DE102014003465A1 - Obtaining brain region-specific neuronal cultures from three-dimensional tissue cultures of stem cells - Google Patents

Abstract

The invention relates to neuronal cell culture methods and their use as well as cell cultures produced therewith. In particular, the invention relates to a method for producing a neuronal planar cell culture in which stem cells, e.g. Human induced pluripotent stem cells, predifferentiated 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 planar carrier, e.g. As an MEA neurochip, further cultivated. The result is a neural planar cell culture in which the cells form a neural network and have electrical or electrophysiological activity that is specific to the particular brain region. Such a cell culture can, for. For example, high-throughput screening may be used to test substances for neurological effect.

Description

The invention relates to neuronal cell culture methods and their use as well as cell cultures produced therewith. In particular, the invention relates to a method for producing a neuronal planar cell culture in which stem cells, e.g. Human induced pluripotent stem cells, predifferentiated 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 planar carrier, e.g. As an MEA neurochip, further cultivated. The result is a neural planar cell culture in which the cells form a neural network and have electrical or electrophysiological activity that is specific to the particular brain region. Such a cell culture can, for. For example, high-throughput screening may be used to test 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 body, the primary neural networks share many of the characteristics of their source tissue, such as the 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.

With dissociated neuronal cell cultures taken from specific areas of the brain (eg, cortex, hippocampus, thalamus, spinal cord), brain region-specific traits 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 ).

As in their tissue of origin, the primary neuronal cell cultures develop from a mixture of different types of neurons and types of glial cells. Primary neuronal networks are composed of different brain region-specific neuron types, such as neuronal networks. As pyramidal cells, motor neurons, various interneurons. Furthermore, they also differentiate various glial cell types, such as astrocytes, microglia and, to a small extent, oligodendrocytes. As in the brain, the glial cells fulfill various functions here. The astrocytes have important auxiliary functions for the metabolism, such as. Maintenance of pH, potassium levels, nutrient supply, and the 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). 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 functional and morphological properties z. In the frontal cortex and the auditory cortex ( Xia et al., 2003b ), in the spinal cord ( Gramowski et al., 2000 ) and in the striatum ( Schock et al., 2010 ) can be examined.

These brain region-specific networks also react in a substance-specific manner to the administered substances ( Gramowski et al., 2004 ). Xia and Gross (2003a) show in their functional electrophysiological studies of ethanol and the antidepressant fluoxetine on primary cortical networks excellent agreement of their results with studies from experimental studies in animals.

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. Thus, 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 a spatial and temporal resolution ( Johnstone et al., 2010 ; Gramowski et al., 2010 ). Thereby, potential mechanisms of action can be elucidated, which can accelerate the preclinical development of drugs acting in the CNS.

NeuroProof technology (Neuroproof GmbH, Rostock, Germany) routinely uses murine neural networks on MEAs to quantify substance effects by measuring the effects of substances on electrophysiological activity patterns. Here, 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. In particular, 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. Since the use of animal, such. B. murine cells but only conditionally allow a good predictability of the effects of substances, it would be desirable to use human stem cells and cells derived therefrom increasingly for drug testing. So far, microcircuits similar to the in vivo situation could not be found in neuronal cultures obtained from human stem cells. For use in the testing of substances for pharmacological purposes, 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.

In 2006, 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 resemble 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.

The process of producing 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 allowed to correct some neuronal-specific anomalies through a pharmacological intervention in tissue cultures. In addition, by comparing iPS cells from healthy individuals and patients, differences in the stages of differentiation of stem cells into functional neurons were identified, which also provides opportunities for pharmacological intervention ( Vaccarino et. al. 2011a, b ).

Current work in neuropsychiatry with patients' iPS cells focuses on diseases that have specific genetic deficits, thus uncovering previously unknown pathologies of little or unknown origin and enabling pharmacological interventions ( Vaccarino et. al. 2011b ).

For drug testing, it is usually necessary to prepare a broad spectrum of differentiated cells separately in different and specialized cell cultures. When using conventional adult stem cells with a limited spectrum of differentiation, this usually requires culturing the differentiated cell cultures from different parent stem cells, often with very different growth conditions. This is usually associated with a great deal of time and expense. This disadvantage can be partly avoided by using pluripotent embryonic stem cells, which can differentiate into different cell types of all three cotyledons, but this is severely limited by ethical reasons already mentioned. Moreover, even with the use of pluripotent embryonic stem cells in these conventional test systems, several different and spatially separate cell cultures are required. Another major disadvantage of these cell monocultures is that the effect of a substance on a composite of different cells, such as is present in a natural tissue or organ, can not be investigated.

In the past, mouse pluripotent stem cells, such as embryonic stem cells, have been widely used for differentiation in in vitro studies. They form complex three-dimensional cell aggregates called embryoid bodies (EBs). This intermediate step allows cells of the neuronal line ( Bain et. al. 1995 ) as well as glial cell lines ( Brüstle et. al. 1999 ) are produced 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. These studies have opened the scientific field of embryoid bodies and more complex organoids, organoid bodies (OBs) from human induced pluripotent stem cells. 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 ( WO 2001053465 A1 ).

Embryoid bodies develop when stem cells are cultured under conditions that provide for three-dimensional contact of the cells, e.g. As the culture in hanging drops, in suspension culture, etc. ( DE 10 2004 025 080 . WO 2005113747 A1 . Wobus et al., 1988 ,). The resulting embryoid bodies contain different cells of all three cotyledons. By using 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 substances ( DE 10 2004 025 080 A1 ).

More complex structures than those in embryoid bodies are formed in organoids, which requires scaffolding for 3D cell culture. Here, the structure and design of the 3D framework are crucial to organize diverse cells. Within this 3D matrix, 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 3D organotypic 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 made it possible to design very specific cocultures and to integrate stem cells ( Haycock 2011 ). However, knowledge of the optimal spatial and temporal microenvironment is critical to the success of organotypic and tissue-specific differentiations such as those of the cortex or other brain regions.

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 temporal 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 ).

In 2D cultures of embryonic pluripotent stem cells it has been shown that it is possible to produce human cerebral neocortical stem cells and functional neurons of all cortical classes To form projection neurons. The process of cortical development is recapitulated. First, the 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 time sequence before they become electrically active and spontaneously active and active synaptogenesis, thus forming new neuronal interconnections ( Gaspard et. al. 2009 ; Shi et. al. 2012 ).

Lancaster et. al. (2013, Nature 501: 373-9) have developed a three-dimensional organoid culture system that has been formed from human pluripotent stem cells and has various discretely developed and interdependent brain regions. These include a cerebral cortex that contains a progenitor cell population that is capable of producing and organizing mature cortical neuron subtypes. In addition, these cerebral organoids recapitulate the features of human cortical development, namely a characteristic organization in zones of precursors, such as the subventricular zone, which contains the in vivo occurring radial radial glial cells. This recent development of a novel three-dimensional culture system for the differentiation of cerebral organoids from human induced pluripotent stem cells allows the "in vitro assembly" of a human embryonic brain equivalent that recapitulates the development of the brain. These organoids contain precursor populations that can develop into cortical neuronal subtypes, which could allow the in vitro study of complex brain diseases that lack appropriate animal models ( Muzio & Consalez 2013 ). Cerebral organoids or cerebral organoid bodies, which can be produced from pluripotent human stem cells, have distinct (at least two) discrete regions in which known brain region specific molecular marker proteins can be detected.

WO 2013063588 A1 describes the generation of three-dimensional organoids from explants of human tissue for long-term culture, and the use of screening for potential therapeutics. EP 1088096 A1 describes the testing of drugs on organoids from muscle and nerve cells.

WO 2001053465 A1 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.

• a) Stammzellen in einem dreidimensionalen Zellaggregat vordifferenziert, welches mindestens zwei Regionen umfasst, die unterschiedlichen Gehirnregionen entsprechen, und
• b) Zellen aus mindestens einer Region, die einer bestimmten Gehirnregion entspricht, entnimmt und
• c) auf einem planaren Träger weiter kultiviert, wobei eine neuronale planare Zellkultur entsteht, bei der die Zellen ein neuronales Netzwerk bilden und eine elektrische Aktivität aufweisen, welche für die bestimmte Gehirnregion spezifisch ist.

In particular, the invention relates to a method of producing a neuronal planar cell culture, comprising steps of

• a) pre-differentiated stem cells in a three-dimensional cell aggregate comprising at least two regions corresponding to different brain regions, and
• b) cells from at least one region that corresponds to a particular brain region, takes 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. As a result, 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. By appropriate measures (eg explained in Lancaster et al., 2013 ) can 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. As different brain regions are referred to in the invention: 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. It can therefore z. B. cells removed which correspond to the 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.

Generally, to produce cells of a neuronal cell culture of the present invention, cells are removed from a region and further cultured corresponding to a particular brain region. Thus, the cell culture produced corresponds to a culture of cells from that one brain region.

Of course, from a cerebral organoid in an experiment parallel cells can be taken from several regions that correspond to two or more brain regions, so that with the help of an organoid z. For example, a cortex cell culture, a hippocampal cell culture, and / or a thalamic cell culture can be established, depending on what regions are represented in the organoid, and as needed.

In one embodiment of the method according to the invention, in 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. B. on 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. Alternatively, a co-culture is also z. B. in different, possibly separate regions of a cell culture vessel possible, if communication is possible. In 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.

In 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.

• – GBX2, PAX2, für das Stammhirn;
• – SHH, Wnt5, Notch für das Mittelhirn;
• – OTX2, PAX6 für das Diencephalon;
• – Cerebrus, Dickkopf, FOXG1, SIX3, NKX2-1 für das Vorderhirn;
• – Marker der Nodal, BMP, Hedgehog, Wnt/ß-Catenin Signalwege und NPY, AGRP, POMC und CART für den Hypothalamus;
• – EMX1, FGF und SHH für die Kortexbildung und -Struktuierung
• – Hox, Pax, Pou, Lim für das Kleinhirn.

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:

• - GBX2, PAX2, for the brain stem;
• - SHH, Wnt5, notch for the midbrain;
• - OTX2, PAX6 for the Diencephalon;
• - Cerebrus, Dickkopf, FOXG1, SIX3, NKX2-1 for the forebrain;
• - markers of nodal, BMP, hedgehog, Wnt / ß-catenin signaling pathways and NPY, AGRP, POMC and CART for the hypothalamus;
• EMX1, FGF and SHH for cortical formation and trituration
• - Hox, Pax, Pou, Lim for the cerebellum.

Furthermore, various 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).

• – Nurr1, PITX3 und Tyrosinhydroxylase für das Mittelhirn;
• – LIM-HD und HOX Gene für die Rückenmarksidentität;
• – Parvalbumin, Cholecystokinin, Somatostatin, Calretinin, NRP2, DZF9, PROX1 für den Hippocampus;
• – Emx1, AUTS2, TSHZ2, LMO4, Calretinin, CTIP2, SATB2 für den Cortex;
• – NPY, AGRP, POMC, CART und Orexin-A für den Hypothalamus;
• – Lhx2/9, Meis1/2, lrx3. LHX1, Math1, Pdelc für das Kleinhirn,
• – GAD65, GAD67, Calbindin, Calretinin, Dopaminrezeptoren 1 und 2, Somatostatin, Parvalbumin, tyrosine hydroxylase für das Striatum;
• – KROX20, ISL1 für das Stammhirn.

Examples of differentiated marker markers include (in addition to the example markers mentioned above), among others:

• - Nurr1, PITX3 and tyrosine hydroxylase for the midbrain;
• - LIM-HD and HOX genes for spinal cord identity;
• - Parvalbumin, cholecystokinin, somatostatin, calretinin, NRP2, DZF9, PROX1 for the hippocampus;
• Emx1, AUTS2, TSHZ2, LMO4, calretinin, CTIP2, SATB2 for the cortex;
• NPY, AGRP, POMC, CART and Orexin-A for the hypothalamus;
• - Lhx2 / 9, Meis1 / 2, lrx3. LHX1, Math1, Pdelc for the cerebellum,
• - GAD65, GAD67, calbindin, calretinin, dopamine receptors 1 and 2, somatostatin, parvalbumin, tyrosine hydroxylase for the striatum;
• - KROX20, ISL1 for the brain stem.

One or more such molecular markers can, for. After staining with fluorescently labeled antibodies, to identify the region of the organoid that corresponds to a particular brain region. The detection of markers can also take place at the RNA level.

Optionally, 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 z. As the green fluorescent protein (GFP), or similar singular proteins, RNA or DNA markers, or a labeled cell type-specific fusion product such. GFP or His or similar markers fused to cell-type specific proteins, RNA or DNA. When using z. B. plasmid constructs, these can be integrated into the undifferentiated iPS cells, z. By viral transduction or transfection, thus resulting in cell type and differentiation specific cell labeling. This marks a defined region within the organoid. Also, multiple regions can be labeled simultaneously, with distinguishable detectable transgenes should be used. In this case, 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.

Alternatively or additionally, the region corresponding to a particular brain region from which the cells in b) are taken may be identified upon removal by infrared spectroscopy.

In another embodiment, 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. B. can be stored in a database, be verified. Alternatively, in this way, the particular brain region can be identified retrospectively.

Preferably, the stem cells used to produce the cerebral organoids are adult stem cells or pluripotent stem cells, more preferably induced pluripotent stem cells. In the context of the invention, the use of human stem cells is basically preferred, in particular human pluripotent, preferably human induced pluripotent stem cells can be used. Preferably, no human embryos are used or destroyed in the production of the stem cells. It may also be non-human, z. B. be mouse, rat or monkey stem cells, z. Non-human embryonic stem cells. Stem cells from umbilical cord blood can also be used by humans.

In one embodiment, the stem cells are stem cells isolated from a preferably human patient, again preferably induced pluripotent stem cells.

The planar support can z. B. be a cell culture dish, wherein an analysis of the electrical activity, eg. B. based on calcium signaling and corresponding imaging method is possible.

The planar support is preferably a microelectrode array (MEA) neurochip. This has the advantage that the electrical activity of the cells can be analyzed simply and accurately both at the cell level and at the population level. The electrical network activity of the neurons is recorded, analyzed and characterized and classified on 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. In addition to the analyzed cells, there are other, non-electrically active cells in the culture, which fulfill important functions for the maintenance of the functions of the neurons. The neurons in the cultures form networks with neighboring neurons. A communication of the neurons of a network via electrical and / or chemical synapses.

The technology was developed in 1972 by Thomas et al. first described and further developed by Guenter Gross ( Gross et al., 1977 ). All electrically active cells can be applied to these MEA chips to register their extracellular action potentials. The derivation of spontaneously active, self-organized neural networks on these MEA chips allows the study of the neuro-active potential of test substances by recording the extracellular action potentials of a large number of individual neurons over several hours. MEA Neurochips are z. From the Center for Network Neuroscience (CNNS) of the University of North Texas. These 5 × 5 cm ² glass chips have a central matrix with a diameter of 2 mm ² , which includes 64 (or in another embodiment 2 times 32) passive electrodes and indium-tin interconnects embedded in silicone and gold plated free electrodes end. The electrode diameter is 20 μm, the minimum distance between the electrodes is 40 μm. 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). After setting the threshold for the noise, 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. As a result, 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 μV. 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 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.

When 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.

• – zweidimensionale Kulturen weisen weniger ischämische Zustände auf, da ein effizienter Gasaustausch mit dem umgebenden Medium gewährleistet ist,
• – es findet eine bessere elektrische Kopplung der Neurone mit den Elektroden der MEA-Chips statt. Daher erhält man ein verbessertes Signal-Rausch-Verhältnis,
• – die Neurone sind pharmakologisch direkt zugänglich für Wirkstoffe. In dreidimensionalen Strukturen müssen die Wirkstoffe zunächst durch den gesamten Gewebeverband diffundieren, um an ihren Wirkort zu gelangen.

However, the 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 an efficient gas exchange with the surrounding medium is ensured,
• There is a better electrical coupling of the neurons with the electrodes of the MEA chips. Therefore, one obtains an improved signal-to-noise ratio,
• - The neurons are pharmacologically directly accessible to drugs. In three-dimensional structures, the active ingredients must first diffuse through the entire tissue network in order to reach their site of action.

Therefore, with physiologically specific cell cultures according to the invention, physiologically better predictive tests can be carried out for pharmacological questions.

Routine screening of substance effects with primary human neuronal cells is not possible. The pharmacological investigation of the properties of neural networks in two-dimensional cell cultures has hitherto been carried out by using dissociated cells from the brains of mice or rats. The use of neuronal cultures according to the invention therefore represents a tremendous advantage over conventional screening methods in early drug testing, because it significantly improves the predictability of the statements on efficacy.

The advantage of using human cells is their significantly higher predictive value compared to animal models that do not adequately reflect human physiology. This saves tremendous costs in drug testing because about 90% of all substances identified in high-throughput screening (HTS) do not exist in the clinical phase. This is partly due to the low transferability of the effectiveness of the substances of test methods in which animal cells are used, in the human physiology and on the other hand to side effects that were unpredictable in animal models.

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. Analysis of spicetrains and a variety of features derived therefrom, describing the general activity, burst structure, regularity of bursts, and synchronicity and connectivity of neurons, allows multiparametric data analysis and subsequent classification of the substances into different classes of compounds, such as anticonvulsants , Antipsychotics, antidepressants, analgesics etc.

• a) mit dem erfindungsgemäßen Verfahren eine neuronale planare Zellkultur herstellt,
• b) die Zellen analysiert,
• c) die zu testende Substanz in Kontakt mit der neuronalen planaren Zellkultur bringt,
• d) die Zellen nochmals analysiert,

wobei eine Wirkung der Substanz sich durch einen Vergleich der Zellen vor und nach In-Kontakt-Bringen mit der Substanz ergibt.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) analyzing the cells,
• c) bringing the substance to be tested into contact with the neuronal planar cell culture,
• d) the cells are analyzed again,

wherein an effect of the substance results from a comparison of the cells before and after bringing into contact with the substance.

A substance may be an already known drug with neuronal effects, but also a substance not previously characterized in this respect. It can be z. 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.

An exemplary method of testing a substance according to the present invention is disclosed in the co-pending application of Neuroproof GmbH "Method of Testing Neuroactive Substances" which comprises neural planar cell cultures derived from stem cells (eg, human induced pluripotent stem cells) prepared according to the invention. can be carried out.

• a) mit dem erfindungsgemäßen Verfahren eine neuronale planare Zellkultur herstellt,
• b) die Zellkultur analysiert.

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) analyzed the cell culture.

Such a method is z. B. 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 continues to allow the Identification of biological markers, as well as their changes in the expression pattern that are formed or modified in the onset of a disease, or lack, and allow the creation of a personalized disease pattern specific to the patient from which the original cells were derived from which the stem cells are derived.

• a) mit dem erfindungsgemäßen Verfahren eine neuronale planare Zellkultur aus Stammzellen eines Patienten mit einem pathologischen Zustand herstellt,
• b) die Zellkultur analysiert, wobei man das Ergebnis der Analyse mit dem Ergebnis der Analyse einer entsprechenden Zellkultur von Patienten mit bekannten pathologischen Zuständen und/oder Gesunden vergleicht.

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,
• b) analyzing the cell culture, comparing the result of the analysis with the result of analyzing a corresponding cell culture of patients with known pathological conditions and / or healthy subjects.

It may be useful to build a database containing appropriate analysis results from patients with known pathological conditions and / or healthy subjects. On a software basis can then z. B. Analysis results of a patient are compared with results from the database to classify the pathological condition or to diagnose the disease.

• a) mit dem erfindungsgemäßen Verfahren eine neuronale planare Zellkultur aus Stammzellen eines Patienten herstellt,
• b) die Zellen analysiert,
• c) ein zu testendes Medikament in Kontakt mit der neuronalen planaren Zellkultur bringt,
• d) die Zellen nochmals analysiert,

wobei eine Wirkung des Medikaments sich durch einen Vergleich der Zellen vor und nach In-Kontakt-Bringen mit dem Medikament ergibt.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,
• b) analyzing the cells,
• c) bringing a drug to be tested into contact with the neuronal planar cell culture,
• d) the cells are analyzed again,

wherein an effect of the medicament results from a comparison of the cells before and after contact with the medicament.

The optimization of the therapy may be an optimization of the dose of a drug and / or a selection of a therapeutically effective drug.

• a) die Morphologie der Zellen analysiert, und/oder
• b) Expressionsmuster der Zellen analysiert, bevorzugt unter Verwendung einer Methode, welche ausgewählt ist aus der Gruppe umfassend, Nachweis von Markern mit Fluoreszenzmikroskopie, spektroskopischem Assay, Immunhistochemischem Assay, Immuncytochemischem Assays, FACS, Immunoassay, enzymatischem Assay, Proteinassay, elektrophoretischem und chromatographischem Assay, HPLC, Westernblot, Northerblot, Southerblot, Lumineszenzdetektionsassay, ELISA-Bindungsassay und kolorimetrischem Assay, und/oder
• c) die elektrische Aktivität der Zellen analysiert, bevorzugt auf einem MEA-Neurochip, mehr bevorzugt mit einem Spike Train Analyseverfahren oder Klassifikationsverfahren.

The analysis can be based on that one

• a) analyzes the morphology of the cells, and / or
• b) 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
• c) analyzing the electrical activity of the cells, preferably on an MEA neurochip, more preferably with a spike train analysis method or classification method.

In a preferred embodiment, 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, in particular for analyzing spike trains or for classification, 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.

In this case, the stem cells can be derived from a human patient with a pathological condition, which for the first time the in vitro analysis and thus systematic testing of patient neurons from a particular brain region, eg. B. on an MEA neurochip allowed.

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, e.g. As 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.

In one embodiment, the cell culture is a human neuronal cell culture present on an MEA neurochip.

Legend

1 Native activity of various tissue-specific neuronal cell cultures from murine embryos on MEAs. A: frontal cortex (FC), B: spinal cord (SC), C: midbrain (Mb + FC), D: hippocampus (Hc). In each case the spicetrains of several neurons (each line corresponds to the activity of a neuron) over a length of 60 s are shown.

2A 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 μM 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.

example 1

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.

From embryonic mice on day 15-19 various brain regions (tissue of the frontal cortex (FC), hippocampus (Hc), midbrain, midbrain + frontal cortex (Mb + FC), spinal cord (SC)) were excised, the cells then enzymatically and mechanically dissociated and on microelectrode array chips for 27 to 35 days, usually over 4 weeks, cultivated.

The culture was carried out in serum-containing cell culture medium with glucose. The cell density was adjusted tissue-specific. In each case, 300 μl of this cell suspension were placed on the electrode field on the MEA neurochips. In addition, 100 μl of feeder cells, which serve to condition the networks but can not form any physical contacts with the neurons to be analyzed, were additionally brought outside the electrode field.

From day 28 in vitro (28 DIV), the native activity of these neuronal cell cultures with extracellular recordings was recorded and analyzed.

The native activity of the cultures from the various embryonic brain regions was analyzed multiparametrically and a classification was performed.

The 204 activity-descriptive features used include features that describe the general activity of the networks, the burst structure, the regularity of the bursts occurring, 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. Table 1: Features that distinguish the different tissues very well FC - Hc FC - Mb + FC FC - SC HC - SC HC - Mc + FC SC - Mb + FC Bursting (SUM) burst duration % Spikes in bursts Peak frequency amplitude Peak frequency amplitude % Spikes in bursts Burst Surprise Burstevent period Peak frequency position Peak frequency position SynAll SynAll Spike contrast Burst duration (SUM) Bursting (SUM) % Spikes in bursts event rate event rate Burst SD Burst form: slow SynAll Spike rate Interspike interval Peak frequency position Peak frequency position Bursting (SD) Spikerate in bursts Maximum spiking rate in bursts

The cultures of the frontal cortex (FC), spinal cord (SC), midbrain + frontal cortex (Mb + FC) and hippocampus (Hc) had tissue-specific patterns (Fig. 1 ). FC cultures are characterized by highly synchronous burst activity and few interburst spikes ( 1A ).

For cross-validation of the 4 tissues FC, SC, Mb + FC and Hc, the native activity of all datasets with 204 activity-descriptive characteristics was analyzed. The classifier was trained with 90% randomly selected records (DS). The remaining 10% of the respective data sets were then classified against it. The respective number of data sets (DS) is given in Table 2. The numbers in the table indicate the allocation to the respective tissues (in%). Table 2: Cross-validation of automatic assignment to specific brain regions FC hc Mb + FC SC DS Fc 88 2 8th 1 2315 hc 2 96 2 1 646 Mb + FC 10 4 85 2 336 SC 1 1 3 95 302

Example 2

According to the method of Lancaster et al., 2013 , cerebral organoids are produced.

Cells that are specific to a particular brain region, eg. B. the hippocampus, are identified, for. By staining with fluorescently labeled antibodies against a marker specific for this region (eg, parvalbumin, cholecystokinin, somatostatin, calretinin, NRP2, DZF9, PROX1 for the hippocampus).

The identified regions are z. B. isolated by microsection of the remaining, unlabeled organoid, the organoid piece removed enzymatically digested to separate the cells. The enzymatic comminution is carried out by incubation in papain and DNase in the CO 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 incubated in the incubator, after about 2 h, the chips with each 3 ml of cell culture medium (such as DMEM or Neurobalsalmedium with the appropriate additives for better growth of the cells) filled. The cultivation takes place in an incubator at 37 ° C. and a CO 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. If the glial cells are confluent, 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.

Optionally, the type of cells or the corresponding brain regions can be confirmed again with the help of markers and / or transgenes.

After formation of the network, 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.

Example 3

In murine networks of the hippocampus, spontaneous long-lasting (> 5 s) highly synchronous, ictagenic bursts may occur whose duration is reduced by application of anticonvulsants, such as carbamazepine or valproate ( 2 ).

In networks of neurons harvested and cultured from organoid hippocampal regions generated from patients' pluripotent stem cells, comparable ictagenic events are to be analyzed. This should make it possible to predict a susceptibility to epilepsy in patients / volunteers, even if epilepsy is not yet manifested or clinically proven by EEG derivations.

Furthermore, it should be possible to make a prediction in vitro about the success of a pharmacological treatment. Especially in temporal lobe epilepsy there is a high number of patients who do not respond adequately to therapies. The method described here would allow for preliminary testing of various drugs before initiating pharmacological therapy on the patient. Thus, a possible emergence of resistances can be avoided or searched for suitable additive or combination therapies. This would have a tremendous advantage for the patients, because a personalized therapy can be developed and side effects occurring due to a lengthy therapy finding are limited or even prevented.

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• DE 10 2004 025 080 . EP 1088096 A1 . WO 2001053465 A1 . WO 2005113747 A1 . WO 2013063588 A1

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2001053465 A1 [0017, 0024]
• DE 102004025080 [0018]
• WO 2005113747 A1 [0018]
• DE 102004025080 A1 [0018]
• WO 2013063588 A1 [0023]
• EP 1088096 A1 [0023]

Cited non-patent literature

• Gramowski et al., 2000 [0004]
• Xia et al. 2003a [0004]
• Xia et al. 2003b [0004]
• Gramowski et al., 2006 [0006]
• Johnstone et al, 2010 [0006]
• Xia et al., 2003b [0006]
• Gramowski et al., 2000 [0006]
• Schock et al., 2010 [0006]
• Gramowski et al., 2004 [0007]
• Xia and Gross (2003a) [0007]
• Johnstone et al., 2010 [0008]
• Gramowski et al., 2010 [0008]
• Takahashi et. al. 2006 [0013]
• Park et. al. 2008 [0013]
• Takahashi et. al. 2007 [0013]
• Yu et. al. 2007 [0013]
• Vaccarino et. al. 2011a, b [0014]
• Vaccarino et. al. 2011b [0015]
• Bain et. al. 1995 [0017]
• Brüstle et. al. 1999 [0017]
• Wobus et al., 1988 [0018]
• Haycock 2011 [0019]
• Franco et. al. 2012 [0020]
• Gaspard et. al. 2009 [0021]
• Shi et. al. 2012 [0021]
• Lancaster et. al. (2013, Nature 501: 373-9) [0022]
• Muzio & Consalez 2013 [0022]
• Lancaster et al., 2013 [0028]
• Gross et al., 1977 [0049]
• Lancaster et al., 2013 [0085]

Claims (15)

A method for producing a neuronal planar cell culture, comprising steps of a) pre-differentiated stem cells in a three-dimensional cell aggregate comprising at least two regions corresponding to different brain regions, and b) cells from at least one region that corresponds to a particular brain region, takes 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 method according to claim 1, characterized in that the cells taken in b) correspond to cells from a brain region selected from a group comprising: hippocampus, cerebellum, midbrain, striatum, brain stem, thalamus, hypothalamus, amygdala, spinal cord and cortex Frontal cortex, auditory cortex, primary motor cortex, primary visual cortex, primary sensory cortex, Broca center, temporal lobe, Wernicke center and pareto occipital association area. Method according to one of claims 1 or 2, characterized in that the region corresponding to a particular brain region and from which the cells are taken in b) comprises at least one molecular marker for this brain region, wherein the stem cells optionally comprise a detectable transgene, which is operably linked to a promoter which causes expression of the transgene in a particular brain region, wherein detection of the transgene is for selection of the cells to be extracted. Method according to one of the preceding claims, characterized in that the region corresponding to a particular brain region and from which the cells are taken in b), is identified in the extraction by means of infrared spectroscopy. Method according to one of the preceding claims, characterized in that in step b) cells from at least two regions corresponding to a particular brain region are removed, wherein these cells are cultured in step c) in co-culture or mixed culture. Method according to one of the preceding claims, characterized in that the stem cells are human stem cells, in particular human pluripotent stem cells. Method according to one of the preceding claims, characterized in that the stem cells from a preferably human patient isolated stem cells. Method according to one of the preceding claims, characterized in that the planar support is a microelectrode array neurochip. Method for testing a substance, characterized in that a) a neuronal planar cell culture is produced by the method according to one of the preceding claims, b) the cells are analyzed, c) the substance to be tested is brought into contact with the neuronal planar cell culture, d) the cells are re-analyzed, e) wherein an effect of the substance results from a comparison of the cells before and after bringing into contact with the substance. Method for evaluating the differentiation of a neuronal planar cell culture, characterized in that a) a neuronal planar cell culture is produced by the method according to any one of claims 1-8, b) the cell culture is analyzed. Method for the diagnosis of a pathological condition, characterized in that a) with the method according to one of claims 1-8, produces a neuronal planar cell culture from stem cells of a patient with a pathological condition, b) analyzes the cell culture, the result of the analysis with the result of analyzing a corresponding cell culture of patients with known pathological conditions. A method for optimizing the medical therapy of a patient, characterized in that a) using the method according to one of claims 1-8 produces a neuronal planar cell culture from stem cells of a patient, b) the cells are analyzed, c) a drug to be tested in contact with the neural planar cell culture, d) re-analyzing the cells, an effect of the drug being obtained by comparing the cells before and after contacting the drug, optimizing the therapy optionally optimizing the dose of a drug or a selection of a therapeutically effective drug. Method according to one of claims 9-12, characterized in that in the analysis a) the morphology of the cells is analyzed, and / or b) expression patterns of the cells are analyzed, preferably using a method which is selected from the group comprising detection of Markers with 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 c) the electrical activity of the cells is analyzed, preferably on an MEA neurochip, more preferably with a spike train analysis method or classification method. A method according to claim 13, characterized in that the cells are 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. Neuronal cell culture producible by a method according to any one of claims 1-8, wherein preferred a) the stem cells are derived from a human patient with a pathological condition, b) the cell culture comprises a co-culture of cells which does not occur physiologically, and / or c) the cell culture is a human neuronal cell culture present on an MEA neurochip.

Images