drum, spray the solution containing the polymer material from the nozzle to the rotating surface of the drum By winding the fiber formed on the rotating drum, a fiber sheet can be manufactured. When manufacturing a fiber sheet having a non-oriented structure, a fiber sheet can be manufactured by spraying a solution containing a polymer material onto a flat plate. When producing a fiber sheet having a mixed structure of an oriented structure and a non-oriented structure, for example, it can be produced by combining the above-described production methods for producing a fiber sheet having an oriented structure and a non-oriented structure. .
　ポリテトラフルオロエチレン（PTFE）シートは、例えば、市販されている住友電工株式会社のポアフロン（登録商標）を使用することができる。 As the polytetrafluoroethylene (PTFE) sheet, for example, commercially available Pouflon (registered trademark) of Sumitomo Electric Industries, Ltd. can be used.
　高分子材料の溶液としては、使用する高分子材料を、室温で10～30重量％で溶解する有機溶媒であればよく、例えば1,1,1,3,3,3-ヘキサフルオロ-2-プロパノール(HFIP)、N,N-ジメチルホルムアミド（DMF）などが挙げられる。 The solution of the polymer material may be any organic solvent that dissolves the polymer material used at room temperature at 10 to 30% by weight. For example, 1,1,1,3,3,3-hexafluoro-2- And propanol (HFIP) and N, N-dimethylformamide (DMF).
　ファイバーシートは、周囲をフレームで固定または保持することができる。フレームをファイバーシートに固定または保持する場合は、細胞培養に影響を及ぼさなければ特に限定されないが、例えば市販の生体適合性粘着剤、例えばシリコーン一液縮合型RVTゴム（信越化学、カタログ番号KE-45）を用いて、フレームとファイバーシートとを接着することができる。 The fiber sheet can be fixed or held around the frame. When the frame is fixed or held on the fiber sheet, it is not particularly limited as long as it does not affect the cell culture. For example, a commercially available biocompatible adhesive such as a silicone one-pack condensation type RVT rubber (Shin-Etsu Chemical, Catalog No. KE- 45) can be used to bond the frame and the fiber sheet.
　フレームの素材は、細胞培養に影響を及ぼさなければ特に限定されない。例えば、ポリジメチルシロキサン（PDMS）、PS、ポリカーボネート、ステンレスなどが例示される。フレームの厚さは、特に限定されないが、0.1～4 mm、好ましくは0.25～3 mm、より好ましくは0.5～2 mmである。フレームの形状は、使用目的によって変えることができ、縦長×横長が、それぞれ2 mm×2 mm～15 mm×15 mmが好ましく、円形または多角形である。 素材 The material of the frame is not particularly limited as long as it does not affect the cell culture. For example, polydimethylsiloxane (PDMS), PS, polycarbonate, stainless steel and the like are exemplified. 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, and the length and width are preferably 2 mm × 2 mm to 15 mm × 15 mm, respectively, and are circular or polygonal.
　神経細胞を播種した細胞足場、例えばファイバーシート、またはファイバーシートの周囲をフレームで固定もしくは保持した該ファイバーシートは、細胞培養ディッシュまたは複数のウェルを有するマルチウェルプレートに含まれるウェルの少なくとも一つにそのまま配置することができる。 A cell scaffold in which nerve cells are seeded, for example, a fiber sheet, or the fiber sheet fixed or held around the fiber sheet by a frame, is attached to at least one of the wells included in a cell culture dish or a multi-well plate having a plurality of wells. It can be placed as it is.
　本明細書において、神経細胞とは、細胞体、樹状突起及び軸索から構成される神経単位を意味し、ニューロンとも呼ばれる。神経細胞は、神経細胞が産生する神経伝達物質の違いにより分類することができ、神経伝達物質としては、ドーパミン、ノルアドレナリン、アドレナリンおよびセロトニンなどのモノアミン、アセチルコリン、γ－アミノ酪酸、グルタミン酸などの非ペプチド性神経伝達物質、また、副腎皮質刺激ホルモン（ACTH）、α－エンドルフィン、β－エンドルフィン、γ－エンドルフィン、バソプレッシンなどのペプチド性神経伝達物質が挙げられる。例えば、ドーパミン、アセチルコリンおよびグルタミン酸を伝達物質とする神経細胞を、それぞれドーパミン作動性ニューロン、コリン作動性ニューロンおよびグルタミン酸作動性ニューロンという。 神 経 In the present specification, a nerve cell means a neuron composed of a cell body, dendrites, and axons, and is also called a neuron. Nerve cells can be classified according to the difference in neurotransmitters produced by the nerve cells. Examples of neurotransmitters include monoamines such as dopamine, noradrenaline, adrenaline and serotonin, and non-peptides such as acetylcholine, γ-aminobutyric acid, and glutamic acid. Sexual neurotransmitters and peptide neurotransmitters such as adrenocorticotropic hormone (ACTH), α-endorphin, β-endorphin, γ-endorphin, vasopressin and the like. For example, neurons that transmit dopamine, acetylcholine and glutamate as transmitters are called dopaminergic neurons, cholinergic neurons and glutamate neurons, respectively.
　神経細胞としては、初代培養細胞を用いることができる。初代培養細胞は、生体内において本来有する細胞機能を多く保持しているため、生体内における薬物などの影響を評価する系として重要である。初代培養細胞としては、哺乳類、例えばマウスもしくはラットのげっ歯類、またはサルもしくはヒトの霊長類の中枢神経系および末梢神経系の神経細胞を使用することができる。これらの神経細胞を調製および培養するに際し、動物の解剖方法、組織採取方法、神経分離・単離方法、神経細胞培養用培地、培養条件などは、培養する細胞の種類および細胞の目的に応じて、公知の方法より選択することができる。市販の初代培養神細胞製品としては、例えばロンザ社（スイス）のラット脳神経細胞およびScienCell Research Laboratories社（米国）のヒト脳神経細胞を用いることができる。 初 Primary cultured cells can be used as nerve cells. Primary cultured cells retain many of the inherent cell functions in vivo, and are therefore important as a system for evaluating the effects of drugs and the like in vivo. As the primary cultured cells, neurons of the central nervous system and peripheral nervous system of mammals such as rodents of mice or rats, or monkeys or human primates can be used. When preparing and culturing these nerve cells, animal dissection methods, tissue collection methods, nerve isolation / isolation methods, nerve cell culture media, culture conditions, etc., depend on the type of cells to be cultured and the purpose of the cells. Can be selected from known methods. As commercially available products of primary cultured myeloid cells, for example, rat brain nerve cells from Lonza (Switzerland) and human brain nerve cells from ScienCell Research Laboratories (USA) can be used.
　神経細胞としては、さらに多能性幹細胞由来の神経細胞を用いることができる。多能性幹細胞としては、例えば胚性幹細胞（ES細胞）やiPS細胞がある。多能性幹細胞を、公知の神経分化誘導方法を用いて分化誘導することにより、様々なタイプの神経細胞を得ることができる。例えば、文献（Honda et al. Biochemical and Biophysical Research Communications 469 (2016) 587-592）に記載の低分子化合物を用いた分化誘導方法によって神経細胞を得ることができる。また、市販の多能性幹細胞由来の神経細胞製品、例えば、セルラーダイナミックスインターナショナル社（米国）のiCellニューロン、Axol Bioscience社（英国）の各種神経幹細胞、BrainXell社（米国）の各種神経細胞の前駆細胞およびXCell Science社（米国）のXCL-1ニューロンを用いることもできる。これらの市販神経細胞は、付属の培養液を使用して培養することができる。 さ ら に As the nerve cell, a neural cell derived from a pluripotent stem cell can be further used. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and iPS cells. Various types of neural cells can be obtained by inducing differentiation of pluripotent stem cells using a known neural differentiation inducing method. For example, nerve cells can be obtained by a differentiation induction method using a low molecular compound described in the literature (Honda et al. Biochemical and Biophysical Research Communications 469 (2016) 587-592). Also, commercially available neural cell products derived from pluripotent stem cells, such as iCell neurons from Cellular Dynamics International (US), various neural stem cells from Axol Bioscience (UK), and various neural cells from BrainXell (US) Cells and XCL-1 neurons from XCell Science (USA) can also be used. These commercially available neurons can be cultured using the attached culture solution.
　神経細胞は、哺乳類の脳由来のグリア細胞または哺乳類のiPS細胞から分化させたグリア細胞と共に培養することができる。グリア細胞としては、アストロサイト、オリゴデンドロサイト、ミクログリアなどが挙げられる。また、アストロサイトを培養したあとの培養液（アストロサイト培養上清）を神経細胞用培養液に、終濃度5～30％で添加し培養することもできる。 Neurocytes can be cultured with glial cells derived from mammalian brain or glial cells differentiated from mammalian iPS cells. Glial cells include astrocytes, oligodendrocytes, microglia and the like. Alternatively, a culture solution after culturing astrocytes (astrocyte culture supernatant) can be added to a culture solution for nerve cells at a final concentration of 5 to 30% for culturing.
　神経ネットワークとは、細長い突起である軸索と複数の複雑に分枝した樹状突起とを有する神経細胞が、これらの神経突起を介して別の神経細胞とつながり、その結果形成された神経回路である。神経回路において、神経細胞（シナプス前細胞）が電気的に発火（活動電位の変化）すると、軸索を伝わった電気信号は、シナプスにおいて、シナプス前細胞から次の神経細胞（シナプス後細胞）の樹状突起に神経伝達物質を介して電気信号を伝達する。軸索と樹状突起とが接続している部位がシナプスである。本明細書において、神経ネットワークの機能的な成熟化とは、神経ネットワークを形成する神経細胞が機能的に成熟化した状態を指す。機能的に成熟化した神経ネットワークでは、神経細胞のスパイク発火頻度の上昇およびスパイクのシナプス伝播による強い同期バースト発火が観察される。ここでスパイク発火とは、神経細胞の活動電位の急激な変化であり、また同期バースト発火とは、神経ネットワークを形成する神経細胞が、シナプス伝播により同時期に発火する現象である。 A neural network is a type of neural network in which axons, which are elongated processes, and a plurality of intricately branched dendrites, are connected to other nerve cells via these neurites, and the resulting neural circuit is formed. It is. In a neural circuit, when a nerve cell (presynaptic cell) fires electrically (changes in action potential), the electrical signal transmitted through the axon is transmitted from the presynaptic cell to the next nerve cell (postsynaptic cell) at the synapse. It transmits electrical signals to dendrites via neurotransmitters. The site where axons and dendrites connect is a synapse. In the present specification, the functional maturation of a neural network refers to a state in which nerve cells forming a neural network are functionally matured. In functionally mature neural networks, increased spike firing of neurons and strong synchronous burst firing due to synaptic propagation of spikes are observed. Here, spike firing is a rapid change in the action potential of a nerve cell, and synchronous burst firing is a phenomenon in which nerve cells forming a neural network fire at the same time due to synaptic propagation.
　本明細書において、神経ネットワークは、幹細胞由来神経細胞、株化神経細胞、初代神経細胞、培養過程で神経細胞へ分化する幹細胞および神経前駆細胞など、ならびにグリア細胞などの神経組織を構成する細胞から選ばれる１種以上の細胞を、単細胞ではなく、複数個の細胞を細胞足場上で培養することにより形成される。 As used herein, a neural network is derived from stem cell-derived neural cells, established neural cells, primary neural cells, stem cells and neural progenitor cells that differentiate into neural cells in the course of culture, and the like, and cells that constitute neural tissue such as glial cells. One or more selected cells are formed by culturing not a single cell but a plurality of cells on a cell scaffold.
　ファイバーシートまたはフレームで周囲を固定または保持したファイバーシートに対して、培養液に懸濁した神経細胞を、1×104～4×106細胞／cm2、好ましくは1×105～4×106細胞／cm2、より好ましくは8×105～4×106細胞／cm2、さらに好ましくは2×106～4×106細胞／cm2の高密度で播種して、培養液を1～7日間隔で交換しながら培養することによって、神経細胞が均一に３次元構造を形成した神経ネットワークを形成させることができる。３次元構造を形成するとは、神経細胞がファイバーシートの片面または両面上およびファイバーシート内に入り込んで生育した状態をいう。配向性構造を有するファイバーシート上に神経細胞を播種した場合、神経細胞は、ファイバーシートを構成するファイバーに沿って接着し、ファイバーの配向方向に沿って伸展する。ファイバーシート上に神経細胞を播種し、培養すると、ファイバーシートを使用せず、培養シャーレに直接播種した場合に認められる培養中の細胞の凝集やスフェロイド形成がなく、また細胞の剥離もなく、均一に広がった状態で細胞が維持される。 A nerve cell suspended in a culture solution is applied to a fiber sheet or a fiber sheet whose periphery is fixed or held by a frame at 1 × 10 4 to 4 × 10 6 cells / cm 2 , preferably 1 × 10 5 to 4 × The cells are seeded at a high density of 10 6 cells / cm 2 , more preferably 8 × 10 5 to 4 × 10 6 cells / cm 2 , and still more preferably 2 × 10 6 to 4 × 10 6 cells / cm 2. By culturing while exchanging at intervals of 1 to 7 days, it is possible to form a neural network in which nerve cells uniformly form a three-dimensional structure. Forming a three-dimensional structure refers to a state in which nerve cells have grown on one or both sides of the fiber sheet and inside the fiber sheet. When a nerve cell is seeded on a fiber sheet having an oriented structure, the nerve cell adheres along a fiber constituting the fiber sheet and extends along the orientation direction of the fiber. When neurons are seeded on a fiber sheet and cultured, there is no cell aggregation or spheroid formation in the culture observed when seeding directly into a culture dish without using a fiber sheet, and there is no cell detachment and uniformity. The cells are maintained in a spread state.
　ファイバーシートは、播種した神経細胞の接着および伸展を促進するため、ポリリジン、ポリオルニチン、ラミニン、フィブロネクチン、マトリゲル（登録商標）もしくはゲルトレックス（登録商標）などの細胞外マトリックスタンパク質、またはカチオン性の水溶性ポリマーであるポリエチレンイミンでコーティングしてもよい。コーティングは、上記細胞外マトリックスタンパク質またはポリエチレンイミンを生理食塩水、リン酸緩衝生理食塩水、細胞培養液などに溶解した溶液に、ファイバーシートを浸漬することにより行うことができる。 Fiber sheets are used to promote adhesion and spreading of seeded nerve cells, extracellular matrix proteins such as polylysine, polyornithine, laminin, fibronectin, Matrigel® or Geltrex®, or cationic aqueous May be coated with polyethyleneimine which is a hydrophilic polymer. Coating can be performed by immersing the fiber sheet in a solution in which the above-mentioned extracellular matrix protein or polyethyleneimine is dissolved in physiological saline, phosphate buffered saline, cell culture solution, or the like.
　本発明において使用する、神経細胞を播種した細胞足場、例えばファイバーシートまたはファイバーシートの周囲をフレームで固定もしくは保持した該ファイバーシートは、微小電極アレイと接触させ、該細胞足場に含まれる神経細胞の細胞外電位を5％CO2、37℃環境下で測定することができる。微小電極アレイには基板上に平面微小電極が多数配置されており、複数の細胞からの電気信号を同時に観測することができる。 In the present invention, a cell scaffold seeded with nerve cells, for example, a fiber sheet or a fiber sheet fixed or held around a fiber sheet with a frame, is brought into contact with a microelectrode array to form a nerve cell contained in the cell scaffold. Extracellular potential can be measured in an environment of 5% CO 2 and 37 ° C. In the microelectrode array, a large number of planar microelectrodes are arranged on a substrate, and electric signals from a plurality of cells can be simultaneously observed.
　神経活動を測定する手段として、微小電極アレイ以外に、カルシウム感受性色素およびカルシウム感受性蛍光タンパク質などの蛍光カルシウムインジケーターならびに電位感受性色素および電位感受性蛍光タンパク質などの蛍光電位インジケーターが用いられる（Grienberger, C. and Konnerth, A., Neuron 73, 862-885, 2012; Antic, S. D., et al. J Neurophysiol 116: 135-152, 2016; Miller, E. W., Curr Opin Chem Biol. 33: 74-80, 2016）。これらのインジケーターを使用して、本発明の神経細胞デバイスに含まれる神経細胞のカルシウムや電位の変動を、セルイメージング装置によって測定することもできる。 As means for measuring nerve activity, in addition to microelectrode arrays, fluorescent calcium indicators such as calcium-sensitive dyes and calcium-sensitive fluorescent proteins and fluorescent potential indicators such as voltage-sensitive dyes and voltage-sensitive fluorescent proteins are used (Grienberger, C. and Konnerth, A., Neuron 73, 862-885, 2012; Antic, S. D., et al. J Neurophysiol 116: 135-152, 2016; Miller, E. W., Curr Opin Chem Biol. 33: 74- 80, 2016). Using these indicators, fluctuations in calcium and potential of nerve cells included in the nerve cell device of the present invention can also be measured by a cell imaging device.
　次に実施例を挙げて本発明を詳細に説明するが、本発明はこれに限定されない。 Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
〔ファイバーデバイスの作製〕
（１）ランダムファイバーシートの作製
　PLGA（SIGMA P1941）またはPSU（SIGMA 182443）を、HFIP（和光純薬、089-04233）によって20重量%濃度になるように室温で溶解し、その溶解液をシリンジ（Norm-Ject Syringes 5 ml容量、大阪ケミカル）に充填後、ナノファイバー電界紡糸装置NANON-03（株式会社メック）に装着し、プレートコレクター上に、PLGAの場合、針直径22 G、電圧：20 kVおよび送り速度：1 ml/hの条件下で、また、PSUの場合、針直径27G、電圧：15 kVおよび送り速度：1 ml/hの条件下でランダムファイバーシートを作製した。
（２）配向性ファイバーシートの作製
　配向性PLGAファイバーシートは、PLGA（SIGMA P1941）をHFIP（和光純薬、089-04233）によって20重量%濃度になるように室温で溶解し、その溶解液をシリンジ（Norm-Ject Syringes 5 ml容量、大阪ケミカル）に充填後、ナノファイバー電界紡糸装置NANON-03（株式会社メック）に装着し、ドラムコレクター上に針直径22 G、電圧：20 kV、送り速度：1 ml/hおよび回転速度：750 rpmの条件下で作製した。配向性PSファイバーシートは、PS（Fluka）をDMF（N,N-ジメチルホルムアミド、和光純薬）によって30重量%濃度になるように室温で溶解し、その溶解液をシリンジ（Norm-Ject Syringes 5 ml容量、大阪ケミカル）に充填後、ナノファイバー電界紡糸装置NANON-03（株式会社メック）に装着し、ドラムコレクター上に針直径25 G、電圧：10 kV、送り速度：1.5 ml/hおよび回転速度：2000 rpmの条件下でファイバーシートを作製した。
（３）ファイバーシートへのフレーム接着
　作製されたファイバーシートに、シリコーン一液縮合型RVTゴム（信越化学、カタログ番号KE-45）を用いて、ポリカーボネート製のフレーム（15 mm×15 mm）またはステンレス製の円形フレーム（外径6 mm、内径3 mm）を接着させ、ファイバーデバイスを作製した。 [Production of fiber device]
(1) Preparation of random fiber sheet PLGA (SIGMA P1941) or PSU (SIGMA 182443) is dissolved at room temperature by HFIP (Wako Pure Chemicals, 089-04233) to a concentration of 20% by weight, and the solution is syringed. (Norm-Ject Syringes, 5 ml capacity, Osaka Chemical), and then mounted on a nanofiber electrospinning device NANON-03 (Mec Co., Ltd.). A random fiber sheet was prepared under the conditions of kV and a feed rate of 1 ml / h, and in the case of PSU, under the conditions of a needle diameter of 27 G, a voltage of 15 kV and a feed rate of 1 ml / h.
(2) Preparation of Oriented Fiber Sheet Oriented PLGA fiber sheet is prepared by dissolving PLGA (SIGMA P1941) at room temperature with HFIP (Wako Pure Chemicals, 089-04233) to a concentration of 20% by weight. After filling into a syringe (Norm-Ject Syringes 5 ml capacity, Osaka Chemical), it was mounted on a nanofiber electrospinning machine NANON-03 (Mec Co., Ltd.), and the needle diameter was 22 G on a drum collector, voltage: 20 kV, feed rate : 1 ml / h and rotation speed: 750 rpm. Oriented PS fiber sheet is prepared by dissolving PS (Fluka) with DMF (N, N-dimethylformamide, Wako Pure Chemical) at room temperature so as to have a concentration of 30% by weight, and dissolving the solution with a syringe (Norm-Ject Syringes 5). After filling into a nanofiber electrospinning device NANON-03 (Mec Co., Ltd.), needle diameter 25 G on a drum collector, voltage: 10 kV, feed rate: 1.5 ml / h and rotation A fiber sheet was produced under the conditions of speed: 2000 rpm.
(3) Adhesion of frame to fiber sheet Polycarbonate frame (15 mm x 15 mm) or stainless steel, using silicone one-component condensation type RVT rubber (Shin-Etsu Chemical, catalog number KE-45), is applied to the prepared fiber sheet. A circular frame (outer diameter 6 mm, inner diameter 3 mm) was bonded to produce a fiber device.
〔ヒトiPS細胞由来神経細胞の培養〕
　実施例１で作製した配向性PLGAファイバーシートに対し、SureBond（登録商標）コーティング溶液（Axol Bioscience社（英国）、カタログ番号ax0052）を加え、5％CO2環境下、37℃で1時間前処理した。コーティング処理した配向性PLGAファイバーシートおよびMEA電極チップMEDプローブ標準4×4配列／MED-PG515Aおよび4×4配列／MED-Q2430M、アルファメッドサイエンティフィック社）を細胞足場として使用し、ヒトiPS細胞由来大脳皮質ニューロン（Axol Bioscience社（英国）、カタログ番号ax0019）を、8×105細胞／cm2の密度で、これらの細胞足場上に播種した。これらを、BrainPhys培地（STEMCELL technologies社、米国）を用いて、5％CO2、37℃のインキュベータ中で2週間培養した。培養後、得られた細胞を光学顕微鏡で観察した結果を、配向性PLGAファイバーシート上の細胞については図１Ａに示し、またMEA電極チップ上の細胞については図１Ｂに示した。MEA電極チップ上では、細胞が凝集してスフェロイド状となり、電極から脱離することが示された。一方、配向性PLGAファイバーシート上では、細胞が均一に広がって生育していることが示された。 (Culture of neurons derived from human iPS cells)
A SureBond (registered trademark) coating solution (Axol Bioscience (UK), catalog number ax0052) was added to the oriented PLGA fiber sheet prepared in Example 1, and pretreated at 37 ° C for 1 hour in a 5% CO 2 environment. did. Using coated PLGA fiber sheet and MEA electrode tip MED probe standard 4 × 4 array / MED-PG515A and 4 × 4 array / MED-Q2430M, Alpha Med Scientific) as a cell scaffold, human iPS cells Derived cerebral cortical neurons (Axol Bioscience (UK), catalog number ax0019) were seeded onto these cell scaffolds at a density of 8 × 10 5 cells / cm 2 . These were cultured for 2 weeks in a 5% CO 2 , 37 ° C. incubator using BrainPhys medium (STEMCELL technologies, USA). After the culture, the obtained cells were observed with an optical microscope. The results on the cells on the oriented PLGA fiber sheet are shown in FIG. 1A, and those on the MEA electrode chip are shown in FIG. 1B. On the MEA electrode chip, it was shown that the cells aggregated into spheroids and detached from the electrode. On the other hand, it was shown that the cells spread uniformly and grew on the oriented PLGA fiber sheet.
〔細胞足場上で培養した神経細胞のMEAによる細胞外電位測定（１）〕
　ヒトiPS細胞由来大脳皮質ニューロン（Axol Bioscience社、英国）を、細胞足場として実施例１で作製した配向性PLGAファイバーシート上に、8×105細胞／cm2または24×105細胞／cm2の密度で播種し、実施例２と同一の条件下で4週間、5％CO2、37℃のインキュベータ中で培養した。得られた細胞シートを微小電極アレイ（MEA）プローブ（MED64システム、アルファメッドサイエンティフィック社）上に載せて、細胞とMEAプローブの電極とを接触させ、神経活動（スパイク発火および同期バースト発火）を測定した。並行して、MEAプローブ上に直接神経細胞を8×105細胞／cm2で播種し、4週間、5％CO2、37℃のインキュベータ中で培養し、同様に神経活動を測定した。それらの結果を図２に示した。MEAのプローブ上に直接播種した細胞（従来方法）に比較して、配向性PLGAファイバーシート上に高密度で播種した細胞では、MEAプローブ上の細胞では神経活動が十分に観察されない時期に、高頻度でスパイクが確認されることが示された。また、強い同期バーストが観察された。配向性PLGAファイバーシート上で、高頻度のスパイクおよび強い同期バーストが生じる現象は、8×105細胞／cm2の密度で播種した場合より、より高密度の24×105細胞／cm2で播種した場合に顕著に観察された。これらのことより、配向性PLGAファイバーシートを用いて神経細胞を培養することにより、形成された神経ネットワークの機能的成熟化が促進されたことが示された。さらに、配向性PLGAファイバーシートに対して神経細胞をより高密度で播種することにより、機能的成熟化が促進されることも示された。 [Measurement of extracellular potential of neurons cultured on cell scaffold by MEA (1)]
Human iPS cell-derived cerebral cortical neurons (Axol Bioscience, UK) were used as cell scaffolds on the oriented PLGA fiber sheet prepared in Example 1 at 8 × 10 5 cells / cm 2 or 24 × 10 5 cells / cm 2. And cultured for 4 weeks in an incubator at 37 ° C. with 5% CO 2 under the same conditions as in Example 2. The obtained cell sheet is placed on a microelectrode array (MEA) probe (MED64 system, Alpha Med Scientific), and the cells are brought into contact with the electrodes of the MEA probe, and neural activity (spike firing and synchronous burst firing) is performed. Was measured. In parallel, nerve cells were seeded directly on the MEA probe at 8 × 10 5 cells / cm 2 , cultured for 4 weeks in a 5% CO 2 , 37 ° C. incubator, and the nerve activity was measured similarly. The results are shown in FIG. Compared to cells seeded directly on MEA probes (conventional method), cells seeded at a higher density on oriented PLGA fiber sheets have higher levels of activity when neural activity is not sufficiently observed on cells on MEA probes. Spikes were confirmed by frequency. Also, strong synchronization bursts were observed. In orientation PLGA fibers sheet, a phenomenon spikes and strong synchronization bursts of the high frequency occurs, at 8 × 10 5 cells / than were seeded at a density of cm 2, higher density of 24 × 10 5 cells / cm 2 It was noticeably observed when seeded. These results indicated that the functional maturation of the formed neural network was promoted by culturing neurons using the oriented PLGA fiber sheet. In addition, it was shown that functional maturation was promoted by seeding neurons at higher density on oriented PLGA fiber sheets.
〔神経細胞のスパイク発火頻度および薬物応答性に対する細胞足場および細胞播種密度の影響〕
　実施例１で作製した配向性PSファイバーシートを、ポリ-D-リジンおよびラミニンによりコーティング処理した。コーティング処理した配向性PSファイバーシート上に、ヒトiPS細胞由来ニューロン（XCL-1ニューロン、XCell Science社、米国）を、3×105細胞／cm2（低密度播種）または12×105細胞／cm2（高密度播種）の密度で播種し、5％CO2、37℃のインキュベータ中で2、4および6週間培養後、実施例３と同様にして神経活動を測定した。並行して、MEAプローブ上に直接神経細胞を3×105細胞／cm2で播種し、同様に神経活動を測定した。6週間培養時の神経活動を測定した後、ニューロンの培養液に100μM 4-アミノピリジン（4-AP）を添加し、引き続き神経活動を測定した。これらの結果を図３Ａ～Ｃに示した。 [Effect of cell scaffolding and cell seeding density on spike firing frequency and drug responsiveness of nerve cells]
The oriented PS fiber sheet produced in Example 1 was coated with poly-D-lysine and laminin. On a coated oriented PS fiber sheet, human iPS cell-derived neurons (XCL-1 neurons, XCell Science, USA) were cultured at 3 × 10 5 cells / cm 2 (low density seeding) or 12 × 10 5 cells / cell. The cells were seeded at a density of cm 2 (high-density seeding), cultured in a 5% CO 2 , 37 ° C. incubator for 2, 4 and 6 weeks, and then the nerve activity was measured in the same manner as in Example 3. In parallel, nerve cells were seeded directly on the MEA probe at 3 × 10 5 cells / cm 2 , and nerve activity was measured similarly. After measuring the nerve activity during the 6-week culture, 100 μM 4-aminopyridine (4-AP) was added to the culture solution of the neurons, and the nerve activity was subsequently measured. These results are shown in FIGS. 3A to 3C.
　神経細胞を播種して所定期間培養した後、MEAプローブ上に直接播種した細胞と、配向性PSファイバーシート上に低密度播種または高密度播種した細胞との間で、神経活動であるスパイク発火の頻度を比較した。2週間培養後では、MEAプローブ上に直接播種した細胞（従来方法）に比較して、配向性PSファイバーシート上に高密度播種した細胞において高頻度でスパイクが確認された（図３Ａ）。一方、4週間培養後では、配向性PSファイバーシート上に低密度播種した細胞において、従来方法による細胞と比較して、高頻度でスパイクが確認された（図３Ｂ）。6週間培養後では、従来方法による細胞（MEAプローブ上に直接播種した細胞）と比較して、播種密度に関わらず、配向性PSファイバーシート上に播種した細胞において高頻度でスパイクが確認された。高頻度のスパイクは、配向性PSファイバーシート上に低密度播種した細胞に比較して、高密度播種した細胞において顕著に観察された（図３Ｃ）。 After seeding the nerve cells and culturing for a predetermined period, the spike firing, which is a neural activity, is performed between the cells directly seeded on the MEA probe and the low-density or high-density seeded cells on the oriented PS fiber sheet. The frequencies were compared. After two weeks of culture, spikes were observed more frequently in cells seeded at high density on oriented PS fiber sheets than in cells seeded directly on MEA probes (conventional method) (FIG. 3A). On the other hand, after culturing for 4 weeks, spikes were observed more frequently in cells seeded at low density on the oriented PS fiber sheet as compared to cells by the conventional method (FIG. 3B). After culturing for 6 weeks, spikes were observed more frequently in cells seeded on oriented PS fiber sheets, regardless of seeding density, compared to cells obtained by the conventional method (cells seeded directly on the MEA probe). . High frequency spikes were significantly observed in cells seeded at high density compared to cells seeded at low density on oriented PS fiber sheets (FIG. 3C).
　6週間培養した神経細胞の薬物応答性を検討するため、4-APを神経細胞の培養液に添加し、スパイク発火の頻度に対する効果を測定した。4-APは、細胞膜上のカリウムチャネルをブロックすることにより、神経細胞の膜電位の変化を誘導する薬物である。4-APの添加により、MEAプローブ上に直接播種した細胞および配向性PSファイバーシート上に低密度播種または高密度播種した細胞は、すべて4-APに対して応答し、4-AP無添加の場合と比較してスパイク数を増加させた（図３Ｃ）。しかしながら、従来方法による細胞（MEAプローブ上に直接播種した細胞）と比較して、配向性PSファイバーシート上に播種した細胞においてスパイク数が多く、この現象は、配向性PSファイバーシート上に高密度で神経細胞を播種した場合に明瞭であった。これらの結果より、配向性PSファイバーシートを用いて神経細胞を培養することにより、神経ネットワークの機能的成熟化が促進されたことが示された。さらに、配向性PSファイバーシートに対して神経細胞をより高密度で播種することにより、機能的成熟化が促進されることが示された。 To examine the drug response of neurons cultured for 6 weeks, 4-AP was added to the culture of neurons and the effect on spike firing frequency was measured. 4-AP is a drug that induces changes in the membrane potential of nerve cells by blocking potassium channels on the cell membrane. Due to the addition of 4-AP, cells seeded directly on the MEA probe and cells seeded at low density or high density on the oriented PS fiber sheet all responded to 4-AP, The number of spikes was increased compared to the case (FIG. 3C). However, compared to cells according to the conventional method (cells seeded directly on the MEA probe), the number of spikes is higher in cells seeded on the oriented PS fiber sheet. It was clear when the neurons were seeded in. These results indicated that the functional maturation of the neural network was promoted by culturing neurons using the oriented PS fiber sheet. Furthermore, it was shown that seeding neurons at higher density on oriented PS fiber sheets promotes functional maturation.
〔細胞足場上で培養した神経細胞のMEAによる細胞外電位測定（２）〕
　実施例１（３）で作製したステンレス製の円形フレームを装着した配向性PSファイバーシートを、卓上プラズマ処理装置（ストレックス社）を用いるプラズマ処理によって親水化した後、ポリ-D-リジンおよびラミニンによりコーティング処理した。コーティング処理した配向性PSファイバーシート上に、ヒトiPS細胞由来神経細胞（XCL-1ニューロン、XCell Science社、米国）を、3×105細胞／cm2または9×105細胞／cm2の密度で、またはヒトiPS細胞由来大脳皮質ニューロン（Axol Bioscience社、英国）を、8×105細胞／cm2または24×105細胞／cm2の密度で播種し、5％CO2、37℃のインキュベータ中で4週間培養した。得られた細胞シートを、微小電極アレイ（MEA）MED64-Presto（アルファメッドサイエンティフィック社）のプローブ上に載せて、細胞とMEAプローブの電極とを接触させ、神経活動電位を測定した。並行して、MEAプローブ上に直接神経細胞を、3×105細胞／cm2（XCell Science社、XCL-1ニューロンの場合）または8×105細胞／cm2（Axol Bioscience社、ヒトiPS細胞由来大脳皮質ニューロンの場合）で播種し、4週間培養した。 [Measurement of extracellular potential of neurons cultured on cell scaffold by MEA (2)]
The oriented PS fiber sheet provided with the stainless steel circular frame prepared in Example 1 (3) was hydrophilized by plasma treatment using a desktop plasma treatment device (Strex), and then poly-D-lysine and laminin were used. Coating treatment. On the coated oriented PS fiber sheet, human iPS cell-derived neurons (XCL-1 neuron, XCell Science, USA) were cultured at a density of 3 × 10 5 cells / cm 2 or 9 × 10 5 cells / cm 2 . Or human iPS cell-derived cerebral cortical neurons (Axol Bioscience, UK) were seeded at a density of 8 × 10 5 cells / cm 2 or 24 × 10 5 cells / cm 2 and 5% CO 2 , 37 ° C. Cultured for 4 weeks in an incubator. The obtained cell sheet was placed on a probe of a microelectrode array (MEA) MED64-Presto (Alpha Med Scientific), and the cell was brought into contact with the electrode of the MEA probe to measure a nerve action potential. In parallel, neural cells were directly plated on the MEA probe at 3 × 10 5 cells / cm 2 (for XCell Science, XCL-1 neurons) or 8 × 10 5 cells / cm 2 (Axol Bioscience, human iPS cells (In case of derived cerebral cortical neurons) and cultured for 4 weeks.
　ヒトiPS細胞由来神経細胞XCL-1ニューロン（XCell Science社）における細胞外電位の測定結果を図４に示した。図４ＡおよびＢの比較により、同じ細胞播種密度であっても、配向性PSファイバーシートを用いて培養した細胞において、MEAプローブ上で培養した細胞より強い同期バーストが観察された。また、配向性PSファイバーシートに対して高密度で細胞を播種し、培養した細胞は、低密度で細胞播種した場合およびMEAプローブ上に直接播種した場合と比較して、高頻度のスパイク発火および強い同期バースト発火が観察された（図４ＣおよびＤ）。 FIG. 4 shows the results of measurement of extracellular potential in human iPS cell-derived neuronal XCL-1 neurons (XCell Science). 4A and 4B, even at the same cell seeding density, a stronger synchronous burst was observed in the cells cultured using the oriented PS fiber sheet than in the cells cultured on the MEA probe. In addition, cells were seeded at a high density on the oriented PS fiber sheet, and the cultured cells showed higher spike firing and higher frequency than when the cells were seeded at a low density and directly on the MEA probe. Strong synchronous burst firing was observed (FIGS. 4C and D).
　ヒトiPS細胞由来大脳皮質ニューロン（Axol Bioscience社）における細胞外電位の測定結果を図５に示した。配向性PSファイバーシート上に播種して培養した細胞では、MEAプローブ上に直接播種して培養した細胞に比較して、高頻度のスパイク発火および強い同期バースト発火が明瞭に観察された。 FIG. 5 shows the measurement results of extracellular potentials in human iPS cell-derived cerebral cortical neurons (Axol Bioscience). In the cells seeded and cultured on the oriented PS fiber sheet, higher frequency spike firing and strong synchronous burst firing were clearly observed compared to the cells seeded and cultured directly on the MEA probe.
　以上の結果より、異なる供給元の神経細胞を用いる場合であっても、ファイバーシートを用いる本発明の方法により、神経ネットワークを形成する神経細胞の機能的な成熟が促進されることが確認された。 From the above results, it was confirmed that the functional maturation of the nerve cells forming the nerve network was promoted by the method of the present invention using the fiber sheet even when the nerve cells from different sources were used. .

Claims (11)

  1. 　細胞足場を用いて神経細胞を培養することを含む、神経ネットワークを機能的に成熟化させる方法。 方法 A method for functionally maturating a neural network, comprising culturing neural cells using a cell scaffold.
  2. 　前記細胞足場が高分子材料で形成されたファイバーシートである、請求項１に記載の方法。 The method according to claim 1, wherein the cell scaffold is a fiber sheet formed of a polymer material.
  3. 　前記ファイバーシートが、配向性構造、非配向性構造または配向性と非配向性との混合構造を有する、請求項２に記載の方法。 The method according to claim 2, wherein the fiber sheet has an oriented structure, a non-oriented structure, or a mixed structure of oriented and non-oriented.
  4. 　前記ファイバーシートが、ポリリジン、ポリオルニチン、ラミニン、フィブロネクチン、マトリゲル（登録商標）およびゲルトレックス（登録商標）から選ばれる細胞外マトリックスタンパク質でコーティングされた、請求項２または３に記載の方法。 The method according to claim 2 or 3, wherein the fiber sheet is coated with an extracellular matrix protein selected from polylysine, polyornithine, laminin, fibronectin, Matrigel (registered trademark) and Geltrex (registered trademark).
  5. 　前記ファイバーシートが、ポリエチレンイミンでコーティングされた、請求項２または３に記載の方法。 The method according to claim 2 or 3, wherein the fiber sheet is coated with polyethyleneimine.
  6. 　前記神経細胞が、細胞足場上および／または細胞足場内で３次元構造を形成した、請求項１～５のいずれか１項に記載の方法。 The method according to any one of claims 1 to 5, wherein the nerve cell has formed a three-dimensional structure on and / or within the cell scaffold.
  7. 　前記神経細胞が、初代培養細胞または多能性幹細胞由来の神経細胞である、請求項１～６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 6, wherein the nerve cell is a primary cultured cell or a neural cell derived from a pluripotent stem cell.
  8. 　前記初代培養細胞または前記多能性幹細胞由来の神経細胞が、哺乳類由来の神経細胞である、請求項７に記載の方法。 8. The method according to claim 7, wherein the primary cultured cells or the pluripotent stem cell-derived nerve cells are mammalian-derived nerve cells.
  9. 　前記神経細胞が、グルタミン酸作動性、ドーパミン作動性、γ-アミノ酪酸作動性、モノアミン作動性、ヒスタミン作動性またはコリン作動性の神経細胞を含む、請求項１～８のいずれか１項に記載の方法。 The method according to any one of claims 1 to 8, wherein the nerve cells include glutamatergic, dopaminergic, γ-aminobutyratergic, monoaminergic, histaminergic or cholinergic neurons. Method.
  10. 　請求項１～９のいずれか１項に記載の方法を使用して機能的に成熟化させた神経細胞を用いる、神経活動の評価方法。 方法 A method for evaluating nerve activity, using a nerve cell functionally matured using the method according to any one of claims 1 to 9.
  11. 　請求項１～９のいずれか１項に記載の方法を使用して機能的に成熟化させた神経細胞を微小電極アレイと接触させ、該神経細胞の細胞外電位を測定することを含む、神経活動の評価方法。 10. A nerve comprising contacting a nerve cell functionally matured using the method of any one of claims 1 to 9 with a microelectrode array and measuring the extracellular potential of said nerve cell. Activity evaluation method.
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