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WO2022154080A1 - Tissu de circuit neuronal in vitro présentant une activité et une plasticité, dispositif d'observation de ladite activité neuronale et procédé de criblage de substances à l'aide dudit dispositif - Google Patents

Tissu de circuit neuronal in vitro présentant une activité et une plasticité, dispositif d'observation de ladite activité neuronale et procédé de criblage de substances à l'aide dudit dispositif Download PDF

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WO2022154080A1
WO2022154080A1 PCT/JP2022/001090 JP2022001090W WO2022154080A1 WO 2022154080 A1 WO2022154080 A1 WO 2022154080A1 JP 2022001090 W JP2022001090 W JP 2022001090W WO 2022154080 A1 WO2022154080 A1 WO 2022154080A1
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activity
neural
organoids
neural circuit
tissue according
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与志穂 池内
達哉 大崎
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University of Tokyo NUC
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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

Definitions

  • the present invention relates to an in vitro induced neural circuit tissue exhibiting complex activity and plasticity, an observation device thereof, and a method for screening a substance that changes the neural activity of the neural circuit tissue using the observation device.
  • the organoid production technique is a technique for three-dimensionally modeling an animal organ in vitro, and can be used for functional analysis of various organs in a state closer to in vivo. So far, brain organoids including the cerebral cortex, thalamus, cerebellum, hippocampus and choroid plexus have been reported (see Non-Patent Documents 1 to 6 and the like), and a new platform for studying human brain regions in vitro has been reported. It is being offered. However, although the structural and morphological characteristics of brain organoids reported so far are similar to those of the human brain at the developmental stage, it is considered that functional improvements are required. There is.
  • a brain organoid model tissue having the property of exchanging activities between regions and having plasticity as a neural circuit is particularly required. Further, in order to obtain an organoid showing a complicated activity pattern, long-term (3 months or more) culture is required, and a method for obtaining an organoid showing a complicated activity in a short culture period is required.
  • Non-Patent Document 7 and Patent Document 1 As an in vitro model of nerve tissue, brain organoids connected by axon bundles have been reported (Non-Patent Document 7 and Patent Document 1).
  • This "connected organoids” model is constructed by connecting two brain organoids via a bundle of axons extending from each organoid. This model reflects the state of the in vivo brain in which brain regions are connected by axon bundles, and is considered to be a very useful model in brain research.
  • “fused organoids” technology has also been developed in which two or more brain organoids are directly fused by adjoining them (Non-Patent Document 8 and the like). However, it was unclear what kind of activity and function these in vitro neural circuit models exert.
  • an object of the present invention to obtain an in vitro induced neural circuit tissue capable of exchanging neural activity between regions and exhibiting complex activity and plasticity. Another object of the present invention is to provide an observation device for neural activity of neural circuit tissues. Furthermore, it is an object of the present invention to provide a method for screening a substance that changes the neural activity of the neural circuit tissue by using the neural network tissue induced in vitro and this observation device.
  • the present inventors cultivate a plurality of brain organoids induced to differentiate from human iPS cells on a multi-electrode array (chip having a plurality of electrodes on the array; MEA), and the brain organoids are connected by an axon bundle.
  • the nerve tissue was prepared and the neuronal activity occurring in the nerve tissue was analyzed.
  • we succeeded in detecting extremely complex neuronal activity from organoids connected by axon bundles hereinafter also referred to as "connected (brain) organoids").
  • the neuronal activity of the connecting brain organoid was detected as spontaneous firing activity after a relatively short period of culture (about 7-8 weeks after the iPS cell culture).
  • the connected organoids performed burst activity, and the coefficient of variation of the frequency of burst activity was 0.2 or more.
  • the local field potential (LFP) detected from the nerve tissue is characterized by the ⁇ wavelength band component (0.5 Hz-4 Hz) and / or the ⁇ wavelength body component (4.0 Hz-8.0 Hz) element. It was something that could be done.
  • Neural circuit tissue induced in vitro in which two or more nerve cell clusters are connected via axons (also referred to as "connecting organoids").
  • the nerve cell mass may be an organoid.
  • the two or more nerve cell clusters exhibit interrelated activities.
  • the interrelated activities can occur with a time lag of less than 500 milliseconds. Also, the interrelated activities can be synchronous activities.
  • Spontaneous ignition activity is performed, and the spontaneous ignition activity may be 50 times or more per minute.
  • Burst activity is performed, and the coefficient of variation of the frequency of the burst activity can be 0.2 or more.
  • the activity of each nerve cell mass connected via the axon exhibits coherence, and the interference may differ from frequency band to frequency band.
  • the local field potential detected from the nerve cell mass includes a ⁇ wavelength band (0.5 Hz-4.0 HZ) component and / or a ⁇ wavelength band (300 Hz-3000 Hz) component. Further, the local field potential may further include a ⁇ wavelength band (4.0 Hz-8.0 Hz) component.
  • the nerve cell mass may be one in which differentiation is induced from pluripotent stem cells, and the nerve cell mass may be formed by culturing pluripotent stem cells for 6 weeks or more. Further, the pluripotent stem cell can be an iPS cell (induced pluripotent stem cell).
  • An in vitro induced neural activity observation device in which two or more nerve cell masses are connected via an axon, and is a substrate and the substrate for accommodating the nerve cell mass.
  • a plurality of wells provided on the surface of the above, and an induction groove provided by connecting the wells for inducing and extending the axons so as to connect the nerve cell masses to each other.
  • Each well is provided with an electrode and further includes an analysis unit that analyzes the correlation of electrical signals from the electrode.
  • the analysis unit separates each of the electric signals for each frequency band and analyzes the correlation by phase amplitude coupling.
  • the analysis unit analyzes by wavelet coherence.
  • the analysis unit classifies the neural activity into any of action potential, burst activity, brain hypothesis, and local field potential.
  • the analysis unit has a signal pattern of the electric signal corresponding to the nerve activity in advance, and classifies the nerve activity by collating with the signal pattern.
  • the nerve cell mass and / or the axon can be externally stimulated to form the nerve activity, and the obtained signal pattern can be given in advance.
  • the stimulus can be any one or more of light irradiation, electrical stimulation or compound administration. Also, the stimulus can enhance or suppress the neural activity.
  • the electrode is formed by arranging a plurality of electrodes in an array at the bottom of the well.
  • the electrode receives the electric signal from the nerve cell mass and electrically stimulates the nerve cell mass.
  • the substrate is transparent so that the nerve cell mass in the well can be optically observed from the bottom.
  • a light irradiation device for optically stimulating the nerve cell mass and / or the axon is provided so as to face the surface of the substrate.
  • the light irradiation device includes an irradiation unit that locally applies light irradiation to the nerve cell mass and / or a part of the axon.
  • An in vitro induced neural activity observation device in which two or more nerve cell masses are connected via an axon, the substrate and the above-mentioned device for accommodating the nerve cell mass.
  • the wells include a plurality of wells provided on the surface of the substrate and a guide groove provided by connecting the wells for guiding and extending the axons so as to connect the nerve cell clusters.
  • Each of the above is provided with an optical system for measuring the light intensity from the nerve cell mass, and further includes an analysis unit for acquiring the correlation of the light intensity signal from each of the nerve cell clusters in the well.
  • (22) Two wells are provided to the substrate, the first objective lens of the optical system is brought close to one of the wells from one surface of the substrate, and the second objective lens is the other of the substrate. It can be provided close to the other side of the well from the surface of the above.
  • the emission from the calcium fluorescent probe can be detected, and the increase in calcium ion concentration in the nerve cell mass caused by the action potential can be detected.
  • the light beam for causing the light emission can be incident from the side end surface of the substrate.
  • a method of screening a substance that changes the neural activity of a neural circuit tissue using an observation device having the above-mentioned characteristics The target neural circuit tissue is set in the observation device, and the substance is used as the nerve. It is characterized in that it is given to a circuit tissue and the fluctuation of the neural activity is observed.
  • the symbol of "-" indicates a numerical range including the values to the left and right of the reference numeral.
  • the neural circuit tissue according to the present invention mimics the function of the brain in vitro, and by combining with the observation device according to the present invention, various useful information can be obtained by observing (analyzing) this. For example, it is possible to screen for substances that may fluctuate the neural activity of the brain by using the neural circuit tissue and the observation device, which is one of the neural circuit tissues according to the present invention and is composed of cerebral organoids. Such findings are useful for searching for therapeutic agents for psychiatric disorders and neurodegenerative diseases as a brain disease model including disorders of higher brain function.
  • the first embodiment of the present invention is a neural circuit tissue induced in vitro, in which two or more nerve cell clusters are connected via an axon, a neural circuit tissue (neural circuit according to the present embodiment).
  • the "cell mass” is a cell population in which cells adhere to each other to form a three-dimensional structure and have a structure similar to the state of existence in a living body.
  • a nerve cell mass is a cell mass composed of nerve cells.
  • An "organoid” is a complex cell cluster consisting of cells derived from or organ-specific cells such as the brain, stomach, liver, and bladder.
  • Organoids can be produced by self-aggregating pluripotent stem cells and the like (see, for example, Non-Patent Documents 1 to 7).
  • organoid means a brain organoid, that is, an organoid containing nerve cells, unless otherwise specified.
  • the neural circuit tissue has a minimum unit of a tissue in which nerve cell clusters such as two brain organoids are connected to each other by a bundle of axons, and three or more nerve cell clusters are mutually axon bundles. It may be connected with.
  • the "neural circuit tissue” is a nerve tissue in which neurons are connected to each other via an axon, and the activity of nerve cells in one brain organoid or nerve cells as a group is the activity of another brain. It is an organization that can interfere with the activity of organoids. This can be defined, for example, by the spontaneous firing activity of each cerebral organoid, the synchrony or asynchrony of neural activity between two or more cerebral organoids, the strength of PAC, and the like.
  • the neural circuit tissue is prepared, for example, by placing a nerve cell mass such as one organoid in a small hole (well) connected by a guide (thin groove) for extending an axon and culturing it. can do.
  • Organoids can be produced by culturing pluripotent stem cells such as iPS (induced pluripotent stem cells) cells for a period of about 4 to 6 weeks under appropriate culture conditions (of the nerve tissue according to this embodiment).
  • pluripotent stem cells such as iPS (induced pluripotent stem cells) cells for a period of about 4 to 6 weeks under appropriate culture conditions (of the nerve tissue according to this embodiment).
  • iPS induced pluripotent stem cells
  • the nerve cell mass becomes "spontaneous firing activity" (from the outside world) 1.5 weeks or 2 weeks after the start of the culture of the nerve cell mass. Even if there is no stimulus, a phenomenon that causes spikes with a short time width) will occur.
  • the number of spontaneous firing activities is, for example, 50 times or more per minute.
  • the "activity" is a change in a nerve cell caused by an action potential generated in the nerve tissue.
  • an action potential Starting from a change in the membrane potential that occurs in the cell membrane due to synaptic activity, the diffusion of sodium ions and potassium ions on the cell membrane by voltage-gated ion channels due to the difference in concentration inside and outside the cell is generally called an action potential.
  • the membrane potential cannot be measured directly by MEA, it is possible to measure the weak change in electrical signal caused by the action potential outside the cell near the nerve cell (extracellular recording).
  • extracellular recording In calcium imaging, the increase in intracellular calcium ion concentration (Ca 2+ ) caused by action potential can be observed by detecting the luminescence from the calcium fluorescent probe with a microscope or the like. Changes caused by nerve action potentials recorded by techniques such as those given in these examples are referred to as "activity" of nerve cells and neural circuit tissues.
  • the neural circuit tissue causes a "burst activity" in which a plurality of action potentials are collectively fired at a high frequency.
  • Burst activity can be triggered by the continuous activity of one nerve cell. It can also be caused by the continuous activity of multiple nerve cells. During burst activity, the frequency of observed activity is significantly higher than during non-burst activity.
  • the characteristic of burst activity in the nervous tissue is that the coefficient of variation of the frequency of burst activity is, for example, 0.2 or more.
  • the coefficient of variation is a value obtained by dividing the standard deviation of the burst frequency by the average.
  • a coefficient of variation of burst activity of 0.2 or more is an indicator that the activity of neural circuit tissue has "complexity".
  • LFP Local field potential
  • This local field potential includes a ⁇ wavelength band (0.5 Hz-4.0 HZ) component, a ⁇ wavelength band (300 Hz-3000 Hz) component, and / or a ⁇ wavelength band (4.0 Hz-8.0 Hz) component.
  • the neural cell masses in the neural circuit tissue show interrelated activities.
  • Interrelated activity refers to the neural activity in each neural mass, where any association is found between the connected neural masses, the neural activity in each, or the neuronal activity.
  • organoid 1 and organoid 2 are connected as an example, when activity or burst activity occurs in organoid 1, when activity or burst activity occurs in organoid 2 at about the same time, that is, organoid 1
  • organoid 1 it can be said that the neural circuit tissue has performed interrelated activities when the neural activities in the organoid 2 and the organoid 2 are synchronized.
  • the activity performed by each nerve cell mass of the two nerve cell masses occurs simultaneously or at short time intervals.
  • the activity (related activity) of each nerve cell mass is an activity performed within 1,000 milliseconds, preferably within 500 milliseconds, and more preferably within 100 milliseconds. Showing interrelated activities with time or phase differences within an organization is also an indicator of "complexity.”
  • the activities performed by each nerve cell mass connected by the axon bundle may interfere with each other (coherence), and this interference is one of the characteristics of the neural circuit tissue. ..
  • This interference may vary from frequency band to activity pattern.
  • This interference for each frequency band is measured as a degree of linear relevance at each frequency by performing wavelet coherence analysis based on the time series data of the two active waves detected from each nerve cell mass. Can be represented.
  • the wavelet coherence analysis has been described in detail in the Examples section, so please refer to it. It is also an index of "complexity" that different sites in the neural circuit tissue show wavelet coherence.
  • the neural circuit tissue according to the present embodiment is characterized by having plasticity against external stimuli (for example, light irradiation).
  • plasticity refers to the property of responding to an external stimulus and changing the activity pattern after the stimulus.
  • the neural circuit tissue is characterized by having plasticity.
  • means for detecting the neuron activity detected from the neural network tissue may be arranged.
  • the “means” includes electrodes as the detection means when the neuron activity is detected as an electric signal.
  • the electrical signal of neuron activity is analyzed, for example, by arranging a nerve cell mass on a substrate on which one or more electrodes are arranged and detecting the electrical signal of the activity wave derived from each nerve cell mass from the electrode. be able to.
  • the nerve cell masses are cultured on a substrate provided with a multi-electrode array, and at the same time, an electrical signal obtained from the nerve cell masses is detected. May be good.
  • small holes small holes for inserting the nerve cell mass and the medium capable of culturing the nerve cell mass are arranged on each array of the multi-electrode array, and each of them. Culturing was performed between the small holes using a device in which a groove was arranged so as to serve as an extension guide (also a flow path for the medium) of the axon extending from the nerve cell mass toward another nerve cell mass. You may.
  • the activity of the nerve tissue according to the present embodiment is to manipulate the activity of the nerve tissue (modify the activity of the nerve tissue) by irradiating a part of the nerve tissue (for example, an axon bundle) with light. can.
  • the device When performing such an operation, the device is provided with an element for giving an external stimulus such as light irradiation, for example, a groove (also serving as a flow path for a medium) for arranging an optical fiber or the like. May be. See FIGS. 1A and 1B, which will be described later, as an example of the device.
  • an optical system for detecting light emission emitted from each nerve cell mass is used.
  • a CCD camera or the like that measures the light intensity is used, and the presence or absence of correlation of the light intensity signals from each of the nerve cell clusters is included and analyzed.
  • the first objective lens (Objective 1) of the optical system is brought close to one side of the nerve cell mass from one surface of the substrate, and the second objective lens (Objective 2) is moved from the other surface of the substrate to the nerve cell. The light intensity is measured by bringing it close to the other side of the mass.
  • the light beam for causing fluorescence emission may be incident from the side end surface of the substrate using a transparent substrate.
  • the second embodiment is a method of analyzing neural activity in vitro, in which the activity of each frequency band obtained from the neural cell mass constituting the neural circuit tissue according to the first embodiment is detected and the presence or absence of correlation is present. It is a method including analyzing the detected activity correlation by a method for analyzing the correlation of neural activity such as Phase-Amplitude-Coupring (PAC) and wavelet coherence.
  • PAC Phase-Amplitude-Coupring
  • wavelet coherence the phase amplitude coupling was established to calculate a value that is an index of the relationship between the phase of low frequency activity and the amplitude of high frequency spikes, and to evaluate the relationship between brain waves in different frequency bands.
  • PAC Phase-Amplitude-Coupring
  • the method (Fell et al., Nat. Rev. Neurosci.
  • a delta-gamma (amplitude) PAC can analyze whether the frequency amplitude intensity in the gamma wave region affects the phase in the delta wave region.
  • a delta (phase) -gamma (amplitude) PAC or ata (phase) -gamma (amplitude) PAC correlation rate (Modulation index) is associated with the cultivation of brain organoids. Elevation can explain the maturity of brain organoids in terms of neural activity.
  • the PAC correlation rate of activities in different frequency bands is preferably 0.05 or higher, which is also an indicator of the "complexity" of neural network tissue.
  • a third embodiment is a method of modulating the neural activity of the neural circuit tissue according to the present embodiment, in which the neural activity of the neural network tissue is recorded and the neural network tissue is stimulated by an arbitrary pattern. It is a method including that.
  • the "nerve activity pattern” is a spatiotemporal feature or relationship of the activity of a neural circuit tissue or a nerve cell.
  • the neural activity pattern includes, for example, the frequency of "burst activity” (or “burst-like activity”).
  • Burst activity means that high-frequency neural activity is concentrated in time during a short period of time (for example, 100 ms) (nerve activity is observed at a significantly higher frequency than during non-burst). It is a characteristic neural activity pattern.
  • the frequency is constant, but also the non-constant frequency (the coefficient of variation of the interval between burst activities is small or large) can be said to be a neural activity pattern. Similar to burst activity, neural activity in a specific frequency band and its correlation can be said to be a neural activity pattern. Not only temporal features and correlations, but also the association of neural activity between electrodes (or between observed data of spatially different sites or positions) is captured as part of the neural activity pattern. Therefore, a neurological avalanche is also one of the neural activity patterns. In addition, spatiotemporal correlation of neural activity between connected neural tissues can also constitute a neural activity pattern.
  • stimulating the neural circuit tissue by an arbitrary pattern means applying stimulation to the neural activity at an arbitrary frequency and place determined spatiotemporally in order to induce neural activity. For example, stimulating at any frequency or stimulating any site. It is also possible to stimulate with a random spatiotemporal pattern. The pattern can be determined in advance as desired, but it can also be calculated and determined based on the observed neural activity pattern.
  • stimulation is not particularly limited, but is, for example, a stimulus given by light, electricity, compound administration, or the like.
  • the neural tissue circuit according to the present embodiment is characterized in that its activity is enhanced or suppressed by these stimuli.
  • the fourth embodiment is a method for screening a substance that may fluctuate the neural activity of the brain, in which the candidate substance is brought into contact with the neural circuit tissue according to the present embodiment to detect the neural activity of the neural tissue. It is a screening method including that.
  • the neural circuit tissue according to this embodiment is spontaneously firing and exhibits an activity pattern similar to that of neural activity in the brain in vivo.
  • an external stimulus is repeatedly applied to the nerve tissue (for example, the axon bundle is stimulated by light irradiation)
  • the time to respond to the stimulus becomes shorter as the number of stimuli is increased.
  • the neural circuit tissue according to the present embodiment has a function of storing (remembering) an external stimulus pattern in the tissue. Since the activity supporting the memory in the nerve tissue is suppressed by the CaM kinase II inhibitor, at least the neural circuit tissue according to the first embodiment is the nerve activity of the brain generated during short-term memory. It was suggested that similar activities would be performed.
  • the neural circuit tissue according to this embodiment can be used as a model of the brain that mimics the function of the brain. Therefore, for example, the neural circuit tissue may be brought into contact with a desired substance (NMDA inhibitor, AMPA inhibitor, psychotropic drug, etc.) to detect the effect on excitatory synaptic transmission or inhibitory synaptic transmission, or By detecting the effect on Phase-Amplitude Coupling (PAC), which is characteristic of various diseases, it is possible to screen for candidate substances that change the neural activity of the brain. Furthermore, it is possible to screen for substances that may change short-term memory or long-term memory (such as K252a and anisomycin), especially substances that may enhance short-term memory.
  • a desired substance NMDA inhibitor, AMPA inhibitor, psychotropic drug, etc.
  • PAC Phase-Amplitude Coupling
  • the duration of neural activity (duration of neural activity after light irradiation) in response to the light stimulation of the axon bundle of the neural circuit tissue according to the present embodiment is in the absence of the substance. If it is longer than in the case of, it can be determined that the substance may function effectively for the sustainability of short-term memory.
  • UV 365 nm, 2.5-3.0 mWcm 2
  • SU-8 was developed with SU-8 developer for 15 minutes and washed 3 times with isopropyl alcohol.
  • the wafer was heat treated in an oven at 150 ° C. for 3 minutes.
  • the thickness of SU-8 was about 150 ⁇ m.
  • Microfluidic devices were made using a polydimethylsiloxane (PDMS) silicone elastomer kit Sylgard184 (Dow Corning).
  • PDMS polydimethylsiloxane
  • Silicone elastomers and hardeners were mixed in a 10: 1 weight ratio, degassed, poured into patterned SU-8 structures and cured in an oven at 80 ° C. for 6 hours. Holes for organoids and reference electrodes were made with biopsy punches (1.5 mm and 2 mm, respectively). The glass ring used to store the medium (inner diameter: 22 mm, outer diameter: 25 mm) was glued to the PMDS device. The prepared PMDS device was sterilized in an autoclave and subjected to 70% ethanol treatment and UV treatment.
  • Human iPS cells Human iPS cells were obtained from Riken Cell Bank (409B2, HPS0076) (Okita et al., Nat Methods 8 409-412 2011). Cells were maintained on ESC-qualified Matrigel-coated 6-well plates on the first day in mTeSR plus medium (STEMCELL Technologies) supplemented with 10 ⁇ M Y-23632 (Wako for the first 24 hours only). Then, using ReLeSR (STEMCELL Technologies), the plants were planted every 5-7 days and then cultured.
  • Riken Cell Bank (409B2, HPS0076) (Okita et al., Nat Methods 8 409-412 2011). Cells were maintained on ESC-qualified Matrigel-coated 6-well plates on the first day in mTeSR plus medium (STEMCELL Technologies) supplemented with 10 ⁇ M Y-23632 (Wako for the first 24 hours only). Then, using ReLeSR (STEMCELL Technologies), the plants were planted every 5-7 days and then cultured.
  • iPS cells were separated using TrypLE Express so as to become a single cell. 20,000 cells were then plated on a round-bottomed low-adhesion surface 96-well plate (Prime surface, Sumitomo bakelite) supplemented with mTeSR medium containing 10 ⁇ M Y-23632.
  • the medium was changed to nerve induction medium (DMEM-F12, 15% (v / v) knockout serum replacement, 1% (v / v) MEM-NEAA, 1% (v / v) Glutamax, 100 nM LDN- It was replaced with 193189 and 10 mM SB431542), and then the medium was replaced every two days.
  • the medium was 0.5% (v / v) N2 supplement, 1% (v / v) B27 supplement (without vitamin A), 1% (v / v) Glutamax, 0.5% (v / v).
  • Multi Microelectrode Array Twenty-four hours before measuring neuronal activity with multiple electrodes, maintenance medium was added to 1% (v / v) B27 supplement (containing vitamin A), 1% (v / v) Glutamax, 20 ng / ml BDNF and 1%. (v / v) Replaced with Brainphys containing Penicillin / Streptomycin.
  • PDMS-MEA was set in the MED64 system (Alpha MED Scientific) and electrical signals from all 64 electrodes were recorded at 37 ° C. for 5-30 minutes at a sampling rate of 20,000 Hz. Noise during recording of electrical signals was removed with a bandpass filter between 0.1-10,000 Hz.
  • the unprocessed (raw) signal is further filtered through a bandpass filter (300-3,000 Hz) for spike analysis, raster plotting, spike clustering, or a low frequency path ( ⁇ 1000) for analysis of local field potentials. Hz). All post-hoc analysis was then performed using MATLAB Signal Processing Toolbox, Curve fitting Toolbox, Deep learning Toolbox, Parallel Computing Toolbox, and Wavelet Toolbox. All analyzes and calculations were performed using MATLAB software. All scripts for the calculations in this example were downloaded from https://github.com/TatsuyaOsaki/Matlab_function.
  • Wavelet coherence and wavelet transform for frequency separation Wavelet coherence is a measure of the correlation between two signals at a particular frequency.
  • Wavelet coherence from LFP records was calculated by Eq. (1).
  • f (t) was calculated using the functions cwt () and icwt () in the "Wavelet Toolbox":
  • a and b represent the center positions of the scale factor (1 / Hz) and the mother wavelet factor, respectively.
  • G (x) represents a complex Morlet function:
  • the frequency bandwidth F B was set to 5 and the center frequency F C was set to 1.
  • Neuronal avalanche A neuronal avalanche is an event characterized by a continuous pattern of neuronal activity within nerve tissue.
  • the time bin ( ⁇ t) calculation was set to 3 milliseconds.
  • the probability was calculated by the following formula: P (S) is the probability of observing a size S avalanche.
  • ⁇ is an exponent, represents the slope of the correlation in log-log coordinates, and ⁇ is a proportional coefficient.
  • AAV-CAG-GFP was provided by Karel Svoboda (Addgene plasmid # 28014).
  • AAV was made with AAVpro 293T (Takara).
  • AAV was recovered using the AAVpro Purification kit midi (Takara) according to the manufacturer's protocol.
  • AAV was infected with the connecting organoid at 4 to 6 weeks, and a photoconversion experiment was performed in the culture at 7 weeks.
  • a 405 nm laser was applied to the axon bundle area and a Nikon confocal microscope (1.5 mm x 0.5 mm x 0.2 mm, 5x, total time: 60 min). , Nikon A1R).
  • the organoids were dissociated by Accumax at 37 ° C. for 10-30 minutes and centrifuged at 200 xg for 5 minutes.
  • For flow cytometry single cells were resuspended in PBS containing 1% BSA.
  • Kaede red positive / Kaede green negative population and Kaede red negative / Kaede green positive population were collected as axon-related neurons and non-axon-related neurons, respectively. After sorting, total RNA was collected for RT-PCR analysis.
  • Human iPS cells were transfected with GFP fluorescent protein or mCherry fluorescent protein to visualize knock-in axonal elongation by electroporation with the CRISPR-Cas9 method of GFP fluorescent protein and mCherry fluorescent protein.
  • the GFP fluorescent protein and mCherry fluorescent protein were inserted into the safe region site of AAVS1 (Adeno-associated virus integration site 1), respectively.
  • the iPS cells were collected by TrypL Eexpress treatment and centrifuged to collect the cells.
  • Cells were fixed with 4% paraformaldehyde for 20 minutes and treated with 0.2% Triton X-100 for 5 minutes for permeabilization of cell membranes. Blocked with 1% bovine serum albumin (BSA) for 2 hours. The cells were then treated with the primary antibody for 2 hours at room temperature. In addition, it was treated with a secondary antibody for 2 hours at room temperature.
  • BSA bovine serum albumin
  • mouse anti-neuron-specific ⁇ III tubulin Biolegend 801202, 1: 1200
  • rabbit anti-neuron-specific ⁇ III tubulin Sigma, ZooMAb, 1: 200
  • mouse anti-human PAX6 DHSB, 1: 1200
  • rabbit anti-human GAD67 Santa Cruz, 1: 100
  • rabbit anti-human vGluT1 Sigma, ZooMAb, 1: 200
  • mouse anti-human CTiP2 Abcam, 1: 100
  • rabbit anti-human SATB2 Abcam, 1: 100
  • Abcam ab51502, 1: 100) or rabbit anti-human MAP2 (Sigma, ZBR2290, 1: 200) was used.
  • Alexa Fluor 555 anti-rabbit IgG H + L
  • Alexa Fluor 405 anti-rabbit IgG H + L
  • Alexa Fluor 488 goat anti-mouse IgG H + L
  • Alexa Fluor 488 goat anti-rabbit IgG H + L
  • hAlexa Fluor 647 goat anti-rat IgG H + L
  • the nuclei were stained with Hoechst dye for 20 minutes at room temperature and rinsed 3 times with Dulbecco's Phosphate-Buffered Saline (D-PBS ++ ) containing Ca 2+ and Mg 2+ . All cells and samples were observed under a fluorescence microscope (Axio Observer, Zeiss) or a confocal laser scanning microscope (Zeiss).
  • RT-PCR Real-time reverse-transcription
  • Total RNA was isolated from tissues using TriPure (Sigma) to measure the biological activity of brain organoids. Reverse transcription was performed using KOD One (Toyobo). The primer sequences are shown in Table 1.
  • RT-PCR was performed at CFX Connec using KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems). In all experiments, the mRNA expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard.
  • RT-PCR was performed at least 3 times using cDNAs prepared from different tissues.
  • Ca 2+ indicator (GCaMP6f controlled by the CAG promoter) was transfected with AAV1 to visualize neuronal activity in fluorescence.
  • pAAV.CAG.GCaMP6f.WPRE.SV4 was donated by Douglas Kim & GENIE Project (Addgene # 100836).
  • the maintenance medium was replaced with medium in PDMS-MEA 3 days prior to measurement.
  • the medium was replaced with fresh maintenance medium.
  • FIG. 1 shows the results of evaluating the formation and characteristics of connecting organoids on a PDMS-MEA chip.
  • A is a schematic diagram of the connecting organoids on the PDMS-MEA chip. The two brain organoids were cultured in two chambers cross-linked by microchannels on the chip.
  • B shows an example of a PDMS-MEA chip.
  • the PDMS-MEA chip is composed of a MEA probe (MEA), a PDMS (PDMS microfldic layer), a glass solution storage ring (Reserviro ring), and a PDMS lid (Lid) (i and ii). Under each cerebral organoid, electrodes (4 ⁇ 4 array) composed of 16 metal thin films and the like are installed (iii).
  • C shows a typical internal structure after 4 weeks (upper figure) and 8 weeks (lower figure). Scale bar: 150 ⁇ m.
  • D shows the gene expression profile of brain organoids cultured for 2 to 10 weeks. The vertical axis shows the gene name, and the horizontal axis shows the time (week) after culturing.
  • E the axon extended from one organoid to another organoid at 5 weeks, and the axon bundle was formed by 6 weeks. From the top, fluorescent images of brain organoids 4 weeks, 5 weeks (axon elongation) and 6 weeks (organoid connection by axon bundles) after culturing iPS cells are shown.
  • F indicates the change over time in the thickness of the axon bundle.
  • G indicates the abundance ratio of excitatory neurons (VGLUT1 antibody staining positive) and inhibitory neurons (GAD67 antibody staining positive) in organoids.
  • H indicates the result of immunohistological analysis of organoids. After culturing iPS cells for 8 weeks, immunohistological analysis revealed the presence of layered structures within the connecting organoids. Immunostaining for PAX6 and CTIP2 shows proliferative and cortical sublayers, respectively.
  • I is a schematic representation of a method of recording neuronal activity from connecting organoids on a PDMS-MEA chip.
  • the raw analog signal from the electrodes was amplified at a sampling rate of 20 kHz and converted to a digital signal (16 bits). After that, the signals are 300-3,000Hz bandpass filter (Bandpass filter) for spike analysis and 1,000Hz low frequency bandpass filter (Lowpass filter) for local field potential (LFP). Processing was performed.
  • J shows a representative example of a connecting organoid 5 weeks after culturing iPS cells.
  • the scale bar is 1 mm (i).
  • examples of filtered signals from four typical electrodes under each organoid are shown.
  • LE is the signal of the left brain organoid
  • RE is the signal of the right brain organoid
  • Wavelet coherence between the signals derived from the left and right organoids is shown (iii).
  • K represents a representative example of the connecting connectoid 5.5 weeks after iPS cell culture.
  • the scale bar is 1 mm (i).
  • Burst-like activity synchronized with dense spikes and left and right organoids was detected from multiple electrodes (ii).
  • Wavelet coherence showed a strong association between the two connecting connectoids (iii).
  • L indicates the result of analyzing the synchronization of activities between organoids. The synchronization of neuronal activity of the two organoids increased during the culture period.
  • the vertical axis shows the synchronicity index
  • the horizontal axis shows the iPS cell culture time (week).
  • M indicates the result of measuring the frequency of burst activity. The frequency of burst-like activity increased significantly depending on the culture period.
  • the vertical axis shows the burst frequency
  • the horizontal axis shows the iPS cell culture time (week).
  • N shows the neuronal activity signals of the two connecting organoids (middle figure) and their enlarged views (lower figure). The figure above shows a raster plot.
  • a time lag (Burst delay) was observed in the burst-like activity synchronized with the left and right organoids.
  • O indicates the time lag between the bursts of the left and right organoids at different culture points.
  • the vertical axis shows the burst delay
  • the horizontal axis shows the iPS cell culture time (week).
  • n 20. * P ⁇ 0.05, ** p ⁇ 0.01; one-way ANOVA, error bar indicates SD (standard deviation).
  • FIG. 2 shows the results of analysis of neuronal activity of brain organoids connected by axon bundles.
  • A shows the local field potential (LFP) signals in the 0.2-0.5 Hz band, 0.5-4 Hz ( ⁇ ) band, and 30-300 Hz ( ⁇ ) band extracted by the inverse continuous wavelet transformation. .. Eight weeks after culturing iPS cells, the connecting organoid generated low frequency oscillations in the 0.5-4 Hz ( ⁇ ) band.
  • B indicates the power integration result for each frequency band.
  • the vertical axis shows the integrated value of wave power, and the horizontal axis shows the time (week) after culturing.
  • C represents a representative example of the signal of neuronal activity of connecting organoids, single organoids and fused organoids.
  • D indicates the burst frequency of various organoids.
  • E shows the result of the inverse continuous wavelet transform in the 0.2-0.5 Hz band, the 0.5-4 Hz ( ⁇ ) band, and the 30-300 Hz ( ⁇ ) band. Oscillations in the ⁇ band were detected in connecting organoids but not in single and fused organoids.
  • the left bar graph shows the results of single organoids
  • the middle bar graph shows the results of fused organoids
  • the right bar graph shows the results of connected organoids.
  • F indicates the coefficient of variation of the burst frequency of the three types of organoids.
  • the vertical axis shows the coefficient of variation of the burst interval
  • the horizontal axis shows the post-culture time (week).
  • G indicates the relationship between the volume of organoids (horizontal axis) and the average burst frequency (vertical axis).
  • FIG. 3 shows the results of genetic analysis of linked brain organoids (connecting organoids) using Single cell RNA seq.
  • A shows 17,636 UMAP plots. Leiden clustering was performed on single, fused, connected organoids.
  • the cell population was classified into 14 clusters.
  • UMAP plots and density plots are shown for each sample.
  • B and D show heatmaps of the top 30 genes that are statistically significant in each cluster. By known markers, the 14 clusters were further divided into four groups (Group 1 "NPC", Group 2 "Intermediate”, Group 3 "Neurons", Group 4 "Other”).
  • C is the result of normalizing the cell proportions of clusters 8, 6, 12, and 4 of single, fused, and connected organoids.
  • E is an annotated UMAP plot with a known marker gene.
  • F is a classification result by cell type by a known marker gene.
  • G shows a UMAP plot of vGlut1 for visualization of excitatory neurons and DLX6 and GAL for visualization of inhibitory neurons.
  • H indicates a UMAP plot of GRIA1 and GRIA2 that are highly expressed in cluster 8.
  • I and J show plots of GRIA2 expression in cluster 8 of single organoids, fused organoids, and connected organoids. The average expression of GRIA2 in the linked brain organoids was higher than that of the single and fused organoids. Among the cluster 8, GRIA2 was highly expressed in DCX-positive cells (J).
  • FIG. 4 shows the results of characterizing axon bundles using a photoconverted fluorescent protein.
  • A shows a plasmid map of a pAAV backbone plasmid expressing Kaede whose fluorescence wavelength is variable by UV irradiation by a CAG promoter.
  • Kaede green fluorescent protein can be converted to Kaede red fluorescent protein by ultraviolet irradiation.
  • .. C shows a confocal microscope image of the articulated organoid before and after irradiation with ultraviolet rays. Kaede green is rapidly converted to Kaede red by UV irradiation. After that, Kaede red rapidly diffused and distributed in the axon bundle in the anteroposterior direction, and a gradation of Kaede green and Kaede red was formed in the axon bundle.
  • D shows a cross-sectional view of the axon bundle and XY cross-sectional views below and above.
  • Kaede red In the central part of the axon bundle, most of the Kaede green was converted to Kaede red. It was also found that Kaede red in the axon bundle spreads not only to the lower part of the linked brain organoid but also to the perikaryon located at the upper part.
  • E shows the distribution of Kaede red in axon-related neurons by three-dimensional reconstruction.
  • F shows the result of quantifying the fluorescence intensities of Kaede green and Kaede red corresponding to the distance from the center of the axon bundle before and after ultraviolet irradiation. Kaede red was concentrated on the closest side of the axon bundle (see G).
  • H indicates the distribution of Kaede green and Kaede red in the z direction of the linked organoids.
  • Kaede red nerve cells extending axon bundles
  • nerve cells with axon extension and nerve cells without axon extension were separated and quantified by flow cytometry.
  • the average proportion of axon-related neurons was 32% and that of non-axon-related neurons was 68%, correlating with confocal microscopy images (see J).
  • K indicates a change in gene expression relative to axon-stretching neurons relative to non-axon-stretching neurons.
  • NEAT1 and GRIA2 co-expressing cells are predominant in cluster 8 and that they are neurons involved in axon bundle formation.
  • N shows a summary of the results obtained from the Kaede experiment and scRNA-seq. * P ⁇ 0.05, ** p ⁇ 0.01; student's t-test, error bar indicates SD (standard deviation).
  • FIG. 5 shows the results of a study on optogenetic inhibition of burst activity between connected organoids and synchronization between both organoids.
  • A shows the configuration of an optogenetic device for inhibiting synaptic interactions between left and right organoids via axons.
  • i shows a schematic diagram of a microfluidic device for optogenetic control
  • ii shows a state in which ArchT is expressed in a connecting organoid by AAV and irradiated with light.
  • the fiber optics can be moved and placed so that the organoids and / or axon bundles can be selectively illuminated via a fiber guide in which the optical fibers are installed, but here the distance between the axon bundles is 100 ⁇ m. It was placed and placed vertically.
  • MEA was measured by connecting a 470 nm or 565 nm LED and a pulse generator (Arduino) to the optical fiber.
  • the timing of light irradiation and the signal from a typical channel from the MEA amplifier were recorded by TTL logger, and the TTL signal and neural activity were synchronized at the time of analysis before analysis (Analysis Department) (see iii). ).
  • the curved structure as a PDMS lens helped to focus the light on the axon bundle (see B).
  • C shows the LFP and raster plots detected from the left and right organoids of the connecting organoids with or without light irradiation.
  • E indicates wavelet coherence between the left and right organoids of the connecting organoid.
  • H is the result of calculating the total number of single spikes for 5 minutes.
  • FIG. 6 shows the results of analysis of complex activities occurring in connected organoids using Phase-Amplitude Coupling and a hidden Markov model.
  • A shows the raw (untreated) LFP plots detected from each connecting organoid at 9 weeks of culture of iPS cells.
  • B indicates wavelet coherence between the two organoids.
  • C indicates the modulation index of the phase amplitude coupling in the ⁇ -phase / ⁇ power and the ⁇ -phase / ⁇ power of the connected organoids at the 5th, 7th, and 9th weeks of culture.
  • D is the delta-phase / ⁇ -power and ⁇ -phase / ⁇ -power PAC modulation index of single organoids (“S” in the figure), fused organoids (“F” in the figure) and connected organoids (“C” in the figure). modulation index) is shown.
  • E indicates the PAC modulation index within or between organoids in the connecting organoid.
  • F outlines the brain hypothesis analysis of the left and right organoids. In addition, the extraction results of the brain hypothesis cascade are shown. The brain hypothesis was calculated based on the signals from 8 electrodes. The single spike cascade was analyzed on a 3 msec scale size. G shows a logarithmic plot of brain avalanche size and probability of occurrence at 5, 5.5 and 8.5 weeks after culture.
  • H indicates the number of hidden patterns in the brain hypothesis at 5, 5.5 and 8.5 weeks after culture.
  • I, J, K and L are the neural activities of connecting organoids treated with various neuromodulatory compounds (CNQX, APV, Bicculline, Baclofen, Buprenorphine, Clozapine and Diazepam). The result of comparing the patterns is shown. The average number of spikes (I), burst frequency (J), integrated power in the ⁇ band (K), and PAC modulation index (L) were calculated. note that. * p ⁇ 0.05, ** p ⁇ 0.01; one-way ANOVA, error bar indicates SD (standard deviation).
  • FIG. 7 shows the analysis results of the short-term memory mechanism in the connecting organoid.
  • A shows a schematic diagram of an optogenic stimulation experiment. Optogenic stimulation of axon bundles at 0.5, 1 and 1.5 Hz with a 470 nm laser source or LED induced synchronized burst activity. The effect of the stimulus persisted even after discontinuation of light irradiation.
  • B and C show the measurement result of the burst frequency modulated by the light stimulus. There was a time lag between the photostimulation and the modulation of the burst frequency.
  • D indicates a logarithmic plot (vertical axis) of the brain hypothesis size (horizontal axis) and appearance probability before stimulation (Before Stim.), During stimulation (During Stim.), And after stimulation (After Stim.).
  • E indicates a time course of burst frequency stimulated at 1 Hz for 20 minutes (i) and 5 minutes (ii).
  • the vertical axis shows the burst frequency
  • the horizontal axis shows the time (minutes) from the start of stimulation.
  • F indicates the result of measuring the duration of burst activity after light stimulation (20 minutes or 5 minutes). The duration is the time until the burst frequency after discontinuation of light stimulation decreases to 75% of the maximum burst frequency.
  • G indicates the time lag (Delay time) from the light stimulation to the burst induction activity.
  • the time lag from the start of the light stimulation to the induction of the burst was significantly reduced compared to the first stimulation, and the time lag after the second and third stimulations was significantly reduced.
  • H indicates the time course of burst frequency in the presence of K252a or anisomycin. I stimulates the duration of burst activity of connecting organoids under conditions without compound treatment (control) or with compound (K252a or Anisomycin) treatment (1st (1st), 2nd (2nd) and Shown every 3rd (3rd)).
  • FIG. K is a diagram showing the relationship between the number of hidden patterns of a nerve avalanche and the size of hidden patterns of a nerve avalanche.
  • L indicates the result of examining the probability of occurrence of a neurological avalanche.
  • M indicates the result of examining the total number of hidden patterns of the brain hypothesis.
  • N and O represent the fractal dimension of the LFP signal.
  • FIG. 8 shows the results of analyzing various burst activity patterns supported by CaMKII-dependent signals.
  • FIG. A is a diagram showing an example of burst activity induced by optogenic stimulation and neuron-derived potential distributions in the right-sided organoid and the left-sided organoid. 891 Burst traces are shown. The light-stimulated spikes and burst waves continued after the photostimulation. Secondary and tertiary waves of burst activity were also observed.
  • B indicates the time lag (ms) after the light stimulation (1st, 2nd and 3rd) until the burst activity is induced. Measured in the presence of control, K252a or anisomycin.
  • E represents a representative example of crosstalk between each organoid of the connecting organoids in self-induced burst activity.
  • FIG. 9 is the result of visualizing and analyzing the elongated axons of the connecting organoids.
  • A shows a schematic view of a method of knocking EGFP and mCherry under the control of a CAG promoter into the AAVS1 region of iPS cells (left figure) and a fluorescent image of a fluorescent protein expressed in iPSC cells (right figure).
  • GFP-labeled or mCherry-labeled brain organoids were generated to visualize axon elongation on PDMS chips.
  • the vertical axis of the graph on the right shows the length of the most elongated axon ( ⁇ m), and the horizontal axis shows the culture time (days) of iPS cells.
  • C is an image of observing the elongation of axon bundles between organoids placed at different intervals (2 mm, 3 mm, 4 mm, 5 mm) at 5 and 6 weeks after culturing iPS cells. show.
  • D indicates the result of measuring the thickness of the axon bundle extending between the organoids placed at different intervals (2 mm, 3 mm, 4 mm, 5 mm). The thickness of the axon bundle was measured at the center of the microchannel.
  • Axon bundle thickness of GFP-labeled connecting organoids (n 3).
  • E shows a 3D confocal microscope image of the GFP-labeled connecting organoid.
  • Brain organoids labeled with GFP and mCherry were ligated in a microfluidic device.
  • Axons labeled with GFP or mCherry extended to mCherry-labeled brain organoids and GFP-labeled organoids, respectively, on the chip after 2 weeks via overlapping thick axons (F).
  • G indicates the result of counting the number of axons that have reached other brain organoids.
  • the vertical axis shows the number of axons reached by the organoid, and the horizontal axis shows the iPS cell culture time (day).
  • H is a graph showing the relationship between the thickness of the axon bundle and the frequency of synchronized burst activity.
  • the frequency of synchronized burst activity increased as the thickness of the axonal bundle increased.
  • I is an image showing the state of axon extension toward the other organoid. Immunostaining of SynI revealed synaptic connections between organoids. ** p ⁇ 0.01; one-way ANOVA or student's t-test, error bar indicates SD (standard deviation).
  • FIG. 10 shows the results of comparing the neural activities of single organoids, fused organoids, and connected organoids.
  • A is a schematic diagram of a procedure for producing a single organoid, a fused organoid, and a connected organoid. All brain organoids were made in the same way until day 21. To make fused organoids, two brain organoids were placed in one well of a poorly adhesive 96-well plate. Then, on day 28, single and fused organoids were placed on the MEA probe. Also, on day 28, organoids were placed on PDMS-MEA probes to generate connecting organoids. After culturing on the MEA probe for 2 weeks, neuronal activity was measured.
  • B shows a representative example of a single organoid placed on the MEA probe (left figure). Periodic and synchronized neuronal activity was detected in a single organoid (right figure).
  • C shows a representative example of the fusion organoid placed on the MEA probe (left figure). More active and synchronized neuronal activity was detected in fused organoids compared to single organoids.
  • D shows an outline of a method for detecting a time difference in neuronal activity within an organoid (Intra-) and between organoids (Inter-) by an electrode.
  • FIG. 11 shows the results of measuring organoids connected by axon bundles of various lengths and their axon velocities.
  • A shows an example of three types of microfluidic chips (5.5 mm, 7.8 mm and 12 mm) with different lengths between channels. Arrows indicate the location of organoids.
  • FIG. 12 shows the results of analysis of neuronal activity in organoids in which axon bundles are physically cleaved. After physically cleaving the axon bundle (A) between the connecting organoids (B), the neuronal activity detected in each organoid (left organoid and right organoid) was measured. C shows the result of measuring the wavelet coherence of the disconnected organoid. From this result, it was clarified that the truncated organoids had almost no synchronized activity.
  • FIG. 13 shows the results of drug treatment for connecting organoids.
  • A indicates the LFP signal of the connecting organoid at different culture periods (5.5 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks and 8.5 weeks).
  • FIG. 14 shows a scalogram of the LFP signal and wavelet transform of the connecting organoid in the presence of various agents.
  • FIG. 15 shows the results of simultaneous measurements of Ca 2+ transients and electrical activity.
  • (I) of A shows the configuration of an optical device for performing Ca 2+ imaging and MEA recording of connecting organoids.
  • the connecting organoid was transiently transfected with the Ca 2+ reporter gene (GCaMP6f) (see ii and iii).
  • GCaMP6f Ca 2+ reporter gene
  • B showed the firing pattern of neurons in the connecting organoid at 7 weeks after culturing iPS cells by a trace image of the calcium response. Synchronized burst activity was observed between the left and right organoids (see C).
  • D shows a plot of Ca 2+ concentration changes and MEA signal recordings.
  • the two signals corresponded to each other.
  • Calcium imaging and MEA records were consistent during burst activity (see E).
  • F and G are the results of plotting the correlation coefficients obtained from the 12 neurons of the organoid on the left side g and the 12 neurons of the organoid on the right side for each nerve.
  • Ca 2+ transient activity was correlated between organoids as well as within each organoid.
  • the signal is intentionally shifted by 50 m sec (1 frame)
  • the correlation between the organoids becomes stronger, indicating that the left and right organoids are active with a delay of about 50 m sec. rice field.
  • the PDMS-MEA chip consists of a multi-electrode array (MEA) layer, a polydimethylpolyolefin (PDMS) microfluidic layer, and a ring and lid of a culture tank.
  • the PDMS layer is provided with a pair of holes for containing and culturing each brain organoid.
  • a spatial flow path structure that guides organoid axons to each other.
  • cerebral organoids express neural markers (eg, DCX and TUBB3) and cortical layer-related genes (eg, TBR1 and SATB2). From this (Fig. 1C and D), it was confirmed that the brain organoids were successfully differentiated.
  • brain organoids were placed on PDMS-MEA. The two brain organoids were connected by a thick axon bundle within 6 weeks (within 2 weeks of placement on the chip) (Fig. 1E).
  • FIGS. 9A-I GFP-expressing brain organoids and mCherry-expressing brain organoids "hand-shake" type connections were shown (FIGS. 9A-I).
  • Two brain organoids were connected to other organoids with as many axons as each other (Fig. 9G).
  • the thickness of the axon bundle was about 75 ⁇ m after 6 weeks and about 120 ⁇ m after 8 weeks (4 weeks after placement on the chip) (Fig. 1F).
  • vGlut1 positive excitatory neurons and GAD67 positive inhibitory neurons were contained in about 70% and 5-10% of the cells in the brain organoid, respectively (Fig. 1G).
  • a layered structure was observed under the cortex (Fig. 1H). As described above, it is considered that the two brain organoids can be connected to each other by axons by culturing, and as a result, the functions of the brain generated during the developmental process can be imitated.
  • neuronal activity was detected from the electrodes installed under each brain organoid (Fig. 1B and I).
  • Action potential spikes and local field potentials (LFPs) were extracted by high frequency filters and low frequency filters, respectively.
  • LFPs local field potentials
  • the activity of connected organoids was concentrated in the ⁇ wavelength band (0.5-4 Hz) compared to single organoids and fused organoids (Fig. 2E).
  • Fig. 2E The coefficient of variation of the frequency of burst-like neuronal activity (CV, showing variability; standard deviation / arithmetic mean) was significantly higher for connected organoids when compared to single and fused organoids (Fig. 2F). ). This suggests that burst-like neuronal activity in connecting organoids is significantly more irregular and complex than burst-like neuronal activity in single and fused organoids.
  • RNA-seq Single-cell (sc) RNA-seq of single organoids, fusion organoids, and connecting organoids at 7 weeks of culture was performed. 17,636 single cells aggregated from all three conditions were subjected to principal component analysis (PCA) and then visualized as UMAP plots (Fig. 3A (i)). The gene expression profiles of single organoids, fused organoids, and connected organoids were found to be generally similar with slight differences (Fig. 3A (ii)). The cells were isolated into 14 clusters divided into 4 groups ("NPC”, "intermediate”, “neurons”, “other” clusters) (Fig.
  • Kaede-green can be immediately converted to red-fluorescent Kaede (Kaede-red) by UV irradiation (Ando et al., Proc. Acad. Natl. Sci., 99 12651-12656 2002).
  • Kaede-expressing virus AAV-CAG-Kaede
  • FIG. 4B Prior to UV irradiation, Kaede green was distributed throughout the connecting organoids, but Kaede red was almost absent (Fig. 4C). The central part of the axon bundle was irradiated with ultraviolet rays for 60 minutes to induce photoconversion. Furthermore, when the converted Kaede red protein was transferred into the cell over 2 hours (Fig. 4C), we succeeded in visualizing the axon bundle-related nerve cells as an organoid. The green and red distribution of Kaede revealed the location of neurons associated with axon bundles within the linked organoids (Fig. 4D).
  • the red Kaede is similarly distributed on the left and right organoids (Fig. 4F). Within the organoid, it was found that there are more axon-related neurons in the region near the axon bundle (Fig. 4G). It was confirmed that the axons in the lowest layer of the connected organoids were distributed almost evenly over the entire z-axis, except that the axons in the lowest layer did not extend much into the bundle (Fig. 4H).
  • Flow cytometry analysis of dissociated neurons of articulated organoids exposed to UV light revealed that from the Kaede green / red ratio (Fig. 4I), approximately 30% of neurons extended axons into the bundles between the organoids. It turned out that it was (Fig. 4J).
  • axon connections between organoids optogenic inhibition of axon bundles between organoids was performed.
  • the microfluidic chip was modified to provide optogenic inhibition of axon bundles (Fig. 5A).
  • ArchT archrhodopsin; excretes H + extracellularly in response to light and suppresses neural activity
  • AAV adeno-associated virus
  • Phase-Amplitude Coupling Culture of neural circuit tissue for more than 8 weeks further increased the LFP frequency and action potential spikes of the connecting organoids (Fig. 13A). In addition, the complexity of the signal has increased, and activity in the ⁇ wavelength band has been frequently observed (Fig. 6A). In addition, a strong correlation was observed between the ⁇ wavelength band and the ⁇ wavelength band, and the ⁇ wavelength band and the ⁇ wavelength band. Therefore, in order to investigate the correlation between these wavelength bands, Phase-Amplitude Coupling (PAC), which is an index of the relationship between low-frequency activity and the amplitude of high-frequency spikes, was calculated.
  • PAC Phase-Amplitude Coupling
  • PAC is an established method for assessing the relationship between electroencephalograms (EEGs) in different frequency bands (Fell et al., Nat. Rev. Neurosci. 12 105-118 2011: Canolty et al., Trends Cogn. Sci. 14 506-515 2010).
  • EEGs electroencephalograms
  • the LFP waves in the ⁇ wavelength band and the ⁇ wavelength band appeared as synchronized burst activity in coordination with the ⁇ wavelength band (Fig. 6B).
  • the delta- ⁇ PAC modulation of the connecting organoid increased with continued culture, followed by an increase in theta- ⁇ PAC modulation (FIG. 6C).
  • ⁇ - ⁇ PAC modulation and the ⁇ - ⁇ PAC modulation that occur in the connected organoids were significantly higher than the PAC modulation in the single or fused organoids (Fig. 6D).
  • ⁇ - ⁇ PAC and ⁇ - ⁇ PAC showed a high degree of modulation (intraorganoid PAC).
  • the ⁇ - ⁇ PAC modulation and ⁇ - ⁇ PAC modulation between organoids connected by axon bundles is higher than the PAC modulation within the organoid, which means that the ⁇ and ⁇ frequencies between the two organoids. It shows that the communication of the band is close (Fig. 6E).
  • Measurements by MEA have made it possible to record extracellular and local neuronal activity at high spatiotemporal resolution.
  • the low density of electrodes compared to the cell density within organoids can make it difficult to understand the spike activity of action potentials from individual neurons.
  • Ca 2+ imaging can measure with higher spatial resolution than the electrode-based method, but it is difficult to obtain high-magnification images from two organoids at the same time with a normal microscope. Therefore, we set up a microscope with two independent optics to simultaneously obtain individual Ca 2+ imaging of two organoids located a few millimeters apart. This system allows the acquisition of Ca 2+ imaging signals from two organoids with high spatiotemporal resolution, even when combined with MEA recording (Fig. 15A).
  • the Ca 2+ indicator GCaMP6f was expressed in organoids connected by AAV for 3-7 days (Fig. 15A).
  • the frames of the camera images of the left and right organoids were synchronized by a trigger signal, and the images were acquired at a speed of 20 frames per second.
  • the change in fluorescence intensity ( ⁇ F / F) in neurons in organoids was calculated. Fluorescent signals showed burst activity in both of the two organoids, synchronized with the activity of individual isolated neurons (Fig. 15B).
  • the interval between bursts was consistent with the burst interval measured by MEA (Fig. 15C).
  • Simultaneous measurements of MEA and Ca 2+ imaging showed a consistent increase in MEA signal and Ca 2+ concentration in organoids (Fig. 15D).
  • Fig. 6F Temporally proximal signals obtained from the electrodes were grouped and quantified as a brain hypothesis.
  • the critical brain hypothesis index is considered a scale-free index of network critical dynamics (Beggs et al., Journal of Neuroscience 23 11167-11177 2003: Bowen et al., Frontiers in Systems Neuroscience 13 45 2019).
  • the distribution of avalanche size increased with continued culture of connecting organoids.
  • CNQX, APV and Bicuculline are antagonists of NMDA-type glutamate receptor (excitatory receptor), AMPA-type glutamate receptor (excitatory receptor) and GABA receptor (suppressive receptor), respectively (Figs. 6I-L, Figures). 13A and FIG. 14). No change in signal propagation rate was observed with antagonist treatment (FIGS. 13B and C). However, each antagonist affected neuronal activity such as action potential spikes and burst-like activity (FIGS.
  • the frequency of burst-like activity was maintained at a high level for 10 minutes or more, and then returned to the frequency before the stimulation (FIGS. 7B and C).
  • the sustained echo-like neuronal activity generated by photostimulation indicates that the temporal activity pattern of the connecting organoid is modulated by the external stimulus, and that the connecting organoid can maintain the temporal information of the stimulus.
  • connecting organoids can maintain temporal information as a primitive (basic) form of memory.
  • Fig. 7C since there is a time lag between the start of stimulation and the occurrence of burst-like activity (Fig. 7C), it is suggested that multiple stimulations are required to modulate the activity of the connecting organoid.
  • the connecting organoid brain hypothesis expanded with periodic stimulation (Fig. 7D). This result indicates that the nerve cells that make up each organoid slowly adapt to the stimulus and establish a local circuit (subcircuit) that is easily excited by the time the activity of the connecting organoid activates in response to the temporal stimulus pattern. Suggest.
  • the burst frequency returned to normal from the stimulus-induced enhanced levels with each repeated stimulus.
  • the delay time to respond to the external stimulus was significantly shorter for each second or third stimulus compared to the first stimulus (Fig. 7G). ..
  • the delay time to respond to the external stimulus did not change.
  • Neuronal plasticity and memory are controlled by synaptic plasticity through a variety of molecular programs, including early and late responses, the calcium-dependent signal pathways required for each, and local protein synthesis.
  • the connecting organoids were treated with the CaM kinase II inhibitor K252a and the protein synthesis inhibitor anisomycin (Fig. 7H).
  • Connected organoids responded to photostimulation and sustained post-stimulation activity was observed under K252a or anisomycin treatment.
  • the duration of sustained activity decay after stimulation was significantly reduced in the presence of K252a after the second and third stimulations (Fig. 7I).
  • anisomycin treatment resulted in a slight reduction in sustained activity only after the third stimulus.
  • the brain hypothesis was also reduced by K252a treatment (Fig. 7K).
  • the number of hidden patterns in the brain hypothesis during light irradiation decreased compared to the nerve avalanche during non-light irradiation (FIG. 7L). This result suggests that during light irradiation, the activity of organoids is immobilized by a stimulus of a certain cycle, and as a result, a brain avalanche is less likely to occur and the amount of information possessed by the neural activity is reduced.
  • the number of hidden patterns gradually increased with each stimulus, suggesting that the neural circuits within the connecting organoids developed and matured due to the stimuli and the proliferation of neural activity by the stimuli. do.
  • the anisomycin treatment suppressed the expansion of the hidden pattern of the brain hypothesis during the light irradiation discontinuation period (Fig. 4M). Therefore, although the conventional long-term potentiation form was not observed, the long-term potentiation (Fig. 4M) was observed. It is suggested that the mechanism of long-term potentiation) was partially activated in the connecting organoids.
  • Higuchi's fractal dimension was calculated.
  • the fractal dimension (FD) is an indicator of the complexity of fine structures and has been used to assess the complexity and temporal changes of EEG signals (Varley et al., PLOS ONE 15 e0223812 2020).
  • the FD increased and the increased FD was maintained even in the absence of the second or third light irradiation (FIGS. 7N and O). This result suggests that the entire network gained complexity on the first stimulus and remained complex thereafter.
  • Fig. 8A In order to deepen the understanding of burst-like activities, the induced activities were sorted and compared (Fig. 8A). Detailed analysis revealed that photostimulation induces multiple brain waves of neuronal activity in burst-like activity. In control, the time lag of burst-like activity became shorter with each repeated stimulus (Fig. 8B). This reduction in time lag was also observed under anisomycin treatment, but not in the presence of K252a. To analyze the induced burst-like activity in more detail, the probabilities of the induced bursts were calculated (FIGS. 8C and D, kernel density estimation). As a result, it was found that a second-order response and a third-order response occur after the sharp first peak.
  • the present invention provides in vitro neural circuit tissues that exhibit complex activity and plasticity, especially neural tissues that mimic brain function.
  • an observation device for the nerve activity and a method for screening a substance using the device are provided. Therefore, it is highly expected to be used as a brain model in the medical field, pharmacy field, and the like.

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Abstract

La présente invention concerne un tissu de circuit neuronal qui est induit in vitro et qui a au moins deux masses de cellules nerveuses (par exemple, des organoïdes) connectées par l'intermédiaire d'axones. La présente invention concerne également un dispositif d'observation de l'activité neuronale du présent tissu de circuit neuronal et un procédé de criblage de substances à l'aide du présent dispositif. Le tissu de circuit neuronal de la présente invention réalise une activité de déclenchement spontanée et est caractérisé en ce que deux masses de cellules nerveuses présentent une activité interdépendante et peuvent en particulier être utilisées en tant que modèle cérébral.
PCT/JP2022/001090 2021-01-15 2022-01-14 Tissu de circuit neuronal in vitro présentant une activité et une plasticité, dispositif d'observation de ladite activité neuronale et procédé de criblage de substances à l'aide dudit dispositif Ceased WO2022154080A1 (fr)

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WO2017187696A1 (fr) * 2016-04-28 2017-11-02 一般財団法人生産技術研究奨励会 Dispositif de culture de cellule nerveuse, procédé de culture d'une cellule nerveuse, cellule nerveuse cultivée, procédé d'analyse et d'identification d'une protéine dans un faisceau d'axone, et procédé d'utilisation d'une cellule nerveuse
JP2020517283A (ja) * 2017-04-25 2020-06-18 イーエムベーアー−インスティテュート フュール モレクラレ バイオテクノロジー ゲゼルシャフト ミット ベシュレンクテル ハフツング 二分化または多分化オルガノイド

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