Generation of adult-like murine brain organoids from adult stem cells

Abstract

The invention relates to an in vitro method of generating a murine cerebral organoid cell culture model, comprising cultivating murine neural stem cells obtained from an adult mouse until neurospheres have formed, and subsequently differentiating said neurospheres, thereby generating a murine cerebral organoid cell culture model. The invention further relates to an in vitro murine cerebral organoid cell culture model comprising mature astrocytes, oligodendrocytes and / or neurons produced according to the method of the invention and its use for drug or toxicity screenings or for studying neurological diseases. In other aspects the invention further relates to kits, cell culture media and supplement compositions comprising components to be used in the context of the present invention.

Description

[0001] GENERATION OF ADULT-LIKE MURINE BRAIN ORGANOIDS FROM ADULT STEM CELLS

[0002] DESCRIPTION

[0003] The invention is the field of cell biology, neurobiology, murine cell culture models and respective methods.

[0004] The invention in general relates to an in vitro method of generating murine brain organoid models from murine neural stem cells, which have been obtained from adult mice, and to cell culture models generated accordingly.

[0005] In particular, the invention relates to an in vitro method of generating a murine cerebral organoid cell culture model, comprising cultivating murine neural stem cells obtained from an adult mouse until neurospheres have formed, and subsequently differentiating said neurospheres, thereby generating a murine cerebral organoid cell culture model. The invention further relates to an in vitro murine cerebral organoid cell culture model comprising mature astrocytes, oligodendrocytes and / or neurons produced according to the method of the invention and its use for drug or toxicity screenings or for studying neurological diseases. In other aspects the invention further relates to kits, cell culture media and supplement compositions comprising components to be used in the context of the present invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Organoids are an important tool for modelling disease mechanisms and screening for new treatment options. Currently, organoids are revolutionizing infection and drug research, among other things, as larger quantities of very comparable, complex tissue can be readily processed in parallel. In addition, organoid research represents a milestone in the reduction of animal testing. The most common technology at the moment is the use of so-called induced pluripotent stem cells (iPSCs) as the starting material for various organoids. This is particularly important for human material, as the extraction of adult stem cells from living humans has high ethical hurdles or is not possible.

[0008] Especially brain organoids demonstrate a promising approach to study neurodegenerative diseases in a manner more physiological than simple monoculture of primary neurons isolated from mouse models. The use of organoids further enables investigating the increased complexity of the brain with many cell types and the associated neuronal performance in situ.

[0009] So far, most brain organoids were generated from human iPSCs, which are able to build a selforganizing three-dimensional structure. Murine organoids have shown to be faster in development than human organoids and by that are efficient systems to study a diverse range of diseases (Ciarpella et al., 2021). However, brain organoids from murine background are rarely found, although many mouse cell lines and mouse strains for modelling neurodegenerative diseases have been established in the past. So far, only few murine organoid systems, for example made from embryonic stem cells (ESC), have been established. At present, the production of murine organoids is dependent either on the availability of iPSCs or from E14.5 embryos obtained from pregnant mice (Ciarpella et al., 2021 and 2023). However, such methods have several disadvantages.

[0010] The production of embryonic progenitor cells (EPCs) from embryos of pregnant mice is an animal experiment that requires official approval, raises ethical concerns (e.g., as embryonic pups are isolated from sacrificed mothers) and constitutes a time-consuming procedure. The use of the tiny E14.5 embryos also results in very small number of progenitor cells per sacrificed mouse.

[0011] In addition, both protocols (Ciarpella et al., 2021 and 2023) start from embryonic stem cells, which are less mature than adult stem cells, and therefore require at least one additional differentiation step, extending the risk and duration of the differentiation protocol, rendering the procedure less reliable. The protocols of the prior art, e.g., Ciarpella et al., 2021 and 2023, involve a first growth phase of neurospheres, followed by a subsequent dissociation and organoid assembly. However, neurosphere cultivation of NSCs has the disadvantage that cell populations inside the neurosphere differ from those outside, due to the varying access to growth factors in the medium. Thus, the resulting cells used for organoid generation in the prior art, e.g., Ciarpella et al., 2021 and 2023, are less uniform. In addition, organoids from embryonic stem cells lead to products with an embryonic maturity and represent a fetal brain environment, likely including epigenetic signatures and lack processes of the adult brain, such as myelination, which occurs after birth.

[0012] In alternative approaches brain organoids are generated from human iPSCs. However, first, the production of iPSCs by viral transduction of pluripotency markers (e.g. Oct and SOX) into differentiated adult cells is not reliable. Often, dedifferentiated teratoma-like populations are obtained or organoids can only differentiate into certain tissues. When using iPSCs for generating organoids, the reliable generation of a tissue type of interest is not guaranteed; therefore, for each batch of reprogrammed cells, it has to be evaluated anew, whether the desired tissue type, e.g., neuronal cells, can be generated. In the prior art, e.g., WO2019 / 174535A1 or KR20210106966A, the use of human iPSCs as starting cells for generating neural organoids is described. However, organoids originating from human reprogrammed iPSCs have the disadvantage that iPSCs are permanently immortalized and retain fetal traits concerning maturity of the product, including concerning epigenetic signatures. For example, genetic and epigenetic instability is a key issue in iPSC cultivation, leading to reduced uniformity and reproducibility of experiments and the obtained organoids.

[0013] On the other hand, cultured embryonic stem cells (ESCs) may also be used to generate genetically modified mice or organoids. However, as ESCs are (almost) omnipotent stem cells, they must be cultured on so-called ‘feeder cells’ to maintain their omnipotency and any irritation or variation in culture condition leads to rapid cell loss. In particular, the protocol to differentiate ESCs towards neuronal differentiation is complex and errors, as with iPSCs, often result in cell populations that can no longer form complex cell types and tissue patterns. Finally, such organoids would again resemble fetal features and not reflect postnatal to adult features. In summary, such factors have so far hindered the use of the promising research tool of murine organoid cultures.

[0014] An alternative to the above approaches would be the use of adult neural stem cells (NSC). However, no NSC-derived organoids have been published so far according to the knowledge of the inventors.

[0015] Hence, considering the prior art, there remains a significant need in the art to provide means for a time-saving and reproducible generation of neural, preferably cerebral cell culture models that may be employed in diverse laboratory experiments and in vitro screening applications thereby reducing the need for animal experiments.

[0016] SUMMARY OF THE INVENTION

[0017] In light of the prior art, a technical problem underlying the present invention was the provision of an improved method for generating a neural cell culture model suitable for in vitro screening applications.

[0018] Another problem underlying the present invention was the provision of a fast and straightforward method for generating murine cerebral neural cell culture models.

[0019] A further problem underlying the invention was the provision of a murine cerebral neural cell culture model.

[0020] Another problem underlying the invention was the reproducible provision of a murine cerebral neural cell culture model from cells obtained from adult mice.

[0021] The above-mentioned problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0022] To overcome the afore-mentioned problems of providing a fast and straightforward method for generating murine cerebral neural cell culture models, the inventors developed a method employing murine neural stem cells obtained from adult mice that enables a faster and simpler generation of in vitro neural organoids than current state of the art methods.

[0023] The invention therefore relates to an in vitro method of generating a murine cerebral organoid cell culture model, comprising cultivating murine neural stem cells obtained from an adult mouse until neurospheres have formed, and subsequently differentiating said neurospheres, thereby generating a murine cerebral organoid cell culture model.

[0024] In other words, in embodiments, the present invention relates to in vitro methods of generating murine brain organoid models from murine neural stem cells, which have been obtained from adult mice. In embodiments the murine brain organoid models according to the invention may be generated according to the present method within 30, 25 or even 21 days.

[0025] The present method allows in advantageous embodiments the fast, reproducible and efficient generation of a large number of organoids, for example, for brain research and from a wide range of established mouse strains / lines. In embodiments, the present method enables the use of established mouse models and mouse strains for brain research without the need for animal experiments.

[0026] In preferred embodiments the method comprises the steps of: a. providing a cell culture comprising murine neural stem cells obtained from an adult mouse and cultivating the cell culture for between 3-7 days until neurospheres have formed, b. replacing at least a fraction of the culture medium with induction medium and cultivating the cell culture for between 1-4 days, c. replacing the induction medium with differentiation medium and cultivating the cell culture for between 9-17 days, wherein the total duration of cell culture is at least 21 days, thereby generating a murine cerebral organoid cell culture model.

[0027] In embodiments, the murine neural stem cells are obtained from the hippocampus, preferably from the subventricular zone (SVZ) and / or the dentate gyrus (DG), of the adult mouse.

[0028] In some embodiments the extraction of neural stem cells from the hippocampus, preferably from the subventricular zone (SVZ) and / or the dentate gyrus (DG), of an adult mouse is performed according to Walker et al., 2014, as described in Kirchenwitz et al., 2022, or similar methods for obtaining adult neural stem cells from mice.

[0029] In some embodiments the extraction of neural stem cells from the hippocampus, preferably from the subventricular zone (SVZ) and / or the dentate gyrus (DG), of an adult mouse is performed as described by Walker et al., 2014 in combination with Babu et al. 2011 .

[0030] In embodiments, the murine neural stem cells are obtained from two neurogenic zones of the hippocampus, namely the subventricular zone (SVZ) and the dentate gyrus (DG) of the adult mouse. Using neural stem cells from two neurogenic zones preferably enables the reliable generation of neurons (mainly from cells of the subgranular zone (SGZ)) and glial cells (mainly from SVZ cells), such that a complex organoid structure can be obtained.

[0031] In preferred embodiments, the present murine neural stem cells or neural precursor cells have already made the neuronal fate decision and / or are not immortalized by (still) expressing sternness factors, thereby enabling a more straightforward and reliable terminal differentiation of the cells.

[0032] Until today, embryonic stem cells from mouse blastocyst have commonly been used for generating murine organoid models. To reduce ethical concerns regarding animal experiments and time-intensive differentiation and validation approaches, the present inventors aimed to provide alternative methods enabling the generation of organoid models from neural precursor cells (NPC) or murine neural stem cells obtained from adult mice. According to the knowledge of the inventors, methods for obtaining NPC-derived organoids have not been published so far. Hence, the aim of the present inventors was to generate brain organoids from adult mice. For this, the inventors established a protocol for the isolation of adult neural precursor cells (NPCs). NPCs can be differentiated at least into neurons and glia cells and therefore in (nearly) all neural cell types present in the mature brain. Moreover, NPCs can be cultivated as adherent cells, but are also able to form 3D-neurosphere structures. The inventors developed a method to differentiate such neurospheres into self-organizing organoids thereby providing a new model for investigating neurodegenerative diseases, neuro-inflammation or infections.

[0033] The inventors found that the methods and other means described herein surprisingly facilitate the generation of murine cerebral organoids in vitro significantly faster compared to prior art methods and in a reproducible manner. The inventors found, that when cultivating murine adult neural stem cells according to the methods described herein, the reproducible and stable murine cerebral organoids might be generated in about a month’s time of cultivation or even less. In embodiments, the present organoid model is obtained already after 21-25 days of total culture time according to the present method.

[0034] In vitro approaches for modeling brain development or disease are an important area of research. The present method enables in embodiments the generation of murine cerebral organoids, which resemble endogenous 3-dimensional brain tissue layering in a single rosette structure. In embodiments, the present method may be employed for developmental studies, as well as for the investigation of a variety of brain diseases and infections. In addition, in embodiments the cerebral organoids according to the invention may be used to study the efficacy and response to drugs in a time-efficient and high-throughput manner. The present organoid model facilitates in embodiments the use of any known and established mouse model for organoid-based research without the need of extensive mouse experiments.

[0035] In addition, compared to the methods of the prior art relying on isolation of ESCs from murine embryos, the present method enables a significant reduction of laboratory animals required, as far fewer cells can be isolated from the quite small E14.5 embryos than from adult brains.

[0036] Protocols of the prior art, e.g., Ciarpella et al., 2021 and 2023, start from embryonic progenitor cells (EPCs), which are less mature, than adult stem cells, and therefore require at least one additional differentiation step, extending the duration of the differentiation protocol and rendering the procedure less reliable. In addition, said prior art protocols involve a first growth phase of neurospheres, followed by a subsequent dissociation and organoid assembly. On the contrary, the method according to the invention preferably enables organoid generation without an interim step, which is faster and easier to perform, and more gentle for the cells, resulting in higher organoid quality.

[0037] Further, neurosphere cultivation of NSCs has the disadvantage that cell populations inside the neurosphere differ from those outside, due to the varying access to growth factors in the medium. Thus, the cells used for organoid generation in the prior art, e.g., Ciarpella et al., 2021 and 2023, are less uniform. In contrast, the present method preferably enables in embodiments the culture of isolated NSCs in a monolayer, where all cells are subjected to the same culture conditions, resulting in uniformity of the obtained organoid. This can be of particular importance, as cell differentiation is complex and the initial cell state has significant relevance for the quality of the obtained product. Moreover, preferred embodiments of the present method enable reliable control of the exact number of cells forming each organoid.

[0038] In addition, embodiments of the present method combine stem cells from two neighboring, yet different, niches in the brain (SGZ and SVZ) enabling a higher complexity, as, for example, compared to NSCs from just one region. Cells from the subgranular zone (SGZ) in the hippocampus preferentially generate neurons, while NSCs from the SVZ, have a higher potential to generate glial cells. The combined use of cells from both regions, as described herein, presents a novel and unique approach for developing a complex organoid structure, as prior art methods have only isolated and used NSCs from the SGZ so far, e.g., Ciarpella et al., 2021 and 2023.

[0039] In addition, organoids from embryonic stem cells lead to products with an embryonic maturity including age related epigenetic signatures. In embodiments the organoids generated according to the present invention are generated from adult stem cells and may even show age-dependent features. For example, Figure 9 shows organoids from younger adult (under 40 weeks) and old (over 70 weeks) mice, wherein organoid size was significantly reduced in organoids derived from old mice compared to those derived from young mice, indicating an apparent aging phenotype that cannot be reproduced using methods described in the prior art, e.g., Ciarpella et al., 2021 and 2023.

[0040] For example, a distinguishing feature between adult neural stem cells (aNSCs) as compared to embryonic neural stem cells (eNSCs) is that they differentiate faster and more reliably into more complex organoids with mature brain-like features. Therefore, neither the cell source, nor the procedures described in the prior art starting from embryonic stem cells, e.g., Ciarpella et al., 2021 and 2023, are suitable to generate mature cerebral organoids, and such procedures are not specific enough for differentiating neurons and glia cells. The same principle applies to other protocols that generate brain organoids based on Lancaster et al., as they merely use slight variations of the neuronal differentiation media, or cells from slightly different sources (e.g., murine versus human).

[0041] On the contrary, the method of the present invention represents a major advance over protocols of the prior art for differentiating mouse cells and, due to the achieved, age-related e.g., epigenetic features, even advances beyond all types of organoids from mammal embryonic or induced / reprogrammed stem cells of the prior art.

[0042] In summary, prior art protocols were not able to make full use of all the established mouse models, yet. The present method, on the contrary, enables a new connection of aspects of traditional mouse experiments in neuroscience with advanced organoid and cell-based approaches. In this regard, the present method also facilitates the application to genetically engineered mice or even other mammals due to its superior versatility.

[0043] In embodiments the murine neural precursor cells obtained from an adult mouse comprise murine neural stem cells (NSCs). In embodiments the murine neural precursor cells (NPCs) obtained from an adult mouse comprise murine neural stem and / or progenitor cells. In embodiments the cell culture is cultivated in a neurobasal medium. The inventors surprisingly found, that in embodiments due to the use of tissue-specific stem cells, such as neural stem cells, obtained from adult mice no specific and expensive stem cell culture medium has to be used. It was entirely unexpected that instead, in embodiments only neurobasal cell culture medium may be used for the generation of a neural organoid model according to the invention. In detail, the inventors surprisingly found, that neural stem cells may be cultured using neurobasal medium instead of the commonly used DMEM / F12-Glutamax, which was considered in the prior art to be the best option for stimulating proliferation and maintaining stem cell identity. However, the inventors unexpectedly found that neurobasal medium, which was until now primarily viewed as supportive for neuronal differentiation of adherent cultures or neural progenitors, may be advantageously used also for preserving and expanding free-floating neural stem cell cultures (e.g., neurospheres).

[0044] In embodiments, the initial cell culture medium in step a. (providing a cell culture comprising murine neural stem cells obtained from an adult mouse and cultivating the cell culture for between 3-7 days until neurospheres have formed) comprises neurobasal medium and one or more of fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), heparin, L-glutamine and B27- supplement is added before cultivating the cell culture for at least 4 days.

[0045] In embodiments, the initial cell culture medium in step a. (providing a cell culture comprising murine neural stem cells obtained from an adult mouse and cultivating the cell culture for between 3-7 days until neurospheres have formed) in which the cell culture is cultivated is proliferation medium, preferably comprising neurobasal medium, B27 supplement, L-glutamine or GlutaMAX, EGF, FGF2, heparin and optionally penicillin / streptomycin.

[0046] In embodiments, the initial cell culture medium in step a. comprises fibroblast growth factor 2 (FGF2). In embodiments, the initial cell culture medium in step a. comprises epidermal growth factor (EGF). In embodiments, the initial cell culture medium in step a. comprises heparin. In embodiments, the initial cell culture medium in step a. comprises L-glutamine. In embodiments, to the initial cell culture medium in step a. B27- supplement is added, preferably before cultivating the cell culture for at least 4 days.

[0047] In embodiments, the induction medium comprises neurobasal medium and / or one or more of heparin, L-glutamine and B27- supplement. In embodiments, the induction medium comprises heparin. In embodiments, the induction medium comprises L-glutamine. In embodiments, the induction medium comprises B27- supplement. In embodiments, the induction medium comprises neurobasal medium and one or more of heparin, L-glutamine and B27- supplement, and lacks FGF2 and / or EGF. In embodiments, the induction medium does not comprise FGF2 and / or EGF.

[0048] In some embodiments, L-glutamine is added / comprised as GlutaMAX (e.g., Gibco GlutaMAX supplement).

[0049] In embodiments, heparin may be essential for the proliferation and / or differentiation of stem cells, supporting their transformation into progenitor cells, e.g., during the induction phase. Thereby heparin preferably enables culture conditions having an impact on the obtained organoid, e.g., by facilitating generating mature organoids, e.g., comprising mature oligodendrocytes and / or myelinated neuronal axons.

[0050] In embodiments, in step c. (replacing the induction medium with differentiation medium and cultivating the cell culture for between 9-17 days) the cell culture is cultivated in a first differentiation medium for between 3-7 days, and subsequently in a second differentiation medium for between 6-10 days.

[0051] In embodiments, in step c. the cell culture is cultivated in a differentiation medium, wherein the culture duration in the differentiation medium is preferably divided into a culture duration in a first differentiation medium and a subsequent culture duration in a second differentiation medium. In embodiments differentiation of the cell culture is induced by the culture in a differentiation medium, optionally by the culture in a first differentiation medium and subsequently a second differentiation medium.

[0052] In embodiments, in step c. the cell culture is cultivated in a first differentiation medium for between 3-7 days. In embodiments, in step c. the cell culture is cultivated, after culture in a first differentiation medium, in a second differentiation medium for between 6-10 days.

[0053] In embodiments, the neurobasal medium is or comprises neurobasal medium plus, preferably, wherein the culture medium is used for differentiating the cultivated cells and / or as a (first or second) differentiation medium.

[0054] In embodiments, two differentiation media (I and II) are used subsequently, comprising a first (I) for inducing differentiation of the cells and a second (II) for maturation and maintenance of the organoid. In embodiments, a first differentiation medium enables the unguided differentiation of neurons and glia cells. In embodiments, a second differentiation medium enables the maturation of organoids, e.g., comprising one or more of GDNF, ascorbic acid, and / or BDNF. In embodiments the second differentiation medium not only fosters optimal neuronal differentiation and maturation, but also promotes glial differentiation and processes, such as myelination, which can be crucial for gaining complex neuron-glia interactions.

[0055] In embodiments, the first differentiation medium comprises neurobasal medium plus. In embodiments, the first differentiation medium comprises neurobasal medium plus and / or one or more of insulin, L-glutamine (e.g., GlutaMAX), non-essential amino acids (NEAA), B27-Plus supplement, N2-supplement and one or more basement membrane and / or extracellular matrix (ECM) component (e.g., Geltrex or Matrigel). In embodiments, the first differentiation medium comprises insulin. In embodiments, the first differentiation medium comprises L-glutamine. In embodiments, the first differentiation medium comprises non-essential amino acids (NEAA). In embodiments, the first differentiation medium comprises B27-Plus supplement. In embodiments, the first differentiation medium comprises N2-supplement. In embodiments, the first differentiation medium comprises on or more basement membrane component and / or extracellular matrix (ECM) component (e.g., laminin, collagen, Geltrex and / or Matrigel).

[0056] In some embodiments, L-glutamine is added / comprised as GlutaMAX (e.g., Gibco GlutaMAX supplement). In embodiments, the one or more basement membrane component is selected from or comprises extracellular matrix (ECM) components, laminin, collagen, collagen type IV, entactin, or Geltrex. In embodiments, basement membrane components comprise one or more of laminin, collagen, collagen type IV, entactin, extracellular matrix (ECM) components, or Geltrex.

[0057] In embodiments, the cell culture is cultivated in one or more of method steps, preferably during the cultivation in first differentiation medium in a culture vessel, wherein at least one basement membrane component is added to the cell culture medium, preferably after 1-10 days.

[0058] In embodiments, after 7-10 days of total culture duration at least one basement membrane component and / or ECM component is added to the cell culture medium.

[0059] In embodiments, after 7-10 days of total culture duration the organoid cell culture is passaged / re- seeded / re-plated, preferably wherein the passaging / re-seeding / re-plating comprises the embedding or coating of the organoids with at least one basement membrane component and / or ECM component.

[0060] In embodiments, only (or primarily) the organoid within the cell culture vessel, preferably not the culture vessel itself, is ‘coated’ by adding at least one basement membrane component and / or ECM component to the cell culture medium, preferably after 7-10 days of total culture duration. In some of such embodiments, a ‘liquid coating’ procedure is performed, wherein at least one basement membrane component and / or ECM component is added to the culture medium.

[0061] In other embodiments organoids are directly embedded in I coated with at least one basement membrane component and / or ECM component, preferably after 7-10 days of total culture duration, and subsequently added (preferably after polymerization of the least one basement membrane and / or ECM component (matrix)), to the cell culture medium, preferably without any additional basement membrane component and / or ECM component being added or present in the medium itself. In some of such embodiments the differentiation medium does not comprise any (additional) basement membrane component and / or ECM component.

[0062] In embodiments, the coating of the organoid is repeated at least once after 7, 8, 9, 10, 11 , 12, 13 or 14 days of total culture duration.

[0063] In embodiments, the second differentiation medium comprises neurobasal medium plus. In embodiments, the second differentiation medium comprises neurobasal medium plus and / or one or more of L-glutamine (e.g., GlutaMAX), NEAA, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, insulin, N2-supplement and B27-Plus supplement.

[0064] In embodiments, at least a fraction of the neural stem cells expresses Sox2 and / or Nestin. In embodiments, at least a fraction of the neural stem cells used as initial cells expresses Sox2 and / or Nestin.

[0065] In embodiments, at least a fraction of the cultured cells expresses Nestin and / or microtubule- associated protein 2a / b (Map2a / b) and / or Tau or glial fibrillary acidic protein (GFAP) and / or oligodendrocyte transcription factor 2 (OLIG2) after 1-10 days of total cell culture duration, preferably after 3-5 days. In embodiments, at least a fraction of the cultured cells expresses Nestin after 1-10 days of total cell culture duration. In embodiments, at least a fraction of the cultured cells expresses microtubule-associated protein 2a / b (Map2a / b) after 1-10 days of total cell culture duration. In embodiments, at least a fraction of the cultured cells expresses Tau after 1-10 days of total cell culture duration. In embodiments, at least a fraction of the cultured cells expresses glial fibrillary acidic protein (GFAP) after 1-10 days of total cell culture duration. In embodiments, at least a fraction of the cultured cells expresses oligodendrocyte transcription factor 2 (OLIG2) after 1-10 days of total cell culture duration.

[0066] In embodiments, at least a fraction of the cultured cells expresses Opalin after 5-14 days, preferably after 8 days, of total cell culture duration, indicating myelinating oligodendrocytes. In embodiments, at least a fraction of the cultured cells expresses CNP after 5-14 days, preferably after 8 days, of total cell culture duration, indicating maturing oligodendrocytes.

[0067] In embodiments, during induction, the expression of Nestin and / or Sox2 decreases (is downregulated), while the expression of GFAP and / or Map2a / b is upregulated. In embodiments, this shift in expression is gradual and depending on the stage of differentiation.

[0068] In embodiments, at least a fraction of the cultured cells are fully differentiated / mature neurons or astrocytes after 5-14 days of total cell culture duration, preferably after 8-12 days. In embodiments, at least a fraction of the cultured cells are fully differentiated / mature neurons after 5-14 days of total cell culture duration, preferably after 8-12 days. In embodiments, at least a fraction of the cultured cells are (fully differentiated / mature) astrocytes after 5-14 days of total cell culture duration, preferably after 8-12 days.

[0069] In embodiments, at least a fraction of the cultured cells are fully differentiated / mature neurons, astrocytes, glial cells or oligodendrocytes after 10-21 days of total cell culture duration.

[0070] In embodiments, at least a fraction of the cultured cells are fully differentiated / mature neurons after 10-21 days of total cell culture duration. In embodiments, at least a fraction of the cultured cells are (fully differentiated / mature) astrocytes after 10-21 days of total cell culture duration. In embodiments, at least a fraction of the cultured cells are (fully differentiated / mature) glial cells after 10-21 days of total cell culture duration. In embodiments, at least a fraction of the cultured cells are (fully differentiated / mature) oligodendrocytes after 10-21 days of total cell culture duration.

[0071] In general, the presence of oligodendrocytes may be considered beneficial within a neuronal organoid, as oligodendrocytes are considered important for normal neuronal function and brain homeostasis.

[0072] The inventors surprisingly found that the present method enables the generation of murine neural cell culture models, comprising mature astrocytes, oligodendrocytes and / or neurons in a time frame, such as within 21-30 days of total culture duration according to the invention, which has not been achieved before in the art.

[0073] In embodiments, the method comprises the steps of: a. providing a cell culture comprising murine neural stem cells obtained from an adult mouse and cultivating the cell culture for between 3-7 days until neurospheres have formed, b. replacing at least a fraction of the culture medium with induction medium and cultivating the cell culture for between 1-4 days, c. replacing the induction medium with differentiation medium and cultivating the cell culture for between 9-17 days, wherein the total duration of cell culture is at least 21 days, thereby generating a murine cerebral organoid cell culture model, and wherein the cells are cultivated in the initial cell culture medium in a. until day 5 of total cell culture duration, wherein the cells are cultivated in the induction medium in b. until day 7 of total cell culture duration, wherein the cells are cultivated in a first differentiation medium in c. until day 12 of total cell culture duration, and wherein subsequently the cells are cultivated in a second differentiation medium in c. at least until day 21 of total cell culture duration.

[0074] One advantage of the method according to the present invention is that the present method, e.g., due to the use of adult stem cells, enables in preferred embodiments the generation of organoids within about 3 weeks, which preferably comprise mature and myelinated neurons, mature astrocytes, and / or mature and myelinating oligodendrocytes. This represents a significant advance as compared to the other protocols of the prior art, e.g., starting from embryonic cells.

[0075] In embodiments, the cell culture is cultivated at least until day 21 , or preferably day 30 of total culture duration. In embodiments, the cell culture is cultivated at least until day 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 200, 250, 300, 350 or 365 or more of total culture duration.

[0076] In embodiments, the cell culture is cultivated at least until day 21 of total culture duration, or even longer. In embodiments the cell culture is cultivated for at least for 1 month of total culture duration. In embodiments the cell culture is cultivated at least for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 36 or 48 months of total culture duration.

[0077] All time ranges disclosed herein may in embodiments also comprise a variation of ±1 day, of ±2, ±3, ±4, ±5, ±6 or ±7 days, of ±10 days, or ±14, or ±28, ±35, or ±40 days, or even more variation.

[0078] An important advantage of the present model over prior art neural organoid cell culture models is that it can be maintained, if desired, for more than 21 or 60 days.

[0079] In embodiments, the cell culture comprises an entirely / fully differentiated murine cerebral organoid cell culture model from day 35, preferably from between day 21 and day 30, of total culture duration on. In embodiments, from day 21 , 30 or 35 of total culture duration on, the fully differentiated murine cerebral organoid cell culture model is stable with regard to (most of) its cellular features and / or neuronal gene expression profile for the subsequent time of cell culture. In other words, in embodiments the present model is, as soon as fully differentiated, stable and reproducible with regard to its relevant cellular features. In embodiments, the murine neural stem cells are obtained from a genetically engineered / modified mouse.

[0080] One advantage of organoids generated according to embodiments of the present invention is that the initial adult stem cells may be isolated from adult mice with a specific age (days post birth), gender, genotype, and health state, which are freely selectable, depending on the research context for which the obtained organoids are intended to be used.

[0081] In embodiments, the murine neural stem cells are genetically engineered / modified cells. In embodiments the initial cell population used in the method of the present invention comprises one or more mutations in comparison to a wildtype cell.

[0082] In embodiments, the present method facilitates the generation of disease-specific murine cerebral organoid cell culture models. For example, the disease-specific murine cerebral organoid cell culture model is generated from murine neural stem cells obtained from an adult mouse, wherein the mouse is a mouse carrying a certain genetic mutation(s) and / or possesses a certain disease phenotype and / or genotype (is a murine model for a certain genotype, disease or condition), such that in vitro murine organoid disease models may be obtained and, e.g., employed in laboratory experiments and screening instead of a mouse model of certain disease.

[0083] In embodiments, diseased cells might also be generated from adult mice or murine stem cells comprising genetically engineered or introduced mutations (e.g., using CRISPR / Cas9 or CRISPR / Cas9 vectors to induce specific disease-associated mutations).

[0084] In embodiments, the present model can be used to study a disease or condition and / or the treatment of a certain disease or condition in a disease-specific background, e.g., from a certain mouse model of disease or condition.

[0085] For example, organoids from adult mice can reveal signs of aging, if desired, which are vital, e.g., for aging research and studying neurodegenerative disorders like Alzheimer's and Parkinson's disease, commonly found in older populations. In the present examples, the inventors show that the method according to the present invention enables successfully producing both, young and aged brain organoids, which are also considered to exhibit distinct epigenetic features. The unique myelination processes which may occur during the present method can also facilitate the study of diseases that involve damaged myelin sheaths and / or defective excitatory signal transmission along axons.

[0086] The present organoids can, for example, be created to investigate the brain at both functional and cellular levels. In addition, the present method additionally allows for an efficient examination of age and gender influences. For example, NSCs from adult donor mice may encode features of the donor such as age related ones that cannot be studied in organoids derived from embryo- derived or reprogrammed starting cells.

[0087] Hence, in embodiments the present method and in vitro murine cell culture model may be generated from ‘diseased’ cells derived from a mouse model of a neurological and / or brain disease, a neurodegenerative disease, brain injuries or any other condition or disease of the brain. In preferred embodiments the respective mouse model is a model for a human disease or condition.

[0088] In an exemplary and non-limiting example, from a euthanized mouse of a certain mouse strain about 30-35 brain organoids, preferably even more, can be generated from the isolated adult neural stem cells. In embodiments, the present method enables the generation of up to 100 brain organoids from the cells obtained from 3 mice.

[0089] In an exemplary and non-limiting example, the present method enables large experimental groups to be compared in parallel (e.g. male versus female or Black6 versus BalbC, etc.) or to produce brain organoids quickly and reliably for research from established genetic models (knockouts and transgenic mice).

[0090] Another advantage of the present method is the genetic accessibility of adult stem cells. Since adult stem cells divide rapidly, they can be transfected in embodiments with plasmids or transformed with viral vectors, allowing genome editing using CRISPR-Cas technology as well as the introduction of fluorescent marker proteins to be efficiently visualized. Consequently, in embodiments a high number of comparable brain organoids with different genotypes or transient transgenes can be produced from a homogeneous population of neural precursor cells according to the present method.

[0091] Hence, the present method may be advantageously employed in infection and cancer research, or in developmental biology. Overall, present method significantly simplifies and accelerates the production of brain organoids in the mouse model system, with far-reaching implications for organoid research based on adult stem cells. In embodiment, the present method may also be transferred to other animal models, such as other mammal animal models.

[0092] In another aspect, the present invention relates to an in vitro murine cerebral organoid cell culture model comprising mature astrocytes, mature (myelinating) oligodendrocytes, and / or mature (myelinated) neurons produced according to the method of the invention.

[0093] Embodiments and features of the invention described herein before with respect to the method according to the invention are also considered to (apply to) be disclosed with respect to the murine cerebral organoid cell culture model according to the invention, and vice versa.

[0094] In embodiments of the in vitro cell culture model, at least a fraction of the astrocytes expresses glial fibrillary acidic protein (GFAP), at least a fraction of the neurons express microtubule- associated protein 2a / b (Map2a / b) and / or comprise myelinated axons, and / or at least a fraction of the oligodendrocytes express Oligodendrocyte transcription factor 2 (OLIG2).

[0095] In embodiments, the in vitro murine cerebral organoid cell culture model comprises mature astrocytes, mature (and myelinating) oligodendrocytes and mature (and myelinated) neurons and wherein at least a fraction of the astrocytes expresses glial fibrillary acidic protein (GFAP), at least a fraction of the neurons express microtubule-associated protein 2a / b (Map2a / b) and / or comprise myelinated axons, and at least a fraction of the oligodendrocytes express Oligodendrocyte transcription factor 2 (OLIG2). For example, see Figures 6 and 8 depicting organoids generated according to the present method comprising oligodendrocytes and the formation of axon-myelination and Figures 6 and 7 evidencing the presence, differentiation, and proliferation of astrocytes and oligodendrocytes as detected by IF and RT-qPCR.

[0096] In embodiments of the in vitro cell culture model, at least a fraction of the astrocytes expresses glial fibrillary acidic protein (GFAP). In embodiments of the in vitro cell culture model, at least a fraction of the neurons expresses microtubule-associated protein 2a / b (Map2a / b). In embodiments of the in vitro cell culture model, at least a fraction of the neurons comprises myelinated axons. In embodiments of the in vitro cell culture model, at least a fraction of the oligodendrocytes express Oligodendrocyte transcription factor 2 (OLIG2).

[0097] In embodiments, the present in vitro murine cerebral organoid cell culture model is used for (high throughput) drug or toxicity screening and / or for studying a neurological disease.

[0098] In embodiments, such use involves methods of performing a (high throughput) drug or toxicity screening comprising exposing the present murine cerebral organoid cell culture model to substances, drugs or other compounds or conditions of interest and determining a respective corresponding readout.

[0099] In embodiments of high throughput screening approaches, the present model may be cultivated in multi-well plates, wherein e.g., each well comprises preferably more than one organoid (e.g., 10 or even 25), thereby simplifying the analysis and increasing the reproducibility and quality of the screening outcome.

[0100] In embodiments, the present murine neural organoid model may serve as a brain model, which may be used for drug screenings, toxicity screenings and / or personalized medicine approaches as a cost-efficient brain model, which may be generated and screened with less effort than previous models. In embodiments the present murine neural organoid model may serve as a brain model for studying the murine brain. In embodiments the present murine neural organoid model may serve as a brain model for studying the mammal, or even the human brain.

[0101] In embodiments, the murine neural stem cells are singularized (separated into single cells) after isolation from an adult mouse and before the cultivation.

[0102] In embodiments, the cell culture is cultivated in at least one, or all of steps a.- c. in a cell culture medium comprising at least one basement membrane component and / or ECM component. In embodiments, the cell culture is cultivated in at least one of steps b.- c. in a cell culture medium comprising at least one basement membrane component and / or ECM component. In embodiments, the cell culture is cultivated from step c. on, or in step c. in a cell culture medium comprising at least one basement membrane component and / or ECM component.

[0103] In some preferred embodiments the cell culture is cultivated from day 7, 8, 9, or 10 (of total culture duration) on in a cell culture medium comprising at least one basement membrane component and / or ECM component. In embodiments, after 7-10 days of total culture duration at least one basement membrane component and / or ECM component is added to the cell culture medium, preferably leading to the embedding or coating of the organoid(s).

[0104] In embodiments, the cell culture is cultivated in a cell culture medium comprising at least one basement membrane component and / or ECM component between day 7 and day 20, preferably between day 7, 8, 9 or 10 and day 20 (of total culture duration), preferably wherein the basement membrane component and / or ECM component is added between day 7 and 10 (of total culture duration) to the cell culture medium. In embodiments, the cell culture is cultivated in a cell culture medium comprising at least one basement membrane component and / or ECM component, wherein the basement membrane component and / or ECM component is added between day 7 and day 10 (of total culture duration) to the cell culture medium.

[0105] An advantage of supplying at least one basement membrane component and / or ECM component to the cell culture, preferably at least once between day 7-10 (of total culture duration), is a stabilization of the organoids during the differentiation, preferably as organoid(s) are embedded in or coated with the at least one basement membrane component and / or ECM component.

[0106] In embodiments, L-glutamine is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0107] In embodiments, L-glutamine is added to and / or comprised within the cell culture medium at a concentration of 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 %, preferably at 1%.

[0108] In some embodiments, L-glutamine is added to and / or comprised within the cell culture medium as GlutaMAX (e.g., Gibco GlutaMAX supplement).

[0109] In embodiments, B27 Plus supplement is added to the cell culture medium. As the B27 Plus contains vitamin A, the inventors consider that its addition in certain embodiments may improve neuronal survival and differentiation.

[0110] In embodiments, B27 supplement is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0111] In embodiments, B27 supplement is added to the cell culture medium at a concentration of 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 %, preferably at 1 %.

[0112] In embodiments, B27 Plus supplement is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0113] In embodiments, B27 Plus supplement is added to the cell culture medium at a concentration of 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 %, preferably at 1 %. In embodiments, Heparin is added to the cell culture medium every day, every other day, or every

[0114] 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0115] In embodiments, Heparin is added to the cell culture medium at a concentration of 0.1 , 0.25, 0.5, 0.75, 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, or 10 pg / ml, preferably at 2 or 5 pg / ml.

[0116] In embodiments, NEAA (non-essential amino acids) are added to the cell culture medium. The inventors considered that the addition of NEAA in certain embodiments may relieve cellular metabolism and / or enhance differentiation, survival, and maintenance of cell culture, which may be particularly advantageous in embodiments, where organoids are generated from disease- associated cells and / or as disease models.

[0117] In embodiments, NEAA (non-essential amino acids) are added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1- 14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0118] In embodiments, NEAA are added to the cell culture medium at a concentration of 0.1 , 0.25, 0.5, 0.75, 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, or 10 %, preferably at 1%.

[0119] In embodiments, Insulin is added to the cell culture medium. The inventors considered that the addition of insulin in certain embodiments may support cell survival and differentiation.

[0120] In embodiments, Insulin is added to the cell culture medium every day, every other day, or every

[0121] 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0122] In embodiments, Insulin is added to the cell culture medium at a concentration of 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11.5, 12, 12.5, 13 or 13.5 pl / 50 ml, preferably at 12.5 pl / 50 ml.

[0123] In embodiments, EGF is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0124] In embodiments, EGF is added to the cell culture medium at a concentration of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 ng / ml, preferably at 20 ng / ml.

[0125] In embodiments, FGF2 is added to the cell culture medium every day, every other day, or every

[0126] 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0127] In embodiments, FGF2 is added to the cell culture medium at a concentration of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 ng / ml, preferably at 20 ng / ml.

[0128] In embodiments, BDNF (brain derived neurotrophic factor) is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days. In embodiments, BDNF is added to the cell culture medium at a concentration of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 ng / ml, preferably at 20 ng / ml.

[0129] In embodiments, GDNF (glia cell line-derived neurotrophic factor) is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0130] In embodiments, GDNF is added to the cell culture medium at a concentration of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 ng / ml, preferably at 20 ng / ml.

[0131] In embodiments, ascorbic acid is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0132] In embodiments, ascorbic acid is added to the cell culture medium at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nM, preferably at 200 nM.

[0133] In embodiments, penicillin / streptavidin (pen / strep) is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0134] In embodiments, penicillin / streptavidin (pen / strep) is added to the cell culture medium at a concentration of 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 %, preferably at 1%.

[0135] In embodiments, N2 is added to the cell culture medium. The inventors considered that the addition of N2 supplement in certain embodiments may support neuronal and glial differentiation.

[0136] In embodiments, N2 is added to the cell culture medium every day, every other day, or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 21 days or between 1-14 days, 1-5 days, between 2-7 or 3-15 days or every 5-10 days.

[0137] In embodiments, N2 is added to the cell culture medium at a concentration of 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 %, preferably at 1%.

[0138] In embodiments a ‘proliferation medium’ comprises neurobasal medium, B27 supplement, L- glutamine or GlutaMAX, penicillin / streptomycin, EGF, FGF2 and heparin. In specific embodiments a ‘proliferation medium’ comprises neurobasal medium, 1% B27 supplement, 1% L- glutamine or GlutaMAX, 1% penicillin / streptomycin, 20 ng / ml EGF, 20 ng / ml FGF2 and 2 pg / ml heparin.

[0139] In embodiments an ‘induction medium’ comprises neurobasal medium, B27 supplement, L- glutamine or GlutaMAX, penicillin / streptomycin and heparin. In specific embodiments an ‘induction medium’ comprises neurobasal medium, 1 % B27 supplement, 1 % L-glutamine or GlutaMAX, 1 % penicillin / streptomycin and 5 pg / ml heparin. In embodiments a ‘first differentiation medium’ comprises neurobasal Plus medium, B27 Plus supplement, L-glutamine or GlutaMAX, penicillin / streptomycin, N2 supplement, NEAA and insulin. In specific embodiments a ‘first differentiation medium’ comprises neurobasal Plus medium, 1% B27 Plus supplement, 1 % L-glutamine or GlutaMAX , 1% penicillin / streptomycin, 1 % N2 supplement, 1% NEAA and 12.5 pl / 50 ml insulin.

[0140] In embodiments a ‘second differentiation medium’ comprises neurobasal Plus medium, B27 Plus supplement, L-glutamine or GlutaMAX, penicillin / streptomycin, N2 supplement, NEAA, insulin, BDNF, GDNF and ascorbic acid. In specific embodiments a ‘second differentiation medium’ comprises neurobasal Plus medium, 1% B27 Plus supplement, 1% L-glutamine or GlutaMAX , 1% penicillin / streptomycin, 1% N2 supplement, 1 % NEAA, 12.5 pl / 50 ml insulin, 20 ng / ml BDNF, 20 ng / ml GDNF and 200 nM ascorbic acid.

[0141] In embodiments, at least one basement membrane component and / or ECM component is added to the cell culture medium, preferably wherein the cell culture medium is a differentiation medium.

[0142] Surprisingly, the inventors found that the method of the present invention enables long-term cell culture of the model beyond 25 days, preferably beyond 30, 50 or even beyond 100 days.

[0143] Any numerical range derived from, using end or intermediate points, or within the aforementioned concentration ranges is also envisaged herein and comprised by the present disclosure. The afore-mentioned concentrations may also apply to the kit and supplement cocktail embodiments described below.

[0144] In embodiments, the cell culture is cultivated initially (the cells are ‘seeded’ / provided in step a.) at a cell-density of at least 20.000 cells / well. In embodiments, the cell culture is cultivated initially (the cells are ‘seeded’ / provided in step a.) at a cell-density of at least 20.000 cells / well in a well of a 96-well plate, preferably in a 96-well round-bottom plate. In embodiments, the cell culture is cultivated / provided initially in step a. (the cells are ‘seeded’) at a cell-density of between 10.000- 70.000 cells / well, preferably in a 96-well (round-bottom) plate. In embodiments the cells are then cultivated for between 3-7 days until neurospheres have formed (e.g., according to step a. of the present method).

[0145] In embodiments, the cell culture is cultivated / provided initially in step a. (the cells are ‘seeded’) at a cell-density of between 10.000-80.000 cells / cm2, preferably between 50.000-70.000 cells / cm2, (e.g., in a well of a 96-well (round-bottom) plate). In embodiments, the cell culture is cultivated / provided initially in step a. (the cells are ‘seeded’) at a cell-density of at least 62.500 cells / cm2(e.g., in a well of a 96-well (round-bottom) plate). In embodiments the cells are then cultivated for between 3-7 days until neurospheres have formed (e.g., according to step a. of the present method).

[0146] In embodiments, the neural stem cells (NSCs) provided in step a. of the present method are cultivated, after isolation from an adult mouse, in advance of step a. in an initial NSC cell culture.

[0147] In embodiments, the initial NSC cell culture (comprising NSCs freshly isolated from an adult mouse) is cultivated in proliferation medium. In embodiments, the initial NSC cell culture (comprising NSCs freshly isolated from an adult mouse) is passaged / re-seeded / re-plated (‘passage 1’) between day 6 and day 8, preferably on or around day 7 after isolation from the mouse. In embodiments, the NSC cell culture may be passaged / re-seeded / re-plated again between day 10 and day 12, preferably on or around day 11 after isolation from the mouse (preferably about 4 days after the first passage / re-seeding). In other words, in embodiments cultivation of the NSCs after isolation from the mouse (and prior to step a.) comprises a first passaging / re-seeding / re-plating step between day 6 and day 8, and optionally a second passaging / re-seeding / re-plating step between day 10 and day 12, preferably on or around day 11 .

[0148] In embodiments, the once or twice passaged initial NSC cell culture may then be cultivated for between 3-7 days until neurospheres have formed (according to step a. of the present method).

[0149] In exemplary non-limiting embodiments, the murine neural stem cells (NSCs) freshly obtained from an adult mouse are initially cultivated (preferably in advance of step a. of the present method) such that (about) 75 % of the cells obtained from one mouse are seeded per well of a standard 24 well-plate (with a surface area of 1.9 cm2). In embodiments, the murine neural stem cells obtained from an adult mouse are initially cultivated (preferably in advance of step a. of the present method) such that 25%, 30%, 40%, 50%, 75 % or 100% or between 30-40% of the cells obtained from one mouse are seeded per cm2. In exemplary, non-limiting embodiments a well of a cell culture vessel has a surface area of between 1-10.5 cm2, e.g., 1.9 cm2in a standard 24 well plate (e.g., Thermo Fischer), wherein between 20-50% or at least 20 %, 30%, 40% or 50% of the cells obtained from one mouse are seeded per cm2(preferably about 40% of the cells per cm2).

[0150] In embodiments, at least a fraction of the neural stem cells used as initial cells in the present method express Sox2 and / or Nestin.

[0151] In embodiments, at least a fraction of the cultured cells of the neurosphere or differentiating brain organoid express Nestin and / or microtubule-associated protein 2a / b (Map2a / b) or glial fibrillary acidic protein (GFAP) or OLIG2 or Tau after 1-10 days of total cell culture duration, preferably after 3-5 days.

[0152] In embodiments, at least a fraction of the cells of the neurosphere or developing murine cerebral organoid are fully differentiated / mature neurons or astrocytes after 5-14 days of total cell culture duration, preferably after 8-12 days.

[0153] In embodiments, at least a fraction of the cultured cells of the neurosphere or developing murine cerebral organoid are fully differentiated / mature neurons, astrocytes, glial cells or oligodendrocytes after 10-21 days of total cell culture duration. In some embodiments, at least a fraction of the cultured cells of the neurosphere or developing murine cerebral organoid are glial cells (neuroglia) after 10-21 days of total cell culture duration.

[0154] In embodiments, from culture day 21 on and after, the murine cerebral organoid cell culture model comprises neurons, astrocytes or oligodendrocytes. In embodiments, after 21 days of culture according to the present invention at least a fraction, preferably at least 1-70%, of the cells comprised within the murine cerebral organoid are cerebral neurons expressing Map2a / b and / or Tau.

[0155] In embodiments, after 12 days of culture (according to step c.) at least a fraction, preferably at least 1-70%, of the cells comprised within the organoid cerebral neurons expressing Map2a / b and / or Tau.

[0156] In some embodiments, the cell culture according to the present method is passaged / re- seeded / re-plated at least once between day 5 and day 21 , preferably at least between day 7-10.

[0157] The murine neural organoid models according to the present invention are preferably highly reproducible and scalable.

[0158] In embodiments, the present organoids have a diameter of about 300-500 pm, preferably 400 pm.

[0159] Examples of the murine cerebral organoid cell culture model and its physical characteristics thereof are presented in Fig. 2, 4, 5.

[0160] In another aspect, the present invention relates to a kit, preferably suitable for carrying out the method of the invention, said kit comprising a culture medium and at least one supplement selected from the group comprising fibroblast growth factor 2, (FGF2), epidermal growth factor (EGF), heparin, insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, , N2-supplement, one or more basement membrane (BM) component and / or extracellular matrix (ECM) component, ascorbic acid, brain-derived neurotrophic factor (BDNF) and glial cell line- derived neurotrophic factor (GDNF).

[0161] In some embodiments, L-glutamine is comprised as GlutaMAX (e.g., Gibco GlutaMAX supplement).

[0162] In embodiments, the kit further comprised the in vitro cell culture model according to the present invention.

[0163] In another aspect, the present invention relates to a culture medium supplement cocktail comprising a. fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), heparin and / or B27 supplement, or b. heparin and B27 supplement, or c. insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2- supplement and / or one or more basement membrane component and / or ECM component, or d. insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2- supplement, one or more basement membrane component and / or ECM component, ascorbic acid, brain-derived neurotrophic factor (BDNF) and / or glial cell line-derived neurotrophic factor (GDNF).

[0164] In embodiments the culture medium supplement cocktail according to a) is suitable for preparing a proliferation medium. In embodiments the culture medium supplement cocktail according to b) is suitable for preparing an induction medium. In embodiments the culture medium supplement cocktail according to c) is suitable for preparing a differentiation medium I. In embodiments the culture medium supplement cocktail according to d) is suitable for preparing a differentiation medium II.

[0165] In one aspect, the present invention relates to a cell culture plate comprising the in vitro cell culture model according to the present invention.

[0166] In preferred embodiments, the in vitro cell culture of NSCs is adherent (attached) to the cell culture vessel or plate, preferably whereas neurospheres and (cerebral) organoids are nonadherent.

[0167] In embodiments, the cell culture plate is a multi-well plate and each well comprises one or more in vitro cell culture models according to the present invention.

[0168] In embodiments, the cell culture plate is a multi-well plate and each well comprises two or more in vitro cell culture models according to the present invention.

[0169] In embodiments, the cell culture plate is a multi-well plate and each well comprises one or more neurosphere(s) according to the present invention.

[0170] In embodiments, the cell culture plate is a 96-well plate comprising one neurosphere / well. The separation of neurospheres in single wells has the advantage, that the differentiation process may be conducted in a controlled manner, as the number of organoids created can be clearly determined.

[0171] In embodiments, when early organoids are re-seeded / passaged / moved from a 96-well plate they are embedded or coated with at least one basement membrane component and / or ECM component such that one or more organoids may subsequently be seeded / plated per well.

[0172] In embodiments, the cell culture plate is a multi-well plate and each well comprises one or more in vitro murine cerebral organoid cell culture model according to the present invention.

[0173] In embodiments, the multi-well plate is a 96 well plate. In embodiments the multi-well plate is a 2, 4, 6, 8, 10, 12, 24 or 96 well plate. In embodiments, the multi-well plate comprises at least 2 or more wells, preferably at least 6, more preferably at least 12 or 96 wells.

[0174] In embodiments, the cell culture plate is a multi-well plate that comprising more than 1 , preferably between 1 and 500 or 1 and 100 in vitro cell culture model according to the present invention.

[0175] In another aspect, the present invention relates to the use of the in vitro model according to the invention for high throughput drug or toxicity screening and / or for studying a neurological disease. In embodiments, the present method facilitates the generation of disease-specific murine cerebral organoid cell culture models. For example, the disease-specific murine cerebral organoid cell culture model is generated from murine neural stem cells obtained from an adult mouse, wherein the mouse is a mouse carrying a certain genetic mutation(s) and / or possesses a certain disease phenotype and / or genotype (is a murine model for a certain genotype, disease or condition), such that in vitro murine organoid disease models may be obtained and, e.g., employed in laboratory experiments and screening instead of a mouse model of certain disease.

[0176] In embodiments, diseased cells might also be generated from adult mice or murine stem cells comprising genetically engineered or introduced mutations (e.g., using CRISPR / Cas9 or CRISPR / Cas9 vectors to induce specific disease-associated mutations).

[0177] In embodiments, the present model can be used to study a disease or condition and / or the treatment of a certain disease or condition in a disease-specific background, e.g., from a certain mouse model of a disease or condition.

[0178] Hence, in embodiments, the present method and in vitro murine cell culture model may be generated from ‘diseased’ cells derived from a mouse model of a neurological and / or brain disease, a neurodegenerative disease, brain injuries, or any other condition or disease of the brain. In preferred embodiments, the respective mouse model is a model for a human disease or condition.

[0179] In another aspect, the present invention relates to the use of the in vitro murine cerebral organoid cell culture model according to the present invention in toxicity screening, drug screening and / or for studying a disease, such as a brain disease, a neurological disease, a neurodegenerative disease or any other disease or condition involving or affecting the brain, preferably of human individuals.

[0180] In another aspect, the present invention relates to a method for testing a candidate molecule, substance or compound, the method comprising: i. providing the in vitro murine cerebral organoid cell culture model according to the invention, ii. bringing into contact a candidate compound with the in vitro murine cerebral organoid cell culture model, iii. detecting a readout, characteristic and / or one or more parameter(s) of the cell culture model, iv. optionally repeating steps a-c with one or more further candidate compounds.

[0181] In embodiments, the readout or parameter in step c. comprises the viability and / or proliferation of said model. A skilled person is familiar with different methods for detecting the viability and / or proliferation of a cell culture. Examples of methods for detecting the viability and / or proliferation of a cell culture are Trypan Blue staining, MTT-assay, BrdU-assay, XTT-assay, EdU-assay, WST-1- assay, Ki67-staining, immunocytochemistry (ICC), immunohistochemistry, immunofluorescence, Hoechst-staining, or Calcein-AM staining.

[0182] In embodiments, detecting a readout or parameter in step c. comprises using one or more of the methods of image analysis (e.g., via a plate reader, microscope or camera), microscopy, (patch) clamp recording, immunocytochemistry (ICC), immunohistochemistry, Immunofluorescence analysis, droplet-based microfluidic technology, Fluorescence Activated Cell Sorting (FACS), ELISA, IHC and / or spectrophotometry.

[0183] In embodiments, the readout or parameter to be detected may be a shape, a characteristic, and / or a pattern of the present organoid model or of certain cells comprised therein. In embodiments, a non-limiting list of characteristics or parameters may be, e.g., a cellular developmental stage, the expression of certain genes or proteins, the length, appearance and / or shape of cells, the length, organization and / or orientation of cells, the transfer and / or excretion of neurotransmitters or other neural functional properties. In embodiments, the assessments are preferably performed in triplicates, to discard non-specific readouts or signals.

[0184] In embodiments, screening methods and systems used for the screening mentioned herein before may employ commonly used software and hardware of (pharmaceutical) compound screening systems. The skilled person is familiar with automated setups that may be adapted accordingly.

[0185] In embodiments, the method for testing a candidate molecule comprises assessing toxicity of the candidate substance, comprising: providing a cell culture model according to the invention, bringing into contact a candidate compound with said cell culture model, and identifying the candidate substance as a substance with low or no toxic effect, if said cell culture mode exhibits no detectable cytotoxic effect.

[0186] In one aspect, the present invention relates to a cell culture medium and / or supplement-cocktail, preferably as described herein, or as derived from the components of cell culture medium or other supplements described herein.

[0187] In another aspect, the present invention relates to a cell culture medium as such, defined by one or more of the embodiments disclosed herein. Such culture medium products may be offered as such, or the components thereof offered in the form of a kit, of separated but combinable compounds or substances.

[0188] In another aspect, the present invention relates to a supplement cocktail as such, defined by one or more of the embodiments disclosed herein. Supplemental cocktail products may be offered as such, or the components thereof offered in the form of a kit of separated but combinable compounds or substances.

[0189] In another aspect, the present invention relates to a culture medium and / or supplement-cocktail comprising a. fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), heparin and / or B27- supplement, or b. heparin and / or B27- supplement, or c. insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2- supplement and / or one or more basement membrane component and / or ECM component, or d. insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2- supplement, one or more basement membrane component and / or ECM component, ascorbic acid, brain-derived neurotrophic factor (BDNF) and / or glial cell line-derived neurotrophic factor (GDNF).

[0190] In some embodiments, L-glutamine is comprised as GlutaMAX (e.g., Gibco GlutaMAX supplement).

[0191] In embodiments the culture medium and / or supplement-cocktail according to a) is, or is suitable for preparing a proliferation medium. In embodiments the culture medium and / or supplementcocktail according to b) is, or is suitable for preparing an induction medium. In embodiments the culture medium and / or supplement-cocktail according to c) is, or is suitable for preparing a differentiation medium I. In embodiments the culture medium and / or supplement-cocktail according to d) is, or is suitable for preparing a differentiation medium II.

[0192] Any range derived from, using end or intermediate points, or within the afore-mentioned numerical concentration ranges is also comprised by the present disclosure. The afore-mentioned concentrations may also apply to the kit embodiments described in further detail below.

[0193] The instant disclosure also includes kits, packages and multi-container units containing the herein described media and / or factors separately or as compositions.

[0194] Each optional or preferred feature of the invention that is disclosed or described in the context of one aspect of the invention is herewith also disclosed in the context of the other aspects of the invention described herein. Embodiments and features of the invention described with respect to the method, the murine cerebral organoid cell culture models, kits and cocktails, are considered to be disclosed with respect to each and every other aspect of the disclosure, such that features characterizing the methods, may be employed to characterize the cerebral organoid cell culture models, cocktails or kit and vice-versa. The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the common and surprising finding of the generation of an in vitro murine cerebral organoid cell culture model from murine neural stem cells obtained from adult mice.

[0195] DETAILED DESCRIPTION OF THE INVENTION

[0196] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

[0197] The invention relates to an in vitro method of generating a murine cerebral organoid cell culture model. The invention relates to an in vitro murine cerebral organoid cell culture model generated from murine neural stem cells obtained from adult mice. The term ‘individual’, ‘subject’ or ‘patient’ includes any mammalian subject or patient, preferably a murine subject or a human subject. In embodiments, the subject or patient is a human or mammal diagnosed with and / or suffering from a certain disease. In embodiments, the subject is a mouse, preferably an adult mouse or any other adult (model) animal or (multicellular) model organism.

[0198] Cell culture

[0199] The term ‘pluripotent stem cells’ in this context comprises any cells having the properties of pluripotency, as known by a skilled person. The term includes pluripotent stem cells (PSCs) derived from an embryo and induced pluripotent stem cells (iPSCs) derived from other cell types such as a somatic cell (such as a skin cell, fibroblast, blood cell, or any other somatic cell). Pluripotent stem cells can generally be derived from a stem cell culture that has been established without destroying a donor embryo. Pluripotent stem cells may be healthy pluripotent stem cells derived from a healthy organism or diseased pluripotent stem cells derived from an organism (e.g., a mouse or mouse model) suffering from a disease.

[0200] In embodiments, the pluripotent stem cell according to the present invention is a pluripotent or multipotent mammalian cell. Preferably, a pluripotent stem cell is murine neural stem cell obtained from an adult mouse.

[0201] ‘Adult stem cells’, such as neural stem cells, are in the context of the present invention preferably stem cells derived from an adult subject, preferably derived from a specific tissue, such as a neuronal tissue, of an adult subject, such as an adult mouse or other model animal. In embodiments, stem cells derived from a specific tissue of an adult subject are also termed ‘tissue-specific stem cells’. Such ‘adult stem cells’ or ‘tissue-specific stem cells’ are preferably multipotent, but not omnipotent or pluripotent. In embodiments herein, ‘adult stem cells’ or ‘tissuespecific stem cells’ are neural stem cells. Commonly, multipotent stem cells are only able to differentiate into certain cell types specific for a certain tissue, e.g., for neural or brain-tissue.

[0202] The term ‘neurosphere’ commonly refers to a spherical aggregation of neural stem cells, which are formed by coalesce of the stem cells in culture. Neurospheres are commonly used for neurogenesis experiments, where single differentiating cells start to attach to the culture dish and “leave” the neurosphere structure.

[0203] Cell culture (also termed tissue culture) is a process by which cells are grown under controlled conditions, generally outside of their natural environment. Generally, cells are cultivated in suitable culture vessel, e.g., plates, wells, well plates or flasks, with a substrate or cell culture medium that is supplied with essential nutrients (such e.g., one or more of amino acids, carbohydrates, vitamins, minerals), and optionally with one or more of growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure etc.). Depending on the aim of the cell cultivation, the medium can be chosen appropriately, and the skilled person is aware of suitable choices of culture conditions, such as vessel, medium, temperature and incubator etc. A ‘progenitor cell’ in general refers to a neural progenitor cell. In embodiments, the radial glia cells are the progenitors of both intermediate progenitors and mature neurons, wherein the intermediate cell state preferably does not have projections.

[0204] In the context of the present invention, ‘NSC’ preferably refers to ‘neural stem cell’. Herein ‘NPC’ preferably refers to ‘neural precursor cells’. In preferred embodiments neural precursor cells (NPCs) comprise neural stem cells (NSCs) and neural progenitor cells. One advantage of NSCs is that such cells are commonly still capable of cell division, while neural progenitors commonly lack such ability.

[0205] A ‘(terminally) differentiated neuron’ may also be referred to as mature neuron or post-mitotic neuron. A differentiated neuronal cell may in embodiments be characterized by the expression of one or more of the genes, including, without limitation, glial fibrillary acidic protein (GFAP; in astrocytes), microtubule-associated protein 2a / b (Map2a / b; in neurons), oligodendrocyte transcription factor 2 (OLIG2; in oligodendrocytes) and / or wherein a mature neuron comprises myelinated axons.

[0206] Oligodendrocytes are myelinating cells of the central nervous system (CNS) and a type of glial cells (neuroglia). Microglia are a further type of glial cell (neuroglia) within the CNS, commonly acting as resident macrophages. Schwann cells are a type of glial cells (neuroglia), which serve as the myelinating cells in the peripheral nervous system (PNS).

[0207] Commonly, the term ‘organoid’ refers to a self-organized, small and three-dimensional tissue culture of cells, which may be generated, e.g., from omnipotent, pluripotent or even multipotent cells, such as (multipotent) neural stem cells. A neural organoid is a small, self-organized and three-dimensional (3D) tissue culture comprising neural and preferably other (assisting) cells commonly present in the brain or neural tissue. In embodiments, a neural organoid may resemble a simplified and miniaturized model of the brain or region of the brain, such as the cerebrum.

[0208] In the context of the present invention a ‘neural organoid’ or ‘neural organoid model’ is preferably a 'cerebral organoid’ or ‘cerebral organoid cell culture model’. Herein, the present ‘murine cerebral organoid cell culture model’ may hence also be referred to as ‘neural organoid model’ or ‘neural organoid cell culture model’. According to the invention the ‘neural organoid model’ or ‘cerebral organoid cell culture model’ is preferably of murine origin, more preferably originates from neural stem cells obtained from an adult mouse according to the present invention.

[0209] In general, a ‘basement membrane’ or ‘extracellular matrix’ (ECM) refers to a network of extracellular macromolecules comprising water, polysaccharides, various glycoproteins, enzymes, tissue hormones, nutrients (e.g. amino acids, glucose) and electrolytes. In general, the extracellular matrix ensures structural and biochemical support to the surrounding cells by comprising fibers / fibrous components and fluid. The extracellular matrix is present in almost every tissue and consist predominantly of collagens that form different types of fibers, and carbohydrates, particularly glycosaminoglycans, long-chain polysaccharides of very specific individual building blocks. The glycosaminoglycans associate with proteins and form even larger macromolecules, the proteoglycans, fibrillin and elastin form elastic fibers. In addition, there is a wide variety of adhesion matrix proteins that connect the cells to the extracellular matrix. The properties of the extracellular matrix are facilitated by a diversity of basement membrane components or ECM components, comprising proteins, glycosaminoglycans, and proteoglycans and their interactions. Hence, in embodiments basement membrane components or ECM components may comprise one or more of proteins (e.g., fibrillin and elastin), glycosaminoglycans, proteoglycans, polysaccharides, glycoproteins, enzymes, tissue hormones, nutrients and electrolytes. In embodiments the terms ‘basement membrane component’ or ‘extracellular matrix component’ may be used interchangeably.

[0210] ‘Geltrex’ is an LDEV-free reduced growth factor basement, namely a soluble form of basement membrane sold by Thermo Fisher Scientific.

[0211] Cell culture materials and supplements

[0212] A ‘neurotrophic factor1refers herein to biomolecules supporting the growth, survival, and differentiation of developing and mature neurons. In preferred embodiments, neurotrophic factors comprises one or more of BDNF, NT3, GDNF, cAMP and ascorbic acid.

[0213] In some embodiments, L-glutamine is added as GlutaMAX (e.g., Gibco GlutaMAX supplement) to the cell culture medium. Gibco GlutaMAX supplement is a dipeptide, L-alanyl-L-glutamine with increased stability.

[0214] In preferred embodiments ‘N2’ or ‘N2 supplement’ refers to a supplement commonly used to support neural cell growth, e.g., of post-mitotic neurons in neural cell culture, and is considered important for neuronal and glial differentiation.

[0215] In general, the B27 Plus supplement contains vitamin A, which may increase antioxidant potential of a culture medium (which optionally further comprises ascorbic acid). At the same time, vitamin A is known to improve neuronal survival and differentiation.

[0216] A high throughput screening (HTS) is a method of scientific experimentation that is particularly relevant to the fields of biology and chemistry. For example, a combination of robotics and other specialized laboratory hardware may be used to provide systems that enable a high throughput screening approach. In the context of the invention, HTS enables a skilled person to effectively screen a great number of organisms or parts thereof in vivo or in vitro, simultaneously or sequentially. Commonly used labware or test vessels of HTS are 6-, 24-, 48-well plates and microtiter plates, which is a container, possibly disposable and made of plastics, having a grid of small, open wells called wells. In some embodiments, microplates are used for HTS and have either 96, 192, 384, 1536, 3456 or 6144 wells. These are all multiples of 96 and reflect the original 96-well microplate with 8 x 12 wells spaced 9 mm apart. In embodiments, 24- or 48-well plates are used to set up the intended high-throughput in vivo or in vitro screening. Most of the wells contain test items, according to the nature of the experiment, which may be adapted according to the nature of the test and / or reference organisms described herein, and the library of substances to be tested as candidate substances. A screening facility will typically have a library of stock plates, the contents of which are carefully catalogued and each of which may have been generated by the laboratory or purchased from a commercial source. For example, to prepare an assay, each well of a plate may be seeded with a biological entity on which the experiment is to be performed, such as the cells, a cell culture model, animals or embryos of the test and / or reference organisms described herein. After an incubation period to allow the biological material to absorb, bind to, or otherwise react (or not) with the substances in the wells, e.g., a candidate drug or compound, measurements are made over the wells of the plate, either manually or by an automated procedure. Manual measurements may be necessary if microscopy is used to look for changes or defects in embryonic development caused by the candidate compounds; software may also be used to identify relevant effects. In some embodiments, a specialized automated analysis machine may be used. In dependence on the results of an HTS, follow-up assays may be performed, for example within the same screen, by identifying wells in which a desired reaction has occurred (also known as ‘hits’), transferring the reaction components to new assay plates and repeating the experiment.

[0217] Means for automating the present invention will also be known to those skilled in the art. Automation is an advantageous element in HTS. Typically, an integrated system consisting of one or more robots will transport assay microplates from station to station for sample and reagent addition, mixing, incubation, and finally readout or detection. An HTS system can usually prepare, incubate, and analyze multiple plates in parallel, further speeding up the process.

[0218] The term ‘candidate substance’ shall mean any substance used for (preferably high-throughput) screening. The compound characteristics, i.e. structure, purity, and quantity, may be stored in a chemical library database for reference. A candidate substance may further refer to any substance that the skilled person finds suitable for the contemplated method and / or for treating a disorder that affects synaptic transmission between a motor neuron and a muscle cell.

[0219] The term ‘toxic effect’ shall mean any adverse effect of the candidate substance produced in the respective organism, cell culture model or part thereof. A candidate substance preferably does not affect the survival of the cell culture model negatively. In embodiments, it may be desired that a candidate substance changes a certain parameter of the cell culture model, e.g., in case of a diseased cell culture model towards a more healthy or normal parameter range. In embodiments the present method may comprise a control cell culture model established from healthy stem cells (e.g., of a healthy donor) and at least one control cell culture models established from diseased stem cells (e.g., of a diseased donor or artificially engineered).

[0220] In the context of the present disclosure the term ‘at least a fraction’ may refer to at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, to between 1-100%, 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90% or to about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of a certain amount, number or population, e.g., a cell population.

[0221] The embodiments disclosed above may also represent an aspect of the invention. Any feature disclosed in the context of any given aspect or embodiment may be combined with other embodiments or aspects of the invention.

[0222] FIGURES The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0223] Brief Description of the Pictures:

[0224] Figure 1 : Isolated NSCs show characteristics of multipotency and are able to differentiate. NSCs show Nestin (upper left) and Sox2 (upper right) expression, two markers of stem cell multi potency.

[0225] Figure 2: Shown is a scheme of an embodiment of the generation of brain organoids from adult NSCs according to the present invention.

[0226] Figure 3: Shown is a scheme of an embodiment of the isolation of adult neural stem cells according to the present invention.

[0227] Figure 4: 12 pm cryosections of cerebral organoids stained with different antibodies.

[0228] Figure 5: 3D organoids stained with different antibodies.

[0229] Figure 6: Adult NSC-derived organoids show progressive maturation over the time course of the generation protocol.

[0230] Figure 7: Adult NSC-derived organoids comprise mature neurons, mature astrocytes, and myelin-producing oligodendrocytes

[0231] Figure 8: Adult NSC-derived brain organoids comprise neurons with myelinated axons.

[0232] Figure 9: Adult NSC-derived organoids show an aging phenotype.

[0233] Detailed Description of the Pictures:

[0234] Figure 1 : Isolated NSCs show characteristics of multipotency and are able to differentiate. NSCs show Nestin (upper left) and Sox2 (upper right) expression, two markers of stem cell multipotency. At day 4 of growth factor deprivation, cells start to express Map2a / b (neurons) and GFAP (astrocytes) additionally to Nestin (lower left). Furthermore, cells elongate and become thinner. After 9 days of differentiation, Map2a / b-positive cells continue elongation and show neuron-like structure, pointing to terminal differentiation with a neuronal fate (lower right). GFAP- positive cells expand and show pronounced expression of this astrocyte marker.

[0235] Oligodendrocytes are not detectable at this point in culture, since they need more time to differentiate. This shows that isolated NSCs are not only expressing markers of multipotency, but are also able to differentiate into neurons and astrocytes. For differentiation experiments, NSCs were detached and seeded on Poly-D-Lysine and Laminin coated glass coverslips. Differentiation was induced by deprivation of growth factors for several days. After fixation with PFA and MeOH, cells were stained with Antibodies for Nestin, Sox2, Map2a / b and GFAP. Scale bar: 10 pm.

[0236] Figure 2: Generation of brain organoids from adult NSCs. (A) Protocol of one embodiment of the present invention showing the time schedule of respective method steps disclosed herein. (B) Adult NSCs were freshly isolated from 12-25 weeks old mice and cultured until confluence was reached. Afterwards, cells were placed in ultra-low attachment, round-bottom 96-well plates for neurosphere formation. Since NSCs already have a neuronal fate, no ectodermal induction is needed. 3 d after the neurospheres formed, differentiation was induced by deprivation of bFGF and EGF. Subsequently, cells start self- organization and build a neurogenic loop, leading to organoid development. (C) Neurospheres show uniform distribution of Sox2+and Nestin+NSCs. After induction of differentiation, cells reorganize and Map2a / b+ neurons start to form at the outer layer of the early organoid. NSCs begin to withdraw to the inner side and thereby forming a cavity between inner and outer layer. Neurospheres and organoids were fixed at day 5 or 11 and embedded for cryo-sectioning. 12 pm sections were prepared and stained via immunofluorescence using the respective antibodies. Scale bar: 100 pm.

[0237] Figure 3: The figure shows an exemplary embodiment according to the present invention, wherein an adult neural stem cell culture is generated from neural precursor cells obtained from an adult mouse. In general, neural stem cells (NSCs) can differentiate into nearly all neuronal and glial cell types. They are present in embryonic and mature adult brains and are linked to learning, memory storage, pattern recognition, and memory clearance. There are two neurogenic zones from which NSCs can be isolated: the subventricular zone (SVZ) and the dentate gyrus (DG). After tissue dissection and enrichment, NSCs can be grown as an adherent monolayer on poly-D- lysine / laminin-coated petri dish for several weeks.

[0238] Figure 4: The Figure depicts 12 pm cryosections of cerebral organoids stained with different antibodies against OLIG2, Map2a / b and Sox2, or GFAP (pictures from bottom to top) at day 4, 8 and 13 (pictures from left to right) of total culture duration according to the method of the present invention.

[0239] Figure 5: The Figure depicts 3D organoids stained with different antibodies against Map2a / b or GFAP at day 20 and 25 of total culture duration according to the method of the present invention.

[0240] Figure 6: Adult NSC-derived organoids show progressive maturation over the time course of the generation protocol. Proliferation markers of stem cells, such as KI67, decrease in expression after day 8 (D8). Notably, SOX2 expression enhances over the time course of the organoid protocol, indicating the presence of progenitor cells that undergo differentiation. The population of OLIG1-expressing oligodendrocyte progenitor cells increases until day 8 (D8), after which it decreases, likely due to their differentiation into Opalin-expressing myelinating oligodendrocytes and CNP-expressing maturing oligodendrocytes, both of which exhibit increased expression post- D8. GFAP-expressing astrocytes show a substantial increase at day 13 (D13), indicating astrocyte differentiation, which decreases again at day 20 (D20), probably due to a regulatory effect in the organoid. DCX-expressing neuronal progenitor cells increase slightly until D13 and then decrease significantly due to ongoing neuronal differentiation.

[0241] Figure 7: Adult NSC-derived organoids comprise mature neurons, mature astrocytes, and myelin-producing oligodendrocytes. A: D20 organoids harbor Tau-positive neurons (top row, picture in the middle), GFAP-positive astrocytes (top row, picture on the right), and 04- (bottom row, picture on the right), and Opalin-positive (bottom row, picture in the middle), early and Opalin-positive late myelinating oligodendrocytes. Pictures on the left in both rows depict a merge of both staining. B: This state is stable for at least five days of culture (day 25; D25), the pictures have the same order as in A.

[0242] Figure 8: Adult NSC-derived brain organoids comprise neurons with myelinated axons. A: D20 brain organoids show colocalizing TUJ1-positive (each panel, picture 2ndfrom left), immature neurons with 04- (each panel, picture on the right), and Opalin-positive (each panel, picture 2nd from right) early and Opalin-positive late myelinating oligodendrocytes, indicating active myelination processes in adult NSC-derived organoids. Pictures on the left in each panel depict a merge of respective staining. B: TEM reveals several axons with a dark ring (see C). D: The dark ring surrounding axons shows several layers, indicating myelin sheaths that wrap around neuronal axons.

[0243] Figure 9: Adult NSC-derived organoids show an aging phenotype. Adult NSCs were isolated from young (under 40 weeks) and old (over 70 weeks) mice, and subsequently, organoids were generated. Organoids generated from old mice exhibited a significantly smaller diameter at nearly all time points compared to organoids generated from young mice, indicating slower proliferation of adult NSCs from old mice and thus an aging phenotype. Such comparative studies are possible, as the present method enables generation of an exact number of cells per organoid and facilitates the isolation of cells from mice of various ages that possess distinct epigenetic characteristics.

[0244] EXAMPLES

[0245] The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0246] Material and methods

[0247] Animals

[0248] C57BL / 6J mice were bred in the animal facility of the Helmholtz Centre for Infection Research (Braunschweig, Germany) under strict SPF conditions. We want to emphasize that our work was conducted in full accordance with national animal welfare law. The mice were handled with care and sacrificed via CO2 inhalation for brain isolation, ensuring their welfare was prioritized at every step.

[0249] Isolation of neural stem cells from adult mice

[0250] Adult neural stem cells (aNSC) were isolated from the subventricular zone and the dentate gyrus of C57BL / 6J mice, as previously reported (Walker et al. 2014). After dissecting the relevant brain regions, the tissue was minced and dissociated in a tube rotator at 37°C for 8 minutes in prewarmed 0.05% Trypsin / EDTA (Gibco) containing 40 U / ml DNase I. The reaction was stopped by addition of Basic medium (NeurobasalTM Medium, 1x B-27TM supplement, 1x L-Glutamine, 1x P / S). After centrifugation (300 xg, 5 min), the pellet was resuspended in Basic medium and filtered with a cell strainer (40 pm, Corning®). To minimize cell loss, the remaining tissue was pushed through the strainer using an injection stamp, followed by flushing Basic medium until it was almost clean. The suspension was centrifuged (300 xg, 5 min), and the pellet was resuspended in 1 ml Proliferation medium (Basic medium, 20 ng / ml FGF2, 20 ng / ml EGF, 2 pg / ml Heparin). As previously described, a density gradient centrifugation was performed to remove debris (Babu et al. 2011). Afterward, cells were resuspended in Proliferation medium and cultured on Poly-D-Lysin / Laminin coated plates at 7.5% CO2 and 37°C in a humidified atmosphere. Every other day, half of the medium was replaced with 2x Proliferation medium (Basic medium, 40 ng / ml FGF2, 40 ng / ml EGF, 4 pg / ml Heparin).

[0251] Generation of cerebral organoids from adult neural stem cells

[0252] Cerebral organoids were generated using a novel protocol based on Rybak-Wolf et al. (2023) and Ciarpella et al. (2021). aNSCs were washed once with PBS-G (PBS, 30mM D-Glucose) before Accutase® was added. After detachment, the reaction was stopped with Proliferation medium, and cells were centrifuged (300 xg, 5 min). The pellet was resuspended, and the number of cells was determined using a Luna II system (Logos Biosystems, USA). 2x104cells were seeded in each well of a 96-well plate (round-bottom, ultra-low attachment). Before incubation at 37°C, cells were centrifuged (300 xg, 5 min) to ensure uniform neurosphere formation. Neurospheres were cultured for 4-5 days, where 50% of the medium was exchanged every other day with 2x Proliferation medium. Induction of neurospheres was facilitated by replacing half the medium with Induction medium (Basic medium, 5 pg / ml Heparin). After two additional days of cultivation, liquid embedding was performed in Differentiation medium I (Neurobasal Plus, 1x N2 supplement, 1x B27 Plus supplement, Ix GlutaMAX, 1x P / S, 1x MEM-NEAA, 12.5 pl / 50 ml Insulin) with 2% Geltrex on an orbital shaker (Celltron, INFORS HT, Germany) at 85 rpm (with a clockwise throw of 25 mm). The medium was exchanged every other day with Differentiation medium I until day 12 to promote further differentiation of the early organoid. Starting from day 13, the organoids were cultured in Differentiation medium II (Differentiation medium I, 20 ng / ml BDNF, 20 ng / ml GDNF, 200 nM Ascorbic acid). The medium was changed every 3-4 days until the organoids reached terminal differentiation after day 20.

[0253] Cryo-sectioning of organoids

[0254] Organoids were washed twice with PBS and fixed using 4% paraformaldehyde (PFA) in PBS for 15 min at 37°C in the dark. After fixation, organoids were washed again and incubated in 40% sucrose in PBS at 4°C until they sank to the bottom. Next, the organoids were embedded in a mold containing 13% gelatin / 10% sucrose / PBS. The mold was then frozen and stored at -80°C until cryo-sectioning was performed. Sectioning was done using a cryostat (Leica, Germany) at - 20°C with a section thickness of 11-14 pm. Sections were transferred on a slide and stored at - 80°C until further use.

[0255] Immunofluorescence staining

[0256] For immunofluorescence staining, organoid sections were fixed with 4% PFA / PBS for 10 min and washed three times with PBS. Sections or 3D-organoids were then incubated in blocking buffer (0.25% Triton X-100, 5% horse serum in PBS) for 1 h and afterward incubated with the primary antibodies (0.1 % Triton X-100 / 5% horse serum in PBS) overnight at 4°C. Subsequently, sections and organoids were washed three times for 10 min in PBS-T, followed by secondary antibody incubation for 2 h at room temperature. DAPI was added to stain the nuclei 10 min before the end of incubation. 3D organoids and sections were washed again and mounted on rectangular coverslips in mounting medium (Prolong Glass Antifade Mountant, Thermo Fisher Scientific). 3D- organoids were imaged in suspension. Confocal microscopy

[0257] Confocal microscopy was performed with a CSU-W1 spinning disk (Yokogawa) mounted on a Ti2 eclipse microscope (Nikon). Data were acquired using a Zyla 4.2 sCMOS camera (Andor), a 405 / 488 / 561 nm laser (Omicron), and a 20x / NA 0.75 objective. Z-stacks were generated with a step size of 0.8 pm. Microscopes were controlled by the NIS Elements software (Nikon) and used for image analysis and montage generation.

[0258] Table 1 : Chemicals, peptides, and recombinant proteins used

[0259] Others

[0260] Example 1

[0261] Proliferation medium

[0262] Neurobasal medium 1% B27 supplement 1% L-glutamine 1% penicillin / streptomycin 20 ng / ml EGF 20 ng / ml FGF2 2 pg / ml heparin

[0263] Induction medium

[0264] Neurobasal medium 1% B27 supplement 1% L-glutamine 1% penicillin / streptomycin 5 pg / ml heparin

[0265] Differentiation medium I

[0266] Neurobasal Plus medium 1% B27 Plus supplement 1% GlutaMAX 1% penicillin / streptomycin 1% N2 1% NEAA

[0267] 12.5 pl / 50 ml Insulin

[0268] Differentiation medium II

[0269] Neurobasal Plus medium 1% B27 Plus supplement 1% GlutaMAX 1% penicillin / streptomycin 1% N2 1% NEAA

[0270] 12.5 pl / 50 ml Insulin 20 ng / ml BDNF 20 ng / ml GDNF 200 nM Ascorbic acid

[0271] Adult tissue-specific stem cells (termed ‘neural stem cells’ or ‘neural precursor cells’) were obtained from the brain of adult mice as disclosed herein above and used as initial (starting) cell culture. In general, neural stem cells (NSCs) can differentiate into nearly all neuronal and glial cell types. They are present in embryonic and mature adult brains and are linked to learning, memory storage, pattern recognition, and memory clearance. There are two neurogenic zones from which NSCs were isolated in the presently tested embodiment: the subventricular zone (SVZ) and the dentate gyrus (DG). After tissue dissection and enrichment, NSCs can be grown as an adherent monolayer on poly-D-lysine / laminin-coated petri dish for several weeks (see for example Fig. 3). Neural stem cells are considered to have already made the neuronal fate decision. Moreover, these cells are not immortalized with sternness factors and, as such, show more straightforward and reliable terminal differentiation.

[0272] The initial NSCs were cultivated in proliferation medium from the begin of the present method until day 5 until neurospheres had formed.

[0273] The present embodiment advantageously used two differentiation media, the first for differentiation and the second for maturation and maintenance. The first medium enabled the unguided differentiation of neurons and glia cells. Thereby the tested embodiment of the method according to the invention differs from the prior art, where only one differentiation medium is used, e.g., Ciarpella et al., 2021 and 2023, which contains BDNF, and will ‘push’ cell differentiation in a neuronal direction. For proper maturation of organoids, the present method preferably additionally uses GDNF, ascorbic acid, and / or insulin in the second differentiation medium. This not only fosters optimal neuronal differentiation and maturation but also promotes glial differentiation and processes such as myelination. This is crucial for gaining complex neuronglia interactions, which are absent in prior art methods, such as those of Ciarpella et al., 2021 and 2023.

[0274] Induction of differentiation is achieved in the presently tested embodiment by a complete exchange from stem cell medium to only heparin-containing medium. Such method step is entirely absent in the prior art, such as Ciarpella et al., 2021 and 2023, where instead a gradual withdrawal of growth factors FGF2 and EGF induces differentiation. In general, heparin is considered essential for the proliferation and differentiation of stem cells, supporting their transformation into progenitor cells during the induction phase (see Figure 7, day 4 (D4) to day 8 (D8)). This presents a substantial difference in the culture conditions and has a strong impact on the product, e.g. gaining mature organoids already after day 20 (D20), which is not achieved by prior art methods.

[0275] The neurospheres were cultured for 4-5 days, where 50% of the medium was exchanged every other day with 2x Proliferation medium. Subsequently cells were cultured in induction medium on day 6-7. Induction of neurospheres was facilitated by replacing half the medium with Induction medium. After two additional days of cultivation, liquid embedding was performed in Differentiation medium I with 2% Geltrex on an orbital shaker. The medium was exchanged / replaced every other day with a first differentiation medium (I) until day 12 to promote further differentiation of the early organoid. Starting from day 13, the organoids were cultured in a second differentiation medium (II). The medium was changed every 3-4 days until the organoids reached terminal differentiation after day 20 and cells were cultured until day 25.

[0276] Different development stages and the final obtained murine cerebral organoid cell culture models were analyzed by immunofluorescence staining and confocal microscopy as described herein above. The results are shown in Figures 1 , 2C and 4-5.

[0277] In brief, as shown in Figure 1 , the initially NSCs isolated from adult mice show characteristics of multipotency (Nestin and Sox2 expression) and were able to differentiate. At day 4 of growth factor deprivation, cells started to express Map2a / b (neurons) and GFAP (astrocytes) additionally to Nestin (lower left panel of Figure 1). Furthermore, cells elongated and became thinner. After 9 days of differentiation, Map2a / b-positive cells showed continued elongation and adapt a neuronlike structure, pointing to terminal differentiation with a neuronal fate (lower right panel of Figure 1). GFAP-positive cells expanded and showed pronounced expression of this astrocyte marker. Oligodendrocytes were not detectable at this point in culture, probably as they need more time to differentiate. Consequently, the inventors were able to show that isolated NSCs were not only express markers of multipotency, but were also able to differentiate into neurons and astrocytes.

[0278] For differentiation experiments, NSCs were detached and seeded on Poly-D-Lysine and Laminin coated glass coverslips. Differentiation was induced by deprivation of growth factors for several days. After fixation with PFA and MeOH, cells were stained with Antibodies for Nestin, Sox2, Map2a / b and GFAP.

[0279] As shown in Figure 2C, neurospheres showed uniform distribution of Sox2+ and Nestin+ NSCs. After induction of differentiation, cells reorganized and Map2a / b+ neurons started to form at the outer layer of the early organoid. NSCs began to withdraw to the inner side, thereby forming a cavity between inner and outer layer. Neurospheres and organoids were fixed at day 5 or 11 and embedded for cryo-sectioning. 12 pm sections were prepared and stained via immunofluorescence using the respective antibodies.

[0280] As shown in Figures 4 and 5 (12 pm cryosections of cerebral organoids stained with different antibodies) between day 4 to day 25 of total culture duration according to the present method the (cerebral) organoids increasingly expressed OLIG2, Map2a / b and GFAP as markers of differentiated cerebral cells whereas stem cell marker expression like Sox 2 is down regulated.

[0281] Figure 7 further evidences that day 20 (D20) organoids harbor Tau-positive neurons (top row, picture in the middle of Fig. 8), GFAP-positive astrocytes (top row, picture on the right of Fig. 8), and 04- (bottom row, picture on the right of Fig. 8), and Opalin-positive (bottom row, picture in the middle of Fig. 8), early and Opalin-positive late myelinating oligodendrocytes.

[0282] Figure 8 further shows that the generated organoids comprise neurons with myelinated axons, wherein (Fig. 9 A) D20 brain organoids show colocalizing TUJ1 -positive (each panel, picture 2nd from left Fig. 9A), immature neurons with 04- (each panel, picture on the right Fig. 9A), and Opalin-positive early and Opalin-positive late myelinating oligodendrocytes (each panel, picture 2nd from right Fig. 9A), indicating active myelination processes in the present organoids. In summary, the present method enable a superior differentiation of starting cells as compared to prior art methods, such as of Ciarpella et al., 2021 and 2023. Whereas Ciarpella et al. could detect RNA of the astrocyte marker GFAP, they failed to detect an increase of astrocytes in the organoid during differentiation by Immunofluorescence. It can be assumed that the astrocytes present in the organoids of Ciarpella et al., are only present and surviving as they were directly isolated from E14.5 brains, and are not differentiated from the NSCs.

[0283] On the other hand, the organoids generated according to the present invention display oligodendrocytes and astrocytes, that apparently differentiate throughout the culture period. These were absent in Ciarpella’s organoids, making it challenging to utilize said structures to mimic complex neuron-glia interactions. In contrast, the structures of the organoids generated according to the present invention additionally display myelin sheaths essential for saltatory signal conduction and correct representation of the brain. Figure 6 shows qPCR data revealing elevated levels of GFAP, Opalin, and CNP expression, alongside reducing OLIG1 expression during organoid maturation. Further, Figure 8 shows TEM images that clearly indicate myelin sheaths surrounding axons.

[0284] The herein provided comparative data show that only embodiments of the present method achieve a significant improvement in differentiation and organoid quality. The present example demonstrates that the culturing steps, along with the respective media, yield organoids that comprise not only mature neurons and astrocytes, but also mature myelinating oligodendrocytes and myelinated neuronal axons within 3 weeks of culture. In contrast, prior art methods, such as those of Ciarpella et al., yield less complex organoids after 4 weeks of culture, indicating that the presently used medium composition, the chosen cell source, and the culture duration and steps differ not only majorly from the prior art, but achieve a significant improvement.

[0285] Furthermore, utilizing stem cells derived from the SVZ alongside DG-derived stem cells to generate a broader variety of neuronal and glial cells constitutes a new and unique approach.

[0286] In summary, the present method starts with a different material (aNSCs of two neurogenic zones) compared to prior art methods, and thereby facilitates obtaining a different product, namely mature brain organoids with features, e.g., related to the donor age. Thereby the present method opens new avenues for age- and / or disease-related research in neuroscience. For the first time, the inventors could generate mature oligodendrocytes and myelinated neuronal axons within a brain organoid from mouse stem cells. Therefore the organoids obtained according to the present invention are more complex and more mature, comprises more cell types and resembles a mature brain more accurately than organoids obtainable by prior art methods.

[0287] Example 2

[0288] In the present example, adult NSCs were isolated from young (under 40 weeks) and old (over 70 weeks) mice, and subsequently, organoids were generated as described above in Example 1 . Organoids generated from ‘old’ mice exhibited a significantly smaller diameter at nearly all time points compared to organoids generated from young mice (see Figure 9), indicating slower proliferation of adult NSCs from old mice and thus an aging phenotype. Such comparative studies are possible, as the present method enables generation of an exact number of cells per organoid and facilitates the isolation of cells from mice of various ages that possess distinct epigenetic characteristics.

[0289] REFERENCES

[0290] Babu, H., Claasen, J.-H., Kannan, S., Riinker, A.E., Palmer, T., and Kempermann, G. (2011). A Protocol for Isolation and Enriched Monolayer Cultivation of Neural Precursor Cells from Mouse Dentate Gyrus. Front Neurosci-switz 5, 89. 10.3389 / fnins.2011.00089. Ciarpella, F., Zamfir, R.G., Campanelli, A., Ren, E., Pedrotti, G., Bottani, E., Borioli, A., Caron, D., Chio, M.D., Dolci, S., et al. (2021). Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity. iScience 24, 103438. 10.1016 / j.isci.2021.103438.

[0291] Kirchenwitz, M., Stahnke, S., Grunau, K. et al. The autophagy inducer SMER28 attenuates microtubule dynamics mediating neuroprotection. Sci Rep 12, 17805 (2022). https: / / doi.Org / 10.1038 / S41598-022-20563-3

[0292] Rybak-Wolf, A., Wyler, E., Pentimalli, T.M., Legnini, I., Martinez, A.O., Glazar, P., Loewa, A., Kim, S.J., Kaufer, B.B., Woehler, A., et al. (2023). Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids. Nat. Microbiol. 8, 1252-1266. 10.1038 / s41564- 023-01405-y. Walker, T.L., and Kempermann, G. (2014). One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice. J Vis Exp Jove, 51225. 10.3791 / 51225.

Claims

CLAIMS1 . An in vitro method of generating a murine cerebral organoid cell culture model, comprising cultivating murine neural stem cells obtained from an adult mouse until neurospheres have formed, and subsequently differentiating said neurospheres, thereby generating a murine cerebral organoid cell culture model.

2. The in vitro method according to claim 1 , comprising the steps of: a. providing a cell culture comprising murine neural stem cells obtained from an adult mouse and cultivating the cell culture for between 3-7 days until neurospheres have formed, b. replacing at least a fraction of the culture medium with induction medium and cultivating the cell culture for between 1-4 days, c. replacing the induction medium with differentiation medium and cultivating the cell culture for between 9-17 days, wherein the total duration of cell culture is at least 21 days, thereby generating a murine cerebral organoid cell culture model.

3. Method according to any one of claims 1-2, wherein the murine neural stem cells are obtained from the hippocampus, preferably from the subventricular zone (SVZ) and / or the dentate gyrus (DG), of the adult mouse.

4. Method according to any one of the preceding claims, wherein the cell culture is cultivated in a neurobasal medium.

5. Method according to any one of the preceding claims 2-4, wherein the initial cell culture medium in a. comprises neurobasal medium and one or more of fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), heparin, L-glutamine and B27- supplement is added before cultivating the cell culture for at least 4 days, and / or, wherein the induction medium comprises neurobasal medium and one or more of heparin, L- glutamine and B27- supplement, and lacks FGF2 and / or EGF.

6. Method according to any one of the preceding claims 2-5, wherein in step c. the cell culture is cultivated in a first differentiation medium for between 3-7 days, and subsequently in a second differentiation medium for between 6-10 days.

7. Method according to the preceding claim, wherein the first differentiation medium comprises neurobasal medium plus and one or more of insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2 supplement, and one or more basement membrane component and / or extracellular matrix (ECM) component (e.g., Geltrex or Matrigel), and / orwherein the second differentiation medium comprises Neurobasal Medium Plus and one or more of L-glutamine, NEAA, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid and B27-Plus and N2 supplement.

8. Method according to any one of the preceding claims, wherein at least a fraction of the neural stem cells expresses Sox2 and / or Nestin, and / or wherein at least a fraction of the cultured cells expresses Nestin and / or microtubule- associated protein 2a / b (Map2a / b) and / or Tau or glial fibrillary acidic protein (GFAP) and / or oligodendrocyte transcription factor 2 (OLIG2) after 1-10 days of total cell culture duration, preferably after 3-5 days.

9. Method according to any one of the preceding claims, wherein at least a fraction of the cultured cells are fully differentiated / mature neurons or astrocytes after 5-14 days of total cell culture duration, preferably after 8-12 days, and / or wherein at least a fraction of the cultured cells are fully differentiated / mature neurons, astrocytes, glial cells or oligodendrocytes after 10-21 days of total cell culture duration.

10. Method according to any one of the preceding claims, wherein the murine neural stem cells are genetically engineered cells and / or have been obtained from a genetically engineered mouse.11 . Method according to any one of the preceding claims 2-10, wherein the cells are cultivated in the initial cell culture medium in a. until day 5 of total cell culture duration, wherein the cells are cultivated in the induction medium in b. until day 7 of total cell culture duration, wherein the cells are cultivated in a first differentiation medium in c. until day 12 of total cell culture duration, and wherein subsequently the cells are cultivated in a second differentiation medium in c. at least until day 21 of total cell culture duration.

12. An in vitro murine cerebral organoid cell culture model comprising mature astrocytes, oligodendrocytes and / or neurons produced according to the method of claims 1-11 , preferably wherein at least a fraction of the astrocytes expresses glial fibrillary acidic protein (GFAP), at least a fraction of the neurons express microtubule-associated protein 2a / b (Map2a / b) and / or comprise myelinated axons, and / or at least a fraction of the oligodendrocytes express Oligodendrocyte transcription factor 2 (OLIG2).

13. Use of the in-vitro model according to claim 12 for (high throughput) drug or toxicity screening and / or for studying a neurological disease.

14. A kit comprising a culture medium and at least one supplement selected from the group comprising fibroblast growth factor 2, (FGF2), epidermal growth factor (EGF), heparin, insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2 supplement, one or more basement membrane component (BM) and / or ECM component, ascorbic acid, brain- derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF).

15. The kit according to claim 14, further comprising the in vitro cell culture model according to claim 12.

16. A culture medium supplement cocktail comprising d. fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), heparin and / or B27- supplement, or e. insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2 supplement and / or one or more basement membrane component and / or ECM component, or f. insulin, L-glutamine, non-essential amino acids (NEAA), B27-Plus supplement, N2 supplement, one or more basement membrane component and / or ECM component, ascorbic acid, brain-derived neurotrophic factor (BDNF) and / or glial cell line-derived neurotrophic factor (GDNF).

17. A cell culture plate comprising the in vitro cell culture model according to claim 12.

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