WO2020013269A1 - Procédé de maturation fonctionnelle de neurones - Google Patents

Procédé de maturation fonctionnelle de neurones 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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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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    • 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

La présente invention concerne un procédé permettant de favoriser la maturation fonctionnelle d'un réseau neuronal par la culture de neurones sur une feuille de fibres servant d'échafaudage cellulaire.
PCT/JP2019/027476 2018-07-12 2019-07-11 Procédé de maturation fonctionnelle de neurones WO2020013269A1 (fr)

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