Miniaturized brain organoid cell culture model

Abstract

The present invention relates to an in vitro method for generating a neural organoid cell culture model. The present invention relates to an in vitro adhesive neural organoid cell culture model comprising an organoid having a defined spatial structure and a regular eccentricity, comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and edges of neuronal and / or glial cell processes. The present invention further relates to an in vitro neural organoid cell culture model prepared according to the method of the present invention, the use of the in vitro model of the present invention for high-throughput drug or toxicity screening and / or for testing neurological diseases, a kit for carrying out the method of the present invention, a cell culture plate or micropattern cell culture chip containing the in vitro neural organoid model of the present invention, and a computer implementation method for analyzing the neural organoid cell culture model of the present invention.

Description

[Technical Field]

[0001] This invention relates to the field of cell biology, specifically to cell culture models and corresponding screening methods.

[0002] The present invention relates to an in vitro method for generating a neural organoid cell culture model. In another embodiment, the present invention relates to an in vitro adhesive neural organoid cell culture model comprising an organoid having a defined spatial structure and regular eccentricity, comprising a core region comprising differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of processes of neuronal cells and / or glial cells.

[0003] The present invention further relates to an in vitro neural organoid cell culture model prepared according to the method of the present invention, the use of the in vitro model of the present invention for high-throughput drug or toxicity screening and / or testing for neurological diseases, a kit for carrying out the method of the present invention, a cell culture plate or micropattern cell culture chip containing the in vitro neural organoid model of the present invention, and a computer implementation method for analyzing the neural organoid cell culture model of the present invention. [Background technology]

[0004] Since the first brain organoids were developed in 2013, Lancaster et al. 1 Based on the initial protocol, a series of such organoids have been developed. The goal is to obtain cell types specific to different regions of the brain, such as dorsal and ventral forebrain or midbrain cells. 2~5 .

[0005] A major drawback of current-generation organoids is the lack of spatial structure and spatial reproducibility. One of the main goals when using organoids is to generate tissues with different cell types that are arranged similarly to their in vivo counterparts. Current brain organoids partially achieve this, but still exhibit high variability between individual organoids, batches, and cell lines.

[0006] For example, in the case of nerve or brain organoids, these organoids grow to different sizes in culture, generating a random number of organizing centers (ventricular zones). This makes phenotypic identification in disease models extremely difficult and elusive, and as a result, early disease phenotypes may not be detectable. In the case of drug screening approaches, this also undermines the use of standard brain organoids for drug or toxicity screening because wild-type (WT) and disease phenotypes cannot be reliably isolated in screening to rule out false positives or false negatives (i.e., potentially successful drugs may be missed, and conversely, drugs that are ineffective may appear potent).

[0007] Currently, organoid production is not easily scalable. In particular, current neural or brain organoids cannot be produced in sufficient numbers (and with sufficient reproducibility) for high-throughput applications. However, even if higher reproducibility is achieved, a large number of organoids are still required to achieve statistically robust assays for screening applications. However, currently, only batches of organoids with yields of tens or at most hundreds of organoids can be produced, which is too few for screening applications. Attempts to scale up organoid production in 96-well or 384-well systems are still limited to one organoid per well. 2、6 Furthermore, they still suffer from a complete lack of spatial structure, and therefore such organoids are of little use in identifying or analyzing disease phenotypes.

[0008] In the context of brain organoids, further difficulties have been observed in obtaining cell culture models that are adhesive and allow for longer-term culture. To date, adhesive culture of brain organoids for more than a few days has not been established.

[0009] Metzger et al. 8 International Publication No. 2020 / 257756 describes a drug screening platform using "neural tube-forming organoids" differentiated for 7 days on a micropattern with a diameter of 500 or 700 μm. In a 96-well plate, 25 organoids per well were analyzed and compared to neural organoids with mutations typical of Huntington's disease. Compound screening was then performed to investigate whether phenotypic rescue of the disease phenotype could be observed. Experiments were performed after 7 days because cell adhesion was not guaranteed in longer cultures.

[0010] Other studies have demonstrated the neuronal differentiation of embryonic stem cells into neuronal rosettes, including culture on micropatterns for up to 7 days. Furthermore, Knight et al. 10 This allowed us to obtain neural rosettes containing neuroepithelial cells that could be cultured on micropatterns for up to 12 days. However, the longest culture period identified in the literature was Tang et al. 11 This took only 18 days. Tang et al. generated neuroepithelial organoids showing dorsal-ventral pattern formation as an early model of the spine.

[0011] Further studies, including those by Miura et al. (2022) and International Publication No. 2022 / 080974, analyzed different culture conditions for brain organoid generation, but these studies also failed to report favorable conditions for long-term adherent organoid cell culture.

[0012] Generally, the main drawbacks of non-adherent organoid cultures, or organoids lacking long-term adhesion, are their lack of spatial reproducibility and their unsuitability for high-throughput applications or screening.

[0013] Furthermore, many other prior art methods, such as International Publication No. 2022 / 080974, cannot generate neuronal organoids with reproducible spatial structures or regular eccentricity, for example, because brain organoids are assembled by combining or fusing separate differentiated organoids of different brain structures. Further studies, such as International Publication No. 2023 / 090427, describe the generation of other neuronal models, such as retinal organoid models, but these have a maximum size of 5 cm and are therefore not very suitable for high-throughput applications.

[0014] In summary, one significant problem remains regarding the difficulty of adherent culture of organoids: the possibility of detachment from the culture substrate or support during culture and differentiation, which has hindered long-term adherent culture. Until now, long-term culture required non-adherent culture, which had the disadvantages of lacking spatial reproducibility and not being high-throughput.

[0015] Therefore, considering the prior art, there remains a great need in this field for an advanced neuronal cell culture model that is suitable for long-term culture and can be produced in the large quantities required for screening purposes. [Overview of the Initiative]

[0016] In light of prior art, the fundamental technical problem underlying the present invention was to provide an alternative or improved neuronal cell culture model suitable for long-term culture.

[0017] Another underlying problem of this invention was to provide a culture model for adherent nerve cells.

[0018] Another object underlying the present invention was to provide an adherent nerve cell culture model that can be produced on a sufficient scale required for screening applications.

[0019] Another object underlying the present invention was to provide an adherent nerve cell culture model having sufficiently regular structural characteristics such as regular eccentricity that enables reliable production and use in high-throughput screening applications.

[0020] The above-mentioned problem is solved by the features of the independent claim. Preferred embodiments of the present invention are provided by the dependent claims.

[0021] To overcome the above-mentioned problems of reproducibility, scalability, lack of adhesiveness, and long-term culture, the present inventors have developed in vitro organoids that are preferably smaller (miniaturized) than current neuronal organoid models, but retain the relevant features of larger organoids and their in vivo counterparts.

[0022] Accordingly, the present invention relates to an in vitro method for generating an adherent neural organoid cell culture model, comprising a. providing a cell culture comprising pluripotent stem cells; b. adding at least one Rho-associated protein kinase inhibitor (ROCKi) and / or at least one extracellular matrix (ECM) protein or component to the cell culture medium; c. culturing the cell culture for at least 18 days; thereby producing an adherent neural organoid having a defined spatial structure and regular eccentricity in cell culture, the organoid comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and a rim of neurites and / or glial cell processes.

[0023] In other words, in the embodiment, the core region of the neural organoid cell culture model is surrounded by the edges of cellular processes of neuronal cells and / or glial cells. In the embodiment, the cellular processes are formed by mature neurons and / or radial glial cells.

[0024] In an alternative embodiment, the method thereby generates an adhesive neural organoid in a cell culture comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of the processes of the neuronal cells and / or glial cells.

[0025] In the embodiment, steps a. to c. of the method generate an adhesive neural organoid in a cell culture, the organoid comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and edges of processes of neuronal cells and / or glial cells.

[0026] In this embodiment, the method generates an adhesive neural organoid having a defined spatial structure in cell culture, comprising a core region with a regular eccentricity, containing differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of the processes of the neuronal cells and / or glial cells. Therefore, the steps of the method and the resulting physical properties of the organoid represent relevant aspects of the present invention.

[0027] In some embodiments, the defined spatial structure is a preferred but not essential characteristic of the organoid.

[0028] In some embodiments, the regular eccentricity of the organoid and / or core region is a preferred but not essential characteristic of the organoid.

[0029] In a preferred embodiment, the neural organoid cell culture model is / remains adhesive (adheres to the cell culture vessel / support) throughout the culture. In the embodiment, the adhesion is maintained for at least 18 days of culture time (culture period).

[0030] In embodiments, after a total culture period of 18 days, the neural organoid cell culture model comprises one or more of differentiated (post-mitotic / mature) neurons, neuroepithelial cells, radial glial cells, progenitor cells, and / or intermediate progenitor cells. In embodiments, the culture in step c. is an adherent cell culture (a cell culture comprising adherent cells).

[0031] Accordingly, the present invention preferably relates to a self-organizing neural organoid cell culture model, preferably a self-organizing cerebral organoid cell culture model, which can be cultured for several months or even more than one year, and to a method for producing said model. The inventors surprisingly found that the method according to the present invention enables the production of "miniaturized" neuronal organoids, for example cerebral organoids, that remain adherent to their respective culture vessels over a long period of time, preferably over 18, 30, 50 days, or over 100 days, respectively.

[0032] Our previous work demonstrated neuronal differentiation of embryonic stem cells into neuronal rosettes on micropatterned structures, which remained adherent for a total culture period of up to 7 days 8 . Other studies 10 have obtained neural rosettes composed of neuroepithelial cells that can be cultured on micropatterns for up to 12 days. To date, the longest culture period reported for neuronal organoids in the prior art has been 18 days 11 However, according to the findings of the inventors, a neuronal organoid model that allows long-term culture time exceeding 18 days and comprises cells at a mature differentiation stage has not yet been described in the prior art.

[0033] One limiting factor in the long-term culture period of neuronal organoid models is the time-limited adhesion of such organoids to the culture vessel or support. This limitation was previously addressed by embedding neuronal organoids in hydrogels, but this itself has further drawbacks, such as irregular organoid growth and limited reproducibility of the generated organoids. Other limiting factors for culture time and adhesion are mechanical shear forces and remodeling processes that occur during organoid differentiation, which result in cellular debris, as well as the detachment of apoptotic and necrotic cells.

[0034] The inventors have found that the methods and other means described herein surprisingly promote the long-term adhesion of the neuronal cell culture model according to the present invention to the surface of the culture vessel, thereby advantageously enabling long-term culture times of more than 50 days, or even more than 100 days. Furthermore, this long-term adhesion of the model according to the present invention is achieved not only on standard cell culture dishes or well plates, but also on so-called "micropatterns" (specific microgrids) or chips (micropattern cell culture chips) that allow differentiation of early pluripotent cells into different neuronal cell types without increasing the mass or size of differentiated organoids (including different cell types and / or brain tissues). Furthermore, in embodiments, the method further enables the generation of different cortical organoid models that resemble different regions of the brain cortex.

[0035] Therefore, in the embodiment, the cell culture is cultured on a micropatterned cell culture chip (or micropatterned coverslip) or micropatterned cell culture dish either from day 0 or from day 10 to 12 in step c.

[0036] In the embodiment, the cell culture is first cultured in a standard cell culture dish or plate for 1 to 12 days, preferably 8 to 12 days, or 10 to 12 days, in step c, and then transferred to a micropattern cell culture chip.

[0037] In the embodiment, the cell culture is first cultured in a cell culture dish or plate for 8 to 12 days in step c, and then transferred to a micropattern cell culture chip for further culture.

[0038] Preferably, such a model is defined by additional characteristics of an organoid, which further comprises one or more astrocytes, superneurons, and / or deep neurons, preferably at least a portion of the deep neurons localized to the core region of the neural organoid and / or at least a portion of the superneurons localized to the edge of the neural organoid, thereby mimicking the inside-out development of the mammalian brain.

[0039] In the embodiment, in step b, at least one WNT pathway inhibitor (WNT(pathway) inhibitor) or a WNT inhibitor is added to the cell culture medium.

[0040] In this embodiment, the cell culture is cultured on a micropattern cell culture chip from day 0 onwards in step c. to generate a neural organoid, which is a neural organoid containing a neural rosette structure in the central region of the neural organoid.

[0041] In the embodiment, at least one WNT pathway inhibitor (WNT(pathway) inhibitor) or a WNT inhibitor is added to the cell culture medium to improve the reproducibility of the neural rosette structure during organoid culture and subsequent processing.

[0042] Culturing cells on a micropatterned cell culture chip (coverslip) offers several advantages for organoid differentiation. In embodiments, the area of ​​a "microspot" (a segment of the micropatterned chip containing one organoid) limits the surface area on which the organoid grows and adheres, thereby allowing the neuronal cell aggregates to grow to a defined and reproducible size. As a result, the resulting organoids have their growth restricted, reaching a maximum size of approximately 300–700 μm in diameter, preferably approximately 500 μm in diameter.

[0043] This, when combined with the present method, offers several advantages. On the one hand, it further promotes the long-term adhesion of neuronal organoids to the support. On the other hand, when multiple organoids are generated in parallel on a micropattern chip according to the present method, all organoids will be essentially of the same size. In contrast, neuronal organoid cultures of prior art grown in hydrogels typically expand continuously during their differentiation, reaching several centimeters in size and often becoming irregularly shaped. When multiple such organoids are grown in parallel in a hydrogel, they often differ significantly in size and structure, thereby reducing their comparability and suitability for (high-throughput) screening or comparative approaches.

[0044] Therefore, one advantage of the neuronal cell culture models of the present invention is that they are suitable for high-throughput screening because they contain diverse types of differentiated neuronal cells, combined with small, reproducible organoid sizes.

[0045] In embodiments of the high-throughput screening approach, the model may be cultured in a multi-well plate, for example, each well preferably containing two or more organoids (e.g., 10 or even 25), thereby simplifying the analysis and improving the reproducibility and quality of the screening results. In embodiments, such analysis may be carried out essentially as previously described by the inventors. 8 (Metzger et al., Cell Reports Methods (2022) Vol.2, 100297). Thereafter, in embodiments, the neuronal organoids of the present invention may function as separate brain models that can be used as cost-effective brain models for drug screening, toxicity screening, and / or personalized medicine approaches, and which can be generated and screened with less effort than previous models.

[0046] In embodiments, the long-term culture time of the neuronal cell culture model of the present invention allows for the formation of neuronal connections between different organoids, for example, when the culture is performed on a micropattern chip, and as a result, a neuronal network may be formed between some or more organoids.

[0047] The inventors have surprisingly found that this method enables the generation of neuronal cell culture models, preferably including cortical neuronal organoids or cortical brain organoids, which remain adherent throughout the entire culture period, up to 100 days or even longer. By applying this method, the inventors have for the first time been able to culture neuronal organoids for a period exceeding three months, and the organoids contain highly differentiated cells of diverse cell types, which had not been previously achieved in the art.

[0048] Therefore, in the embodiments, the cell culture is cultured for a total period of at least 18 days, or preferably 30 days. In some embodiments, the cell culture is cultured for a total period of at least 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 days, or longer. In the embodiments, the cell culture is cultured for at least 18 days.

[0049] In the embodiment, the cell culture is cultured for a total period of at least 100 days, or longer. In the embodiment, the cell culture is cultured for a total period of at least 3 months. In the embodiment, the cell culture is cultured for a total period of at least 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.

[0050] All time ranges disclosed herein may include variations of ±1 day, ±2, ±3, ±4, or ±5 days, ±10, or ±14, or ±28, ±35, or ±50 days, or even more.

[0051] A key advantage of this model over prior art neural organoid cell culture models is that it can be maintained for more than 100 days, if desired.

[0052] In this embodiment, neuronal differentiation is achieved according to a standard protocol such as Shi et al., 2012 (Shi et al., Nat. Prot. (2012) Vol. 7, 1836).

[0053] In the embodiment, step b. includes adding at least one Rho-related protein kinase inhibitor (ROCKi) and at least one extracellular matrix (ECM) protein or component to the cell culture medium.

[0054] In this embodiment, pluripotent cells are single-cell isolated (separated into single cells) before culturing in step c.

[0055] In the embodiment, the cell culture is cultured in a culture vessel coated with at least one extracellular matrix (ECM) protein or component in step c.

[0056] In the embodiment, the cell culture is cultured in a culture vessel coated with at least one extracellular matrix (ECM) protein or component in step c, and / or at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every 5 to 10 days.

[0057] In the embodiments, at least one extracellular matrix (ECM) protein or component is added to the cell culture once or more times during the culture period, for example, every 1 to 14 days, or during / with the change of culture medium. In the embodiments, at least one extracellular matrix (ECM) protein or component is added to the culture medium as a supplement or is included in the culture medium.

[0058] In one embodiment, at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every 5 to 10 days. In another embodiment, at least one extracellular matrix (ECM) protein or component is added to the cell culture medium daily, every other day, or every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 21 days, or every 1 to 14 days, 1 to 5 days, 2 to 7, or 3 to 15 days, or every 5 to 10 days.

[0059] In embodiments, at least one extracellular matrix (ECM) protein or component is selected from laminin, collagen, type IV collagen, entactin, or Geltrex®. In embodiments, at least one extracellular matrix (ECM) protein or component comprises laminin, collagen, type IV collagen, entactin, and / or Geltrex®.

[0060] In the embodiment, the cell culture is cultured in a culture vessel coated with at least one extracellular matrix (ECM) protein or component in step c, and / or at least one extracellular matrix (ECM) protein or component is added to the cell culture medium every 5 to 10 days, wherein the ECM protein or component is laminin.

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

[0062] In the embodiment, at least one Rho-related 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 μM.

[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 improved the adhesion of the cell culture model to the support, thereby supporting long-term cell culture of the model for more than 18 days, preferably more than 30 days, more than 50 days, or even more than 100 days.

[0064] In the embodiment, preferably for at least the first 18 days of culture, at least one Rho-related protein kinase inhibitor (ROCKi) is added to the cell culture medium daily, 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, 2-7 days, or 3-15 days, or every 5-10 days.

[0065] Non-exclusive examples of Rho-related protein kinase inhibitors (ROCKi) include WAY-624704, Y27632, Y-27632 2HCl, thiazovibin, GSK429286A, fasudil (HA-1077) HCl, H-1152 dihydrochloride, Y-27632, azaindole 1 (TC-S 7001), RKI-1447, AT13148, GSK269962A HCl, Y-39983 HCl, bellamsudil (KD025), ripasudil (K-115) hydrochloride dihydrate, hydroxyfasudil (HA-1100) HCl, netalusdil (AR-13324) 2HCl, ZINC00881524 (ROCK inhibitor), and chroman 1.

[0066] In this embodiment, at least one Rho-related protein kinase inhibitor (ROCKi) is chroman 1.

[0067] In the embodiment, a CEPT cocktail is used instead of, or in addition to, at least one Rho-related protein kinase inhibitor (ROCKi), the CEPT cocktail comprising chroman 1, emricasan, a polyamine solution, and trans-ISRIB.

[0068] In embodiments, Rho-related protein kinase inhibitors (ROCKi) may be included in the culture medium and / or supplement cocktail at concentrations of 10 μM, or 0.01–200 μM, 0.1–100 μM, 5–50 μM, 5–25 μM, 5–15 μM, or 10–25 μM, or 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, or 200 μM. The concentration may vary depending on the specific compound used.

[0069] In embodiments, the CEPT cocktail (containing chroman 1, emricasane, polyamine solution, and trans-ISRIB) may be included in the culture medium and / or supplement cocktail at concentrations of 50 nM chroman 1, 5 μM emricasane, 1 × polyamine solution, and 0.7 μM trans-ISRIB, or at concentrations of 25–75 nM chroman 1, 1–10 μM emricasane, 0.5–1.5 × polyamine solution, and 0.5–1.5 μM trans-ISRIB. The concentrations may vary depending on the specific compounds used.

[0070] In some embodiments, the in vitro method of the present invention is a. To provide cell cultures containing pluripotent stem cells, b. In the cell culture medium, Adding one or more of the following: at least one Rho-related 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; c. The cell cultures are cultured for at least 18 days. This includes generating an adhesive neural organoid in cell culture having a defined spatial structure and a regular eccentricity, comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of the processes of neuronal cells and / or glial cells.

[0071] In some embodiments, step b. includes adding to the cell culture medium at least one Rho-related protein kinase inhibitor (ROCKi) and / or a CEPT cocktail (containing Chroman 1, Emricasan, polyamine solution, and trans-ISRIB), at least one WNT (pathway) inhibitor, and at least one extracellular matrix (ECM) protein or component.

[0072] In the embodiment, a SMAD inhibitor, a CEPT cocktail comprising chroman 1, emricasan, a polyamine solution, and trans-ISRIB, or chroman 1 alone, at least one WNT (pathway) inhibitor, and at least one further supplement selected from the group comprising one or more neurotrophic factors, such as BDNF, NT3, GDNF, cAMP, and ascorbic acid, is added to the cell culture medium.

[0073] SMAD (or Smad) is a family of structurally similar proteins that are major signaling molecules for the transforming growth factor beta (TGFb; TGF beta) superfamily receptors.

[0074] Non-exclusive examples of SMAD signaling inhibitors ("SMAD inhibitors") or TGFβ pathway inhibitors include SB431542, LDN-193189, SB525334, ginsenoside Rh4, TGFβRI-IN-3, Lycops, AUDA, sulfasalazine, SB505124, garnicertib, BIBF-0775, LY2109761, GW788388, LY364947, pirfenidone, SIS3, PD, ITD-1, TA-02, DMH1, and LDN. -212854, ML347, RepSox, Dorsomorphine, K02288, Bactocertib, SD-208, LDN-214117, A-83-01, R-268712, Dorsomorphine, SIS3, Halofudinone, TP0427736, LY, Garnicertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, Allantoin Lactone, Hesperetin, SRI-011381, SB 4, Isoxazole 9, Cartogenin, L-Kebrasitol, SJ000291942, LY-3200882, and TP0427736 HCl.

[0075] In embodiments, SMAD signaling inhibitors or TGFβ pathway inhibitors, such as SB431542, may be included in the culture medium and / or supplement cocktail at concentrations of 10 μM, or 1–100 μM, 5–50 μM, 5–25 μM, 5–15 μM, or 10–25 μM, or 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, or 50 μM. The concentration may vary depending on the specific compound used.

[0076] In embodiments, SMAD signaling inhibitors or BMP pathway inhibitors, such as LDN-193189, may be included in the culture medium and / or supplement cocktail at concentrations of 0.2 nM, or 0.1–10 nM, or 0.1–5 nM, or 0.1, 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, and 15 nM. The concentration may vary depending on the specific compound used.

[0077] In embodiments, the SMAD inhibitor SB431542 may be used at a concentration of 10 μM, or at concentrations of 1–100 μM, 5–50 μM, 5–15 μM, or 10–25 μM, or at concentrations of 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, or 50 μM. The concentration may vary depending on the specific compound used.

[0078] In the embodiment, the SMAD inhibitor LDN193189 may be used at concentrations of 0.2 μM, or 0.1 to 10 μM, or 0.1 to 5 μM, or 0.1, 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, and 15 μM. The concentration may vary depending on the specific compound used.

[0079] In the embodiment, at least one WNT (pathway) inhibitor (or WNT pathway inhibitor or WNT signaling inhibitor) is IWR-1-endo (IWR1e), IWP-2, JW74, IWP-4, CCT251545, KY1220, iCRT14, iCRT3, LF3, PNU-74654, KYA1797K, KY02111, Zamaporvint (RXC004), M2912, A group of substances may be selected that includes, but is not limited to, M435-1279, KY-05009, prodigiosin, ginsenoside Rh4, heparan sulfate, RCM-1, recibugenin, MSAB, PRI-724, tegatrabetan (BC-2059), JW55, adavivint (SM04690), tryptonide, isoquercitrin, IQ-1, sarinomycin, and FH535.

[0080] In embodiments, at least one WNT inhibitor, for example, IWR1e, may be included in the culture medium and / or supplement cocktail at a concentration of 3 μM, or 1 to 10, or 1 to 5 μM, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. The concentration may vary depending on the specific compound used.

[0081] In embodiments, at least one WNT inhibitor, for example, IWR1e, may be used at a concentration of 3 μM, or 1 to 10, or 1 to 5 μM, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. The concentration may vary depending on the specific compound used.

[0082] While the addition of WNT inhibitors to the cell culture medium is not inherently necessary for generating adhesive neural organoid models having defined spatial structures and regular eccentricity according to the present invention, in some specific embodiments, the addition of at least one WNT inhibitor (e.g., step b.) may further improve the reproducibility of the generated neural organoids, including central nervous system rosette structures.

[0083] Any range derived from, using, or within the aforementioned numerical concentration ranges is also included in this disclosure. The aforementioned concentrations may also apply to embodiments of the kit described in more detail below.

[0084] In this embodiment, the pluripotent stem cells are induced pluripotent stem (iPS) cells.

[0085] In the embodiment, the cell culture is first prepared in step c. to contain at least 50,000 cells / cm³. 2 The cell culture is cultured at a cell density of 50,000 to 150,000 cells / cm³ in step c. In the embodiment, the cell culture is first cultured at a cell density of 50,000 to 150,000 cells / cm³. 2 The cell culture is cultured at a cell density of 100,000 to 150,000 cells / cm³ in step c. In the embodiment, the cell culture is first cultured at a cell density of 100,000 to 150,000 cells / cm³. 2 The cells are cultured at the above cell density. In the embodiment, the cell culture is first cultured at a density of at least 100,000 cells / cm³ in step c. 2 The cell culture is cultured at a cell density of . In the embodiment, the cell culture is first cultured at a cell density of at least 100,000 cells / cm³ in step c. 2 The cells are cultured at the above cell density. In the embodiment, the cell culture is first cultured at a density of at least 150,000 cells / cm³ in step c. 2 They are cultured at this cell density.

[0086] In some embodiments, the cell culture initially contains at least 150,000 cells / cm³ in step c. 2 They are cultured at a cell density, thereby generating a model that includes a neural rosette structure at the center of the neural organoid.

[0087] In the embodiment, from day 18 of culture onward, the organoid includes a defined spatial structure and a regular eccentricity, including a defined core region containing differentiated (mature) neurons, progenitor cells, and glial cells, surrounded by the edges of the processes of neuronal cells and / or glial cells.

[0088] In the embodiment, from day 18 of culture onward, the organoid preferably contains one or more of the following: cortical progenitor neurons, deep neurons, superior neurons, astrocytes, and / or basal progenitor cells.

[0089] In some embodiments, after 18 days of culture, the organoids preferably include neural rosette structures.

[0090] In the embodiment, after 18 days of culture (according to step c), at least a portion of the cells contained within the organoid, preferably at least 1-70%, are cortical progenitor neurons expressing Foxg1 or Sox2 and / or phosphovimentin (p-vim).

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

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

[0093] In the embodiment, after 18 days of culture (according to step c), at least a portion of the cells contained within the organoid, preferably at least 1-70%, are astrocytes expressing S100 beta and / or GFAP.

[0094] In the embodiment, after 18 days of culture (according to step c), at least a portion of the cells contained within the organoid, preferably at least 1-70%, are basal progenitor cells, also known as outer radial glia (oRGs), that express HOPX.

[0095] In this embodiment, the method is for obtaining an organoid having a neural rosette structure in its center or in the central region of the organoid, and the method includes, in step c. culturing an initial cell culture (including pluripotent cells) on a micropatterned cell culture chip from (immediately after) day 0. Therefore, in this embodiment of the method, the cell culture is cultured on a micropatterned cell culture chip from day 0 in step c. to generate an organoid having a neural rosette structure in its center or in the central region of the neural organoid.

[0096] In other words, in some embodiments, a method for generating a neural organoid cell culture model includes, in step c, culturing a cell culture containing pluripotent stem cells on a micropatterned culture chip for an initial period (from day 0 of culture onwards), thereby generating a neural organoid that includes a neural rosette structure in the center or central region of the neural organoid.

[0097] In some embodiments, cell cultures are subcultured / re-seeded / re-plated on days 6 to 14, preferably on days 10 to 12.

[0098] In embodiments, the method is for obtaining organoids lacking a neural rosette structure, and the method includes, in step c, culturing an initial cell culture containing pluripotent cells in a standard cell culture dish or plate for 5 to 13 days, preferably 8 to 12 days, or 9 to 11 days, and then transferring the cell culture to a micropatterned culture chip around 6 to 14 days, preferably 8 to 12 days, or 10 to 12 days.

[0099] In other words, in some embodiments, a method for generating a neural organoid cell culture model includes, in step c, culturing a cell culture containing pluripotent stem cells on a (standard / conventional) cell culture dish or plate for 5 to 13 days, preferably 8 to 12 days, or 9 to 11 days for initial (from day 0 of culture), and then transferring the cell culture to a micropattern culture chip (re-seeding / seeding) between days 6 and 14, preferably around days 8 to 12, or around days 10 to 12, or at that point, thereby generating neural organoids lacking neural rosette structures.

[0100] In the embodiment, the cell culture is first cultured in a standard cell culture dish or plate for at least 1 day, at least 1 to 6 days, at least 1 to 8 days, at least 1 to 12 days, at least 1 to 18 days, or 6, 12, or 18 days in step c., and then transferred to a micropattern cell culture chip.

[0101] In the embodiment, the cell culture is first cultured in a standard cell culture dish or plate in step c., and then transferred to a micropattern cell culture chip between days 1 to 18 of the culture, or on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 of the culture, or even later.

[0102] In embodiments, the method includes a step of transferring the culture to a micropattern after neural induction (approximately 8-12 days, or 10-12 days of culture), following a step of initial differentiation of pluripotent cells in a normal dish. In these embodiments, the method preferably includes a “feeding” protocol with minimal disruption to keep the organoids adhered for an extended period (e.g., >100 days). The protocol preferably includes permanent immersion of the organoids, specific aspiration and pipetting of the culture medium, and stepwise loading and removal of various differentiation media. In embodiments, the method preferably further includes replenishing the ECM coating at regular intervals and adding specific compounds at optimized times and concentrations to increase cell adhesion to the support (e.g., CEPT).

[0103] In the embodiment, a uniform neuroepithelial sheet appears during the total culture period from day 8 to day 12. In the embodiment, cells may be re-seeded (transferred to a new culture device) and / or divided (seeded with fewer cells than previously grown).

[0104] In the embodiment, after approximately 12 to 20 days, preferably 18 days of culture, at least a portion of the cells form a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, or a marginal structure containing neuronal and / or glial cell processes.

[0105] In the embodiment, after culturing for about 12 to 20 days, preferably 18 days, at least a portion of the cells form a neuronal rosette structure, which includes a defined core region containing differentiated (mature) neurons, progenitor cells, and glial cells, or a (thin) marginal structure containing neuronal and / or glial cell processes.

[0106] In the embodiment, cortical identity of stem cells / progenitor cells is preferably indicated / characterized by the expression of Foxg1 by at least a portion of the cells around day 20.

[0107] In the embodiments, cortical progenitor cells are defined by the expression of Sox2 and phosphovimentin (p-vim), and in the embodiments, they may also be identified as apical progenitor cells.

[0108] In the embodiment, the cell population preferably produces, from around day 20, intermediate progenitor cells characterized by at least a portion of cells that are positive for Tbr2+ staining, and / or newly formed postmittal neurons characterized by at least a portion of cells that express MAP2+.

[0109] In the embodiment, deep neurons (characterized by at least a portion of cells expressing Tbr1+, Tle4+, and Ctip2+) begin to emerge from day 20 onward, and preferably become the main population around day 60.

[0110] In the embodiment, “late-born” upper layer neurons (characterized by at least a portion of cells expressing Satb2+, Cux1+, and Brn2+ cells) begin to be produced around day 40, and their proportion preferably increases over time (for example, between days 60, 90, and 100).

[0111] In the embodiment, the astrocytes are preferably characterized by at least a portion of the cells expressing S100 beta and / or GFAP around day 70.

[0112] In this embodiment, basal progenitor cells, also known as outer radial glial cells (oRGs), are characterized by at least a portion of cells expressing HOPX, which is known to be a specific oRG marker (Pollen et al., Cell 163, 56-67, 2015).

[0113] In the embodiment, around day 50, at least a portion of the cells express HOPX, and preferably, the number of cells expressing HOPX increases over time.

[0114] In another aspect or embodiment of the present invention as described herein, the present invention is a method for generating an adhesive neural organoid cell culture model, a) Coating a cell culture vessel, such as a plate, dish, or micropattern chip, with at least one extracellular matrix (ECM) protein or component, preferably laminin, b) Preferably, a cell culture medium comprising at least one Rho-related protein kinase inhibitor (ROCKi) and optionally at least one extracellular matrix (ECM) protein or component is added. c) Adding pluripotent stem cells to the culture medium, d) The cell cultures are cultured for at least 10 days. e) Optionally, change the culture medium every 2 to 10 days. e) Optionally, if the initial cell culture vessel in step a) is a plate or dish, the cell culture is transferred to a micropattern chip after a total culture period of 1 to 12 days. The present invention relates to a method for which the total duration of cell culture is at least 18 days, preferably at least 30 days, or more preferably 50 days or longer.

[0115] In the embodiment, in step b), the cell culture medium further comprises at least one WNT(pathway) inhibitor.

[0116] In the embodiment, in step b), the cell culture medium comprises at least one Rho-related protein kinase inhibitor (ROCKi) and / or a CEPT cocktail.

[0117] In the embodiment, once the neural rosette structure appears, the method may change the culture medium every other day. In the embodiment, around 16 to 20 days of the total culture period, the cells may be divided and further propagated, preferably by subculturing with Accutase and / or a mild dissociation reagent for organoids.

[0118] In this embodiment, substantial neurogenesis occurs between day 20 and day 30, and the culture medium is optionally changed every other day from that point onward. In this embodiment, a cocktail of neurotrophic factors BDNF, NT3, GDNF, cAMP, and ascorbic acid is optionally added to NMM medium supplemented with VA to promote cell survival and differentiation.

[0119] In this embodiment, the cell culture medium is Stem Flex medium.

[0120] In the embodiment, the cell culture medium is a nerve maintenance medium (NMM) containing a 1:1 mixture of N-2 and B-27.

[0121] In the embodiment, the cell culture medium is an N-2 medium comprising DMEM / F-12 GlutaMAX, 1×N2 supplement, insulin (e.g., 5 μg / mL), L-glutamine (e.g., 1 mM), MEM non-essential amino acids (e.g., 100 μM), 2-mercaptoethanol (e.g., 100 μM), and / or an antibiotic-antifungal agent (e.g., 0.5×).

[0122] In the embodiment, the cell culture medium is B-27 medium comprising neurobasal medium, 1× B-27 (containing or not containing vitamin A), L-glutamine (e.g., 2.5 mM), and / or 0.5× antibiotic-antifungal agent (e.g., 0.5×).

[0123] In this embodiment, the cell culture medium is a neuroinduction medium (NI) containing NMM, SB431542 (e.g., 10 μM), and / or LDN-193189 (e.g., 0.2 μM).

[0124] In another aspect, the present invention relates to an in vitro neural organoid cell culture model prepared according to the method described herein.

[0125] In embodiments, the present invention relates to an in vitro neural organoid cell culture model prepared according to the method of the present invention, wherein the model includes an adhesive neural organoid having a defined spatial structure and a regular eccentricity, comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of the processes of neuronal cells and / or glial cells in cell culture.

[0126] In embodiments, the model is prepared according to the method of the present invention and includes a defined core region having differentiated neurons, progenitor cells, and glial cells, surrounded by the edges of the processes of neuronal cells and / or glial cells.

[0127] In embodiments, the model further includes one or more astrocytes, superneurons, and deep neurons. In embodiments, at least a portion of the deep neurons are localized to the core of the neural organoid, and / or at least a portion of the superneurons are localized to the edge of the neural organoid, thereby mimicking the inside-out development of the mammalian brain.

[0128] In this embodiment, the organoid model self-organizes into a structure having a compact core of nuclei at the center and neuronal and glial cell processes at the ends. This organoid preferably mimics the inside-out development of the mammalian brain, exhibiting pattern formation towards deep neurons at the center and towards higher-layer neurons at the ends.

[0129] In the embodiments, the model is prepared according to the method of the present invention, and the model includes a neural rosette structure in the center or central region of the neural organoid.

[0130] In embodiments of cell culture models, the neuronal organoid includes a neuronal rosette structure at its center, the center includes one or more cell types, comprising one or more different types of progenitor cells and / or glial cells, and / or the cell culture model includes thin / inconspicuous edges of the neuronal and / or glial cell processes.

[0131] In the embodiments, thin / less prominent edges of nerve cell and / or glial cell processes refer to edges that constitute less than 2.5%, 5%, 10%, 15%, 20%, 25%, or 30% of the size of the organoid or the area covered by the organoid.

[0132] In this embodiment, the organoid model self-organizes into a structure having a neural rosette in the center or central region of a neural organoid. This mimics the ventricular zone of a developing brain.

[0133] The organoid model according to the present invention is preferably highly reproducible and scalable.

[0134] In the embodiment, the cell culture model includes cortical progenitor neurons, and at least a portion of the cells, preferably at least 1-70%, express Foxg1 or Sox2 and / or phosphovimentin (p-vim).

[0135] In the embodiment, the cell culture model includes deep neurons, and at least a portion of the cells, preferably at least 1-70%, express Tbr1, Tle4, and / or Ctip2.

[0136] In the embodiment, the cell culture model includes upper layer neurons, and at least a portion of the cells, preferably at least 1-70%, express Satb2, Cux1, and / or Brn2.

[0137] In the embodiment, the cell culture model includes astrocytes, and at least a portion of the cells, preferably at least 1-70%, express S100 beta and / or GFAP.

[0138] In the embodiment, the cell culture model includes basal progenitor cells, also known as outer radial glia (oRGs), in which at least a portion of the cells express HOPX.

[0139] In this embodiment, at least a portion of the neurons in the cell culture model are capable of induced and / or spontaneous neuronal activity.

[0140] In the embodiment, at least two organoids are cultured in the same culture dish or well, and at least a portion of the organoids forms neural connections with adjacent organoids in the same culture dish or well.

[0141] In one embodiment, the present invention relates to an in vitro adhesive neural organoid cell culture model comprising an organoid having a defined spatial structure and a regular eccentricity, comprising a core region comprising differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of processes of neuronal cells and / or glial cells.

[0142] In one embodiment, the present invention relates to an in vitro adhesive neural organoid cell culture model comprising an organoid having a core region with a regular eccentricity, comprising differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of processes of neuronal cells and / or glial cells.

[0143] In a preferred embodiment, the organoid includes astrocytes, superneurons, and deep neurons, wherein at least a portion of the deep neurons are localized to the center of the organoid, and / or at least a portion of the superneurons are localized to the edge of the organoid.

[0144] In a preferred embodiment, the organoid includes a nerve rosette structure in the center or central region of the nerve organoid.

[0145] In embodiments, the organoid of the present invention, particularly the core region of the organoid of the present invention, exhibits a regular eccentricity, preferably a shape having an eccentricity of 0.8 or less. In embodiments, the eccentricity of the core region 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 region is 0.1 to 0.8, preferably 0.2 to 0.7, more preferably 0.3 to 0.7, or any value within that range. In embodiments, the eccentricity of the organoid and / or core region is determined, for example, based on the area of ​​the core region as viewed from above in an adherent culture.

[0146] In embodiments relating to a cell culture plate or micropatterned cell culture chip (or micropatterned coverslip) including an organoid model according to the present invention, preferably at least 30%, 40%, 50%, 60%, or 70% or more of the organoids contained on the plate or micropatterned chip represent core regions having an eccentricity of 0.8, 0.7, or preferably less than 0.6.

[0147] In the embodiments, preferably, in the embodiments relating to a cell culture plate or micropatterned cell culture chip (or micropatterned coverslip) including the organoid model according to the present invention, the organoid has a maximum size of approximately 300 to 700 μm in diameter, preferably approximately 500 μm in diameter.

[0148] In this embodiment, the core region of the organoid is approximately 10 to 200 μm. 2 Preferably, 20-100 μm 2 , more preferably 30-60 μm 2 It has the area of ​​[this area]. In the embodiment, the area of ​​the organoid core is evaluated from above in adherent culture.

[0149] Examples of organoids and their physical properties are shown in Figure 2 and described in the following examples. For organoid type 1 (for example, in step c, the cell culture is first cultured in a cell culture dish or plate for 8 to 12 days, and then transferred to a micropattern cell culture chip for further culture), the inventors have found that the size is 0.02 to 0.08 mm. 2 The physical characteristics of the organoids were determined, including the core area and an eccentricity cutoff of <0.8 (i.e., organoids with an optimally non-round core area were excluded). These characteristics may also be relevant to type 2 organoids, which in embodiments include neural rosette structures.

[0150] In embodiments, the cell culture model includes a central core and outer processes after 12 to 30 days of culture, preferably after about 25 days. In embodiments, the central cells of the cell culture model include progenitor cells and neurons (TBR2 and / or MAP2 positive) after 12 to 30 days of culture, preferably after about 25 days, which are preferably positive for p-vimentin. In embodiments, in a later stage, the cell culture model includes upper layer neurons (BRN2 positive) at the ends of the core after 50 to 70 days of culture, preferably after about 60 days, and astrocytes (GFAP and S100b positive) from 50 to 70 days of culture onward, preferably from about 60 days of culture onward.

[0151] In another embodiment, the present invention preferably relates to a kit suitable for carrying out the method of the present invention, the kit comprising a culture medium and 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, emricasane, polyamine solution, and trans-ISRIB, The present invention relates to a kit comprising at least one supplement selected from the group comprising one or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP, and ascorbic acid.

[0152] In the embodiment, the kit optionally includes a micropattern cell culture chip.

[0153] In the embodiments, the kit optionally includes an in vitro neural organoid model according to the present invention.

[0154] In one embodiment, the kit further includes an in vitro neural organoid model according to the present invention.

[0155] In another embodiment, 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, a polyamine solution, and trans-ISRIB, at least one WNT (pathway) inhibitor, and one or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP, and ascorbic acid.

[0156] In one embodiment, the present invention relates to a cell culture plate or a micropatterned cell culture chip (or micropatterned coverslip) containing an in vitro neural organoid model according to the present invention. In a preferred embodiment, the in vitro neural organoid model is attached to (adhered to) the cell culture plate or micropatterned cell culture chip.

[0157] In the embodiment, the cell culture plate is a multi-well plate, and each well contains at least two (adhered) organoid models. In the embodiment, the multi-well plate is a 96-well plate. In the embodiment, the multi-well plate is a 2, 4, 6, 8, 10, 12, 24, or 96-well plate. In the embodiment, the multi-well plate contains two or more wells, preferably at least six, more preferably at least twelve, or 96 wells.

[0158] In one embodiment, the cell culture plate is a multiwell plate containing 1 to 700, or about 600 to 700, for example, 670 (adhesive) organoid models (organoids). In another embodiment, the cell culture plate is a multiwell plate, for example, a 96-well plate, where each well contains 1 to 100, 1 to 25, or 12 to 15 (adhesive) organoid models (organoids).

[0159] In the embodiment, the cell culture plate is a micropatterned cell culture chip (or micropatterned coverslip) containing an in vitro neural organoid model according to the present invention, and the plate contains at least 10, 25, 50, 100, or preferably 10 to 1000, more preferably 100 to 800 (adhesive) 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 (adhesive) in vitro neural organoid models (organoids).

[0160] In the embodiment, the cell culture plate includes a micropattern structure. In the embodiment, the cell culture plate is a multi-well plate, and each well includes a micropattern structure.

[0161] In one embodiment, the present invention is preferably a computer-implemented method for analyzing a neural organoid cell culture model according to the present invention, wherein the method is i. To provide multiple spatially distinct neural organoids in cell culture, ii. Determine at least one spatial parameter of at least one organoid provided in i. iii. Using machine learning, preferably deep learning, to analyze at least one spatial parameter of at least one organoid to determine the presence, location, spatial ratio, shape, and / or size of at least one region and / or cell type of at least one organoid. iv. A computer implementation method comprising optionally providing and / or displaying on a screen a data summary including the results of the analysis in step iii, and optionally providing a graph or image of the analyzed organoids.

[0162] As shown in the following example, Figure 3(E) shows a deep learning classification network trained on a portion of an image, demonstrating that this network can separate WT states from mutant states with high accuracy, and illustrating the effectiveness of the method, for example, in determining neuronal phenotypes for screening purposes.

[0163] In embodiments of the present invention, a computer-implemented method for analyzing a neural organoid cell culture model enables accurate and reproducible measurement and characterization of the organoid model described herein, and is therefore suitable for identifying potentially small differences in the phenotypic or structural properties or function of the organoid model after any given treatment or between potentially two different disease states (disclosed in more detail below and in the examples).

[0164] By enabling the training of deep learning classification networks on images of organoid models, potential parameters, markers, or other characteristics of organoids can be identified in any given treatment group, and even small changes between groups can be identified with high accuracy. This is particularly important in screening applications where treatment versus non-treatment should be carefully evaluated to identify the potential therapeutic effects of candidate substances. This is also important when evaluating disease states, thereby enabling the accurate identification of small changes between healthy organoid models and diseased organoid models (as in the examples).

[0165] In another aspect, the present invention relates to the use of an in vitro model according to the present invention for high-throughput drug or toxicity screening and / or for testing neurological disorders.

[0166] In this embodiment, the method facilitates the generation of patient-specific individual organoid cell culture models.

[0167] A non-limiting example is the generation of induced pluripotent stem cells (IPSCs) from patients with neuronal or neurodegenerative diseases for the creation of personalized organoid disease models.

[0168] In embodiments, disease cells may also be generated from stem cells containing artificially introduced or genetically engineered mutations (e.g., using CRISPR / Cas9 or a CRISPR / Cas9 vector to induce specific disease-associated mutations in (patient-derived) cells). In embodiments, this approach may also be applied to patient-specific iPS cells, which may be combined with specific mutations that could not be obtained from the patient by biopsy, such as tissue-specific mutations, to generate patient-specific models that include a patient-specific genetic background.

[0169] In other embodiments, the model may be developed from patient-specific iPS cells and may also be used to test diseases and / or treatments for diseases in a patient-specific background, for example, in the case of patient-specific germline mutations, so that the model can function as a personalized neural organoid model for affected or susceptible patients.

[0170] In embodiments, the present invention may be a means for testing diseases affecting the human brain. The model comprises a first differentiated neural organoid model or (cortical) brain organoid model generated from human PSCs.

[0171] In one embodiment, the method of the present invention is suitable for generating miniaturized organoids for phenotypic determination of neurological disorders.

[0172] In embodiments, PSCs containing (preferably heterologous) mutations in DYRK1A can be used to generate disease-specific, potentially individualized organoid models. Mutations in DYRK1A typically result in DYRK1A syndrome, a severe neurodevelopmental disorder associated with autism spectrum disorder, among other things.

[0173] In this embodiment, disease-specific, potentially individualized organoid models can be generated using PSCs containing mutations in apolipoprotein E (APOE), for example, allowing comparison between WT cells with APOE3 / 3 and mutant cells with APOE3 / 4. Mutations in apolipoprotein E (APOE) are a common risk factor for sporadic Alzheimer's disease.

[0174] The method in the embodiments described herein constitutes a novel procedure / workflow of the present invention for the continuous addition and removal of specific factors and / or signaling molecules to and from the cell culture medium of early pluripotent stem cells, thereby enabling the generation of differentiated neural organoid models or (cortical) brain organoid models suitable for long-term culture of at least 30 days, preferably at least 100 days.

[0175] In the embodiment, the first cell culture comprises the patient's pluripotent stem cells, preferably the patient's iPS cells, and the patient has been diagnosed with and / or suffers from a brain disease, neurological disease, neurodegenerative disease, brain injury, or any other disease or condition affecting the patient's brain.

[0176] Therefore, the method and model may, in embodiments, be applied using “susceptible” cells derived from patients diagnosed with and / or suffering from brain disease, neurological disease, neurodegenerative disease, brain injury, or any other disease or condition affecting the brain.

[0177] In embodiments of the in vitro cell culture model according to the present invention, the model is established from a patient's pluripotent stem cells, preferably the patient's iPS cells, and the patient has been diagnosed with and / or suffers from a brain disease, neurological disease, neurodegenerative disease, brain injury, or any other disease or condition affecting the patient's brain.

[0178] Prior art cell culture models cannot reflect the overall development of the brain and the diversity of cell types found in the human brain. This model may be further used for patient stratification and / or to analyze the individual disease outcomes, treatments, and / or circumstances of patients, such as in the context of personalized medicine. Accordingly, in embodiments, this method and / or model may be used in the context of personalized medicine for the development / selection of a suitable treatment for an individual patient, from which the initial pluripotent starting cell culture of this model, as iPS cells, is derived.

[0179] This is particularly advantageous for patients diagnosed with diseases such as brain diseases, neurological diseases, neurodegenerative diseases, brain injuries, or any other diseases or conditions affecting the brain of the patient, each patient exhibiting individual physiological patterns of disease outcomes, such as individual outcomes and disease progression in neurodegenerative diseases. The method and model facilitate the generation of cell culture models that mimic individual phenotypes of, for example, brain diseases, neurological diseases, neurodegenerative diseases, brain injuries, or any other diseases or conditions affecting the brain of an individual patient.

[0180] In another aspect, the present invention relates to the use of an in vitro cell culture model according to the present invention for drug screening, toxicity screening, and / or testing for diseases, such as brain diseases, neurological diseases, neurodegenerative diseases, brain injuries, or any other diseases or conditions affecting the brain of an individual patient.

[0181] In another aspect, the present invention relates to a method for testing a candidate compound, wherein the method is a. To provide a cell culture model according to the present invention. b. Contacting the candidate compound with the cell culture model, c. To read out, characterize, and / or detect one or more parameters of a cell culture model, d. The present invention relates to a method comprising, optionally, repeating steps a to c using one or more further candidate compounds.

[0182] In embodiments, the readout or parameter in step c. includes the viability and / or proliferation of the model. Those skilled in the art are aware of different methods for detecting the viability and / or proliferation of cell cultures. Examples of methods for detecting the viability and / or proliferation of cell cultures include 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.

[0183] In the embodiment, step c. involves detecting the readout or parameter by using one or more of the following methods: microscopy, image analysis (e.g., by plate reader, microscope, or camera), current clamp recording, optogenetic activation analysis, immunocytochemistry (ICC), immunohistochemistry, immunofluorescence analysis, droplet-based microfluidic techniques, fluorescence-activated cell sorting (FACS), IHC, ELISA, and / or spectrophotometric methods.

[0184] In embodiments, the readout or parameter may be a characteristic, shape, and / or pattern of the model or a specific cell contained therein. In embodiments, an indefinite list of parameters or characteristics may be, for example, the expression of a specific gene or protein, developmental stage, cell length, appearance, and / or shape, cell length, organization, and / or orientation, neurotransmitter migration and / or efflux, or other functional characteristics of neurons.

[0185] In embodiments, the screening method and system may use software and hardware commonly used in (pharmaceutical) compound screening systems. Automated setups are known to those skilled in the art and can be adapted accordingly. Thus, screening formats already established in the art may be used to carry out the method and system of the present invention.

[0186] In the embodiment, the evaluation is preferably performed in triplicate to discard nonspecific readouts or signals.

[0187] In another aspect, the present invention relates to a method for evaluating the toxicity of a candidate substance, a. To provide a cell culture model according to the present invention. b. Contacting the candidate compound with the cell culture model, c. The method includes identifying a candidate substance as a safe candidate substance (a substance with low or no toxic effect) if the cell culture mode does not exhibit a cytotoxic effect, preferably a detectable effect.

[0188] Another aspect of the present invention is a system for identifying candidate substances, a. To provide a cell culture model according to the present invention. b. Optionally, at least one substance or substance library, or means for providing and / or utilizing such substance or substance library, c. The system comprises means for determining (screening) the effect of a substance or substance library on a cell culture model, for example, one or more compounds or compositions, a device and / or system configured (optionally for high-throughput screening) to identify the readout, characterization, and / or one or more parameters of a cell culture model.

[0189] In one embodiment, the present invention relates to a cell culture medium and / or supplement cocktail, preferably as described herein or derived from components of a cell culture medium or other supplement described herein.

[0190] In another aspect, the present invention relates to cell culture media as defined by one or more embodiments disclosed herein. Such culture media products may be provided as themselves, or their components may be provided in the form of kits of separated but combinable compounds or substances.

[0191] In another aspect, the present invention relates to a supplement cocktail as defined by one or more embodiments disclosed herein. The supplement cocktail product may be provided as a whole, or its components may be provided in the form of a kit of separate but combinable compounds or substances.

[0192] In another embodiment, the present invention relates to a culture medium and / or supplement cocktail comprising a ROCK inhibitor, Chroman 1, a CEPT cocktail (containing Chroman 1, Emricasan, a polyamine solution, and trans-ISRIB), at least one SMAD inhibitor, BDNF, NT-3, GDNF, cAMP, ascorbic acid (tbd), at least one WNT (pathway) inhibitor, and at least one ECM protein or component.

[0193] In one embodiment, the present invention relates to a cell culture medium and / or supplement cocktail, 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, emricasane, polyamine solution and trans-ISRIB, and / or One or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP, and ascorbic acid, This relates to a cell culture medium and / or supplement cocktail comprising, optionally, at least one WNT (pathway) inhibitor.

[0194] In the embodiment, the cell culture medium includes Stem Flex medium. In the embodiment, the cell culture medium is a neuronal maintenance medium (NMM) containing a 1:1 mixture of N-2 and B-27 medium. In the embodiment, the cell culture medium is an N-2 medium containing DMEM / F-12 GlutaMAX, 1×N2 supplement, insulin (e.g., 5 μg / mL), L-glutamine (e.g., 1 mM), MEM non-essential amino acids (e.g., 100 μM), 2-mercaptoethanol (e.g., 100 μM), and / or an antibiotic-antifungal agent (e.g., 0.5×). In the embodiment, the cell culture medium is a B-27 medium containing neurobasal medium, 1×B-27 (with or without vitamin A), L-glutamine (e.g., 2.5 mM), and / or a 0.5× antibiotic-antifungal agent (e.g., 0.5×). In this embodiment, the cell culture medium is a neuroinduction medium (NI) containing NMM, SB431542 (e.g., 10 μM), and / or LDN-193189 (e.g., 0.2 μM).

[0195] Any range derived from, using, or within the aforementioned numerical concentration ranges is also included in this disclosure. The aforementioned concentrations may also apply to embodiments of the kit described in more detail below.

[0196] This disclosure also includes kits, packages, and multi-container units containing the culture media and / or factors described herein, either separately or as a composition.

[0197] Any optional or preferred features of the Invention disclosed or described in the context of one aspect of the Invention are also disclosed in the context of other aspects of the Invention described herein. Embodiments and features of the Invention described with respect to the Method, cell culture model, and kit are deemed to be disclosed in respect to any other aspect of the Disclosure. Thus, features characterizing the Method may be used to characterize the cell culture model or kit, and vice versa. Various aspects of the Invention are preferably smaller (miniatured) than current neuronal organoid models but maintain the relevant features of larger organoids and their in vivo counterparts, thereby being integrated, benefiting from, based on, and / or related to a common, remarkable finding of generating in vitro adhesive neuronal organoid cell culture models, including a defined spatial structure and regular eccentricity, including a defined core region containing differentiated (mature) neurons, progenitor cells, and glial cells surrounded by the edges of neuronal and / or glial cell processes. [Modes for carrying out the invention]

[0198] All cited patent and non-patent documents are incorporated herein by reference in their entirety.

[0199] The present invention relates to an in vitro method for generating a neural organoid cell culture model. The present invention relates to an in vitro adhesive neural organoid cell culture model comprising an organoid having a defined spatial structure and a regular eccentricity, including a core region comprising differentiated (mature) neurons, progenitor cells, and glial cells, as well as the edges of neuronal and / or glial cell processes.

[0200] The terms “subject” or “patient” include 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 particular disease.

[0201] In this specification, “neuronal activity” generally refers to the ability of a neuron cell to spontaneously or induced generate in vitro action potentials and intracellular calcium transients. Neuron activity may also, in embodiments, involve electrical and / or chemical synaptic transmission and / or functional axonal connectivity.

[0202] cell culture In this context, the term “pluripotent stem cell” encompasses any cell possessing pluripotent properties, as understood by those skilled in the art. This term includes pluripotent stem cells derived from embryos, and induced pluripotent stem cells derived from other cell types, such as somatic cells (e.g., skin cells, fibroblasts, blood cells, or any other somatic cells). Pluripotent stem cells may generally be derived from stem cell cultures established without the destruction of a donor embryo. Pluripotent stem cells may be healthy pluripotent stem cells derived from a healthy organism, or disease-affected pluripotent stem cells derived from an organism suffering from a disease.

[0203] In embodiments, the early pluripotent stem cells according to the present invention are pluripotent or multipotent mammalian cells. Preferably, the early pluripotent stem cells are human induced pluripotent stem cells (iPSCs). iPSCs are pluripotent stem cells that are directly generated from adult cells. iPSCs can give rise to any other cell type in the body or in individual mammalian organisms and have the ability to proliferate indefinitely in cell culture. Advantageously, iPSCs can be directly derived from adult cells or tissues and do not require the use of embryo-derived cells. iPSCs can be generated from patient cells and thus have the advantage of being patient-specific. Furthermore, iPSCs and iPSC-derived cells can be used for personalized drug discovery, toxicity screening, and disease research to understand the patient-specific basis of disease. In embodiments, this also applies to the cell culture model of the present invention, which may also be derived from human patient-specific iPSCs. iPSCs are generally induced by introducing the product of a specific set of pluripotency-related genes, or "reprogramming factors," into a specific cell type, such as the original reprogramming transcription factors Oct4(Pou5f1), Sox2, c-Myc, and Klf4. In embodiments of this common combination, each factor may be functionally replaced by a related transcription factor, or even other genes such as small molecules, miRNAs, or lineage designators.

[0204] Cell culture (also called tissue culture) is the process of growing cells under controlled conditions, generally outside their natural environment. Generally, cells are cultured in suitable culture vessels, such as plates, wells, well plates, or flasks, along with a substrate or cell culture medium that provides essential nutrients (e.g., one or more amino acids, carbohydrates, vitamins, minerals, etc.) and, optionally, one or more growth factors, hormones, and gases (CO2, O2), and regulates the physicochemical environment (pH buffer, osmotic pressure, etc.). Depending on the purpose of cell culture, the medium may be appropriately selected, and those skilled in the art recognize suitable selections of culture conditions, such as containers, media, temperature, and incubators.

[0205] "Progenitor cells" generally refer to neuronal progenitor cells. In embodiments, radial glial cells are progenitor cells of both intermediate and mature neurons, and the intermediate cell state preferably does not have projections.

[0206] (Terminal) differentiated neurons may also be called mature neurons or postmittal neurons. Differentiated neurons may, in embodiments, be characterized by nuclear higher-order structures and / or the expression of one or more genes, including, but not limited to, NeuN, MAP2, vGLUT1 (e.g., in excitatory glutamatergic neurons), GAD67 (e.g., in inhibitory neurons), and / or GFAP or S100 beta (e.g., in astrocytes).

[0207] "Neuron and / or glial cell processes" generally refer to potentially long (extended) cytoplasmic processes that extend from the cell body of individual cells. Neuron processes can be formed by mature neurons and radial glial cells. In this model, so-called "intermediate progenitor cells" preferably do not form processes.

[0208] In this specification, the term “neuronal rosette structure” refers to an organoid structure comprising one or more progenitor cells and / or glial cells in a central region, and thin or inconspicuous margins of nerve and / or glial cell processes. As used herein, thin or inconspicuous margins preferably refer to margins that constitute 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 less than 2.5%, 5%, 10%, 15%, 20%, 25%, or 30% of the area of ​​the organoid as evaluated from above in adherent culture. The organoids of the present invention, in particular the neural rosette structures that may occur in the second model, are characterized by a radial organization of cells surrounding a central lumen, which is identified by the expression of the apical junction marker ZO-1. A second model referred to herein relates to an embodiment in which, in step c., cell cultures are cultured on a micropattern cell culture chip from day 0 onward to generate a neural organoid, wherein the neural organoid includes a neural rosette structure in the central region of the neural organoid.

[0209] Generally, the term “organoid” refers to a small, self-organized, three-dimensional tissue culture of cells, preferably generated from pluripotent cells. Neuronal organoids are small, self-organized, three-dimensional tissue cultures containing neuronal cells and, preferably, other (auxiliary) cells commonly found in the brain, and neuronal organoids are preferably generated from pluripotent cells and, in the context of the present invention, preferably resemble a simplified, miniaturized model of the brain.

[0210] As used herein, “defined spatial structure” refers to an organoid comprising a core region and a marginal region having cellular processes. In preferred embodiments, “defined spatial structure” refers to an organoid comprising a core region comprising differentiated (mature) neurons, progenitor cells, and glial cells, and a marginal region comprising processes of neuronal cells and / or glial cells.

[0211] As used herein, “regular eccentricity” preferably refers to a circular or substantially circular shape. The eccentricity can be determined by routine technical means, such as those described in the following embodiments. Quantitative measurement of the eccentricity is provided in the embodiments. As is commonly used in the art, the eccentricity is typically in the range of 1 to 0, where an eccentricity of 1 results in a straight line shape, and an eccentricity of 0 results in a circular shape. In embodiments, the formula for determining the eccentricity of an ellipse, or the shape of the organoid of the present invention, is the distance between the foci of the ellipse divided by the length of the major axis. In embodiments, the “regular eccentricity” of the organoid of the present invention is 0.8 or less. Further embodiments are provided herein.

[0212] In this specification, the term “adhesion” refers to in vitro adhesive neural organoid cell culture models. The term “adhesion” relates to the property of a neural organoid model to adhere to a cell culture vessel or support during culture. In preferred embodiments, “adhesion” refers to a consistent state of adhesion of cells or organoids to the cell culture vessel / support throughout the entire culture in the cell culture vessel / support. In preferred embodiments, the model is adhesive during the culture period, for example, during cell culture for at least 18 days. In some embodiments, the term “adhesion” refers to the direct and consistent adhesion of organoids to the cell culture vessel / support itself, rather than merely “embedding” the organoids to a gel or matrix (e.g., Geltrex) in the cell culture vessel. In embodiments, the model is adhesive to the cell culture vessel / support itself and / or to the ECM, for example, the model is adhesive to a vessel coated with at least one extracellular matrix (ECM) protein or component.

[0213] Generally speaking, the "extracellular matrix" (ECM) refers to a network of extracellular macromolecules that, in addition to water, includes various glycoproteins, polysaccharides, enzymes, nutrients (e.g., amino acids, glucose), tissue hormones, and electrolytes. The ECM provides structural and biochemical support to the surrounding cells.

[0214] In detail, the extracellular matrix (ECM) consists of fibrous components (fibers) and fluids (matrix containing soluble components and nutrients). The major protein family present in the ECM is collagen, which forms different types of fibers and is found in almost all tissues. Elastic fibers are formed from the proteins fibrillin and elastin. Furthermore, there are a wide variety of adhesive matrix proteins that connect cells to the ECM. A second large group is carbohydrates, particularly glycosaminoglycans, the highly specific individual components of long-chain polysaccharides. Glycosaminoglycans bind to proteins to form even larger macromolecules called proteoglycans. The diversity and interactions of proteins, glycosaminoglycans, and proteoglycans give rise to the characteristics of the ECM.

[0215] Geltrex (trademark) is an LDEV-free Reduced Growth Factor Basement, a soluble form of basement membrane marketed by Thermo Fisher Scientific.

[0216] Cell culture materials and supplements LDN-193189 (DM3189; LDN) is a selective and highly potent small molecule BMP receptor signaling inhibitor that inhibits BMP type I receptors ALK1 (TGF-B superfamily type I receptor), ALK2 (TGF-B superfamily type I receptor), ALK3 (BMP type 1A receptor), and ALK6 (BMP type 1B receptor) with IC50 values ​​of 0.8 nM, 0.8 nM, 5.3 nM, and 16.7 nM, respectively, in kinase assays. LDN-193189 inhibits the transcriptional activity of BMP type I receptors ALK2 and ALK3. LDN-193189 is considered a TGF-beta / Smad signaling inhibitor.

[0217] SB431542 (SB) is a potent and selective inhibitor of ALK5 (transforming growth factor beta receptor 1) and therefore a TGFβ pathway inhibitor with an IC50 of 94 nM. SB431542 is considered an inhibitor of TGF-beta / Smad signaling (TGF-beta / Smad inhibitor).

[0218] In the context of the present invention, any SMAD signaling inhibitor may be used instead of, or in lieu of, either of the two compounds exemplified herein, SB431542 and LDN-193189.

[0219] SMAD (or Smad) is a family of structurally similar proteins that are major signaling molecules for transforming growth factor beta (TGFb) superfamily receptors, and they are important for regulating cell development and growth.

[0220] In the embodiments described herein, any compound, inhibitor, and / or activator compound may be a small molecule compound such as the SMAD inhibitor LDN-193189.

[0221] In general, the (canonical) Wnt / β-catenin pathway (also abbreviated as the WNT pathway) is well-characterized in the art, and the WNT gene family comprises structurally related genes encoding secreted signaling proteins, which are involved in different developmental processes, such as the regulation of cell fate and embryonic pattern formation. The (canonical) Wnt / β-catenin pathway is thought to contribute to the stabilization of cytoplasmic β-catenin and, in combination with transcription factors and coactivators CBP, p300, TCF, LEF, PYGO, or BCL9, activates the transcription of target genes. An inactive Wnt pathway generally leads to the degradation of β-catenin, thereby making it phosphorylated, ubiquitinated, and susceptible to proteasomal degradation. β-catenin degradation is generally mediated by APC, AXINcasein kinase 1α (CK-1α), and glycogen synthase kinase 3β (GSK3β).

[0222] Examples of "WNT inhibitors," or inhibitors of the WNT pathway (inhibitors of WNT signaling), include, in embodiment, IWR-1-endo (IWR1e), IWP-2, JW74, IWP-4, CCT251545, KY1220, iCRT14, iCRT3, LF3, PNU-74654, KYA1797K, KY02111, Zamaporvint (RXC004), M2912, and M43. The group may be selected from, but is not limited to, 5-1279, KY-05009, prodigiosin, ginsenoside Rh4, heparan sulfate, RCM-1, recibugenin, MSAB, PRI-724, tegatrabetan (BC-2059), JW55, adavivint (SM04690), tryptonide, isoquercitrin, IQ-1, sarinomycin, and FH535.

[0223] In the context of the present invention, the terms "WNT inhibitor," "WNT pathway inhibitor," "inhibitor of the WNT pathway," and "inhibitor of WNT signaling" may be used interchangeably.

[0224] Generally, "CEPT" or "CEPT cocktail" refers to a mixture of chroman 1, emricasane, polyamine solution, and trans-ISRIB. 24 H 28 N4O4 (also referred to herein as "Kroman") is a potent and selective ROCK2 inhibitor thought to improve cell survival. Emricasane is a potent pancaspase inhibitor thought to promote hPSC survival in combination with other small molecules. In the context of this invention, both "Kroman 1" and "Kroman" refer to the ROCK2 inhibitor "Kroman 1", CAS number: 1273579-40-0, C 24 H 28 This refers to N4O4, https: / / pubchem.ncbi.nlm.nih.gov / compound / 66577033. Trans-ISRIB, in combination with other small molecules, is thought to promote hPSC survival and act as an integrated stress response (ISR) inhibitor. Polyamine solutions / supplements are culture medium supplements that are thought to enhance cell growth and stem cell viability.

[0225] "Neurotrophic factors" as used herein refer to biomolecules that support the growth, survival, and differentiation of developing and mature neurons. In preferred embodiments, the neurotrophic factors include one or more of BDNF, NT3, GDNF, cAMP, and ascorbic acid.

[0226] A “micropatterned cell culture coverslip,” “micropatterned chip,” or “micropatterned cell culture dish,” or simply “micropattern” (these terms may be used interchangeably herein) refers to a culture vessel or support patterned with micrometer-sized sections of a predetermined geometric feature. Micropatterns are thought to improve (spatially defined) positioning and / or cell adhesion and / or promote the growth of cells cultured thereon.

[0227] High-throughput screening (HTS) is a scientific experimental method particularly relevant to the fields of biology and chemistry. For example, a system can be provided that enables a high-throughput screening approach using a combination of robotics and other specialized laboratory hardware. In the context of this invention, HTS allows those skilled in the art to effectively screen a large number of organisms or parts thereof simultaneously or sequentially, in vivo or in vitro. Commonly used laboratory equipment or test containers for HTS are 6, 24, and 48-well plates and microtiter plates, which are sometimes disposable, plastic containers 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, which has 8 × 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 wells contain test items according to the nature of the experiment, which may be adapted according to the properties of the test organisms and / or reference organisms described herein, as well as a library of substances to be tested as candidate substances. Screening facilities typically have a library of stock plates, the contents of which are carefully cataloged, each of which may be laboratory-produced or purchased from a commercial supplier.

[0228] For example, to prepare the assay, biological entities to be subjected to the experiment, such as cells, cell culture models, animals, or embryos of the test organisms and / or reference organisms described herein, may be seeded in each well of the plate. After an incubation period that allows the biomaterial to absorb, bind to, or otherwise react with (or not react with) the substances in the wells, e.g., candidate drugs or compounds, measurements are performed on the wells of the plate by either a manual or automated procedure. Manual measurements may be required 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 dedicated automated analyzer may be used. Depending on the results of the HTS, a follow-up assay may be performed, for example, within the same screening, by identifying the wells in which the desired reaction occurred (also known as "hits"), transferring the reactive components to a new assay plate, and repeating the experiment.

[0229] Means for automating the present invention are also known to those skilled in the art. Automation is an advantageous element in HTS. Typically, an integrated system consisting of one or more robots transfers assay microplates between stations for the addition, mixing, incubation, and final readout or detection of samples and reagents. HTS systems can typically prepare, incubate, and analyze multiple plates in parallel, further accelerating the process.

[0230] The term “candidate substance” means any substance used for (preferably high-throughput) screening. The properties of the compound, i.e., structure, purity, and quantity, may be stored in a chemical library database for reference. Candidate substances may further refer to any substance that a person skilled in the art would find suitable for the intended method and / or for the treatment of disorders affecting synaptic transmission between motor neurons and muscle cells.

[0231] The term "toxic effect" means any adverse effect of a candidate substance produced in an individual organism, a cell culture model, or a part thereof. The candidate substance preferably does not negatively affect the survival of the cell culture model. In embodiments, it may be desirable that the candidate substance alters certain parameters of the cell culture model toward a healthier or normal parameter range, for example, in a diseased cell culture model. In embodiments, the method may include a control cell culture model established from healthy stem cells (e.g., from a healthy donor) and at least one control cell culture model established from diseased stem cells (e.g., from a diseased donor or artificially manipulated).

[0232] In the context of this disclosure, the term “at least a portion” means a specific amount, number, or group, for example, 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%, 1 to 100%, 1 to 1 It can refer to 0%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90%, or approximately 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%.

[0233] The embodiments disclosed above may represent one aspect of the present invention. Any feature disclosed in the context of any given aspect or embodiment may be combined with other aspects or embodiments of the present invention.

[0234] drawing The present invention is further described by the following embodiments. These are not intended to limit the scope of the invention, but represent preferred embodiments of the invention provided for further illustration of the invention as described herein. [Brief explanation of the drawing]

[0235] [Figure 1] This involves the generation of two types of brain organoids.

[0236] [Figure 2] This is a QC measurement of organoids.

[0237] [Figure 3-1] This involves the use of miniaturized organoids for determining the phenotypic characteristics of neurological disorders. [Figure 3-2] This involves the use of miniaturized organoids for determining the phenotypic characteristics of neurological disorders.

[0238] [Figure 4] This is an exemplary protocol for generating reproducible neural organoids, including neural rosette structures.

[0239] Detailed explanation of the diagram Figure 1 shows two different brain organoid types produced in Example 1 at different time points. Type 1 refers to a first type (Type 1) in vitro neural organoid cell culture model including astrocytes, superior neurons, and deep neurons, where at least a portion of the deep neurons were localized to the center of the neural organoid and / or at least a portion of the superior neurons were localized to the edge of the neural organoid. In embodiments, the Type 1 organoid is prepared by a method in which the cell culture is first cultured in a cell culture dish or plate for, for example, 8-12 days, and then transferred to a micropatterned cell culture chip for further culture. The second type (Type 2) in vitro neural organoid cell culture model includes a defined spatial structure with a regular eccentricity, and the model includes a neural rosette structure at the center of the neural organoid. In the embodiment, Type 2 organoids are produced by a method in which cell cultures are cultured on a micropattern cell culture chip from day 0 onward to generate neural organoids that include a neural rosette structure in the central region of the neural organoid.

[0240] Figure 2 shows the QC measurements of the organoids. (A) An overview of a portion of a chip containing 80 organoids (stained gray for DAPI and green for p-vimentin). (B) Images of individual organoids are analyzed using deep learning to accurately segment the core region. (C) The area and eccentricity of the core organoid region are measured based on B and used for quality control.

[0241] Figure 3 shows the use of miniaturized organoids for phenotypic determination of neurological disorders. (A) Comparison of d20 organoids from WT-derived organisms with d20 organoids from an isogenic hESC strain with heterologous mutations in DYRK1A. This mutation results in an increase in the progenitor cell pool (TBR2) and a decrease in mature neurons (MAP2), suggesting a delay in cell cycle exit in the disease. (B) Quantification of markers shown in Panel A. Each data point corresponds to one organoid, highlighting the high statistical power of phenotypic determination that can be achieved with organoids. (C) Comparison of d15 organoids from WT-derived organisms with d15 organoids from an isogenic hESC strain with mutations in apolipoprotein E (APOE), WT: APOE3 / 3, mutation: APOE3 / 4, the most common risk factor for sporadic Alzheimer's disease. As quantified in (D), an increase in early-born neurons (DCX) and mature neurons (MAP2) can be observed in mutants. (E) A deep learning classification network trained on a portion of the image can separate the WT state from the mutant state with high accuracy, demonstrating the phenotypic effectiveness for screening purposes. Scale bar: 200 μm. *** : p-value < 0.001, **** : p-value < 0.0001.

[0242] Figure 4 shows that in this exemplary embodiment, cells were seeded at 450,000 cells per coverslip (19.5 mm × 19.5 mm), and the CEPT cocktail was added to the culture medium for 3 days starting on day 0 (d0). Subsequently, only component C (Kroman 1, a ROCK2 inhibitor) was added to the culture medium. Kroman 1 is a ROCK inhibitor similar to Y-27632 (a ROCK1 and ROCK2 inhibitor). Furthermore, a Wnt (pathway) inhibitor (IWR1e) was added at the start of the protocol, resulting in reproducible rosette formation at the center of the generated organoids. In detail, Figure 4A: A protocol according to one aspect of the present invention generates reproducible neural rosettes at the center of micropattern organoids. Cells at days 7, 10, and 20 are shown. B: Characterization of an organoid at day 9 (d9). A clear neural rosette can be seen at the center of the organoid. Cells stained with DAPI (from left to right) are shown for NCAD (adhesive junctions), SOX2 (neural progenitor cells), and Ki-67 (proliferation). [Examples]

[0243] The present invention is further illustrated by the following embodiments. These are not intended to limit the scope of the invention, but represent preferred embodiments of the invention, provided for further illustrative purposes of the invention as described herein.

[0244] Example 1 - Overview Similar to neural tube-forming organoids, the inventors fabricated neural organoids (brain organoids) with a diameter of approximately 500 μm. The protocol for growing the associated brain cells was adapted from previous protocols for larger brain organoids.

[0245] The main challenge in growing these organoids was keeping the cells attached to the micropatterns for the long period (up to 100 days or more) required for neural development. Since cells eventually tend to detach from the pattern, the longest time reported to date for growing cells on a micropattern is for neural tube-forming organoids (7 days).

[0246] Therefore, the inventors have developed several new strategies for keeping cells adhered for extended periods, which include, for example, Initial differentiation in a normal dish, and migration to micropatterns after neural induction (approximately 10-12 days),

[0247] A specific supply protocol with minimal disruption (keeping organoids constantly immersed, specific aspiration and pipetting of the culture medium, stepwise addition and removal of various differentiation media), Regular interval replenishment of laminin coating. The method includes the step of adding a specific compound (e.g., CEPT) at an optimized time and concentration to increase cell adhesion.

[0248] These steps have enabled the inventors to keep the organoids adhered for extended periods (up to 100 days, and potentially beyond 100 days).

[0249] The inventors were able to generate two distinct types of neural (brain) organoids, each comprising a defined spatial structure and regular eccentricity, including a defined core region containing differentiated (mature / post-mitotic) neurons, progenitor cells, and glial cells, surrounded by the edges of neuronal and / or glial cell processes.

[0250] The first type (Type 1) in vitro neural organoid cell culture model included astrocytes, superior neurons, and deep neurons, with at least a portion of the deep neurons localized to the center of the neural organoid and / or at least a portion of the superior neurons localized to the edge of the neural organoid.

[0251] The second type (Type 2) in vitro neural organoid cell culture model includes a defined spatial structure with a regular eccentricity, and the model includes a neural rosette structure at the center of the neural organoid.

[0252] To hierarchize the generated models, the inventors applied the following measurements for quality control (QC).

[0253] 1) Presence of central nuclei and lateral processes after 25 days. The cells initially produce progenitor cells and neurons (positive for TBR2 and MAP2 on day 25) in the center and have a positive process for p-vimentin. In the later stages, superior neurons at the periphery of the core (positive for BRN2 on day 60) and astrocytes appear (positive for GFAP and S100b, starting from day 60).

[0254] 2) Regarding organoid type (2), the presence of neural rosettes (easily visible with bright-field imaging, and capable of automated quality control using deep learning classifiers).

[0255] 3) Area and eccentricity of the central core region, which can be measured quantitatively using DAPI imaging or staining. This is shown in Figure 2 for organoid type 1, where the inventors determined that 0.02 mm was obtained for core region A. 2 <A<0.08mm 2 And use a QC value with an eccentricity cutoff e < 0.8 (i.e., exclude core areas that are not sufficiently rounded).

[0256] Example 2 - Protocol The protocol for obtaining the organoid model of the present invention is shown below.

[0257] Materials and culture medium: Stem Flex culture medium for stem cells.

[0258] Nerve maintenance medium (NMM): A 1:1 mixture containing N-2 and B-27.

[0259] ○N-2 medium consists of DMEM / F-12 GlutaMAX, 1×N2 supplement, 5 μg / mL insulin, 1 mM L-glutamine, 100 μM MEM non-essential amino acids, 100 μM 2-mercaptoethanol, and 0.5× antibiotic-antifungal agent.

[0260] ○ B-27 medium consists of Neurobasal medium, 1× B-27 (either with or without vitamin A), 2.5 mM L-glutamine, and 0.5× antibiotic-antimycotic.

[0261] Neural Induction Medium (NI): NMM + 10 μM SB431542 + 0.2 μM LDN.

[0262] Add 2-mercaptoethanol after N2 and B27 supplements and medium filtration.

[0263] Store the medium at 4°C and use it within 3 weeks.

[0264] Small molecule Y-27632 ROCK inhibitor (10 mM stock solution, working concentration 10 μM / 5 μM).

[0265] CEPT cocktail (Chroman 1 1 50 nM, Emricasan 5 μM, polyamine solution 1×, trans-ISRIB 0.7 μM).

[0266] SB431542 (10 mM stock solution, working concentration 10 μM).

[0267] LDN-193189 (2 mM stock solution, working concentration 0.2 μM).

[0268] BDNF (20 μg / mL stock solution, working concentration 20 ng / mL).

[0269] NT-3 (20 μg / mL stock solution, working concentration 20 ng / mL).

[0270] GDNF (tbd).

[0271] cAMP (tbd).

[0272] Ascorbic acid (tbd).

[0273] Method: Coating, 2D culture Coat a 35mm dish (or 6-well plate) with 10 μg / mL laminin and incubate at 37°C for at least 2 hours.

[0274] Wash once with PBS+ / + and add culture medium (SF+10μM ROCKi).

[0275] Chip, 500μm CYTOO chip Prepare a Petri dish with a Parafilm piece sealed at the bottom and place the tip facing upwards.

[0276] By adding approximately 500 μl per tip, the tip is coated with 20 μg / mL laminin.

[0277] Place them in an incubator at 37°C for at least 2 hours (or alternatively, at +4°C overnight).

[0278] Prepare 5 mL of PBS+ / + and place the tip into the wells of a 6-well plate (or 35 mm dish).

[0279] Perform five partial washes with PBS+ / + (remove 3 mL and add another 3 mL).

[0280] Perform two complete washes with PBS+ / + (remove all 5 mL, then add another 5 mL).

[0281] Add culture medium (SF + 10 μM ROCKi).

[0282] 500μm 4D cell chip Prepare a Petri dish with a Parafilm piece sealed at the bottom and place the tip facing upwards.

[0283] By adding approximately 500 μl per tip, the tip is coated with 20 μg / mL laminin.

[0284] Incubate for at least 2 hours (or alternatively, overnight at +4°C).

[0285] Seed cells directly onto the chip; no washing is required.

[0286] Seeding of stem cells Remove the medium and wash twice with PBS- / -.

[0287] Treat with 1 mL of Accutase at 37°C for 3 to 5 minutes.

[0288] Prepare stem cell medium supplemented with 10 μM ROCKi.

[0289] Add 1 mL of medium containing 10 μM ROCKi to stop the Accutase reaction.

[0290] Collect the cells into a 15 mL Falcon tube.

[0291] Add 2 to 3 mL of medium and centrifuge at 200×g for 5 minutes.

[0292] Add 1 mL of medium containing 10 μM ROCKi and resuspend the cells.

[0293] Count the cells with a cell counter (Cell Countess, ThermoFisher).

[0294] 2D culture and 500 μm CYTOO chips Seed a desired number of cells in a drop-wise manner onto a pre-coated and pre-warmed dish.

[0295] 500 μm 4D cell chips Dispense cells only onto the chip. Incubate the cells for 1 hour to allow them to adhere. After 1 hour, remove non-adherent cells and add fresh medium.

[0296] Number of cells to be seeded: 35mm dish (or one well in a 6-well plate): 1 million cells / well, Tip: 100,000-200,000 cells / well.

[0297] Neural induction on a 2D plane (or directly on a chip) 2D culture The cells are checked the day after plating (day 0). If the cells have reached approximately 100% confluence, the cells are washed once with PBS and 3 mL of nerve induction medium is added per well.

[0298] The nerve induction medium is changed and replaced daily by removing old medium (100% medium replacement).

[0299] Between day 8 and day 12 after plating, a uniform neuroepithelial sheet will appear. On day 10, neuroepithelial cells are harvested using Accutase, and organoids are generated on the chip by seeding small clumps while maintaining a 2D culture (see protocol above) and a mild dissociation reagent. Wash once with PBS- / -. Add 1 mL of mild dissociation reagent at room temperature for 2 minutes. Remove it and add 1 mL of NI medium + 5 μM ROCKi. Detach the cells using a scraper. Collect the cells and centrifuge at 200 × g for 3 minutes. Resuspend the cells in 1 mL of NI medium and seed 300 μl of the cell suspension onto a chip (1:3 ratio, approximately 1.2 × 10⁶). 6 Cells). Incubate overnight, then the next day, change the culture medium (NMM + 5μM ROCKi) and remove any suspended cells.

[0300] 500μm tip After directly seeding stem cells onto the chip, the protocol is the same except for the method of changing the culture medium. The organoids are maintained in culture until further processing or experimentation at the desired time.

[0301] Micropatterned culture medium exchange To avoid delamination, omit washing with PBS- / - and do not remove 100% of the old medium (3 mL). Instead, discard only 60% and replace it with fresh medium.

[0302] Using a p1000 pipette, remove 60% of the old medium (2 mL). Add fresh medium to one side of the well by slowly pipetting with a 5 mL serological pipette (Caution: Avoid direct rough surface fluid dynamics). Gently agitate the dish to homogenize the medium.

[0303] Neural differentiation and maturation in 2D After the appearance of the rosette, 16 to 20 days after nerve induction, the culture medium is changed every other day, the cells are divided, and further proliferation is carried out by subculturing using Accutase for 2D culture and a mild dissociation reagent for organoids.

[0304] The next day, change the culture medium (2 mL of NMM). Maintain the cells for another 4-6 days. The cells can proliferate further at this point by repeating the above steps. Seed the cells onto a micropattern (third division).

[0305] Substantial neurogenesis occurs between days 20 and 30, and medium changes may be made every other day. (Optional: Addition of a cocktail of neurotrophic factors BDNF, NT3, GDNF, cAMP, and ascorbic acid promotes cell survival and differentiation - NMM medium supplemented with VA). Seed cells onto a micropattern (fourth division).

[0306] From here, seed more cells for 2D culture: Resuspend 400 μl of cells from a 1 × 35 mm dish in 1 mL of culture medium.

[0307] 27–31 days after the initial plating, the culture is subcultured on a laminin-coated 35mm dish using Accutase as described above. Every 10 days, 5 μg / mL of laminin is directly added to the culture medium to maintain micropatterned organoids attached to coverslips for long-term culture.

[0308] Continue culturing the cells, subculturing them, and seeding them onto the micropattern, changing the culture medium every other day.

[0309] result: Cortical identity of stem cells / progenitor cells is highlighted by widespread Foxg1 expression around day 20. Cortical progenitor cells, also identified as apical progenitor cells, are defined by the expression of Sox2 and phosphovimentin (p-vim). From around day 20, this cell population gives rise to intermediate progenitor cells, mainly characterized by Tbr2+ staining, which then give rise to newly formed postmittal neurons (MAP2+).

[0310] From day 20 onwards, deep neurons (Tbr1+, Tle4+, Ctip2+) begin to emerge and become the dominant population around day 60. Later-developing superficial neurons (Satb2+, Cux1+, and Brn2+ cells) begin to be produced around day 40, but their proportion increases over time (days 60, 90, and 100).

[0311] To detect astrocytes, the inventors co-stained cells with S100 beta and GFAP around day 70. Furthermore, to detect basal progenitor cells, also known as outer radial glial cells (oRGs), the inventors checked the expression of HOPX, which is known to be a specific oRG marker (Pollen et al., Cell 163, 56-67, 2015). Around day 50 (not shown), HOPX expression began to increase over time (staining on day 92).

[0312] Single rosette micropattern Organoids characterized by prominent neural rosettes can be generated by directly seeding stem cells onto the chip from day 0, or by seeding early neural progenitor cells after neural induction on day 10. Importantly, the inventors observed that the timing of seeding on the microchip is a critical determinant of patterning. Furthermore, while increasing the number of seeded cells by 1.5 times can induce neural rosette formation, the timing of microchip seeding appears to be decisive.

[0313] Improvement of organoid adhesion To promote better organoid adhesion to the chip, the inventors use small molecules such as Y-27632 (ROCK inhibitor) or CEPT cocktail, which are known to improve cell viability and reduce cellular stress. Exogenous ECM components reduce cell / organoid detachment for long-term organoid culture. From day 20 after seeding, 500 μl of laminin-521 (5 μg / mL) is added directly to 2.5 mL of culture medium every 10 days.

[0314] Example 3 - Miniaturized organoid for phenotypic determination of neurological disorders.

[0315] As shown in Figure 3, the method of the present invention is suitable for generating miniaturized organoids for phenotypic determination of neurological disorders. Figure 3(A) shows a comparison of d20 organoids derived from wild-type organisms (WT) and d20 organoids derived from an isogeneic human pluripotent stem cell (hESC) line with heterologous mutations in DYRK1A. Mutations in DYRK1A typically result in DYRK1A syndrome, a severe neurodevelopmental disorder associated with autism spectrum disorder, among other things. This mutation results in an increase in the progenitor cell pool (TBR2) and a decrease in mature neurons (MAP2), suggesting a delay in cell cycle exit in the disease. Figure 3(B) shows the quantification of markers shown in panel A. Each data point corresponds to one organoid, highlighting the high statistical power of phenotypic determination that can be achieved with organoids.

[0316] Figure 3(C) shows a comparison between d15 organoids derived from wild-type (WT) cells and d15 organoids derived from an isogenic human pluripotent cell (hESC) strain with a mutation in apolipoprotein E (APOE) (WT: APOE3 / 3, Mutant: APOE3 / 4, the most common risk factor for sporadic Alzheimer's disease). As quantified in (D), an increase in prematurely born neurons (DCX) and mature neurons (MAP2) can be observed in the mutant. Furthermore, Figure 3(E) shows a deep learning classification network trained on a portion of the image, demonstrating that this network can separate the WT state from the mutant state with high accuracy, and showing the effectiveness of the method, for example, in determining neuronal phenotypes for screening purposes.

[0317] Example 4 - Optional use of additional Wnt inhibitors Depending on the context and the specific scientific question to be investigated, reproducible organoids containing neural rosette structures in the central region can be generated according to different embodiments of the present invention.

[0318] In this example, pluripotent stem cells were seeded on day 0 (d0) at a rate of 450,000 cells per coverslip (19.5 mm × 19.5 mm). Furthermore, on d0, a CEPT cocktail (containing chroman 1, emricasan, polyamine solution, and trans-ISRIB) was added to the cell culture medium for 3 days of culture.

[0319] Subsequently, the ROCK2 inhibitor Chroman 1 (one component of the CEPT cocktail), similar to the ROCK1 / ROCK2 inhibitor Y-27632, was added to the cell culture medium. Furthermore, the Wnt inhibitor (IWR1e) was added to the cell culture medium at the start of the culture protocol. Cell culture resulted in the reproducible formation of neural rosette structures in the central region of the obtained neural organoids, which remained adhered to the coverslip well beyond 18 days of culture. References

[0320] 1. Lancaster,M.A.et al.Cerebral organoids model human brain development and microcephaly.Nature 501,373-379(2013).

[0321] 2. Renner,M.et al.Self-organized developmental patterning and differentiation in cerebral organoids.The EMBO Journal 36,1316-1329(2017).

[0322] 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).

[0323] 4. Birey,F.et al.Assembly of functionally integrated human forebrain spheroids.Nature 545,54-59(2017).

[0324] 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).

[0325] 6. Sirenko,O.et al.Functional and Mechanistic Neurotoxicity Profiling Using Human iPSCDerived Neural 3D Cultures.Toxicological Sciences 167,58-76(2019).

[0326] 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).

[0327] 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).

[0328] 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).

[0329] 10. Knight,G.T.et al.Engineering induction of singular neural rosette emergence within hPSC-derived tissues.eLife 7,e37549(2018).

[0330] 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

1. An in vitro method for generating an adhesive neural organoid cell culture model, a. To provide cell cultures containing pluripotent stem cells, b. Adding at least one Rho-related protein kinase inhibitor (ROCKi) and / or at least one extracellular matrix (ECM) protein or component to the cell culture medium, c. The cell culture is cultured for at least 18 days. A method comprising generating an adhesive neural organoid in cell culture having a defined spatial structure and a regular eccentricity, comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of the processes of neuronal cells and / or glial cells.

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

3. The method according to claim 1 or 2, wherein at least one Rho-related protein kinase inhibitor (ROCKi) is added to the cell culture medium every 5 to 10 days, preferably at a concentration of 1 to 10 μM, for at least the first 18 days of culture.

4. The method according to any one of claims 1 to 3, wherein in step c, the cell culture is first cultured in a cell culture dish or plate for 8 to 12 days, and then transferred to a micropattern cell culture chip for further culture.

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

6. The method according to any one of claims 1 to 5, wherein the cell culture is cultured for a total culture period of at least 30 days.

7. The method according to any one of claims 1 to 6, wherein at least one further supplement is added to the cell culture medium, the supplement being a SMAD inhibitor, chroman, a CEPT cocktail comprising chroman, emricasan, a polyamine solution, and trans-ISRIB, and one or more neurotrophic factors selected from the group consisting of BDNF, NT3, GDNF, cAMP, and ascorbic acid.

8. The method according to any one of claims 1 to 7, wherein the pluripotent stem cells are induced pluripotent stem (ips) cells.

9. In step c, the cell culture initially contains at least 50,000 cells / cm². ２ Preferably, at least 100,000 cells / cm² ２ The method according to any one of claims 1 to 8, wherein the cells are cultured at the cell density.

10. An in vitro neural organoid cell culture model produced according to the method of any one of claims 1 to 9, wherein the model comprises an adherent neural organoid in cell culture having a defined spatial structure and a regular eccentricity, comprising a core region comprising differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of processes of neuronal cells and / or glial cells.

11. a. The model is generated according to the method of claim 4, wherein the organoid further comprises one or more astrocytes, upper neurons, and / or deep neurons, preferably at least a portion of the deep neurons localized in the core region of the neural organoid, and / or at least a portion of the upper neurons localized in the edge of the neural organoid, thereby mimicking the inside-out development of the mammalian brain, or b. The in vitro model according to claim 10, wherein the model is generated according to the method of claim 5, and the organoid includes a neural rosette structure in the central region of the neural organoid.

12. The aforementioned organoid, a. Cortical progenitor neurons in which at least a portion of the cells express Foxg1 or Sox2 and / or phosphovimentin (p-vim), b. Deep neurons in which at least a portion of the cells express Tbr1, Tle4, and / or Ctip2, c. A superlayer neuron, wherein at least a portion of the cell expresses Satb2, Cux1, and / or Brn2. d. Astrocytes in which at least a portion of the cell expresses S100beta and / or GFAP, and / or e. The in vitro cell culture model according to claim 10 or 11, comprising basal progenitor cells, wherein at least a portion of the cells express HOPX.

13. The in vitro model according to any one of claims 10 to 12, wherein at least two organoids are cultured in the same culture dish or well, and at least a portion of the organoids forms neural connections with adjacent organoids in the same culture dish or well.

14. An in vitro adhesive neural organoid cell culture model comprising an organoid having a defined spatial structure and a regular eccentricity, comprising a core region containing differentiated (mature) neurons, progenitor cells, and glial cells, and the edges of the processes of neuronal cells and / or glial cells, wherein the organoid is a. Astrocytes, superior neurons, and deep neurons, wherein at least a portion of the deep neurons is localized to the core of the neural organoid, and / or at least a portion of the superior neurons is localized to the edge of the neural organoid, b. An in vitro adhesive neural organoid cell culture model comprising a neural rosette structure in the 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 testing neurological disorders.

16. A kit for carrying out the method according to any one of claims 1 to 9, wherein the kit comprises a culture medium and At least one ROCK inhibitor, At least one ECM protein or component, At least one SMAD inhibitor, A CEPT cocktail comprising at least chroman, or emricasane, a polyamine solution, and trans-ISRIB, and A supplement comprising at least one neurotrophic factor selected from the group consisting of one or more neurotrophic factors selected from BDNF, NT3, GDNF, cAMP, and ascorbic acid, The kit optionally includes a micropattern cell culture chip. The kit optionally includes an 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 an 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, wherein the method is v. To provide multiple spatially distinct neural organoids in cell culture, Determine at least one spatial parameter of at least one organoid provided in vi. i., vii. Using machine learning, preferably deep learning, to analyze at least one spatial parameter of at least one organoid to determine the presence, location, spatial ratio, shape, and / or size of at least one region and / or cell type of at least one organoid. viiii. A computer implementation method comprising optionally providing and / or displaying on a screen a data summary including the results of the analysis in step iii, and optionally providing a graph or image of the analyzed organoid.

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