A miniaturized brain organoid cell culture model

Abstract

The invention relates to an in vitro method of generating a neural organoid cell culture model. The invention relates to an in vitro adherent neural organoid cell culture model, comprising organoids with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections. The invention further relates to an in vitro neural organoid cell culture model, produced according to the method of the invention, to the use of the in-vitro model of the invention for high throughput drug or toxicity screening and / or for studying a neurological disease, to a kit for carrying out the method of the invention, to a cell culture plate or micropattern cell culture chip comprising the in vitro neural organoid model of the invention and to a computer implemented method for analyzing a neural organoid cell culture model of the invention.

Description

[0001] A MINIATURIZED BRAIN ORGANOID CELL CULTURE MODEL

[0002] DESCRIPTION

[0003] The invention is the field of cell biology, cell culture models and corresponding screening methods.

[0004] The invention relates to an in vitro method of generating a neural organoid cell culture model. In other aspects the invention relates to an in vitro adherent neural organoid cell culture model, comprising organoids with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0005] The invention further relates to an in vitro neural organoid cell culture model, produced according to the method of the invention, to the use of the in-vitro model of the invention for high throughput drug or toxicity screening and / or for studying a neurological disease, to a kit for carrying out the method of the invention, to a cell culture plate or micropattern cell culture chip comprising the in vitro neural organoid model of the invention and to a computer implemented method for analyzing a neural organoid cell culture model of the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Since the first brain organoids were developed in 2013, a range of such organoids has been developed based on the original protocol from Lancaster et al1. The aim is usually to obtain specific cell types for different regions of the brain, e.g. dorsal and ventral forebrain or midbrain cells2-5.

[0008] Significant drawbacks of the current generation of organoids are the lack of spatial structure and the lack of spatial reproducibility. One of the main goals in using organoids is to generate tissues with different cell types that are arranged similar to their in vivo counterpart. While current brain organoids partially achieve this, they still show a high variability between individual organoids, batches and cell lines.

[0009] In the case of neural or brain organoids, for example, present organoids grow in culture to different sizes and generate random numbers of organizing centers (the ventricular zones). This renders the identification of phenotypes in disease models very challenging, and more subtle, such that early disease phenotypes may not be detectable. For drug screening approaches, this also impairs the use of standard brain organoids for drug or toxicity screenings, as the wild type (WT) and disease phenotypes cannot reliable be separated to rule out false positives or negatives in the screening (i.e. potentially successful drugs may be missed and conversely, drugs that do not have an effect may appear potent).

[0010] At present, the generation of organoids is not easily scalable. Particularly, current neural or brain organoids cannot be produced in high enough numbers (and with enough reproducibility) to be used for high throughput applications. However, even if higher reproducibility would be achieved, still a large number of organoids would be required to achieve statistically robust assays for screening applications. However, currently only batches of organoids with a yield of a few tens, or maximally a few hundreds of organoids can be generated, which is too few for screening applications. Attempts to scale up the generation of organoids in 96 or 384 wells have yet been limited to one organoid per well2 6, and additionally still suffer from a complete absence of spatial structure, such that such organoids are barely useful for identifying or analyzing disease phenotypes.

[0011] In the context of brain organoids, additional difficulties have been observed in obtaining cell culture models that are adherent and enable culturing over longer periods of time. To date, adherent cultivation of brain organoids beyond a few days has not been established.

[0012] Metzger et al.8and WO2020 / 257756 describe a drug screening platform using "neurulation stage organoids" differentiated for seven days on micropatterns with a diameter of 500 or 700 pm. In 96-well plates, 25 organoids per well were analyzed, and compared against neural organoids that had mutations typical of Huntington Disease. Subsequently, compound screening was performed, and it was investigated whether a phenotypic rescue of the disease phenotype could be observed. Experiments were performed after seven days because cell adherence was not guaranteed with longer cultivation.

[0013] Other studies have demonstrated neuronal differentiation of embryonic stem cells into neuronal rosettes, comprising cultivation on micropatterns for up to seven days. Further, Knight et al10were able to obtain neural rosettes comprising neuro-epithelial cells that could be cultured on micropatterns for up to twelve days. However, the longest cultivation period identified in the literature was only 18 days by Tang et al.11. Tang et al. generated neuro-epithelial organoids that exhibited dorsal-ventral patterning, as an early model of a spine.

[0014] Further studies from Miura et al., 2022 and WO 2022 / 080974 analyzed different culture conditions for the generation of brain organoids, however, also those studies failed to report suitable conditions for long-time adherent organoid cell culture.

[0015] In general, a major disadvantage of non-adherent organoid cultures or of organoids lacking a long-time adherence is their lack of spatial reproducibility and their unsuitability for high- throughput applications or screenings.

[0016] Moreover, many other prior art methods, such as WO 2022 / 080974, fail to generate neuronal organoids comprising a reproducible spatial structure or a regular eccentricity, e.g., as brain organoids are assembled by combining or fusing separate differentiated organoids of different brain structures. Further studies, e.g., WO 2023 / 090427, describe the generation of other neuronal models, such as retinal organoid models, which, however, have a size of up to 5 cm and are therefore less suitable for high throughput applications.

[0017] In summary, one significant problem remains regarding the difficulty in adherent cultivation of organoids due to a possible detachment from the culture substrate or support during cultivation and differentiation, which has so far prevented long-term adherent cultivation. Until now, longterm culture has required non-adherent culture, with the disadvantages of a lack of spatial reproducibility and lack of high-throughput. Hence, considering the prior art, there remains a significant need in the art to provide advanced neuronal cell culture models that are suitable for long-term cultivation and can be generated in large numbers required for screening applications.

[0018] SUMMARY OF THE INVENTION

[0019] In light of the prior art, a technical problem underlying the invention was the provision of an alternative or improved neural cell culture model suitable for long-term cultivation.

[0020] Another problem underlying the invention was the provision of an adherent neural cell culture model.

[0021] Another problem underlying the invention was the provision of an adherent neural cell culture model that can be generated at a sufficient scale as required for screening applications.

[0022] Another problem underlying the invention was the provision of an adherent neural cell culture model with sufficiently regular structural characteristics, such as a regular eccentricity, enabling reliable production and use in high-throughput screening applications.

[0023] 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.

[0024] To overcome the afore-mentioned problems of reproducibility, scalability, lack of adherence and long-term culturing, the inventors have developed in vitro organoids that are preferably smaller (miniaturized) than present neuronal organoid models but maintain relevant features of larger organoids and their in vivo counterparts.

[0025] The invention therefore relates to in-vitro method of generating an adherent neural organoid cell culture model, comprising the steps of: a. providing a cell culture comprising pluripotent stem cells, b. adding to the cell culture medium at least one Rho-associated protein kinase inhibitor (ROCKi) and / or at least one extracellular matrix (ECM) protein or component, and c. cultivating the cell culture for at least 18 days, thereby generating an adherent neural organoid in cell culture with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0026] In other words, in embodiments, the core area of the neural organoid cell culture model is surrounded by a rim of cell projections of neurons and / or glia cells. In embodiments, the cell projections are formed by mature neurons and / or radial glia cells. In alternative embodiments, the method thereby generates an adherent neural organoid in cell culture, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0027] In embodiments, the method steps a. to c. generate an adherent neural organoid in cell culture, said organoid comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0028] In embodiments, the method generates an adherent neural organoid in cell culture with a defined spatial structure, comprising a core area with a regular eccentricity and comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections

[0029] The method steps and the resulting physical characteristics of the organoid therefore represent related aspects of the invention.

[0030] In some embodiments, the defined spatial structure is a preferable but non-essential characteristic of the organoid.

[0031] In some embodiments, the regular eccentricity of the organoid and / or of the core area is a preferable but non-essential characteristic of the organoid.

[0032] In preferred embodiments, the neural organoid cell culture model is / stays adherent (attached to the cell culture vessel / support) throughout cultivation. In embodiments, the adherence is maintained for the cultivation time (culture duration) of at least 18 days.

[0033] In embodiments, the neural organoid cell culture model comprises after 18 days of total culture duration one or more of differentiated (mature I postmitotic) neurons, neuroepithelial cells, radial glia cells, progenitor cells and / or intermediate progenitors. In embodiments, the culture in step c. is an adherent cell culture (a cell culture comprising adherent cells).

[0034] The invention therefore relates to a self-organizing neural organoid cell culture model, preferably a self-organizing brain organoid cell culture model, which preferably may be cultivated for several months, or even a year and longer, and to methods of generating said models. The inventors surprisingly found that the methods according to the invention enable the generation of ‘miniaturized’ neuronal, e.g., brain, organoids that are adherent to the respective culture vessel over a prolonged period of time, preferably over 18, 30, 50 or over 100 days.

[0035] Previous work of the inventors has demonstrated neuronal differentiation of embryonic stem cells into neuronal rosettes on micropattern structures, which were adherent for a total culture duration of up to 7 days8. Other studies10were able to obtain neural rosettes consisting of neuro-epithelial cells that could be cultured on micropatterns for up to 12 days. So far, the longest cultivation period reported for neuronal organoids in the prior art was 18 days11. However, in the prior art, neuronal organoid models capable of a prolonged cultivation time exceeding 18 days and comprising cells of mature differentiation stages have, according to the knowledge of the inventors, not yet been described. One of the limiting factors of extended cultivation duration of neuronal organoid models has been the time-limited adherence of such organoids to culture vessels or support. This limitation was previously addressed by embedding neuronal organoids in hydrogel, which itself has further disadvantages, such as irregular organoid growth and limited reproducibility of the generated organoids. Other limiting factors of cultivation time and adherence are mechanical shear forces and remodeling processes occurring during organoid differentiation, which lead to cell debris and detachment of apoptotic and necrotic cells.

[0036] The inventors found that the methods and other means described herein surprisingly facilitate an extended adherence of the neuronal cell culture model according to the invention to the surface of culture vessels, such that an advantageously prolonged cultivation time of more than 50 or even more than 100 days is possible. Moreover, this prolonged attachment of the inventive model is not only achieved on standard cell culture dishes or well-plates, but also on so-called ‘micropatterns’ (specific microgrids) or chips (micropattern cell culture chips) enabling the differentiation of initially pluripotent cells into different neuronal cell types, without an unlimited increase in mass or size of the differentiating organoids (comprising different cell types and / or brain tissues). Moreover, in embodiments, the present methods further enable the generation of different cortical organoid models resembling different regions of the brain’s cortex.

[0037] Hence, in embodiments, the cell culture is cultivated in step c. on a micropattern cell culture chip (or micropattern coverslip) or micropattern cell culture dish either from day 0, or from day 10-12 onwards.

[0038] In embodiments, the cell culture is cultivated in step c. initially in a standard cell culture dish or plate for between 1 and 12 days, preferably 8 to 12, or 10 to 12 days, and subsequently transferred onto a micropattern cell culture chip.

[0039] In embodiments, the cell culture is cultivated in step c. initially in a cell culture dish or plate for 8 to 12 days and subsequently transferred to a micropattern cell culture chip for further culture.

[0040] Preferably, such models are defined by additional characteristics of the organoids, wherein the organoid further comprises one or more astrocytes, upper-layer neurons and / or deeper-layer neurons, preferably wherein at least a fraction of the deeper-layer neurons is localized in the core area of the neural organoid and / or wherein at least a fraction of the upper-layer neurons are localized at the rim of the neural organoid, thereby mimicking an inside-out development of a mammalian brain.

[0041] In embodiments, in step b., at least one inhibitor of the WNT pathway (WNT (pathway) inhibitor) or WNT inhibitor is added to the cell culture medium.

[0042] In embodiments, the cell culture is cultivated in step c. on a micropattern cell culture chip from day 0 onwards, to generate a neural organoid comprising a neural rosette structure in a central region of the neural organoid.

[0043] In embodiments, at least one inhibitor of the WNT pathway (WNT (pathway) inhibitor) or WNT inhibitor is added to the cell culture medium, and provides improved reproducibility of a neural rosette structure during cultivation and subsequent treatment of the organoid. The cultivation on micropattern cell culture chips (coverslips) has several advantages for the differentiation of the organoids. In embodiments, the area of the ‘microspots’ (a segment of the micropattern chip that comprises one organoid) limits the surface area upon which the organoids grow on and are attached to, thereby allowing the neural cell agglomerate to grow to a defined and reproducible size. The generated organoids are thereby limited in their growth and reach a maximum size of about 300-700 pm in diameter, preferably about 500 pm in diameter.

[0044] This has several advantages in combination with the present method; on one hand it further promotes the prolonged attachment of the neuronal organoids to the support. On the other hand, if multiple organoids are generated according to the present method, in parallel on a micropattern chip, all organoids comprise an essentially equal size. On the contrary, the neuronal organoid cultures of the prior art that are grown in hydrogel, typically expand continuously during their differentiation and can reach a size of several centimeters and often an irregular shape. When multiple such organoids are grown in hydrogel in parallel, they often significantly differ in their size and structure, thereby reducing their comparability and their suitability for (high-throughput) screening or comparative approaches.

[0045] Therefore, one advantage of the present neuronal cell culture models is their suitability for high- throughput screenings, as they comprise diverse types of differentiated neuronal cells in combination with a small and reproducible organoid-size.

[0046] 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. In embodiments, such analyses may be performed essentially as described by the inventors previously8(Metzger et al., Cell Reports Methods (2022) Vol. 2, 100297). Thereby, in embodiments, the present neuronal organoids may serve as separate brain models, 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.

[0047] In embodiments, the extended cultivation time of the present neuronal cell culture model enables, e.g., when cultivation is performed on micropattern chips, the formation of neuronal connections between different organoids, such that neuronal networks may be formed between several or multiple organoids.

[0048] The inventors surprisingly found that the present method enables the generation of neuronal cell culture models, comprising preferably cortical neuronal organoids or cortical brain organoids, which surprisingly are adherent throughout the entire cultivation time of up to 100 days of culture or even longer. Applying the present method, the inventors were able to cultivate neuronal organoids for more than three months for the first time, wherein the organoids comprised highly differentiated cells of diverse cell types, which has not been achieved before in the art.

[0049] Therefore, in embodiments, the cell culture is cultivated at least until day 18, or preferably day 30 of total culture duration. In embodiments, the cell culture is cultivated at least until day 18, 19, 20, 21 , 25, 27, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63, 70, 75, 80, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 200, 250, 300, 350, 365, 400, 450, 500 of total culture duration, or even longer. In embodiments, the cell culture is cultivated until at least day 18.

[0050] In embodiments, the cell culture is cultivated at least until day 100 of total culture duration, or even longer. In embodiments the cell culture is cultivated for at least for 3 months 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 or 36 months of total culture duration.

[0051] All time ranges disclosed herein may in embodiments also comprise a variation of ±1 day, of ±2, ±3, ±4 or ±5 days, of ±10 days, or ±14, or ±28, ±35, or ±50 days, or even more variation.

[0052] 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 100 days.

[0053] In embodiments, the differentiation of the neuronal cells is achieved according to standard protocols, such as Shi et al., 2012 (Shi et al., Nat. Prot. (2012) Vol. 7, 1836).

[0054] In embodiments, step b. comprises adding to the cell culture medium at least one Rho-associated protein kinase inhibitor (ROCKi) and at least one extracellular matrix (ECM) protein or component.

[0055] In embodiments, the pluripotent cells are singularized (separated into single cells) before the cultivation in step c.

[0056] In embodiments, the cell culture is cultivated in step c. in a culture vessel coated with at least one extracellular matrix (ECM) protein or component.

[0057] In embodiments, the cell culture is cultivated in step c. in a culture vessel coated with at least one extracellular matrix (ECM) protein or component, and / or wherein at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every 5-10 days.

[0058] In embodiments, at least one extracellular matrix (ECM) protein or component is added to the cell culture during the time course of cultivation, either once or more than one time, e.g., every 1-14 days or during / with a culture media exchange. In embodiments, the at least one extracellular matrix (ECM) protein or component is added to the culture media as a supplement or is comprised within the culture media.

[0059] In embodiments, the at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every 5-10 days. In embodiments the at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every day, every other day, or every 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.

[0060] In embodiments, the at least one extracellular matrix (ECM) protein or component is selected from laminin, collagen, collagen type IV, entactin, or Geltrex®. In embodiments, the at least one extracellular matrix (ECM) protein or component comprises laminin, collagen, collagen type IV, entactin, and / or Geltrex®. In embodiments, the cell culture is cultivated in step c. in a culture vessel coated with at least one extracellular matrix (ECM) protein or component, and / or wherein at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every 5-10 days, wherein the ECM protein or component is laminin.

[0061] In embodiments, at least for the first 18 days of culture, at least one Rho-associated protein kinase inhibitor (ROCKi) is added to the cell culture medium every 5-10 days, preferably at a concentration between 1-10 pM.

[0062] In embodiments, at least one Rho-associated protein kinase inhibitor (ROCKi) 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 or 20 pM.

[0063] Surprisingly, the inventors found that the addition of at least one ECM component and / or at least one ROCK inhibitor and / or a CEPT cocktail further increased the adherence of the present cell culture model to the support, thereby supporting a long-term cell culture of the model beyond 18 days, preferably beyond 30, 50 or even beyond 100 days.

[0064] In embodiments, and preferably for at least for the first 18 days of culture, at least one Rho- associated protein kinase inhibitor (ROCKi) is added to the cell culture medium every day, every other day, or every 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.

[0065] Non-limiting examples of Rho-associated protein kinase inhibitors (ROCKi) are WAY-624704, Y27632, Y-27632 2HCI, Thiazovivin ,GSK429286A, Fasudil (HA-1077) HCI, H-1152 dihydrochloride, Y-27632, Azaindole 1 (TC-S 7001), RKI-1447, AT13148, GSK269962A HCI, Y- 39983 HCI, Belumosudil (KD025), Ripasudil (K-115) hydrochloride dihydrate, Hydroxyfasudil (HA- 1100) HCI, Netarsudil (AR-13324) 2HCI, ZINC00881524 (ROCK inhibitor) and Chroman 1 .

[0066] In embodiments, the at least one Rho-associated protein kinase inhibitor (ROCKi) is Chroman 1.

[0067] In embodiments, instead of or in addition to the at least one Rho-associated protein kinase inhibitor (ROCKi), a CEPT cocktail comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB, is used.

[0068] In embodiments, Rho-associated protein kinase inhibitors (ROCKi) may be comprised in a culture medium and / or supplement-cocktail at a concentration of 10 pM, or a concentration between 0.01-200 pM, 0.1-100pM, 5-50 pM, 5-25 pM, 5-15 pM or 10-25 pM or at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200 pM. The concentration may depend on the specific compound used.

[0069] In embodiments, a CEPT cocktail (comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB) may be comprised in a culture medium and / or supplement-cocktail at a concentration of 50 nM Chroman 1 , 5 pM Emricasan, 1x Polyamine Solution, and 0.7 pM Trans- ISRIB, or a concentration of between 25-75 nM Chroman 1 , between 1-10 pM Emricasan, between 0.5-1 .5 x Polyamine Solution, and between 0.5-1 .5 pM Trans-ISRIB. The concentration may depend on the specific compounds used. In some embodiments, the present in-vitro method comprises: a. providing a cell culture comprising pluripotent stem cells, b. adding to the cell culture medium one or more of: at least one Rho-associated protein kinase inhibitor (ROCKi), and / or a CEPT cocktail comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB, at least one WNT (pathway) inhibitor and at least one extracellular matrix (ECM) protein or component, and c. cultivating the cell culture for at least 18 days, thereby generating an adherent neural organoid in cell culture with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0070] In some embodiments, step b. comprises adding to the cell culture medium at least one Rho- associated protein kinase inhibitor (ROCKi), and / or a CEPT cocktail (comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB), at least one WNT (pathway) inhibitor and at least one extracellular matrix (ECM) protein or component.

[0071] In embodiments, at least one further supplement is added to the cell culture medium selected from the group comprising SMAD-inhibitors, a CEPT cocktail comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB, or Chroman 1 by itself, at least one WNT (pathway) inhibitor, one or more neurotrophic factors, such as BDNF, NT3, GDNF, cAMP, and ascorbic acid.

[0072] SMADs (or Smads) are a family of structurally similar proteins that are the major signal transducers for transforming growth factor beta (TGFb; TGFbeta) superfamily receptors.

[0073] Non-limiting examples of inhibitors of SMAD signaling (‘SMAD-inhibitors’) or the TGFp pathway are SB431542, LDN-193189, SB525334, Ginsenoside Rh4, TGF RI-IN-3, Lycopus, AUDA, Sulfasalazine, SB505124, Galunisertib, BIBF-0775, LY2109761 , GW788388, LY364947, Pirfenidone, SIS3, PD, ITD-1 , TA-02, DMH1 , LDN-212854, ML347, RepSox, Dorsomorphin, K02288, Vactosertib, SD-208, LDN-214117, A-83-01 , R-268712, Dorsomorphin, SIS3, Halofuginone, TP0427736, LY, Galunisertib (LY2157299), LY2109761 , SB525334, SB505124, GW788388, LY364947, Alantolactone, Hesperetin, SRI-011381 , SB 4, Isoxazole 9, Kartogenin, L-Quebrachitol, SJ000291942, LY-3200882 and TP0427736 HCI.

[0074] In embodiments, an inhibitor of SMAD signaling or the TGFp pathway, e.g., SB431542, may be comprised in a culture medium and / or supplement-cocktail at a concentration of 10 pM, or a concentration between 1-100 pM, 5-50 pM, 5-25 pM, 5-15 pM or 10-25 pM or at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50pM. The concentration may depend on the specific compound used.

[0075] In embodiments, an inhibitor of SMAD signaling or a BMP pathway inhibitor, e.g., LDN-193189, may be comprised in a culture medium and / or supplement-cocktail at a concentration of 0.2 nM, or a concentration between 0.1-10 nM, or between 0.1-5 nM, or at O.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 nM.

[0076] The concentration may depend on the specific compound used.

[0077] In embodiments, the SMAD inhibitor SB431542, may be used at a concentration of 10 pM, or a concentration between 1-100 pM, 5-50 pM, 5-15pM, or 10-25 pM or at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50pM. The concentration may depend on the specific compound used.

[0078] In embodiments, the SMAD inhibitor LDN193189, may be used at a concentration of 0.2 pM, or a concentration between 0.1-10 pM, or between 0.1-5 pM, or at O.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15pM. The concentration may depend on the specific compound used.

[0079] In embodiments, the at least one WNT (pathway) inhibitor (or inhibitor of the WNT pathway, or inhibitor of WNT signaling) may be selected from the group comprising, without limitation thereto, IWR-1-endo (IWR1e), IWP-2, JW74, IWP-4, CCT251545, KY1220, iCRT14, iCRT3, LF3, PNU- 74654, KYA1797K, KY02111 , Zamaporvint (RXC004), M2912, M435-1279, KY-05009, Prodigiosin, Ginsenoside Rh4, Heparan Sulfate, RCM-1 , Resibufogenin, MSAB, PRI-724, Tegatrabetan (BC-2059), JW55, Adavivint (SM04690), Triptonide, Isoquercitrin, IQ-1 , Salinomycin and FH535.

[0080] In embodiments, the at least one WNT inhibitor, e.g., IWR1 e, may be comprised in a culture medium and / or supplement-cocktail at a concentration of 3 pM, or a concentration between 1-10, or 1-5 pM, or at 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 pM. The concentration may depend on the specific compound used.

[0081] In embodiments, the at least one WNT inhibitor, e.g., IWR1 e, may be used at a concentration of 3 pM, or a concentration between 1-10, or 1-5 pM, or at 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 pM. The concentration may depend on the specific compound used.

[0082] Although it is not essentially required for the generation of the adherent neural organoid model with a defined spatial structure and a regular eccentricity according to the present invention to add a WNT inhibitor to the cell culture medium, in some specific embodiments the addition of at least one WNT inhibitor (e.g., in step b.) may further increase the reproducibility of generated neural organoids comprising a central neural rosette structure.

[0083] 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.

[0084] In embodiments, the pluripotent stem cells are induced pluripotent stem (IPS) cells.

[0085] In embodiments, the cell culture is cultivated in step c. initially at a cell-density of at least 50.000 cells / cm2. In embodiments, the cell culture is cultivated in step c. initially at a cell-density of between 50.000 and 150.000 cells / cm2. In embodiments the cell culture is cultivated in step c. initially at a cell-density of between 100.000 to 150.000 cells / cm2or higher. In embodiments, the cell culture is cultivated in step c. initially at a cell-density of at least 100.000 cells / cm2. In embodiments the cell culture is cultivated in step c. initially at a cell-density of at least 100.000 cells / cm2or more. In embodiments the cell culture is cultivated in step c. initially at a cell-density of at least 150.000 cells / cm2.

[0086] In some embodiments, the cell culture is cultivated in step c. initially at a cell-density of at least 150.000 cells / cm2thereby generating a model comprising a neural rosette structure at the center of the neural organoid.

[0087] In embodiments, from culture day 18 on and after, the organoid comprises a defined spatial structure and a regular eccentricity that comprises a defined core area comprising differentiated (mature) neurons, progenitor cells and glia cells, surrounded by a rim of neuronal and / or glia cell projections.

[0088] In embodiments, from culture day 18 on and after, the organoid preferably comprises one or more of cortical progenitor neurons, deeper-layer neurons, upper-layer neurons, astrocytes and / or basal progenitors.

[0089] In some embodiments, from culture day 18 on and after, the organoid preferably comprises a neural rosette structure.

[0090] In embodiments, after 18 days of culture (according to step c.) at least a fraction, preferably at least 1-70%, of the cells comprised within the organoid are cortical progenitor neurons expressing Foxgl or Sox2 and / or Phospho-vimentin (p-vim).

[0091] In embodiments, after 18 days of culture (according to step c.) at least a fraction, preferably at least 1-70%, of the cells comprised within the organoid are deeper-layer neurons expressing Tbr1 , Tle4 and / or Ctip2.

[0092] In embodiments, after 18 days of culture (according to step c.) at least a fraction, preferably at least 1-70%, of the cells comprised within the organoid are upper-layer neurons expressing Satb2, Cux1 and / or Brn2.

[0093] In embodiments, after 18 days of culture (according to step c.) at least a fraction, preferably at least 1-70%, of the cells comprised within the organoid are astrocytes expressing S100beta and / or GFAP.

[0094] In embodiments, after 18 days of culture (according to step c.) at least a fraction, preferably at least 1-70%, of the cells comprised within the organoid are basal progenitors also known as outer Radial Glia (oRG), expressing HOPX.

[0095] In embodiments, the present method is for obtaining an organoid comprising a neural rosette structure at its center, or in a central region of the organoid, the method comprising cultivating the initial cell culture (comprising pluripotent cells) in step c. from day 0 on (immediately) on a micropattern culture chip. Hence, in embodiments of the present method the cell culture is cultivated in step c. on a micropattern cell culture chip from day 0 on to generate an organoid comprising a neural rosette structure at the center, or in a central region of the neural organoid. In other words, in some embodiments, the method of generating a neural organoid cell culture model comprises in step c. the initial (from culture day 0 on) cultivation of a cell culture comprising pluripotent stem cells on a micropattern culture chip, thereby generating a neural organoid comprising a neural rosette structure at the center, or in a central region of the neural organoid.

[0096] In some embodiments, the cell culture is passaged / re-seeded / re-plated between day 6 and day 14, preferably between day 10-12.

[0097] In embodiments the present method is a method for obtaining an organoid lacking a neural rosette structure, the method comprising the cultivation of the initial cell culture comprising pluripotent cells in step c. for 5-13, preferably 8-12 or 9-11 , days in a standard cell culture dish or plate, followed by a transfer of the cell culture to a micropattern culture chip between day 6-14, preferably around day 8-12 or 10-12.

[0098] In other words, in some embodiments, the method of generating a neural organoid cell culture model comprises in step c. the initial (from culture day 0 on) cultivation of a cell culture comprising pluripotent stem cells on a (standard I conventional) cell culture dish or plate for 5-13, preferably 8-12 or 9-11 , days, and subsequently transferring (re-plating / seeding) the cell culture to a micropattern culture chip between day 6-14, preferably around or at day 8-12 or 10-12, thereby generating a neural organoid lacking a neural rosette structure.

[0099] In embodiments, the cell culture is cultivated in step c. initially in a standard cell culture dish or plate for at least 1 day, for at least 1-6 days, for at least 1-8 days, for at least 1-12 days, for at least 1-18 days, or for 6, 12 or 18 days and is subsequently transferred onto a micropattern cell culture chip.

[0100] In embodiments, the cell culture is cultivated in step c. initially in a standard cell culture dish or plate and is subsequently transferred onto a micropattern cell culture chip between day 1 and day 18 of culture, or on day 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18 of culture or even later.

[0101] In embodiments, the present method comprises the steps of initial differentiation of the pluripotent cells in regular dishes, followed by a transfer of the culture to micropatterns after neural induction (approx, after 8-12 or 10-12 days of culture). In said embodiments, the method preferably comprises ‘feeding’ protocols with minimal disruption to keep the organoids attached for long period of time (e.g., >100 days). Said protocols preferably comprise permanent submerging of the organoids, specific aspiration and pipetting of media, phasing in and out of the various differentiation media). In embodiments, the method further preferably comprises replenishing the ECM-coating at regular intervals und the addition of specific compounds at optimized times and with optimized concentrations to increase cell attachment to the support (e.g. CEPT).

[0102] In embodiments, between days 8 and 12 of total culture duration, a uniform neuroepithelial sheet appears. In embodiments the cells may be re-seeded (transferred to a new culture device) and / or split (seeded at a lower cell number than grown at before). In embodiments, after about 12-20 days, preferably 18 days, of culture at least a fraction of the cells forms a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, or a rim structure comprising neuronal and / or glia cell projections.

[0103] In embodiments, after about 12-20 days, preferably 18 days, of culture at least a fraction of the cells forms a neuronal rosette structure comprising a defined core area comprising differentiated (mature) neurons, progenitor cells and glia cells, or a (thin) rim structure comprising neuronal and / or glia cell projections.

[0104] In embodiments, the cortical identity of the stem / progenitor cells is indicated / characterized by the expression of Foxgl by at least a fraction of the cells, preferably around Day 20.

[0105] In embodiments, the cortical progenitors are defined by the expression of Sox2 and Phospho- vimentin (p-vim) and may in embodiments also be identified as apical progenitors.

[0106] In embodiments, the cell population will give rise to intermediate progenitor cells, preferably characterized by at least a fraction of the cells being positive for Tbr2+ staining from around Day 20 and / or to newly born, postmitotic neurons, characterized by at least a fraction of the cells expressing MAP2+.

[0107] In embodiments, from day 20 on, deeper-layer neurons (characterized by at least a fraction of the cells expressing Tbr1+, Tle4+, Ctip2+) start to be evident, while preferably becoming the major population around Day 60.

[0108] In embodiments, ‘late-born’ upper-layer neurons (characterized by at least a fraction of the cells expressing Satb2+, Cux1+ and Brn2+ cells) begin to be produced around Day 40, while their proportions preferably increase over time (e.g., between Day 60, 90 and 100).

[0109] In embodiments, astrocytes are characterized by at least a fraction of the cells expressing S100beta and / or GFAP, preferably around day 70.

[0110] In embodiments, basal progenitors, also known as outer Radial Glia (oRG), are characterized by at least a fraction of the cells expressing HOPX, which is known to be a specific oRG marker (Pollen et al., Cell 163, 56-67, 2015).

[0111] In embodiments, around Day 50, at least a fraction of the cells express HOPX, preferably with the number of cells expressing HOPX increasing over time.

[0112] In another aspect or in embodiments of the invention described herein, the invention relates to a method of generating an adherent neural organoid cell culture model, comprising the steps of: a) coating a cell culture vessel, e.g., a plate, dish or micropattern chip, with at least one extracellular matrix (ECM) protein or component, preferably laminin, b) adding cell culture media, preferably comprising at least one Rho-associated protein kinase inhibitor (ROCKi) and optionally at least one extracellular matrix (ECM) protein or component, c) adding pluripotent stem cells to the media, d) cultivating the cell culture for at least 10 days, e) optionally changing the media every 2-10 days, e) optionally, when the initial cell culture vessel in step a) is a plate, dish, transfer the cell culture to a micropattern chip after 1-12 days of total culture duration, wherein the total duration of cell culture is at least 18 days, preferably at least 30 or more preferably 50 days or longer.

[0113] In embodiments, in step b) the cell culture media (further) comprises at least one WNT (pathway) inhibitor).

[0114] In embodiments, in step b) the cell culture media comprises at least one Rho-associated protein kinase inhibitor (ROCKi) and / or a CEPT cocktail.

[0115] In embodiments, upon appearance of neural rosette structures, the method can change the medium every other day. In embodiments, around days 16-20 of total culture duration, cells may be split and expanded further, preferably by passaging with Accutase and / or with gentle dissociation reagent for organoids.

[0116] In embodiments, between days 20 and 30, substantial neurogenesis should occur, optional from thereon the media is changed every other day. In embodiments, a cocktail of neurotrophic factors BDNF, NT3, GDNF, cAMP, Ascorbic acid is added to promote cell survival and differentiation, optionally in NMM medium supplemented with VA.

[0117] In embodiments, the cell culture media is a Stem Flex media.

[0118] In embodiments, the cell culture media is a Neural maintenance medium (NMM) comprising a 1 :1 mixture of N-2 and B-27-containing media.

[0119] In embodiments, the cell culture media is a N-2 media comprising DMEM / F-12 GlutaMAX, 1 * N2 supplement, insulin (e.g., at 5 pg / ml), L-glutamine (e.g., at 1 mM), MEM non-essential amino acids (e.g., at 100 pM), 2-mercaptoethanol (e.g., at 100 pM), and / or Antibiotic-antimycotic (e.g., 0.5X).

[0120] In embodiments, the cell culture media is a B-27 medium comprising neurobasal medium, 1 * B- 27 (with or w / o Vitamin A), L-glutamine (e.g., at 2.5 mM), and / or 0.5X Antibiotic-antimycotic (e.g., 0.5X).

[0121] In embodiments, the cell culture media is a Neural Induction medium (Nl) comprising NMM, SB431542 (e.g., at 10pM) and / or LDN-193189 (e.g., at 0.2pM).

[0122] In another aspect, the present invention relates to an in vitro neural organoid cell culture model produced according to the method as described herein.

[0123] In embodiments, the invention relates to an in vitro neural organoid cell culture model produced according to the method of the invention, said model comprising an adherent neural organoid in cell culture with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0124] In embodiments, the model is produced according to the method of the invention, and the model comprises a defined core area with differentiated neurons, progenitors and glia cells that is surrounded by a rim of neuronal and / or glia cell projections.

[0125] In embodiments, the model further comprises one or more of astrocytes, upper-layer neurons and deeper-layer neurons. In embodiments, at least a fraction of the deeper-layer neurons is localized in the core of the neural organoid and / or wherein at least a fraction of the upper-layer neurons are localized at the rim of the neural organoid, thereby mimicking an inside-out development of a mammalian brain.

[0126] In embodiments, the present organoid model self-organizes into a structure with a compact core of nuclei at the center, and projections of neurons and glia cells at the edge. This organoid preferably shows patterning into more deeper layer neurons at the center and more upper-layer neurons at the edge, mimicking the inside-out development of the mammalian brain.

[0127] In embodiments, the model is produced according to a method of the invention, and the model comprises a neural rosette structure at the center, or in a central region of the neural organoid.

[0128] In embodiments of a cell culture model comprising a neural rosette structure at the center of the neural organoid, the center contains one or more cell types, comprising one or more different types of progenitor cells and / or glia cells and / or the cell culture model comprises a thin I less pronounced rim of a neural and / or glia cell projections.

[0129] In embodiments a thin I less pronounced rim of a neural and / or glia cell projections refers to a rim that makes up less than 2.5%, 5%, 10%, 15%, 20%, 25% or 30% of the size of the organoid or the area covered by the organoid.

[0130] In embodiments, the present organoid model self-organizes into a structure with a neural rosette at the center, or in a central region of the neural organoid. This mimics the ventricular zone of the developing brain.

[0131] The present organoid models according to the invention are preferably highly reproducible and scalable.

[0132] In embodiments, the cell culture model comprises cortical progenitor neurons, and wherein at least a fraction, preferably at least 1-70%, of the cells expresses Foxgl or Sox2 and / or Phospho- vimentin (p-vim).

[0133] In embodiments, the cell culture model comprises deeper-layer neurons, and wherein at least a fraction, preferably at least 1-70%, of the cells expresses Tbr1 , Tle4 and / or Ctip2.

[0134] In embodiments, the cell culture model comprises upper-layer neurons, and wherein at least a fraction, preferably at least 1-70%, of the cells expresses Satb2, Cux1 and / or Brn2. In embodiments, the cell culture model comprises astrocytes, and wherein at least a fraction, preferably at least 1-70%, of the cells expresses SWObeta and / or GFAP.

[0135] In embodiments, the cell culture model comprises basal progenitors also known as outer radial glia (oRG), and wherein at least a fraction of the cells expresses HOPX.

[0136] In embodiments, at least a fraction of the neurons of the cell culture model are capable of induced and / or spontaneous neuronal activity.

[0137] In embodiments, wherein at least two organoids are cultured in the same culture dish or well, and wherein at least a fraction of the organoids form neural connections to a neighboring organoid in the same culture dish or well.

[0138] In one aspect, the present invention relates to an in vitro adherent neural organoid cell culture model comprising organoids with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0139] In one aspect, the present invention relates to an in vitro adherent neural organoid cell culture model comprising organoids, comprising a core area with a regular eccentricity comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0140] In preferred embodiments, the organoid comprises astrocytes, upper-layer neurons and deeper- layer neurons, wherein at least a fraction of the deeper-layer neurons is localized at the center of the neural organoid and / or wherein at least a fraction of the upper-layer neurons are localized at the rim of the neural organoid.

[0141] In preferred embodiments, the organoid comprises a neural rosette structure at the center of, or in a central region of, the neural organoid.

[0142] In embodiments, the organoids, and in particular the core area of the organoids of the invention, exhibit a regular eccentricity, preferably a shape that has an eccentricity of 0.8 or smaller. In embodiments, the eccentricity of the core area is 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or less. In some embodiments, the eccentricity of the organoid and / or core area is 0.1 -0.8, preferably 0.2- 0.7, more preferably 0.3-0.7 or any value within the range. In embodiments, the eccentricity of the organoid and / or core area is determined based on the area of the core region, for example when viewed from above in adherent culture.

[0143] In embodiments relating to a cell culture plate or micropattern cell culture chip (or micropattern coverslip) comprising the organoid model according to the present invention, preferably at least 30%, 40%, 50%, 60% or 70% or more of the organoids comprised on the plate or micropattern chip exhibit core areas with an eccentricity of below 0.8, 0.7 or preferably 0.6.

[0144] In embodiments, preferably embodiments relating to a cell culture plate or micropattern cell culture chip (or micropattern coverslip) comprising the organoid model according to the present invention, the organoids exhibit a maximum size of about 300-700 m in diameter, preferably about 500 pm in diameter.

[0145] In embodiments, the core area of an organoid has an area of about 10-200 pm2, preferably 20- 100 pm2, more preferably 30-60 pm2. In embodiments, the area of the core of an organoid is assessed from above in adherent culture.

[0146] Examples of organoids and physical characteristics thereof are presented in Fig. 2 and described in the Examples below. For organoid type 1 (wherein for example the cell culture is cultivated in step c. initially in a cell culture dish or plate for 8 to 12 days and subsequently transferred to a micropattern cell culture chip for further culture), the inventors have determined physical features of the organoid including a core area of 0.02 to 0.08 mm2and an eccentricity cutoff of < 0.8 (i.e., excluding organoids with core areas that are not optimally round). These features may, in embodiments, also relate to the organoids of Type 2, including neural rosette structures.

[0147] In embodiments, the present cell culture model comprises a central core and outside processes after 12-30 days, preferably after about 25 days of culture. In embodiments, the cells in the center of the present cell culture model comprise progenitors and neurons (TBR2 and / or MAP2 positive) after 12-30 days, preferably after about 25 days of culture, which are preferably positive for p- vimentin. In embodiments, the present cell culture model comprises at later stages upper layer neurons at the edge of the core (BRN2 positive), after 50-70 days, preferably after about 60 days of culture, and astrocytes (GFAP and S100b positive), from culture day 50-70 on, preferably from about 60 days of culture on.

[0148] 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: at least one ROCK-inhibitor, at least one ECM protein or component, at least one SMAD-inhibitor at least Chroman 1 , or a CEPT cocktail comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB, and one or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP and ascorbic acid.

[0149] In embodiments, the kit optionally comprises a micropattern cell culture chip.

[0150] In embodiments, the kit optionally comprises the in vitro neural organoid model according to the invention.

[0151] In embodiments the kit further comprises the in vitro neural organoid model according to the invention. In another aspect, the present invention relates to a kit comprising a culture medium and at least one supplement selected from the group comprising at least one ROCK-inhibitor, at least one ECM protein or component, at least one SMAD-inhibitor, at least Chroman 1 or a CEPT cocktail comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB, at least one WNT (pathway) inhibitor, one or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP and ascorbic acid.

[0152] In one aspect, the present invention relates to a cell culture plate or micropattern cell culture chip (or micropattern coverslip) comprising the in vitro neural organoid model according to the present invention. In preferred embodiments, the in vitro neural organoid model(s) is / are adherend (attached) to the cell culture plate or micropattern cell culture chip.

[0153] In embodiments, the cell culture plate is a multi-well plate and each well comprises at least 2 (adherend) organoid models. 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 2 or more wells, preferably at least 6, more preferably at least 12 or 96 wells.

[0154] In embodiments, the cell culture plate is a multi-well plate that comprises between 1 and 700, or around 600-700, e.g., 670, (adherent) organoid models (organoids). In embodiments, the cell culture plate is a multi-well plate, for example a 96 well plate, wherein each well comprises between 1 and 100, between 1- 25, or between 12-15 (adherent) organoid models (organoids).

[0155] In embodiments, the cell culture plate is a micropattern cell culture chip (or micropattern coverslip) comprising the in vitro neural organoid model according to the present invention, wherein the plate comprises at least 10, 25, 50, 100 or preferably between 10-1000, more preferably between 100-800 (adherent) in vitro neural organoid models (organoids), or at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 (adherent) in vitro neural organoid models (organoids).

[0156] In embodiments, the cell culture plate comprises micropattern structures. In embodiments the cell culture plate is a multi-well plate, wherein each well comprises micropattern structures.

[0157] In one aspect, the present invention relates to a computer implemented method for analyzing a neural organoid cell culture model, preferably according to the present invention, said method comprising the steps of: i. Providing a plurality of spatially distinct neural organoids in cell culture, ii. Determining at least one spatial parameter of at least one organoid provided in i., iii. Analyzing the at least one spatial parameter of the at least one organoid using machine learning, preferably deep learning, to determine the presence, location, spatial proportion, shape and / or size of at least one region and / or cell type of the at least one organoid, iv. Optionally providing and / or displaying on a screen a data summary comprising the results of the analysis in step iii., and optionally a graphical representation or image of the analyzed organoid.

[0158] As shown in the Examples below, Figure 3 (E) demonstrates a deep-learning classification network trained on a part of the images, indicating that this network can separate WT and mutated conditions with high accuracy, indicating the effectiveness of the method in determining neuronal phenotypes, for example for screening purposes.

[0159] In embodiments of the invention, the computer implemented method for analyzing a neural organoid cell culture model enables accurate and highly reproducible measurements and characterization of the organoid model described herein, and is thus suitable to identify potentially small differences in phenotype or structural characteristics or function of the organoid model, after any given treatment or potentially between two different disease states (as disclosed in more detail below and in the Examples).

[0160] By allowing a deep-learning classification network to be trained on images of the organoid model, potential parameters, markers or other characteristics of the organoids can be identified in any given treatment group, and even small changes between groups may be identified with high accuracy. This is particularly important in screening applications, where treatment vs nontreatment should be carefully assessed to identify a potential therapeutic effect of candidate substances. This is also important in assessing disease states, whereby (as in the examples) small changes between healthy and disease state organoid models can be accurately identified.

[0161] 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.

[0162] In embodiments, the present method facilitates the generation of patient-specific individual organoid cell culture models.

[0163] A non-limiting example is the generation of induced pluripotent stem cells (IPSCs) from patients with neuronal or neurodegenerative diseases for the production of personalized organoid disease models.

[0164] In embodiments, diseased cells might also be generated from stem cells comprising artificially introduced or genetically engineered mutations (e.g., using CRISPR / Cas9 or CRISPR / Cas9 vectors to induce specific disease-associated mutations in (patient-derived) cells). In embodiments, this approach can also be applied for patient-specific IPS cells to generate a patient-specific model comprising the patient specific genetic background in combination with a certain mutation, e.g., a tissue-specific mutation, that could not be obtained from the patient by biopsy.

[0165] In other embodiments, the present model can be used to study a disease and / or the treatment of a disease in a patient-specific background, e.g., also in case of patient-specific germline mutations, such that developed from patient-specific IPS cells the present model can serve as a personalized neural organoid model for a diseased or predisposed patient. In embodiments, the invention can be instrumental to study diseases that affect the human brain. The present models constitute the first differentiated neural organoid model or (cortical) brain organoid model generated from human PSCs.

[0166] In one embodiment, the methods of the present invention are suitable to generate miniaturized organoids for phenotyping neural disorders.

[0167] In embodiments, PSCs comprising a (preferably heterogeneous) mutation in DYRK1A may be employed to generate disease-specific and potentially individualized organoid models. Mutations in DYRK1 A typically lead to DYRK1A syndrome, a severe neural developmental disorder associated with autism spectrum disorder, among others.

[0168] In embodiments, PSCs comprising a mutation in Apolipoprotein E (APOE) may be employed to generate disease-specific and potentially individualized organoid models, for example WT cells with APOE3 / 3, and mutated cells with APOE3 / 4, may be compared. Mutations in Apolipoprotein E (APOE) are a common risk factor for sporadic Alzheimer’s disease.

[0169] The present method in its herein described embodiments constitutes a new and inventive procedure / workflow of successive addition and removal of specific factors and / or signaling molecules to the cell culture medium of initially pluripotent stem cells, which enables the generation of differentiated neural organoid model or (cortical) brain organoid model that are suitable for long term cultivation of at least 30, preferably of at least 100 days of culture.

[0170] In embodiments, the first cell culture comprises pluripotent stem cells of a patient, preferably iPS cells of a patient, and wherein the patient has been diagnosed with and / or is suffering from a brain disease, a neurological disease, a neurodegenerative disease, a brain damage or any other disease or condition affecting the brain of said patient.

[0171] The present method and model may in embodiments therefore be applied using ‘diseased’ cells derived from patients that has been diagnosed with and / or is suffering from a brain disease, a neurological disease, a neurodegenerative disease, brain damage or any other disease or condition affecting the brain.

[0172] In embodiments of the in vitro cell culture models according to the invention, the model is established from pluripotent stem cells of a patient, preferably iPS cells of a patient, and wherein the patient has been diagnosed with and / or is suffering from a brain disease, a neurological disease, a neurodegenerative disease, brain damage or any other disease or condition affecting the brain of said patient.

[0173] The cell culture models of the prior art are not able to reflect the entire development of the brain, as well as the diversity of cell types found in the human brain. The present model can further be used for patient stratification and / or for analyzing an individual disease outcome of a patient, treatment and / or context, such as in the context of personalized medicine context. Therefore, in embodiments, the present method and / or model can be used in a personalized medicine context for the diagnosis, or the development / selection of a suitable treatment for an individual patient, from whom the initial pluripotent starting cell culture of the present model, as IPS cells, is derived. This is especially advantageously for patients having been diagnosed with diseases such as a brain disease, a neurological disease, a neurodegenerative disease, brain damage or any other disease or condition affecting the brain of said patient, where each patient develops an individual physiological pattern of disease outcome with e.g., individual outcomes and disease progression in neurodegenerative diseases. The present methods and models facilitate the generation of cell culture models which mimic, for example, the individual phenotype of a brain disease, a neurological disease, a neurodegenerative disease, brain damage or any other disease or condition affecting the brain of an individual patient.

[0174] In another aspect, the present invention relates to the use of the in vitro cell culture model according to the invention in drug screening, toxicity screening and / or for studying a disease, such as a brain disease, a neurological disease, a neurodegenerative disease, brain damage or any other disease or condition affecting the brain of an individual patient.

[0175] In another aspect the present invention relates to a method for testing a candidate compound, the method comprising: a. providing a cell culture model according to the invention, b. bringing into contact a candidate compound with said cell culture model, c. detecting a readout, characteristic and / or one or more parameter(s) of the cell culture model, d. optionally repeating steps a-c with one or more further candidate compounds.

[0176] In embodiments, the readout or parameter in step c. comprises the viability and / or proliferation of said model. A skilled person is aware of 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 BrdU-assay, EdU-assay, MTT-assay, XTT-assay, WST-1 -assay, Ki67-staining, immunocytochemistry (ICC), immunohistochemistry, immunofluorescence, Hoechst-staining, Calcein-AM staining or Trypan Blue staining.

[0177] In embodiments, detecting a readout or parameter in step c. comprises using one or more of the methods of microscopy, image analysis (e.g., via a plate reader, microscope or camera), current clamp recording, analysis of optogenetic activation, immunocytochemistry (ICC), immunohistochemistry, Immunofluorescence analysis, droplet-based microfluidic technology, Fluorescence Activated Cell Sorting (FACS), IHC, ELISA and / or spectrophotometry.

[0178] In embodiments, the readout or parameter may be a characteristic, a shape and / or a pattern of the present model or of certain cells comprised therein. In embodiments a non-limiting list of parameters or characteristics may be, e.g., the expression of certain genes or proteins, the developmental stage, 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 neuronal functional properties. In embodiments, screening methods and systems may employ software and hardware as commonly used in (pharmaceutical) compound screening systems. Automated setups are known to a skilled person and may be adapted accordingly. Thus, screening formats, as already established in the art, may be employed to implement the methods and systems of the present invention.

[0179] In embodiments, the assessments are preferably performed in triplicates, to discard non-specific readouts or signals.

[0180] In another aspect, the present invention relates to a method assessing toxicity of a candidate substance, comprising: a. providing a cell culture model according to the invention, b. bringing into contact a candidate compound with said cell culture model, c. identifying the candidate substance as a safe candidate substance (a substance with low or no toxic effect) when said cell culture mode exhibits no cytotoxic effect, preferably no detectable effect.

[0181] Another aspect of the present invention relates to a system for identifying a candidate substance, comprising: a. providing a cell culture model according to the invention, b. optionally at least one substance or a substance library, or means for providing and / or utilizing such a substance or substance library, c. means for determining (screening) an effect of said substance or substance library on the cell culture model, such as one or more compound(s) or composition(s), a device and / or a system configured (optionally for high-throughput screening) for identifying a readout, characteristic and / or one or more parameter(s) of the cell culture model.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] In another aspect, the present invention relates to a culture medium and / or supplement-cocktail comprising one or more of a ROCK Inhibitor, Chroman 1 , CEPT-cocktail (comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB), at least one SMAD inhibitor, BDNF, NT-3, GDNF, cAMP, Ascorbic acid (tbd), at least one WNT (pathway) inhibitor, at least one ECM protein or component.

[0186] In one aspect, the present invention relates to a cell culture medium and / or supplement-cocktail comprising: at least one ROCK-inhibitor, at least one ECM protein or component, at least one SMAD-inhibitor at least Chroman 1 or a CEPT cocktail comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB, and / or one or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP and ascorbic acid, optionally at least one WNT (pathway) inhibitor.

[0187] In embodiments, the cell culture media comprises Stem Flex media. In embodiments the cell culture media is a Neural maintenance medium (NMM) comprising a 1 :1 mixture of N-2 and B-27- containing media. In embodiments the cell culture media is a N-2 media comprising DMEM / F-12 GlutaMAX, 1 x N2 supplement, insulin (e.g., at 5 pg / ml), L-glutamine (e.g., at 1 mM), MEM non- essential amino acids (e.g., at 100 pM), 2-mercaptoethanol (e.g., at 100 pM), and / or Antibiotic- antimycotic (e.g., 0.5X). In embodiments the cell culture media is a B-27 medium comprising neurobasal medium, 1 x B-27 (with orw / o Vitamin A), L-glutamine (e.g., at 2.5 mM), and / or 0.5X Antibiotic-antimycotic (e.g., 0.5X). In embodiments the cell culture media is a Neural Induction medium (Nl) comprising NMM, SB431542 (e.g., at 10pM) and / or LDN-193189 (e.g., at 0.2pM).

[0188] 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.

[0189] The instant disclosure also includes kits, packages and multi-container units containing the herein described media and / or factors separately or as compositions.

[0190] 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 cell culture models and kits, 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 cell culture models, 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 adherent neural organoid cell culture model, that is preferably smaller (miniaturized) than present neuronal organoid models, but maintain relevant features of larger organoids and their in vivo counterparts, thereby comprising a defined spatial structure and a regular eccentricity that comprises a defined core area comprising differentiated (mature) neurons, progenitor cells and glia cells, surrounded by a rim of neuronal and / or glia cell projections.

[0191] DETAILED DESCRIPTION OF THE INVENTION

[0192] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

[0193] The invention relates to an in vitro method of generating a neural organoid cell culture model. The invention relates to an in vitro adherent neural organoid cell culture model, comprising organoids with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

[0194] The term "subject" or “patient” includes any mammalian subject or patient, preferably a human patient. In embodiments the subject or patient is a human or mammal diagnosed with and / or suffering from a certain disease.

[0195] “Neuronal activity” herein generally refers to the ability of a neuronal cell to generate in vitro action potentials and intracellular calcium transients, either spontaneously or induced. Neuronal activity may also involve, in embodiments, electrical and / or chemical synaptic transmission and / or functional axonal connectivity.

[0196] Cell culture

[0197] The term "pluripotent stem cells" in this context covers any cells with the property of pluripotency, as understood by a skilled person. The term includes pluripotent stem cells derived from an embryo and induced pluripotent stem cells 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 the 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 suffering from a disease.

[0198] In embodiments, the initial pluripotent stem cell according to the present invention is a pluripotent or multipotent mammalian cell. Preferably, said initial pluripotent stem cell is a human induced pluripotent stem cell (iPSC). iPSCs are pluripotent stem cell that are generated directly from adult cells. iPSCs can give rise to any other cell type in the body or in the respective mammalian organism and have the capability of indefinite proliferation in cell culture. Advantageously iPSCs can be derived directly from adult cells or tissue and do not require the use of cells derived from embryos. iPSCs have the advantage that they can be generated from patient cells, such that they are patient-specific. In addition, iPSCs and iPSC-derived cells can be used for personalized drug discovery, toxicity screenings and disease research to understand the patient-specific basis of disease. In embodiments this also applies to the cell culture models of the present invention, which can be derived from human patient-specific iPSC. iPSCs are generally derived by introducing products of specific sets of pluripotency-associated genes, or "reprogramming factors", into a particular cell type, such as the original reprogramming transcription factors Oct4 (Pou5f1), Sox2, c-Myc and Klf4. In embodiments in this common combination each of the factors can be functionally replaced by related transcription factors, or even small molecules, miRNAs, or other genes such as lineage specifiers.

[0199] Cell culture (also termed or 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 media 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.

[0200] A “progenitor cell” in general refers to a neuronal 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.

[0201] A (terminally) differentiated neuron may also be referred to as mature neuron or post-mitotic neuron. A differentiated neuron may in embodiments be characterized by a nuclear higher-order structure and / or the expression of one or more of the genes, including, without limitation, NeuN, MAP2, vGLUTI (for example in excitatory glutamatergic neurons), GAD67 (for example in inhibitory neurons), and / or GFAP or S Obeta (for example in astrocytes).

[0202] “Neuronal and / or glia cell projections” commonly refer to potentially long (extended) cytoplasmic projections, which extend from the soma of the respective cell. Neuronal cell projections may be formed by mature neurons and radial glia cells. In the present model, the so-called “intermediate progenitors” preferably don’t form projections.

[0203] Herein the term “neural rosette structure” refers to an organoid structure comprising, in a central region, one or more progenitor cells and / or glia cells, and a thin, or less pronounced, rim of neural and / or glia cell projections. As used herein, the thin, or less pronounced, rim preferably refers to a rim that makes up less than 2.5%, 5%, 10%, 15%, 20%, 25%, or 30% of the size of the organoid or the area covered by the organoid, preferably the area of the organoid when assessed from above in adherent culture. The neural rosette structure as may occur in the organoid of the present invention, in particular in the second model, is characterized by the radial organization of cells surrounding a central lumen identified by the expression of apical junction marker ZO-1 . The second model, as referred to herein, relates to embodiments where the cell culture is cultivated in step c. on a micropattern cell culture chip from day 0 onwards, to generate a neural organoid comprising a neural rosette structure in a central region of the neural organoid.

[0204] In general, the term “organoid” refers to small, self-organized and 3D (three-dimensional) tissue cultures of cells, which are preferably generated from pluripotent cells. A neural organoid is a small, self-organized and 3D (three-dimensional) tissue culture comprising neuronal and preferably other (assisting) cells that are commonly present in the brain, wherein the neuronal organoid is preferable generated from pluripotent cells and resembles, in the context of the invention, preferably a simplified and miniaturized model of the brain. As used herein, a “defined spatial structure” relates to an organoid comprising a core area and a rim area with cell projections. In preferred embodiments, the “defined spatial structure” relates to an organoid comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim area comprising neuronal and / or glia cell projections.

[0205] As used herein, a “regular eccentricity” preferably refers to a circular or an approximately circular shape. Eccentricity may be determined by routine technical means, such as those described in the examples below. Quantitative measurements of eccentricity are provided in the Examples. As commonly used in the art, eccentricity values typically range between one and zero, whereby if the eccentricity is one, the shape will be a straight line, and if eccentricity is zero, the shape will be a circle. In embodiments, the formula to determine the eccentricity of an ellipse, or the shape of the inventive organoid, is the distance between the foci of the ellipse divided by the length of the major axis. In embodiments, the “regular eccentricity" of the inventive organoid is equal to or less than 0.8. Further embodiments are provided herein.

[0206] Herein, the term “adherent” is relevant to an in vitro adherent neural organoid cell culture model. The term “adherent" relates to the property of the neural organoid model to attach to the cell culture vessel or support during cultivation. In preferred embodiments, “adherent” refers to a state of constant attachment of a cell or an organoid to the cell culture vessel / support throughout cultivation within said cell culture vessel / support. In preferred embodiments, the model is adherent for the culture duration, such as during cultivation in cell culture for at least 18 days. In some embodiments, the term “adherent” does not simply refer to an “embedding” of the organoid into a gel or matrix (e.g., Geltrex) within the cell culture vessel, but to a direct and constant attachment of an organoid to the cell culture vessel / support itself. In embodiments, the model is adherent to the cell culture vessel / support itself and / or the ECM, for example the model is adherent to a vessel coated with at least one extracellular matrix (ECM) protein or component.

[0207] In general, an “extracellular matrix” (ECM) refers to a network of extracellular macromolecules comprising, in addition to water, various glycoproteins, polysaccharides, enzymes, nutrients (e.g. amino acids, glucose), tissue hormones and electrolytes are also present. ECM provides structural and biochemical support to the surrounding cells.

[0208] In detail, the ECM comprises fibrous components (fibers) and fluid (ground substance comprising the soluble components and nutrients). The predominant protein family present in ECM are collagens, which form different types of fibers and are present in almost every tissue. Elastic fibers are formed from the proteins fibrillin and elastin. In addition, there is a wide variety of adhesion matrix proteins that connect the cells to the ECM. The second large group are carbohydrates, especially glycosaminoglycans, long-chain polysaccharides of very specific individual building blocks. The glycosaminoglycans associate with proteins and form even larger macromolecules, the proteoglycans. The diversity and interactions of proteins, glycosaminoglycans and proteoglycans result in the properties of the ECM.

[0209] “Geltrex™” is a LDEV-free Reduced Growth Factor Basement, namely a soluble form of basement membrane sold by Thermo Fisher Scientific.

[0210] Cell culture materials and LDN-193189 (DM3189; LDN) is a selective and highly potent small molecule BMP receptor signaling inhibitor, which inhibits the BMP type I receptors ALK1 (TGF-B Superfamily Receptor Type I), ALK2 (TGF-B Superfamily Receptor Type I), ALK3 (BMP Type-1 A Receptor) and ALK6 (BMP Type-1 B Receptor) with IC50s of 0.8 nM, 0.8 nM, 5.3 nM and 16.7 nM in the kinase assay, respectively. LDN-193189 inhibits the transcriptional activity of the BMP type I receptors AL K2 and ALK3. LDN-193189 is considered an inhibitor of TGF-beta / Smad signaling (TGF-beta / Smad Inhibitor).

[0211] SB431542 (SB) is a potent and selective inhibitor of ALK5 (Transforming Growth Factor Beta Receptor 1) and is therefore a TGFp pathway inhibitor, with IC50 of 94 nM. SB431542 is considered an inhibitor of TGF-beta / Smad signaling (TGF-beta / Smad Inhibitor).

[0212] In the context of the present invention, any SMAD-signaling inhibitor may be used instead or alternatively to any one of the two herein exemplarily named compounds SB431542 and LDN- 193189.

[0213] SMADs (or Smads) are a family of structurally similar proteins that are the major signal transducers for transforming growth factor beta (TGFb; TGFbeta) superfamily receptors, which are critical for regulating cell development and growth.

[0214] In embodiments herein, any compound, inhibitor and / or activator compound may be a small molecule compound, such as the SMAD inhibitor LDN-193189.

[0215] In general, the “(canonical) Wnt / p-catenin pathway” (also termed briefly “WNT pathway”) is well- characterized in the art, wherein the WNT gene family comprises structurally related genes encoding secreted signalling proteins, which have been implicated, for example, in different developmental processes, such as the regulation of cell fate and embryonal patterning. The (canonical) Wnt / p-catenin pathway is considered to contribute to the stabilization of cytoplasmic P-catenin, which in combination with transcription factors and coactivators CBP, p300, TCF, LEF, PYGO, or BCL9 activates the transcription of target genes. An inactive Wnt-pathway commonly leads to the degradation of p-catenin, whereby p-catenin is phosphorylated and rendered susceptible to ubiquitination and proteasomal degradation. The degradation of p-catenin is commonly mediated by APC, AXINcasein kinase 1 a (CK-1 a), and glycogen synthase kinase 3p (GSK3P).

[0216] Exemplary “WNT inhibitors” or inhibitors of the WNT pathway (inhibitor of WNT signalling) may in embodiments be selected from the group comprising, without limitation thereto, IWR-1-endo (IWR1 e), IWP-2, JW74, IWP-4, CCT251545, KY1220, iCRT14, iCRT3, LF3, PNU-74654, KYA1797K, KY02111 , Zamaporvint (RXC004), M2912, M435-1279, KY-05009, Prodigiosin, Ginsenoside Rh4, Heparan Sulfate, RCM-1 , Resibufogenin, MSAB, PRI-724, Tegatrabetan (BC- 2059), JW55, Adavivint (SM04690), Triptonide, Isoquercitrin, IQ-1 , Salinomycin and FH535.

[0217] In the context of the present invention the terms “WNT inhibitor”, “WNT pathway inhibitor”, “inhibitor of the WNT pathway” and “inhibitor of WNT signalling” may be used interchangeably.

[0218] Commonly “CEPT” or “CEPT cocktail” refers to a mixture of Chroman 1 , Emricasan, Polyamine solution and Trans-ISRIB. “Chroman 1” (C24H2sN4O4.;herein also abbreviated also as “Chroman”) is a potent and selective ROCK 2 inhibitor that is considered to improve cell survival. Emricasan is a potent pan-caspase inhibitor considered to promote survival of hPSCs in combination with other small molecules. In the context of the present invention both terms “Chroman 1” and “Chroman” refer to the ROCK2 inhibitor “Chroman 1”, CAS No.: 1273579-40-0, C24H28N4O4; https: / / pubchem.ncbi.nlm.nih.gov / compound / 66577033). Trans-ISRIB is considered to promote survival of hPSCs in combination with other small molecules and to be an integrate stress response (ISR) inhibitor. Polyamine solution / supplement is a media supplement considered to enhance cell growth and stem cell viability.

[0219] A “neurotrophic factor” refers 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.

[0220] A “micropattern cell culture coverslip”, “micropattern chip” or “micropattern cell culture dish” or simply “micropattern” (said terms may be used interchangeably herein) refers to a culture vessel or support patterned with micrometer-sized sections of predefined geometric features. Micropatterns are considered to improve (spatially defined) positioning and / or cell adhesion and / or to foster the proliferation of cells cultivated thereon.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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 which 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.

[0225] 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 models 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).

[0226] 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.

[0227] 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.

[0228] FIGURES

[0229] 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.

[0230] Brief Description of the Pictures:

[0231] Figure 1 : Generation of two brain organoid types.

[0232] Figure 2: QC measures for organoids. Figure 3: Using miniaturized organoids for phenotyping neural disorders.

[0233] Figure 4: Example protocol for generating reproducible neural organoids comprising a neural rosette structure.

[0234] Detailed Description of the Pictures:

[0235] Figure 1 : Two different brain organoid types generated in Example 1 , at different time points. Type 1 refers to an in vitro neural organoid cell culture model of a first type (Type 1), which comprises astrocytes, upper-layer neurons and deeper-layer neurons, wherein at least a fraction of the deeper-layer neurons was localized at the center of the neural organoid and / or wherein at least a fraction of the upper-layer neurons was localized at the rim of the neural organoid. The type 1 organoids are, in embodiments, produced by a method, wherein the cell culture is cultivated initially in a cell culture dish or plate, for example for 8 to 12 days, and subsequently transferred to a micropattern cell culture chip for further culture. The in vitro neural organoid cell culture model of a second type (Type 2) comprises a defined spatial structure with a regular eccentricity, wherein the model comprises a neural rosette structure at the center of the neural organoid. The type 2 organoids are, in embodiments, produced by a method, wherein the cell culture is cultivated on a micropattern cell culture chip from day 0 onwards, to generate a neural organoid comprising a neural rosette structure in a central region of the neural organoid.

[0236] Figure 2: QC measures for organoids. (A) An overview of part of a chip with 80 organoids (stained for DAPI in grey and pVimentin in green). (B) Images of individual organoids are analyzed using deep learning to precisely segment the core region. (C) Area and eccentricity of the core organoid area are measured based on B and used as quality control.

[0237] Figure 3: Using miniaturized organoids for phenotyping neural disorders. (A) Comparison of d20 organoids derived from WT and from an isogenic hESC line with a heterogeneous mutation in DYRK1A. The mutation leads to an increased progenitor pool (TBR2) and less mature neurons (MAP2), suggesting a delayed exit from the cell cycle in the disease. (B) Quantification of the markers shown in panel A. Each data point corresponds to one organoid, highlighting the high statistical power for phenotyping that can be achieved with the organoids. (C) Comparison of d15 organoids derived from WT and from an isogenic hESC line with a mutation in Apolipoprotein E (APOE), WT : APOE3 / 3, mutation: APOE3 / 4, the most common risk factor for sporadic Alzheimer’s disease. An increase in early born neurons (DCX) and in mature neurons (MAP2) can be observed in the mutant, as quantified in (D). (E) A deep-learning classification network trained on a part of the images can separate WT and mutated condition with high accuracy, indicating effectiveness of the phenotype for screening purposes. Scale bars: 200pm. ***: p-value < 0.001 , ****■. p-value < 0.0001 .

[0238] Figure 4: In this exemplary embodiment cells were seeded at 450.000 cells per coverslip (19.5 mm x 19.5 mm) and already at day 0 (dO), a CEPT cocktail was added to the media for 3 days. Subsequently, only C component (Chroman 1 ; ROCK2 inhibitor,) was added to the media. Chroman 1 is a ROCK-inhibitor similar to Y-27632 (ROCK1 and ROCK2 inhibitor). In addition, a Wnt (pathway) inhibitor (IWR1e) was added at the beginning of the protocol, resulting in reproducible rosette formation at the center of generated organoids. In detail; Fig. 4 A: The protocol according to one embodiment of the present invention generates reproducible neural rosette at the center of the micropatterned organoids. Shown are cells at day 7, 10 and 20. B: Characterization of organoids at day 9 (d9). A clear neural rosette can be seen at the center of the organoids. Shown are cells stained (from left to right) with DAPI, for NCAD (adherens junctions), SOX2 (neural progenitors) and Ki-67 (proliferation).

[0239] EXAMPLES

[0240] 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.

[0241] Example 1 - Outline

[0242] Similar to the neurulation organoids, the inventors generated neural organoids (brain organoids) with a diameter of about 500 pm. The protocol for growing the relevant brain cells was adapted from prior protocols for larger brain organoids.

[0243] The main difficulty of growing these organoids was to keep the cells attached to the micropatterns for the long periods of time that are required for neurodevelopment (up to 100 days and longer). Since the cells have a tendency to eventually detach from the patterns, the longest reported time so far for growing cells on micropatterns are the neurulation organoids (7 days).

[0244] The inventors therefore developed several new strategies to keep the cells attached for long periods of time, comprising, by way of example, the steps of:

[0245] - Initial differentiation in regular dishes, and transfer to micropatterns after neural induction (approx. 10-12 days).

[0246] - Specific feeding protocols with minimal disruption (always keeping organoids submerged, specific aspiration and pipetting of media, phasing in and out of the various differentiation media)

[0247] - Replenishing the Laminin coating at regular intervals

[0248] - Adding of specific compounds at optimized times and with optimized concentrations to increase cell attachment (e.g. CEPT).

[0249] These steps have allowed the inventors to keep the organoids attached for long periods of times (up to and potentially beyond 100 days).

[0250] The inventors were able to generate two different types of neural (brain) organoids comprising a defined spatial structure and a regular eccentricity that comprises a defined core area comprising differentiated (mature / post-mitotic) neurons, progenitor cells and glia cells, surrounded by a rim of neuronal and / or glia cell projections.

[0251] The in vitro neural organoid cell culture model of a first type (Type 1) comprises astrocytes, upper-layer neurons and deeper-layer neurons, wherein at least a fraction of the deeper-layer neurons was localized at the center of the neural organoid and / or wherein at least a fraction of the upper-layer neurons was localized at the rim of the neural organoid.

[0252] The in vitro neural organoid cell culture model of a second type (Type 2) comprises a defined spatial structure with a regular eccentricity, wherein the model comprises a neural rosette structure at the center of the neural organoid.

[0253] For stratifying the generated models, the inventors applied the following measures for quality control (QC).

[0254] 1) Existence of a central core and outside processes after 25 days. Cell fates in the center initially progenitors and neurons (TBR2 and MAP2 positive at day 25) with processes positive for p- Vimentin. At later stages upper layer neurons at the edge of the core (BRN2 positive at day 60) and appearance of astrocytes (GFAP and S100b positive starting from day 60).

[0255] 2) For organoid type (2), existence of a neural rosette (can be easily seen in brightfield imaging, and automatic quality control can be done using a deep learning classifier).

[0256] 3) Area and eccentricity of the central core region, which can be measured using imaging or stains for DAPI in a quantitative way. This is shown in Fig. 2 for organoid type 1 , where the inventors use QC values for the core area A of 0.02 mm2< A < 0.08 mm2, and an eccentricity cutoff e < 0.8 (i.e., excluding core areas that are not round enough).

[0257] Example 2 - Protocol

[0258] Presented below is a protocol to obtain the inventive organoid models.

[0259] Materials and Media:

[0260] - Stem Flex medium for stem cells.

[0261] - Neural maintenance medium (NMM): 1 :1 mixture of N-2 and B-27-containing media. o N-2 medium consists of DMEM / F-12 GlutaMAX, 1 x N2 supplement, 5 pg / ml insulin, 1 mM L-glutamine, 100 pM MEM non-essential amino acids, 100 pM 2- mercaptoethanol, 0.5X Antibiotic-antimycotic. o B-27 medium consists of Neurobasal medium, 1 x B-27 (with orw / o Vitamin A), 2.5 mM L- glutamine, 0.5X Antibiotic-antimycotic.

[0262] - Neural Induction medium (Nl): NMM + 10 pM SB431542 + 0.2 pM LDN.

[0263] - N2 and B27 supplement, 2-Mercaptoethanol added after medium filtration.

[0264] - Store the medium at 4 °C and use it within 3 weeks.

[0265] Small molecules

[0266] - Y-27632 ROCK Inhibitor (10 mM stock - working concentration 10 pM / 5p M). - CEPT cocktail (Chroman 1 1 50 nM, Emricasan 5 pM, Polyamine Solution 1x, Trans- ISRIB 0.7 pM).

[0267] - SB431542 (10 mM stock - working concentration 10 pM).

[0268] - LDN-193189 (2 mM stock - working concentration 0.2 pM).

[0269] - BDNF (20 pg / ml stock - working concentration 20 ng / ml).

[0270] - NT-3 (20 pg / ml stock - working concentration 20 ng / ml).

[0271] - GDNF (tbd).

[0272] - cAMP (tbd).

[0273] - Ascorbic acid (tbd).

[0274] Methods:

[0275] Coating - 2D culture

[0276] - Coat 35 mm dishes (or 6 wells plates) w / 10 pg / ml Laminin, incubate for at least 2 h at 37 C.

[0277] - Wash 1x with PBS+ / + and add the medium (SF + 10 pM ROCKi).

[0278] - Prepare a petri dish with a piece of parafilm sealed to the bottom and place the chips face-up.

[0279] - Coat the chips w / 20 pg / ml Laminin by adding approximately 500 pl / chip.

[0280] - Let them in the incubator for at least 2 h at 37 C (or, alternatively, at +4 C O / N).

[0281] - Prepare 5 ml of PBS+ / + and place a chip in a well of a 6 well plate (or 35mm dishes).

[0282] - Perform 5x partial washes with PBS+ / + (remove 3 ml and add 3 ml more).

[0283] - Perform 2x complete washes with PBS+ / + (remove all 5 ml and add 5 ml more).

[0284] - Add the medium (SF + 10 pM ROCKi).

[0285] Prepare a petri dish with a piece of parafilm sealed to the bottom and place the chips face-up.

[0286] Coat the chips w / 20 pg / ml Laminin by adding approximately 500 pl / chip.

[0287] Incubate for at least 2 h (or, alternatively, at +4 C O / N).

[0288] The cells are directly seeded on the chips and no washes are needed. Seeding stem cells

[0289] - Remove medium, wash 2x w / PBS- / -.

[0290] - Treat with 1 ml Accutase for 3-5 min at 37 C.

[0291] - Prepare stem cell medium + 10 pM ROCKi.

[0292] - Add 1 ml of medium + 10 pM ROCKi to stop the Accutase reaction.

[0293] - Collect cells in a 15 ml falcon tube.

[0294] - Add 2-3 ml of medium and spin at 200xg for 5 min.

[0295] - Add 1 ml of medium + 10 pM ROCKi and resuspend the cells.

[0296] - Count the cells with the cell counter (Cell Countess, ThermoFisher).

[0297] 2D culture and 500pm CYTOO chips

[0298] - Seed the desired number of cells in a dropwise manner on the pre-coated and prewarmed dish.

[0299] 500pm 4D cell chips

[0300] - Distribute the cells only on the chip. Incubate them for 1 h to let the cells attach. After 1 h remove the unattached cells and add fresh medium.

[0301] Number of cells to seed:

[0302] - 35 mm dish (or 1 well of a 6 well plate): 1 million cells / well, Chips: 100.000 - 200.000 cells / well.

[0303] Neural induction on 2D (or directly on the

[0304] 2D culture

[0305] - Check the cells on the day after plating (day 0). If the cells have reached ~100% confluence, wash the cells once with PBS and add 3 ml of neural induction medium per well.

[0306] - Change the neural induction medium every day by removing the old medium and replace (100% medium exchange).

[0307] - Between days 8 and 12 after plating, a uniform neuroepithelial sheet should appear. At day 10, collect the neuroepithelial cells with Accutase to keep the 2D culture (see protocol above) and gentle dissociation reagent to generate organoids on the chips by seeding small clumps:

[0308] Wash 1xw / PBS- / -. Add 1 ml gentle dissociation reagent for 2 min at RT. Remove it and add 1 ml of Nl medium + 5 pM ROCKi. Use the scraper to allow cells detachment. Collect the cells and centrifuge at 200xg for 3 min. Resuspend the cells in 1 ml Nl medium and seed 300 l of cell suspension on a chip (1 :3 ratio, ca 1.2 x 106cells). Incubate overnight and change the medium (NMM + 5 pM ROCKi) the next day to remove floating cells.

[0309] 500

[0310] After seeding the stem cells directly on the chips, the protocol is the same except for the medium change method. The organoids will be kept in culture until further processing or experiments at desired time point.

[0311] Medium change for mi

[0312] To avoid detachment, skip the wash with PBS- / - and do not remove 100% of old medium (3 ml). Instead, just discard 60% and replace with fresh medium.

[0313] - Use a p1000 pipette and remove 60% of old medium (2 ml). Add fresh medium by slowly pipetting with a 5 ml serologic pipette on one side of the well (CAUTION Z!\ avoid direct and rough fluid dynamics). Gently stir the dish in order to homogenize the medium.

[0314] Neural differentiation and maturation on 2D

[0315] - Upon appearance of rosettes change the medium every other day, at days 16-20 after neural induction, cells should be split and expanded further by passaging with Accutase for 2D culture and with gentle dissociation reagent for organoids.

[0316] - Replace the medium the next day (2ml of NMM). Maintain the cells for a further 4-6 d. Cells can be expanded further at this point by repeating the steps above. Seed cells on the micropatterns (3rdsplit).

[0317] - Between days 20 and 30, substantial neurogenesis should occur and medium change can be every other day. (OPTIONAL: addition of a cocktail of neurotrophic factors BDNF, NT3, GDNF, cAMP, Ascorbic acid promotes cell survival and differentiation - NMM medium supplemented with VA). Seed cells on the micropatterns (4thsplit).

[0318] - From now on, seed more cells for the 2D culture: from 1x35 mm dish, resuspension in 1 ml of medium and plate 400 pl of cells.

[0319] - On days 27-31 after initial plating, passage the culture using Accutase onto laminin- coated 35-mm dishes as described above. Top up with 5 pg / ml laminin directly to the medium every 10 days to maintain the micropatterned organoids attached to the glass coverslip for long-term culture.

[0320] - Continue to culture cells for up and proceed with passaging the cells and seeding them on micropatterns, with medium changes every second day.

[0321] Results: The cortical identity of the stem / progenitor cells is highlighted by the widespread expression of Foxgl around Day 20. The cortical progenitors are defined by expression of Sox2 and Phospho-vimentin (p-vim), which are also identified as apical progenitors. This cell population will give rise to intermediate progenitor cells mainly characterized by Tbr2+ staining from around Day 20 and to newly born, postmitotic neurons (MAP2+).

[0322] From day 20 on, deeper- layer neurons (Tbr1 +, Tle4+, Cti p2+) start to be evident while becoming the major population around Day 60. Late-born upper-layer neurons (Satb2+, Cux1 + and Brn2+ cells) begin to be produced around Day 40, but their proportions increase over time (Day 60, 90, 100).

[0323] To detect astrocytes, we co-stained for S100beta and GFAP around day 70. Moreover, to detect basal progenitors also known as outer Radial Glia (oRG), we check for the expression of HOPX which is known to be a specific oRG marker (Pollen et al., Cell 163, 56-67, 2015).

[0324] Around Day 50 (not shown), HOPX started to be expressed while increasing over time (Day 92 staining).

[0325] Single rosette micropatterns

[0326] Organoids featuring a prominent neural rosette can be generated by seeding stem cells directly on the chip from Day 0 or by seeding early neural progenitors after neural induction at Day 10. Importantly, we observed that the timing of seeding on microchips is a critical determinant of patterning. Furthermore, increasing the seeding cell number by a factor of 1.5 can result in neural rosette formation, although timing of microchip seeding appears to be determinative.

[0327] Improvement of organoid attachment

[0328] To promote better organoids attachment to the chips, we employ small molecules like Y- 27632 (ROCK Inhibitor) or the CEPT cocktail which are known to improve cell viability and reduce cell stress. Exogenous ECM components reduce cell / organoid detachment for longterm organoid culture. We add 500 pl of laminin-521 (5pg / ml) every 10 days directly into 2.5 ml of medium from 20 days after seeding onwards.

[0329] Example 3 - Miniaturized organoids for phenotyping neural disorders.

[0330] As shown in Figure 3, the methods of the present invention are suitable to generate miniaturized organoids for phenotyping neural disorders. Figure 3 (A) shows a comparison of d20 organoids derived from WT and from an isogenic human pluripotent cell (hESC) line with a heterogeneous mutation in DYRK1 A. Mutations in DYRK1 A typically lead to DYRK1 A syndrome, a severe neural developmental disorder associated with autism spectrum disorder, among others. The mutation leads to an increased progenitor pool (TBR2) and less mature neurons (MAP2), suggesting a delayed exit from the cell cycle in the disease. Figure 3 (B) shows a quantification of the markers shown in panel A. Each data point corresponds to one organoid, highlighting the high statistical power for phenotyping that can be achieved with the organoids.

[0331] Figure 3 (C) shows a comparison of d15 organoids derived from WT and from an isogenic human pluripotent cell (hESC) line with a mutation in Apolipoprotein E (APOE), WT: APOE3 / 3, mutation: APOE3 / 4, the most common risk factor for sporadic Alzheimer’s disease. An increase in early born neurons (DCX) and in mature neurons (MAP2) can be observed in the mutant, as quantified in (D). Additionally, Figure 3 (E) demonstrates a deep-learning classification network trained on a part of the images, indicating that this network can separate WT and mutated conditions with high accuracy, indicating the effectiveness of the method in determining neuronal phenotypes, for example for screening purposes.

[0332] Example 4 - Optional use of an additional Wnt Inhibitor

[0333] Depending on the context and specific scientific questions that are to be investigated, reproducible organoids comprising a neural rosette structure in a central region may be generated according to different embodiments of the present invention.

[0334] In the present example, pluripotent stem cells were seeded at day 0 (dO) at a number of 450.000 cells per coverslip (19.5 mm x 19.5 mm). In addition, at dO, a CEPT cocktail (comprising Chroman 1 , Emricasan, Polyamine Solution and Trans-ISRIB) was added to the cell culture medium for 3 days of culture.

[0335] Subsequently, only the ROCK2 inhibitor Chroman 1 (one component of the CEPT cocktail) was added to the cell culture media, which is similar to the ROCK1 / ROCK2 inhibitor Y-27632. In addition, a Wnt inhibitor (IWR1e) was added to the cell culture medium at the beginning of the culture protocol. The cultivation of the cells resulted in a reproducible formation of a neural rosette structure in the central region of the obtained neural organoids, which remained adherend to the coverslips for significantly more than 18 days of culture.

[0336] REFERENCES

[0337] 1 . Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501 , 373-379 (2013).

[0338] 2. Renner, M. et al. Self-organized developmental patterning and differentiation in cerebral organoids. The EMBO Journal 36, 1316-1329 (2017).

[0339] 3. Velasco, S., Paulsen, B. & Arlotta, P. 3D Brain Organoids: Studying Brain Development and Disease Outside the Embryo. Annual Review of Neuroscience 43, 375-389 (2020).

[0340] 4. Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54-59 (2017).

[0341] 5. Jo, J. et al. Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. Cell Stem Cell 19, 248- 257 (2016).

[0342] 6. Sirenko, O. et al. Functional and Mechanistic Neurotoxicity Profiling Using Human iPSCDerived Neural 3D Cultures. Toxicological Sciences 167, 58-76 (2019).

[0343] 7. Haremaki, T. et al. Self-organizing neuruloids model developmental aspects of Huntington’s disease in the ectodermal compartment. Nature Biotechnology 37, 1198- 1208 (2019).

[0344] 8. Metzger, J. J. et al. Deep-learning analysis of micropattern-based organoids enables highthroughput drug screening of Huntington’s disease models. Cell Reports Methods 2, 100297 (2022).

[0345] 9. Wang, Y. et al. Modeling human telencephalic development and autism-associated SHANK3 deficiency using organoids generated from single neural rosettes. Nat Commun 13, 5688 (2022).

[0346] 10. Knight, G. T. et al. Engineering induction of singular neural rosette emergence within hPSC- derived tissues. eLife 7, e37549 (2018).

[0347] 11. Tang, C., Wang, X., D'Urso, M., van der Putten, C., & Kurniawan, N. A. (2022). 3D Interfacial and Spatiotemporal Regulation of Human Neuroepithelial Organoids. Advanced Science, 9(22), [2201106],

Claims

CLAIMS1 . An in-vitro method of generating an adherent neural organoid cell culture model, comprising the steps of: a. providing a cell culture comprising pluripotent stem cells, b. adding to the cell culture medium at least one Rho-associated protein kinase inhibitor (ROCKi) and / or at least one extracellular matrix (ECM) protein or component, and c. cultivating the cell culture for at least 18 days, thereby generating an adherent neural organoid in cell culture with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.

2. The method according to claim 1 , wherein the cell culture is cultivated in step c. in a culture vessel coated with at least one extracellular matrix (ECM) protein or component, and / or wherein at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every 5-10 days.

3. The method according to any one of the preceding claims, wherein at least for the first 18 days of culture at least one Rho-associated protein kinase inhibitor (ROCKi) is added to the cell culture medium every 5-10 days, preferably at a concentration between 1-10 pM.

4. The method according to any one of the preceding claims, wherein the cell culture is cultivated in step c. initially in a cell culture dish or plate for 8 to 12 days and subsequently transferred to a micropattern cell culture chip for further culture.

5. The method according to any one of claims 1 to 3, wherein the cell culture is cultivated in step c. on a micropattern cell culture chip from day 0 onwards, to generate a neural organoid comprising a neural rosette structure in a central region of the neural organoid.

6. The method according to any one of the preceding claims, wherein the cell culture is cultivated for at least 30 days of total culture duration.

7. The method according to any one of the preceding claims, wherein at least one further supplement is added to the cell culture medium, said supplement selected from the group consisting of SMAD-inhibitors, Chroman, a CEPT cocktail comprising Chroman, Emricasan, Polyamine Solution and Trans-ISRIB, one or more neurotrophic factors, such as BDNF, NT3, GDNF, cAMP, and ascorbic acid.

8. The method according to any one of the preceding claims, wherein the pluripotent stem cells are induced pluripotent stem (IPS) cells.

9. The method according to any one of the preceding claims, wherein the cell culture is cultivated in step c. initially at a cell-density of at least 50.000 cells / cm2, preferably at least 100.000 cells / cm2.

10. An in vitro neural organoid cell culture model produced according to the method of any one of the of the preceding claims, said model comprising an adherent neural organoid in cell culture with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections.11 . The in vitro model according to claim 10, wherein: a. the model is produced according to the method of claim 4, and wherein the organoid further comprises one or more astrocytes, upper-layer neurons and / or deeper-layer neurons, preferably wherein at least a fraction of the deeper-layer neurons is localized in the core area of the neural organoid and / or wherein at least a fraction of the upperlayer neurons are localized at the rim of the neural organoid, thereby mimicking an inside-out development of a mammalian brain, or b. the model is produced according to the method of claim 5, and wherein the organoid comprises a neural rosette structure in a central region of the neural organoid.

12. The in vitro cell culture model according to any one of claims 10 or 11 , wherein the organoid comprises: a. cortical progenitor neurons, and wherein at least a fraction of the cells expresses Foxgl or Sox2, and / or Phospho-vimentin (p-vim), b. deeper-layer neurons, and wherein at least a fraction of the cells expresses Tbr1 , Tle4 and / or Ctip2, c. upper-layer neurons, and wherein at least a fraction of the cells expresses Satb2, Cux1 and / or Brn2, d. astrocytes, and wherein at least a fraction of the cells expresses S100beta and / or GFAP, and / or e. basal progenitors, and wherein at least a fraction of the cells expresses HOPX.

13. The in vitro model according to any one of claims 10-12, wherein at least two organoids are cultured in the same culture dish or well, and wherein at least a fraction of the organoids form neural connections to a neighboring organoid in the same culture dish or well.

14. An in vitro adherent neural organoid cell culture model comprising organoids with a defined spatial structure and a regular eccentricity, comprising a core area comprising differentiated (mature) neurons, progenitor cells and glia cells, and a rim of neuronal and / or glia cell projections, wherein the organoids comprise: a. Astrocytes, upper-layer neurons and deeper-layer neurons, wherein at least a fraction of the deeper-layer neurons is localized in the core of the neural organoid and / or wherein at least a fraction of the upper-layer neurons are localized at the rim of the neural organoid, orb. a neural rosette structure in a central region of the neural organoid.

15. Use of the in-vitro model according to any one of claims 10 to 14 for high throughput drug or toxicity screening and / or for studying a neurological disease.

16. A kit for carrying out the method of claims 1 to 9, said kit comprising a culture medium and at least one supplement selected from the group comprising: at least one ROCK-inhibitor, at least one ECM protein or component, at least one SMAD-inhibitor at least Chroman, or a CEPT cocktail comprising Chroman, Emricasan, Polyamine Solution and Trans-ISRIB, and one or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP and ascorbic acid,- wherein the kit optionally comprises a micropattern cell culture chip,- wherein the kit optionally comprises the in vitro neural organoid model according to any one or more of claims 10 to 15.

17. A cell culture plate or micropattern cell culture chip comprising the in vitro neural organoid model according to any one of claims 10 to 14.

18. A computer implemented method for analyzing a neural organoid cell culture model according to any one of claims 10 to 14, said method comprising the steps of: v. Providing a plurality of spatially distinct neural organoids in cell culture, vi. Determining at least one spatial parameter of at least one organoid provided in i., vii. Analyzing the at least one spatial parameter of the at least one organoidusing machine learning, preferably deep learning, to determine the presence, location, spatial proportion, shape and / or size of at least one region and / or cell type of the at least one organoid, viii. Optionally providing and / or displaying on a screen a data summary comprising the results of the analysis in step iii., and optionally a graphical representation or image of the analyzed organoid.

Images