EP3137598A1 - Obtention de cultures neuronales spécifiques à une région cérébrale à partir de cultures tissulaires tridimensionnelles de cellules souches - Google Patents

Obtention de cultures neuronales spécifiques à une région cérébrale à partir de cultures tissulaires tridimensionnelles de cellules souches

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Publication number
EP3137598A1
EP3137598A1 EP15708820.4A EP15708820A EP3137598A1 EP 3137598 A1 EP3137598 A1 EP 3137598A1 EP 15708820 A EP15708820 A EP 15708820A EP 3137598 A1 EP3137598 A1 EP 3137598A1
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European Patent Office
Prior art keywords
cells
cell culture
neuronal
stem cells
cell
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German (de)
English (en)
Inventor
Olaf Schröder
Corina Ehnert
Alexandra Voss
Benjamin Bader
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Neuroproof GmbH
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Neuroproof GmbH
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Publication of EP3137598A1 publication Critical patent/EP3137598A1/fr
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2513/00—3D culture
    • 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
    • C12N2531/00—Microcarriers
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    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/32—Polylysine, polyornithine
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the invention relates to neuronal cell culture methods and their use as well as cell cultures produced therewith.
  • the invention relates to a method of producing a neuronal planar cell culture comprising the use of stem cells, e.g. human induced pluripotent stem cells pre-differentiated in a three-dimensional cell aggregate comprising at least two regions corresponding to different brain regions, referred to as cerebral organoid, and taking progenitor cells from a region corresponding to a particular brain region and on a planar support , eg an MEA neurochip, further cultivated.
  • the result is a neural planar cell culture in which the cells form a neural network and have an electrical or electrophysi logical activity that is specific to the particular brain region.
  • Such a cell culture may e.g. in high throughput screening for the testing of substances for neurological effect.
  • a test of drugs in terms of effects on neuronal cells is of great interest for the development of new drugs against neurological disorders such as epilepsy, Parkinson's, Alzheimer's, depression, etc.
  • In vivo studies are usually limited by the complexity of conducting electrical leads from multiple sites and the influence of interconnections with other brain regions.
  • Neuronal cell cultures can be obtained from primary cultures in which cells are taken from a late embryonic stage or postnatally. These are already programmed quite far in their neural development. The in vivo situation is defined by a local neuronal morphological and functional circuit structure. Regardless of the lack of the genetically defined, layered network architecture of the cell bodies, the primary neural networks share many of the characteristics of their source tissue, e.g. phenotypic cell types, receptors, ion channels, intrinsic electrical membrane properties, synaptic development and plasticity. Numerous individual findings have shown that cultures obtained from primary cultures in many ways correspond to the situation of the original tissue.
  • Brain region-specific networks With dissociated neuronal cell cultures taken from specific areas of the brain (eg, cortex, hippocampus, thalamus, spinal cord), brain region specific characteristics and pathological situations can be well mapped.
  • Various working groups have confirmed in animal models that substances in these Brain region-specific networks also electrically induce brain region-specific changes in network activity relevant to the in vivo situation (Gramowski et al., 2000, Xia et al., 2003a, Xia et al., 2003b).
  • the primary neuronal cell cultures develop from a mixture of different types of neurons and types of glial cells.
  • Primary neural networks are composed of different brain region-specific neuron types, such as e.g. Pyramidal cells, motor neurons, various interneurons. Furthermore, they also differentiate various glial cell types, such as astrocytes, microglia and, to a small extent, oligodendrocytes.
  • the glial cells fulfill various functions here.
  • the astrocytes have important auxiliary functions for the metabolism, e.g. Maintaining pH, potassium levels, nutrient supply and recycling and delivery of neurotransmitters.
  • the microglia cells are macrophages located in the brain and act here as the first and main immune system in the central nervous system.
  • the neuronal properties of substances can be studied by evaluating and characterizing the specific changes in neuronal network activity on microelectrode arrays (MEAs).
  • MEAs microelectrode arrays
  • Such network cell cultures are spontaneously active and pharmacologically accessible for several months. It has been shown that these primary cell cultures can be stably reproduced with their brain region-specific receptors (Gramowski et al., 2006, Johnstone et al, 2010) and so have functional and morphological properties e.g. in the frontal cortex and auditory cortex (Xia et al, 2003b), in the spinal cord (Gramowski et al., 2000), and in the striatum (Schock et al., 2010).
  • brain region-specific networks also respond to the applied substances in a substance-specific manner (Gramowski et al., 2004).
  • Xia and Gross (2003a) in their functional electrophysiological studies of ethanol and the antidepressant fluoxetine on primary cortical networks, show excellent agreement between their results and experimental animal studies.
  • the neuronal network activity patterns specific to brain region as well as to substance enable the generation of effect profiles of substances or pathological states with the help of pattern recognition methods and similarity analyzes.
  • the pharmacological and neurotoxic effects of new drug candidates in CNS drug development can be predicted earlier and more rapidly by analyzing functional changes in the networks in spatial and temporal resolution (Johnstone et al., 2010, Gramowski et al., 2010).
  • potential mechanisms of action can be elucidated, which can accelerate the preclinical development of drugs acting in the CNS.
  • NeuroProof technology routinely uses murine neural networks on MEAs to quantify substance effects by measuring the effects of substances on electrophysiological activity patterns.
  • various phenotypic assays of brain region-specific networks such as the frontal cortex, hippocampus, midbrain, thalamus, hypothalamus and spinal cord are used. These are usually cultured in vitro for 4 weeks until the networks and their network activity are differentiated.
  • These complex microcircuits of mature cortical cultures (4 weeks in vitro) have been shown to exhibit functionally very similar network activity in terms of activity levels, synchrony and oscillatory behavior, such as cortical in vivo network activity.
  • Neuronal primary cell cultures of human origin are very limited possible.
  • the use of primary embryonic cell cultures is of ethical concern and their availability is limited. Therefore, these human primary cell cultures are not suitable for routine screening of drugs.
  • animal e.g.
  • murine cells permit only limited predictability of the effects of substances, it would be desirable to increasingly use human stem cells and cells derived therefrom for drug tests. So far, microcircuits similar to those in the vzvo situation could not be found in neuronal cultures derived from human stem cells.
  • such cultures are not suitable because they are extremely limited in their predictive value.
  • Stem cells have a strong self-renewal capacity that allows these cells to regenerate damaged tissue. Because of this, they are widely recognized as an important biological source in regenerative medicine and medical device development. Stem cell-based technologies are already a very valuable new industry subgroup in the future of the medical / clinical industry and will have a major impact on other industries. In particular, induced stem cell differentiation is used to obtain functionally superior, tissue-specific differentiated cells from stem cells, which is already recognized as the core technology of the stem cell industry and will continue to evolve significantly.
  • Pluripotent human stem cells retain the ability to differentiate into every cell type of the body and are thus considered an attractive source of differentiated cells.
  • Tissue-specific differentiated cells from human stem cells provide a great opportunity for basic research in areas of human embryonic development, cell therapy, efficacy testing, cytotoxicity and especially for testing drug candidates.
  • the group of Yamanaka (Takahashi et al., 2006) successfully developed a de-differentiation strategy with transcription factors. Since then, it has been possible to generate these human induced pluripotent stem cells (iPS), which have been shown to be similar to human embryonic stem cells, from human body cells (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007). This advance enabled the use of patient derived iPS cells and resulted in rapid growth of this technology. In addition, tissue-specific differentiation offers great opportunities for testing the efficacy, cytotoxicity and function of drug candidates.
  • iPS human induced pluripotent stem cells
  • pluripotent stem cells induced from already differentiated body cells enabled the development of specific cells from patients, which can be observed in real time. In addition, they have made it possible to correct some neuronal-specific anomalies through pharmacological intervention in tissue culture.
  • the comparison of iPS cells from healthy individuals and patients identified differences in the stages of differentiation of stem cells into functional neurons, which also offers opportunities for pharmacological intervention (Vaccarino et al 201la, b).
  • mouse pluripotent stem cells such as embryonic stem cells
  • embryonic stem cells have been widely used for differentiation in in vitro studies. They form complex three-dimensional cell aggregates called embryoid bodies (embryoid bodies, EBs).
  • EBs embryoid bodies
  • cells of the neuronal line (Bain et al., 1995) and glial cell lines (Brüst le et al., 1999) can be generated from murine pluripotent stem cells.
  • Some of the early brain development processes are present in this microenvironment of an EB, allowing for an arbitrary collection of precursors and differentiated cells from different lineages.
  • OBs organoid bodies
  • Cell cultures derived from these human embryoid bodies contain cells that express different protein or mRNA markers that are characteristic of at least two different cell types (WO2001053465A1).
  • Embryoid bodies develop when stem cells are cultured under conditions that provide three-dimensional contact of the cells, e.g. the culture in hanging drops, in suspension culture, etc. (DE 102004025080, WO 20051 13747 AI, Wobus et al, 1988,).
  • the resulting embryoid bodies contain different cells of all three cotyledons.
  • differentiation factors it is possible to deliberately produce larger amounts of a specific cell type or to produce embryoid bodies with a specific cell type composition. For example, networks of neuronal cells and glial cells, but also simple neuromuscular networks could be produced. It is proposed to use such embryoid bodies for the testing of active ingredients (DE 102004025080 Al).
  • More complex structures than those in embryoid bodies are formed in organoids, which requires scaffolding for 3D cell culture.
  • the structure and design of the 3D framework are crucial to organize diverse cells.
  • the biological interaction between the cells and the scaffold is controlled by the material properties and scaffolding properties required for optimal cell adhesion, proliferation and differentiation, and the three-dimensional cellular architecture required for organotypic SD cultures to build. Therefore, compared to in vitro stem cell cultures differentiated and cultured in 2D, it is of considerable advantage to reproduce the tissue anatomy in a 3D culture.
  • the recent advancement of 3D cultural techniques has therefore allowed to design very specific cocultures and to integrate stem cells (Haycock 201 1).
  • knowledge of the optimal spatial and temporal microenvironment is critical to the success of organo-typical and tissue-specific differentiations such as those of the cortex or other brain regions.
  • radial glial cells For example, during development of the mammalian cortex, a particular family of glial cells, the so-called radial glial cells, produces layer-specific subtypes of excitatory neurons in a defined chronological order. Here, the neurons of the lower layers are formed first, followed by those of the upper layers, which then migrate through the lower layers of the cerebral cortex (Franco et al., 2012).
  • human pluripotent stem cells are predifferentiated in vitro to form first cortical stem and progenitor cells, which then form cortical projection neurons in a defined temporal order, before they become electrically active and spontaneously active, have active synaptogenesis, and thus form new neuronal interconnections (Gaspard et al., 2009; Shi et al., 2012).
  • WO2013063588 A1 describes the generation of three-dimensional organoids from explants of human tissue for long-term culture, and the use for the screening of possible therapeutics.
  • EP 1088096 A1 describes the testing of active substances on organoids from muscle and nerve cells.
  • WO2001053465 Al describes cells that can be obtained from human embryoid bodies. Among other things, the production of neural networks and their use is disclosed, for example, for drug testing. On the other hand, the inventors have set themselves the task of providing a new method for the production of cell cultures, which is a better test system for active substances, as it can be adapted more specifically to certain pathological conditions and therefore better results on an in vivo situation transmissible results , This object is solved by the subject of the claims.
  • the invention relates to a method of producing a neuronal planar cell culture, comprising steps of
  • pre-differentiated stem cells in a three-dimensional cell aggregate comprising at least two regions corresponding to different brain regions, and
  • c) further cultured on a planar support to produce a neuronal planar cell culture in which the cells form a neural network and have electrical activity specific to the particular brain region.
  • a planar cell culture results from the cells being cultured on a planar support.
  • the cells grow in one plane, that is to say essentially two-dimensionally, the cells obviously having a discrete height.
  • Three-dimensional means in connection with the invention that the cells not only grow in one plane, but also form three-dimensional structures. As a result, morphologically and physiologically different structures are formed as explained above. Appropriate measures (e.g., as outlined in Lancaster et al., 2013) can be used to produce three-dimensional cell aggregates comprising at least two regions corresponding to different brain regions. Simple three-dimensional cell aggregates are the spheres known in the art, but more complex organoids can also be used which are also known in the art. Both are referred to as cerebral organoid in the context of the invention. The cell aggregates may also comprise three, four, five, six, seven, eight, nine, ten or more regions corresponding to different brain regions.
  • hippocampus cerebellum, midbrain, striatum, amygdala, brain stem, thalamus, hypothalamus, basal ganglia, spinal cord, cortex, subcortical nuclei, olfactory bulb, optic nerve, retina, dorsal root ganglia and pituitary gland.
  • the cell aggregate comprises a plurality of cells.
  • cells are removed from a region and further cultured corresponding to a particular brain region.
  • the cell culture produced corresponds to a culture of cells from that one brain region.
  • cells from several regions corresponding to two or more brain regions can also be taken from a cerebral organoid in an experiment in parallel so that, for example, by means of an organoid, e.g. can create a cortex cell culture, a hippocampus cell culture, and / or a thalamic cell culture, depending on what regions are represented in the organoid, and as needed.
  • an organoid e.g. can create a cortex cell culture, a hippocampus cell culture, and / or a thalamic cell culture, depending on what regions are represented in the organoid, and as needed.
  • step b) cells from at least two regions which correspond to a particular brain region are removed (preferably initially separately), wherein these cells are cultured in coculture, in particular in mixed culture, in step c).
  • a co-culture is a culture of different cell types from different brain regions on a planar support, which facilitates communication of cell types from different brain regions, e.g. via neuronal compounds and / or soluble messengers that can be delivered into the medium allowed.
  • a mixed culture is a preferred co-culture in which the different brain regions are mixed and then cultured in appropriate cells.
  • a co-culture is also e.g. in different, possibly separate regions of a cell culture vessel possible, if communication is possible.
  • a co-culture or mixed culture preferably not all cells of the organoid are co-cultured. In particular, cells corresponding to another brain region and present in the organoid can be separated.
  • step b) the cells are removed at a stage which is already terminally differentiated neurons, and also their direct progenitor cells, since in order to form a neural network with electrical activity, the plasticity is increased.
  • the neuron type is already defined.
  • the region corresponding to a particular brain region, from which the cells in b) are taken, has at least one molecular marker for this brain region.
  • Examples of molecular markers for particular brain regions are distinguishable between the developing but differentiable status and the differentiated status.
  • Examples of developing area markers include:
  • markers can be used for specific dividing lines of brain areas, such as FGF8 (brain stem / midbrain border), SHH (diencephalon / midbrain border); BMP, Wnts, SHH (Local Forebrain Limits and Organizer).
  • differentiated marker markers include (in addition to the example markers mentioned above), among others:
  • NPY NPY, AGRP, POMC, CART and Orexin-A for the hypothalamus
  • One or more such molecular markers may be, e.g. after staining with fluorescently labeled antibodies, to identify the region of the organoid corresponding to a particular brain region.
  • the detection of markers can also take place at the RNA level.
  • the stem cells may comprise a detectable transgene, wherein the transgene is operably linked to a promoter that effects expression of the transgene in a particular brain region.
  • a suitable detectable transgene is, for example, the green fluorescent protein (GFP), or similar singularly existing proteins, RNA or DNA markers, or a labeled cell type-specific fusion product such as GFP or His or similar markers which bind to cell-type specific proteins, RNA or DNA were fused.
  • GFP green fluorescent protein
  • plasmid constructs these can be integrated into the undifferentiated iPS cells, for example by viral transduction or transfection, which thus leads to a cell type and differentiation stage-specific labeling of cells. This marks a defined region within the organoid.
  • multiple regions can be labeled simultaneously, with distinguishable detectable transgenes should be used.
  • a detection of the transgene is used to select the cells to be removed.
  • the promoters of the aforementioned markers can be used for this purpose, for example.
  • the region corresponding to a particular brain region from which the cells in b) are taken may be identified upon removal by infrared spectroscopy.
  • the region may be identified microscopically visually by morphological and anatomical structural relationships.
  • the selection of the correct region after collection and culture can be made by comparing the electrical activity of the cell culture with typical neuronal cell cultures from the particular region, e.g. be stored in a database, be verified. Alternatively, in this way, the particular brain region can be identified retrospectively.
  • the stem cells used to produce the cerebral organoids are adult stem cells or pluripotent stem cells, more preferably induced pluripotent stem cells.
  • the use of human stem cells is basically preferred, in particular human pluripotent, preferably human induced pluripotent stem cells can be used.
  • no human embryos are used or destroyed in the production of the stem cells.
  • It may also be non-human, e.g. to be mouse, rat or monkey stem cells, e.g. to non-human embryonic stem cells.
  • Stem cells from umbilical cord blood can also be used by humans.
  • the stem cells are stem cells isolated from a preferably human patient, again preferably induced pluripotent stem cells.
  • the planar support may e.g. a cell culture dish, wherein an analysis of the electrical activity, e.g. based on calcium signaling and corresponding imaging techniques is possible.
  • the planar support is preferably a microelectrode array (MEA) neurochip.
  • MEA microelectrode array
  • the electrical network activity of the neurons is recorded, analyzed and characterized and classified at the level of action potential patterns.
  • Neuroactive substances change the tissue-specific activity patterns in a specific way and can thus be characterized by changes in their electrical properties in the network.
  • a corresponding profile can be created by a multivariate data analysis developed and available from NeuroProof GmbH (Rostock, DE), which analyzes more than 200 features describing electrical activity.
  • the outstanding advantage of MEA technology over other neuronal cell electrical assessment techniques is the simultaneous recording of electrical signals in multi-cellular networks, which also allow communication between neurons to be studied.
  • the population of neurons analyzed when analyzed with an MEA chip typically comprises between at least 6 and a maximum of 256, usually between 10 and 50 cells.
  • the neurons in the cultures form networks with neighboring neurons. A communication of the neurons of a network via electrical and / or chemical synapses.
  • MEA Neurochips are available from the University of North Texas Center for Network Neuroscience (CNNS).
  • These 5x5 cm 2 glass chips have a central matrix with a diameter of 2 mm 2 , which includes 64 (or in another embodiment 2 x 32) passive electrodes and indium-tin interconnects embedded in silicone and as gold-plated free electrodes end up.
  • the electrode diameter is 20 ⁇ , the minimum distance between the electrodes is 40 ⁇ .
  • the MEA chips are deposited in a dissipation chamber (CNNS) on the preamplifier station (Plexon Inc, Dallas TX, USA), which contains a temperature control and 64 pre-amplifiers. The extracellular action potentials are recorded using a computer-controlled 64-channel amplifier system (Plexon Inc, Dallas TX, USA).
  • action potentials of up to four different neurons can be derived from each electrode.
  • the individual signals are separated and recorded using their action potential form using the recording software.
  • signals from up to 256 neurons can be recorded on a 64-electrode MEA neurochip.
  • the amplitude of the extracellular signals is in the range of 15 to 1800 ⁇ .
  • the sampling frequency of the signals is 40 kHz. This makes it possible to analyze not only the temporal sequence of the action potentials but also their course (waveform).
  • the times of the occurrence of the action potentials are continuously recorded in chronological order (spiketrains).
  • Action potentials are generated individually or in bursts (groups of rapidly successive action potentials).
  • the characteristics of this network Work communication can be analyzed with a variety of features based on the electrical activity of individual neurons. These features describe the synchrony and connectivity of the networks and their pharmacological influence.
  • the bursts can be defined with the software NPWaveX developed by NeuroProof (NeuroProof GmbH, Rostock, DE).
  • the networks of different tissues can be distinguished based on their electrical fingerprint, which is expressed in the activity of the neurons, the burst structure, the regularity of the occurrence of the bursts, and the synchronicity.
  • the temporal sequence of action potentials is characteristic for the various brain regions. These networks can be used to study the function of individual brain regions that are relevant to certain diseases.
  • the brain region-specific activity patterns may also be pharmacologically influenced.
  • stem cells predifferentiate in three-dimensional cultures, biologically relevant cellular signaling pathways and intercellular communication are formed. Compared with the two-dimensional differentiation of stem cells into different types of neurons, this technology has the advantage that the in vivo organ situation is better mapped. The complex morphology and interconnection of cells and regions allow biologically relevant cell-cell contacts and cell-matrix interactions. This allows similar physiological response patterns to be achieved as in vivo.
  • planar cultures produced according to the invention are more suitable for the investigation of drugs on their physiological activity as three-dimensional cell clusters which can be used according to the prior art:
  • Two-dimensional cultures have less ischemic conditions, since efficient gas exchange with the surrounding medium is ensured.
  • physiologically specific cell cultures according to the invention physiologically better predictive tests can be carried out for pharmacological questions.
  • the isolation of specific neurons, neuronal groups or regions from the cerebral organoids and their further cultivation on microelectrode arrays allows the individual electrical analysis of individual neurons and neuron groups in their network in their region-specific properties.
  • Dissociated neurons from different brain regions of the mouse exhibit typical patterns of activity resulting from the specific composition of various excitatory and inhibitory types of neurons. These patterns and their changes can be characterized after application of substances.
  • These two-dimensional cell cultures are particularly useful for the pharmacological study of the networks because Substance effects can be examined directly on the cells in concentration-response curves.
  • the present invention thus also provides a method for testing a substance comprising steps of:
  • a) produces a neuronal planar cell culture with the method according to the invention, b) analyzes the cells,
  • a substance may be an already known drug with neuronal effects, but also a substance not previously characterized in this respect. It may be, for example, a Protein, a peptide, a lipid, a sugar, a nucleic acid, an antibody or a low molecular weight (up to 900 g / mol) molecule.
  • the invention also provides a method for evaluating the differentiation of a neuronal planar cell culture comprising steps of:
  • a) produces a neuronal planar cell culture with the method according to the invention, b) analyzes the cell culture.
  • Such a method is e.g. useful to study neuronal developmental disorders, or to gain further insight into the physiological and / or pathological development of the brain and / or cerebral organoids. It can also be used to optimize cerebral organoid culture conditions, particularly with respect to the preparation of planar neuronal cell cultures of the invention that correspond to particular brain regions. This method further allows the identification of biological markers, as well as their changes in the expression pattern that are formed or modified in the development of a disease, or may lack and allow the creation of a personalized disease pattern specific to the patient from which the source cells were taken, from which the stem cells are derived.
  • the invention also provides a method for diagnosing or classifying a pathological condition, comprising steps of:
  • a) with the method according to the invention produces a neuronal planar cell culture from stem cells of a patient with a pathological condition
  • the invention also provides a method for optimizing the drug therapy of a patient comprising steps of: a) using the method according to the invention to produce a neuronal planar cell culture from stem cells of a patient,
  • the optimization of the therapy may be an optimization of the dose of a drug and / or a selection of a therapeutically effective drug.
  • the analysis can be based on that one
  • a) analyzes the morphology of the cells, and / or
  • analyzing expression pattern of the cells preferably using a method selected from the group comprising detection of markers by fluorescence microscopy, spectroscopic assay, immunohistochemical assay, immunocytochemical assay, FACS, immunoassay, enzymatic assay, protein assay, electrophoretic and chromatographic assay, HPLC, Western blot, Northerblot, Southerblot, luminescence detection assay, ELISA binding assay and colorimetric assay, and / or
  • the cells can be analyzed in a high-throughput screening by photometric measurements of the activity in multiwell format or, preferably, by recording the electrical activity of the neurons with microelectrode arrays in multiwell format.
  • Suitable software for analyzing electrical activity is available from Neuroproof GmbH, Rostock, Germany (NPWaveX).
  • the invention also relates to a neuronal planar cell culture which can be produced by a method according to the invention.
  • the stem cells may be derived from a human patient with a pathological condition, for the first time in vitro analysis and thus systematic testing of patient neurons from a particular brain region, e.g. allowed on a MEA neurochip.
  • the cell culture according to the invention may also comprise a co-culture or mixed culture of cells which does not occur physiologically in this form, eg a co-culture or Mixed culture of cells from cortex and hippocampus, amygdala and hippocampus, striatum and frontal cortex, midbrain and cortex and other combinations.
  • the cell culture is a human neuronal cell culture present on an MEA neurochip.
  • Fig. 1 Native activity of various tissue-specific neuronal cell cultures from murine embryos on MEAs.
  • C midbrain
  • Hc hippocampus
  • FIG. 2 A Native activity of a murine hippocampal network in which the hippocampus was prepared on embryonic day E17. A shows a synchronous, ictal activity in the form of population bursts lasting> 5 s. An application of 50 ⁇ carbamazepine to the same network prevented the long population bursts. All neurons are still active and generate short bursts (B). Shown are the timestamps (spikes) of 10 neurons over a period of 80 s each.
  • the inventors were able to show that networks from certain brain regions express specific patterns of activity. These can be clearly differentiated on the basis of the native Spike Trains.
  • the culture was carried out in serum-containing cell culture medium with glucose.
  • the cell density was adjusted tissue-specific.
  • 300 ⁇ of this cell suspension were transferred to the electrode field on the MEA neurochips.
  • 100 ⁇ of feeder cells were added outside the electrode field, which serve to condition the networks but can not form any physical contacts with the neurons to be analyzed.
  • the 204 activity-descriptive features used include features that describe the general activity of the networks, the burst structure, the regularity of the occurrence of the bursts, and the synchronicity of the networks.
  • the four tissues may differ in some of these features. All datasets were used to compare the native activity of the networks. Cross-validation was performed to check if the different culture types differ from each other. For this purpose, a classifier with 90% each randomly selected data sets was trained. Subsequently, the remaining 10% of these records were then classified against the learning dataset. The% allocation of the native activity in the respective tissues was determined. It could be shown that all native activities of the tissue-specific networks with a high recognition rate (85% -96%) can be classified in the activity of the respective brain regions. The activity patterns of the different tissues can therefore be quantified.
  • a feature score was identified to evaluate descriptive features for tissue differentiation.
  • the features listed in the table are features that are among the top 15 descriptive features of all 204 features.
  • FC frontal cortex
  • SC spinal cord
  • Mb + FC midbrain + frontal cortex
  • Hc hippocampus
  • Cells that are specific to a particular brain region e.g. the hippocampus, are identified, e.g. by staining with fluorescently labeled antibodies against a marker specific for this region (e.g., parvalbumin, cholecystokinin, somatostatin, calretinin, NRP2, DZF9, PROX1 for the hippocampus).
  • a marker specific for this region e.g., parvalbumin, cholecystokinin, somatostatin, calretinin, NRP2, DZF9, PROX1 for the hippocampus.
  • the identified regions are isolated from the remaining, unlabeled organoid by means of microsections, and the organoid piece removed is enzymatically digested in order to separate the cells.
  • the enzymatic comminution should be carried out by incubation in papain and DNase in a C0 2 incubator at 37 ° C. Thereafter, the tissue-enzyme mixture is centrifuged for 4 min at 800 rpm. The supernatant is carefully withdrawn with a serological pipette and discarded. The cell pellet is titrated with a serological pipette until the cell suspension appears cloudy. Thereafter, the centrifuge tube is allowed to stand for 5 minutes to allow larger cell aggregates to settle.
  • the supernatant is then transferred to a new 50 ml centrifuge tube and processed further.
  • the cell count is determined by means of a Neubauer counting chamber and the cell number is set to an optimized value.
  • Each electrode field is given a drop of 300,000 cells each.
  • the cell suspension should then be applied to the electrode pads of the MEA neurochips previously treated with poly-D-lysine and laminin for better attachment of the cells.
  • the MEA Neurochips with the Cell Suspension Drops are in the Incubated incubator, after about 2 h, the chips are filled with 3 ml of cell culture medium (such as DMEM or Neurobalsalmedium with the appropriate additives for better growth of the cells).
  • the cultivation is carried out in an incubator at 37 ° C and a C0 2 content, which is adapted to the cell culture medium.
  • the cells are fed every 3-4 days by replacing 1/3 of the medium with fresh medium.
  • an antimitotic treatment may be performed by adding FDU (fluoro-deoxy-uridines) and uridines to the culture medium. This prevents the further proliferation of the glial cells. The two antimitotics were gradually removed with each further change of medium. The time of stable electrical activity is determined and determined for substance screening.
  • FDU fluoro-deoxy-uridines
  • uridines uridines
  • the type of cells or the corresponding brain regions can be confirmed again with the help of markers and / or transgenes.
  • the electrical activity - as described in Example 1 for murine cells - can be analyzed with MEA neurochips. If testing of a substance is intended, the electrical activity can be analyzed before and after exposure to the substance.
  • Haycock 2011 3D cell culture a review of current approaches and techniques. Methods Mol Biol. 695: 1-15

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Abstract

L'invention concerne des procédés de culture de cellules neuronales et leur utilisation ainsi que les cultures de cellules ainsi préparées. En particulier, l'invention concerne un procédé de préparation de cultures de cellules planes neuronales, qui consiste à prédifférencier les cellules souches, par exemple les cellules souches pluripotentes induites humaines, dans un agrégat de cellules tridimensionnel, qui comprend au moins deux régions, qui correspondent à des régions différentes du cerveau, et qui est appelé organoïde cérébral, et à retirer les cellules progénitrices provenant d'une région qui correspond à une région déterminée du cerveau et à poursuivre leur culture sur un support plane, par exemple une neuropuce MEA. On obtient alors une culture de cellules planes neuronales dans laquelle les cellules forment un réseau neuronal et présentent une activité électrique ou électro-physiologique, qui est spécifique à la région déterminée du cerveau. Une telle culture cellulaire peut être utilisée par exemple dans le criblage haut débit pour tester l'action neurologique de substances.
EP15708820.4A 2014-03-11 2015-03-10 Obtention de cultures neuronales spécifiques à une région cérébrale à partir de cultures tissulaires tridimensionnelles de cellules souches Pending EP3137598A1 (fr)

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PCT/EP2015/054879 WO2015135893A1 (fr) 2014-03-11 2015-03-10 Obtention de cultures neuronales spécifiques à une région cérébrale à partir de cultures tissulaires tridimensionnelles de cellules souches

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Publication number Priority date Publication date Assignee Title
US9719068B2 (en) 2010-05-06 2017-08-01 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
CN113249297A (zh) 2014-05-28 2021-08-13 儿童医院医疗中心 用于经由定向分化将前体细胞转化为胃组织的方法和系统
EP3186632A1 (fr) 2014-08-28 2017-07-05 Stemonix Inc. Procédé de fabrication d'ensembles de cellules et utilisations de ceux-ci
EP3207123A1 (fr) 2014-10-17 2017-08-23 Children's Hospital Center D/b/a Cincinnati Children's Hospital Medical Center Modèle in vivo d'intestin grêle humain faisant intervenir des cellules souches pluripotentes et ses procédés de fabrication et d'utilisation
US11248212B2 (en) 2015-06-30 2022-02-15 StemoniX Inc. Surface energy directed cell self assembly
US10760053B2 (en) 2015-10-15 2020-09-01 StemoniX Inc. Method of manufacturing or differentiating mammalian pluripotent stem cells or progenitor cells using a hollow fiber bioreactor
CN109996870B (zh) * 2016-03-14 2024-08-13 新加坡科技研究局 从人多能干细胞产生中脑特异性类器官
US11066650B2 (en) 2016-05-05 2021-07-20 Children's Hospital Medical Center Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same
WO2018075890A1 (fr) * 2016-10-21 2018-04-26 StemoniX Inc. Test électronique de douleur neuronale
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same
KR101948396B1 (ko) * 2016-12-26 2019-02-14 동국대학교 산학협력단 특정 전자기파 처리를 통한 효율적 3d 중뇌 유사 오가노이드 제조 방법
CN111065732A (zh) 2017-09-11 2020-04-24 Imba-莫利库尔生物技术研究所 肿瘤类器官模型
CN111849770B (zh) * 2020-07-31 2023-07-18 深圳市博塔生物科技有限公司 一种建立体外神经网络的方法、体外神经网络及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69927685T2 (de) 1998-02-18 2006-07-13 Vandenburgh, Herman H. Verfahren zum screenen von verbindungen mit bioaktivität in organgeweben
US7795026B2 (en) 2000-01-21 2010-09-14 The Johns Hopkins University School Of Medicine Methods for obtaining human embryoid body-derived cells
DE102004025080B4 (de) 2003-06-23 2007-05-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Multizelluläre Testsysteme
WO2005113747A2 (fr) 2004-05-21 2005-12-01 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Systemes de culture tissulaire et organique multicellulaires
WO2013063588A1 (fr) 2011-10-28 2013-05-02 The Board Of Trustees Of The Leland Stanford Junior University Culture ex vivo, prolifération et expansion d'organoïdes tissulaires primaires

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Press release ESSENCE project - Drug Development With Neuronal Stem Cells On Neurochips", 15 May 2010 (2010-05-15), XP055653825, Retrieved from the Internet <URL:https://cdn.pressebox.de/a/5ff0a51ff7aeee46/attachments/0293485.attachment/filename/NeuroProof%20ESSENCE%20PressRelease170510%20eng.pdf> [retrieved on 20191218] *
ANONYMOUS: "Primary and Stem Cell Cultures as Functional Neuronal Networks - NeuroProof", 28 July 2014 (2014-07-28), XP055653837, Retrieved from the Internet <URL:https://www.neuroproof.com/en/Neuronal-Cultures.html> [retrieved on 20191218] *
See also references of WO2015135893A1 *

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