Automated Generation and Analysis of Organoids

Abstract

The present invention relates to a method for producing organoids, the method comprising or consisting of: (a) seeding a plurality of tissue-specific progenitor cells into a container; (b) (i) aggregating the cells; and (ii) allowing the aggregates formed in (i) to mature into a single organoid, wherein the method does not include embedding the cells or the aggregates in a gel.

Description

Technical Field

[0001] The present invention relates to a method for generating organoids comprising or consisting of the steps of: (a) seeding a plurality of tissue-specific progenitor cells in a container; (b) causing (i) aggregation of the cells; and (ii) maturation of the aggregates formed in (i) into a single organoid, wherein the method does not include embedding the cells or the aggregates in a gel.

[0002] In this specification, several documents are cited, including patent applications and manufacturer manuals. The disclosures of these documents are not considered relevant to the patentability of the present invention, but are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] Three-dimensional (3D) cell cultures in the form of organ-like microtissues ("organoids") have rapidly found the following over the past few years. The potential of organoids to mimic cellular niches more closely than 2D cell cultures holds promise for developing next-generation high-throughput screening (HTS) that can provide more relevant predictions of drug efficacy and toxicity. These may enable better modeling of specific human diseases such as Parkinson's disease, Alzheimer's disease, and other disorders involving complex interactions of several cell types in a particular cellular niche.

[0004] However, while the current state of the art enables the generation of many types of organoids, the quantification methods required for precise, standardized organoid generation and screening are becoming less clear. Most established protocols result in complex and highly heterogeneous organoids without predictable morphology, cell composition, and local cellular organization. The obligatory and extensive manual handling, including a cumbersome matrix embedding step, makes most protocols difficult for industrial scale-up. Furthermore, existing analytical methods either do not scale (e.g., sectioning and immunostaining, RNA sequencing) or rely mostly on measurements of size and morphology (Hou, Y., Konen, J., Brat, D.J., Marcus, A.I. & Cooper, L.A.D. TASI: A software tool for spatial temporal quantification of tumor spheroid dynamics. Sci Rep 8, 7248 (2018); and Kang, A., Seo, H.I., Chung, B.G. & Lee, S.H. Concave microwell array-mediated three dimensional tumor model for screening anticancer drug-loaded nanoparticles. Nanomedicine 11, 1153-1161 (2015)) or overall cell viability (Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359, 920-926 (2018)) and do not provide information with cellular resolution. Finally, data revealing cell-cell interactions with respect to the three-dimensional niche are lacking.

[0005] In view of the limitations of the prior art, the technical problem underlying the present invention may be seen in providing improved means and methods for the generation, handling and / or analysis of organoids.

[0006] This technical problem is solved by the subject matter of the claims.

[0007] Accordingly, in a first aspect, the present invention relates to a method for generating an organoid, the method comprising or consisting of: (a) seeding a plurality of tissue-specific progenitor cells into a container; (b) causing (i) aggregation of the cells; and (ii) maturation of the aggregate formed in (i) into a single organoid, wherein the method does not include embedding the cells or the aggregate in a gel.

[0008] The term "organoid" has the meaning established in the art. Thus, the term relates to a miniaturized version of an organ generated three-dimensionally in vitro. Organoids exhibit microanatomy and cellular functions similar to those of natural tissues in vivo. Typically, an organoid contains a plurality of organ-specific cell types, where the cell types are spatially organized in a defined manner, typically in the case of neural organoids in layers. Generally, the defined spatial organization of the plurality of cell types is the result of self-organization that occurs during organoid formation. An organoid preferably contains different cell types that interact spatially and / or functionally with each other in a self-organizing matrix. The term "self-organizing matrix" refers to the spatial arrangement of cells having different cellular functions and identities, which arrangement is partially or wholly similar to the cell arrangement found in natural tissues in vivo. Also, the arrangement can be maintained for an extended period in culture. In this context, an "extended period" is typically longer than about 100 days or longer than about 200 days.

[0009] Functionally, an organoid can generally recapitulate one or more specific functions of the corresponding organ.

[0010] Therefore, organoids are different from spheroids and aggregates. Spheroids are typically cell aggregates that are smaller in size (about 10 to about 200 μm in diameter) than organoids and lack distinct cellular organization such as distinct layers. According to the present invention, "spheroid" and "aggregate" refer to the same subject. Preferably, the organoid is at least 2 times, at least 5 times, more preferably at least 10 times larger than the spheroid in terms of size. A preferred criterion for size is the diameter of the largest cross-section.

[0011] Regarding the generation of organoids, there is a general understanding in the art that cells or their aggregates need to be cultured in a three-dimensional (3D) medium. A prominent feature of such a 3D medium is that the medium is solid or semi-solid, typically gel-like, and / or composed of natural or artificial hydrogels. The use of a 3D medium or gel is considered essential for the culture of organoids. The terms "3D medium" and "gel" are used interchangeably in the present disclosure. The gel can be selected from a basement membrane-like matrix, Matrigel, collagen, dextran, and extracellular matrix. Such materials are well-known in the art and are described, for example, in Semin Cancer Biol. 2005 Oct;15(5):378-86, Matrigel: basement membrane matrix with biological activity; Kleinman HK1, Martin GR; and Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560-564 (2016).

[0012] Surprisingly, the inventors have found, contrary to general understanding, that the above-mentioned 3D medium and gel are not necessary. Eliminating the 3D medium or gel includes several advantages. First, the handling of materials during culturing becomes less cumbersome and more compliant with automation. In fact, the high degree of automation of the procedure according to the present invention is unprecedented. Second, the avoidance of gel is understood to make the generated organoids more homogeneous. As described in more detail below, the aspect of homogeneity includes, but is not limited to, size. The organoids according to the present invention preferably have a size of about 500 μm to about 2 mm and a standard deviation of less than about 20% from the average.

[0013] The step of seeding cells according to (a), followed by step (b), provides for the formation of a single organoid within the container. Note that, as described in more detail below and that the method of the present invention is compliant with automation and high-throughput, a plurality of containers, such as the wells of a microwell plate, can be handled in an automated manner and simultaneously, thereby enabling the format of the method of the present invention, where a plurality of containers are handled and, as a result of steps (a) and (b), each container contains a single organoid.

[0014] Otherwise, the steps of seeding cells (a) and step (b) can follow procedures established in the art. Its preferred embodiments according to the present invention are further described below.

[0015] Another important feature is the use of tissue-specific progenitor cells. While many methods established in the art use embryonic stem cells or pluripotent cells in many examples, the use of tissue-specific progenitor cells is a means to ensure and / or increase the homogeneity of the resulting organoids.

[0016] The seeding step can be performed, for example, by adding a predetermined number of cells to a medium that causes aggregation. Suitable media are known in the art and are described in more detail in the examples. The preferred number of cells seeded in a given container is further detailed below.

[0017] None of the steps occurring during the implementation of the method of the present invention are embedded in a 3D medium or gel. In particular, the aggregates are not embedded in the gel. This is different from the conventional protocol that typically defines the embedding of aggregates into the gel after a period of 10 to 15 days. Contrary to the established opinion, the inventors have shown that embedding into the gel is not necessary and provides obvious advantages. These advantages include the possibility of performing the generation and handling of organoids in an automated manner that is compliant with scaling up and high throughput.

[0018] In a preferred embodiment, (i) the organoid is a neural organoid, preferably a midbrain organoid or a non-patterned homogeneous brain organoid; the tissue-specific progenitor cells are neuron tissue-specific progenitor cells, preferably small molecule neuron progenitor cells (smNPC); (ii) the organoid has a reproducible or homogeneous size and / or cell composition, and homogeneous preferably means a standard deviation of less than or equal to 20% of the average; (iii) step (b) includes (b-i) culturing in an aggregation medium preferably for about 2 days, the aggregation medium preferably containing polyvinyl alcohol; (b-ii) culturing in a maturation medium; and (b-iii) preferably culturing in a ventral patterning medium for about 4 days between (b-i) and (b-ii); (iv) the plurality of cells is about 100 to about 1,000,000, preferably about 10,000 cells; and / or (v) the container is a well of a multi-well plate, where preferably a plurality of the cells are seeded into a plurality of wells or each well of the multi-well plate, and a multi-well plate is obtained in which a plurality of wells or each well contains a single organoid.

[0019] Thus, in a preferred embodiment (i) regarding the generation of neural organoids, suitability is imparted to specific types of neuronal progenitor cells, namely small molecule neuronal progenitor cells (smNPCs). These specific neuronal progenitor cells (see, for example, Reinhardt, P. et al. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One 8, e59252 (2013)) have the advantage that small molecules are sufficient with respect to the factors required in the culture medium. In particular, protein-based growth factors are not required. The term "small molecule" has the meaning established in the art. Thus, small molecules typically relate to low molecular weight organic compounds having a molecular weight of less than 1000 Da, preferably less than 900 Da. Biological macromolecules such as nucleic acids, proteins, polypeptides, and polysaccharides are not included in the term "small molecule".

[0020] The midbrain organoid is also referred to as an "Automated Midbrain Organoid (AMO)" for its convenient and automated generation according to the present invention.

[0021] As described above, a second preferred aspect of the nerves according to the present invention is a non-patterned homogeneous brain organoid (NABO). When preparing NABO, differentiation is carried out without a patterning factor. As a result, NABO is not directed to a specific fate or brain region such as the midbrain. Instead, NABO is a general neuronal organoid or a general brain-like organoid. Preferably, after 28 days of culture, NABO contains early postmitotic and mature neurons characterized by the markers DCX, MAP2, and Tubb3 as well as synapses (synapsin). Over time, NABO further matures to include glial cells including astrocytes as demonstrated by GFAP and S100 expression. Overall, NABO has a high degree of structural homogeneity in both size and internal organization. Except for the absence of patterning factors during maturation, the preparation of NABO corresponds to the preparation of AMO.

[0022] When used in the above-defined smNPC, the term "medium" refers to the medium used for the two-dimensional culture of the smNPC. For further explanation, the two-dimensional culture serves to generate a sufficient amount of smNPC by cell division. Thus, the two-dimensional culture typically occurs prior to the method according to the first aspect. That is, the method of the first aspect may or may not include the aforementioned two-dimensional culture as a step occurring prior to step (a).

[0023] In view of the above, when used for the method according to the first aspect of the present invention, it is an important feature of smNPC that step (a) of the method of the first aspect requires only small molecules and does not require a protein or peptide as a factor. This is also apparent from the list of components of the aggregation medium given below. Preferred small molecules are Smoothened agonist (SAG) and CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile).

[0024] Certain preferred midbrain organoids according to the present invention contain dopaminergic neurons. The organoids mimic, inter alia, the behavior of the substantia nigra of the midbrain. To generate organoids having such properties, the ventral patterning steps disclosed herein are preferred.

[0025] The step of embedding in gels established in the art is a cumbersome process that requires manual intervention. Further, and noting that it is difficult to reproduce the exact placement of the aggregates within the gel drops, each aggregate after being placed within the gel drops typically experiences a different environment. These limitations and unwanted variations are overcome by the method according to the first aspect. As a direct result, and in accordance with item (ii) of the preferred embodiments disclosed above, the organoids obtained by the method of the present invention are highly reproducible with respect to their properties. Such homogeneity refers to both intra-batch variation and inter-batch variation. With respect to the former, the typical standard deviation from the mean is less than 5%, and with respect to the latter, the typical standard deviation from the mean is less than 25%, less than 20% or less than 15%. Preferred parameters used to determine the degree of homogeneity are size including diameter and / or cell composition. The experimental data included herein provide evidence of such homogeneity. The improved homogeneity is associated with improved reproducibility and improved predictability.

[0026] A further feature of the method of the first aspect is that it does not require agitation as generally practiced in organoid culture within bioreactors established in the art. Thus, in a preferred embodiment, the method does not include agitation.

[0027] In addition to obviating the use of gels, the inventors further optimized the conditions for aggregation and maturation. More specifically, and in accordance with item (iii) of the previously disclosed embodiments, culturing in an aggregation medium (the preferred composition of which is further described below) is carried out for about 2 days. This is a shorter period compared to the protocols established in the art, which dictate leaving the cells in the same medium herein referred to as the "aggregation medium" for an extended period. After culturing in the aggregation medium, culturing is continued in a maturation medium, where any intervening steps preferred in the context of generating midbrain organoids define culturing in a ventral patterning medium. A preferred early switch from the aggregation medium to the maturation medium or optionally to the ventral patterning medium is a further means of enhancing the homogeneity of the generated organoids.

[0028] In a further preferred embodiment, fetal calf serum (FCS) is not used for aggregation. To increase aggregation, it is preferred to add polyvinyl alcohol (PVA) at a concentration preferably of 0.1% (w / v) to 1% (w / v).

[0029] The preferred cell number of the plurality of cells seeded in accordance with item (a) of the method of the first aspect is given in item (v). An exemplary cell number constituting the plurality of cells is about 9000 cells.

[0030] In accordance with preferred automated and / or high-throughput implementation, item (vi) defines the use of multiwell plates. Importantly, preferably each well of the multiwell plate contains one single organoid.

[0031] This is advantageous in that batch effects such as effects for paracrine signaling do not occur. The workflow can be easily changed to place more than one organoid in a given container or well to the extent that paracrine signaling is desired.

[0032] A particularly preferred implementation of the protocol for the generation of organoids according to the invention is given in the corresponding subsections of Example 1.

[0033] In a second aspect, the invention relates to an organoid or a plurality of organoids obtained by the method according to any of the preceding claims.

[0034] For a separate method of generating organoids, the resulting organoids are different from prior art organoids.

[0035] This includes a high degree of homogeneity; see the section title "Homogeneity" in Example 2 and Figures 1 and 8 - 11. These figures include microscopic images that emphasize the morphological and size heterogeneity of a plurality of prior art or organoids compared to the organoids of the invention, which are characterized by a very narrow distribution of geometric parameters such as well - defined spherical shapes and radii.

[0036] Thus, considering the unprecedented homogeneity across the population of organoids in particular, it is clear that the organoids according to the invention are essentially different from any prior art organoids. This homogeneity enables uses that are not possible with prior art organoids, and such uses include drug screening and toxicology screening (see also the examples).

[0037] Parameters for quantifying the size and / or shape homogeneity are as follows:

Number

[0038] In a preferred embodiment, the organoids according to the invention have a sphericity of 0.85 to 1.0, more preferably 0.9 to 1.0 and even more preferably 0.95 to 1.0.

[0039] The two-dimensional counterpart of the previously defined sphericity can be used to the extent that the homogeneity is determined based on the two-dimensional image. By way of example, the circularity, which is the ratio between the maximum and minimum sizes of the inscribed circle and the circumscribed circle, i.e., the maximum and minimum sizes of the circle that just fits inside and encloses the shape under consideration, can be used. Thus, the circularity of the two-dimensional object can be defined as R = r i / r c where r i and r c are the radii of the inscribed circle and the circumscribed circle, respectively.

[0040] Preferred values of circularity are 0.85 to 1.0, more preferably 0.9 to 1.0 and even more preferably 0.95 to 1.0.

[0041] Further suitable parameters are the width of the distribution of the maximum diameter d, the coefficient of variation (CV) of the maximum diameter, the width of the distribution of the maximum cross-sectional area A max and the CV of the maximum cross-sectional area. Exemplary data for the CV of A max for the NABOs of the present invention are given in FIG. 13.

[0042] Regarding the width of the distribution of the maximum diameter d, it is preferred that 90% of the maximum diameters measured in a plurality of organoids obtained according to the invention are within + / -20%, more preferably + / -10% of the average of the maximum diameters. For the distribution of A max the same applies with the necessary modifications.

[0043] For both d and A max the preferred range of the CV is less than 10%, less than 5%, less than 4% or about 3%.

[0044] Importantly, such homogeneity is achieved without relying on specific shapes such as molds and / or any kind of device that can impose homogeneity. For further illustration, it is conceivable to utilize a mold, such as a mold generated by a 3D printer, during and / or after the generation of the organoid. Such a mold can impart shape and / or homogeneity to the organoid, where the organoid can be an organoid that does not exhibit the shape and / or homogeneity imparted by a device, such as a mold, in the absence of the device, such as a mold. Consequently, in a preferred embodiment of the method of the first aspect, it is understood that no mold is used. Similarly, the organoid according to the present invention is preferably an organoid that is not shaped by a mold during or after generation. The absence of such a device or mold is a preferred embodiment of all aspects of the present invention.

[0045] It is understood that the container according to the first aspect does not act as a mold or shaping device. Preferably, the dimensions of the container are, for example, at least twice, at least five times, at least ten times or at least one hundred times larger than the size of the organoid. The "dimensions" can be the diameter and / or the depth of the container.

[0046] In a preferred embodiment, (a) the organoid(s) is / are (a) neural organoid(s), preferably (a) midbrain organoid(s) or (a) homogeneous brain organoid(s) that are not pattern-formed; (b) the organoid(s) exhibits (i) a plurality of concentric regions, preferably at least three regions, where each region is different from any of the other regions with respect to cell composition and organization; and / or (ii) tissue-specific cell activity, preferably, in the case of a neural organoid, electrical activity in neurons; and / or (c) the plurality of organoids are homogeneous with respect to structure and / or size; where the organoid or the plurality is preferably obtained by the method according to the first aspect.

[0047] In connection therewith, in a second aspect, the present invention provides an organoid or a plurality of organoids, wherein (a) the organoid(s) is / are (a) a neural organoid, preferably (a) a midbrain organoid(s) or (a) homogeneous brain organoid(s) that has not been pattern-formed; (b) the organoid(s) shows (i) a plurality of concentric regions, preferably at least three regions, each region being different from any of the other regions with respect to cell composition and organization; and / or (ii) the organoid(s) shows tissue-specific cell activity, preferably in the case of a neural organoid, electrical activity in neurons.

[0048] The above-mentioned concentric regions are described in more detail in the examples. The presence of four concentric regions is particularly preferred. This can be seen in FIGS. 2a and 2c. The preferred cell composition of the above-mentioned four concentric regions is described for the midbrain organoids according to the present invention in the subsection title "Automated midbrain organoids express typical neural and midbrain markers and show structural organization" of Example 2. In connection with the above, the organoids produced according to the present invention are preferably spherical or show radial symmetry.

[0049] Item (ii) defines the above-mentioned recaptulation of organ-like behavior. Surprisingly, and as described in more detail in the subsection title "Calcium imaging reveals spontaneous and synchronous activity across the entire organoid" of Example 2, the organoids according to the second and third aspects of the present invention are functionally connected across the entire organoid. In the context of a preferred neural organoid, the tissue-specific cell activity observed across the entire organoid is electrical activity, more specifically, preferably electrical activity including a plurality of neurons in a synchronous manner. It is also preferred that the organoids according to the present invention produce and optionally secrete tissue-specific proteins.

[0050] Regarding the monitoring of tissue-specific proteins, antibodies directed against SOX2 can be used to detect changes in the amount of neural progenitor cells present within the organoids. Antibodies directed against MAP2 can be used to detect changes in the amount of more mature neural cells within the organoids.

[0051] In a fourth aspect, the invention provides a multi-well plate, wherein a plurality of wells each contain one single organoid or each well contains one single organoid, preferably a plurality of organoids or each organoid is defined according to the second or third aspect and / or obtained by the method according to the first aspect.

[0052] As noted above, a preferred implementation of the method of the invention involves the use of a plurality of containers, typically the wells of a multi-well plate. When carrying out the method of the invention in such a format, the multi-well plate according to the fourth aspect results from carrying out such a method.

[0053] In relation to the first aspect, the invention provides, in a fifth aspect, the use of tissue-specific progenitor cells for organoid generation, wherein a gel for embedding cells or aggregates is not utilized, preferably, the tissue-specific progenitor cells are neuron tissue-specific progenitor cells, preferably small molecule neuron progenitor cells (smNPC).

[0054] As described in the background section of the present specification above, the workflow associated with organoids suffers from several drawbacks. One of these drawbacks is embedding in gels established in the art. This is addressed by the aspects of the invention disclosed above. A further drawback of prior art procedures is the size of the organoids and their opaque appearance under the microscope. As a result, conventional staining or labeling approaches enable the analysis of the surface of organoids having a penetration depth of approximately 50 - 100 μm, but fail to enable a comprehensive analysis of all cells contained within the organoids.

[0055] In a sixth aspect, the present invention addresses this drawback by providing a method for preparing an organoid or spheroid for analysis, the method comprising: (a) staining the organoid or spheroid; (b) performing tissue clearing using the organoid or spheroid, or consisting of this step.

[0056] Steps (a) and (b) of this method of the sixth aspect can be performed in any order. Having said that, preference is given to performing step (a) before step (b). This applies in particular to the preferred embodiments of benzyl alcohol and benzyl benzoate (BABB)-based clearing described in more detail below.

[0057] Particularly preferred implementations of staining and tissue clearing are given in the corresponding subsections of Example 1.

[0058] In a preferred embodiment, (a) the staining is carried out using (i) an antibody, preferably a primary antibody and a secondary antibody, wherein the staining using the primary antibody and / or the secondary antibody is carried out for about 5 to about 10 days, preferably about 6 days; the staining is carried out using (ii) a fluorescent label; (iii) a luminescent label; (iv) a radioactive label; and / or (b) the clearing is (BABB)-based clearing, wherein preferably the clearing is carried out in a cycloolefin container, more preferably a cycloolefin multiwell plate.

[0059] As described in the examples, for the purpose of staining with an antibody, a solution containing Triton-X 100 at a concentration of preferably 0.1% (w / v) to 1.0% (w / v), more preferably 0.5% (w / v) is used. This is similar to the specific adaptation for staining of the organoids developed according to the present invention.

[0060] The preferred duration of the staining procedure, namely about 5 to about 10 days, preferably about 6 days, is a further specific adaptation for staining of the organoids.

[0061] BABB clearing is described, for example, in Dent, J.A., Poison, A.G. & Klymkowsky, M.W. A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105, 61-74 (1989).

[0062] Although less preferred, other clearing methods may also be used. Examples are X-Clarity (Logos biosystems (2015): X-CLARITY TM Tissue Clearing System, User Manual, LBSM-0005 Ver 1.8, 205.05.28, www.logosbio.com), CUBIC (Susaki, E.A. et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10, 1709-1727 (2015), ScaleSQ (Hama, H. et al. ScaleS: an optical clearing palette for biological imaging. Nat Neurosci 18, 1518-1529 (2015) and Clear T (Kuwajima, T. et al. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140, 1364-1368 (2013)).

[0063] Suitable labels include nuclear counterstains such as phalloidin and DAPI, luciferase, and live / dead stains, i.e., dyes that indicate whether a cell is dead or alive. The latter is preferred for the toxicity screening described below.

[0064] For the combination of staining and clearing, sectioning of the organoids is no longer necessary to align all cells for analysis. That is, in a preferred embodiment, (a) the method does not include sectioning of the organoid or spheroid, and / or the staining is whole mount staining; and / or (b) the organoid is a second or third aspect organoid or is obtained by the method of the first aspect.

[0065] In connection with the method of the sixth aspect, in a seventh aspect the present invention provides a method of analyzing an organoid or spheroid, the method comprising the method of the sixth aspect; and (c) analysis of the stained and cleared organoid or spheroid, preferably (c-i) optical analysis, where the optical analysis preferably includes microscopy and / or image analysis; (c-ii) genetic analysis such as RNA sequencing; and / or (c-iii) protein analysis including or consisting of mass spectrometry or Western blot. Thanks to the clearing step, it is possible to analyze all cells of the organoid, preferably at the single cell resolution level, and preferably make them visible.

[0066] Considering the methods of generating organoids according to the first aspect and analyzing organoids according to the seventh aspect of the present invention, both of which are preferably automated and high-throughput compliant, the present invention enables an automated and integrated method of preparing and analyzing organoids. This is the subject of an eighth aspect regarding methods of preparing and analyzing organoids, the method comprising or consisting of the method of the first aspect and the method of the seventh aspect.

[0067] As described in the background section, organoids are increasingly recognized as valuable tools in the medical field, including the screening of lead compounds, toxicity testing, disease models, and personalized medicine. Thus, in a ninth aspect, the present invention provides a method for identifying regulators of organoids, organoid formation, and / or organoid-specific functions, the method comprising: (a) (i) adding a test compound to an organoid as described preferably in the second or third aspect or an organoid obtained by the method of the first aspect; (ii) adding a test compound to tissue-specific progenitor cells and then performing the method of the first aspect; or (iii) performing the method of the first aspect, wherein the test compound is added at one or more time points during the performance of the method of the first aspect; (b) performing the method of the seventh aspect; (c) comparing the results of the analysis in the presence of the test compound with the results of the analysis in the absence of the test compound, wherein the difference indicates a regulator.

[0068] In a preferred embodiment, (a) if the analysis indicates an improvement in the function of the organoid, organoid formation, and / or organoid-specific function, the test compound is a lead compound and the method optionally further comprises or consists of developing the lead compound to yield a drug; or (b) if the analysis indicates a decrease in the function of the organoid and / or a negative interference with organoid formation and / or organoid-specific function, the analysis indicates that the test compound is toxic.

[0069] The term "lead compound", as used herein, refers to a compound that can optionally be subjected to optimization for eventual use as a drug. Alternatively, the lead compound itself can be a drug. The optimization described above can include optimization of stability, pharmacokinetics, and pharmacodynamics. Further, a lead compound can be associated with a specific molecular target, i.e., the lead compound can be a molecule that occurs in cells contained in an organoid that is recognized to be a conjugate, preferably an inhibitor of the target molecule, preferably one associated with a disease.

[0070] Perhaps not necessarily relevant thereto, but the above aspects of the toxicity test may also be relevant in the context of drug development. That is, one shows a lead compound that is a useful drug candidate, and the other is likely to have the simultaneous presence of two readouts that show toxicity, perhaps at high doses. Under such circumstances, that is, under beneficial effects at low concentrations and toxic effects at high concentrations, the lead compound or drug has a therapeutic window.

[0071] In a further aspect, the present invention provides for the use of one or more organoids produced by the method of the first aspect or defined as a disease model according to the second or third aspect. The disease to be modeled can be a genetic disease. Also, the disease can be induced by the addition of a pathogen and / or a disease-inducing compound.

[0072] In a preferred embodiment of all the methods of the present invention, the method is carried out (a) in an automated manner; and / or (b) in a high-throughput format, preferably using a multi-well plate, pipetting robot, automated liquid handler, plate reader and / or means for plate transport.

[0073] Multi-well plates established in the art, for example plates having 96, 384 or 1536 wells, can be used.

[0074] In certain preferred embodiments, commercially available high-throughput equipment can be used directly when carrying out the methods of the present invention, i.e., no hardware adjustment is required.

[0075] In the tenth aspect, the present invention (a) Tissue-specific progenitor cells, preferably neuron tissue-specific progenitor cells, more preferably smNPCs; (b) a culture medium, the culture medium comprising: (b-i) an aggregation medium, the aggregation medium preferably containing polyvinyl alcohol; (b-ii) a maturation medium; and (b-iii) optionally, a ventral patterning medium, or consisting of these, to provide a kit comprising or consisting of.

[0076] The aggregation medium preferably consists of a 1:1 ratio of DMEM-F12 and Neurobasal Medium enriched with 1:400 diluted N2 supplement, 1:200 diluted B27 supplement without vitamin A, 1% penicillin / streptomycin / glutamine, 200 μM ascorbic acid, and small molecule SAG (0.5 μM) and CHIR 99021 (3 μM).

[0077] The preferred ventral patterning medium is as follows: it is the same as the aggregation medium except that CHIR is removed and 0.5 ng / mL brain-derived neurotrophic factor (BDNF) and 1 ng / mL glial cell line-derived neurotrophic factor (GDNF) are added.

[0078] The preferred maturation medium is as follows: it is the same as the ventral patterning medium except that SAG is removed and 0.5 - 1 ng / mL transforming growth factor β3 (TGF-β3) and 100 μM dibutyryl cyclic adenosine monophosphate (dbcAMP) are added.

[0079] With respect to the aspects characterized in this specification, particularly in the claims, each aspect described in a dependent claim is intended to be combined with each aspect of the respective claim (independent or dependent) to which the dependent claim depends. For example, in the case of independent claim 1 that describes three options A, B, and C, dependent claim 2 that describes three options D, E, and F, and claim 3 that depends on claims 1 and 2 and describes three options G, H, and I, unless specifically stated otherwise, the specification is understood to clearly disclose aspects corresponding to the combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I.

[0080] Similarly, and also when the independent and / or dependent claims do not describe options, when a dependent claim refers back to multiple preceding claims, any combination of the subject matter covered by them is understood to be considered as explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 that refers back to claim 1, and dependent claim 3 that refers back to both claims 2 and 1, the combination of the subject matter of claim 3 and 1 is considered to be as clearly and explicitly disclosed as the combination of the subject matter of claims 3, 2, and 1. If there is a further dependent claim 4 that refers to any one of claims 1 - 3, the combinations of the subject matter of claim 4 and 1, claim 4, 2, and 1, claim 4, 3, and 1, and claim 4, 3, 2, and 1 are considered to be clearly and explicitly disclosed.

Brief Description of the Drawings

[0081] The drawings show the following:

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Mode for Carrying Out the Invention

[0082] The present invention will be described by way of examples.

Examples

[0083] Example 1 Method smNPC culture Unless otherwise indicated, all cells and organoids were maintained at 37 °C and 5% CO2. The inventors cultured human small molecule progenitor cells (smNPCs) with only minor modifications as previously described. Briefly, the inventors grew smNPCs in 0.0125% (v / v) Matrigel (BD)-coated 6-well plates (Sarstedt) in N2B27 medium supplemented with small molecule smoothened agonist (0.5 μM, SAG, Cayman Chemical) and CHIR 99021 (3 μM, Axon MedChem). N2B27 consisted of DMEM-F12 (Thermo Fisher) and Neurobasal medium (Thermo Fisher) at a 1:1 ratio enriched with 1:400-diluted N2 supplement (Thermo Fisher), 1:200-diluted B27 supplement without vitamin A (Thermo Fisher), 1% penicillin / streptomycin / glutamine (Thermo Fisher), and 200 μM ascorbic acid (Sigma-Aldrich). Typically, the inventors changed the medium every other day. Cells were split every 5-7 days at a split ratio of 1:10-1:20 via accutase (Sigma-Aldrich) treatment for approximately 15 minutes at 37 °C to yield a single cell solution. To stop digestion, cells were diluted in DMEM-F12 supplemented with 0.1% BSA (Thermo Fisher) and centrifuged at 1200 g for 2 minutes. The cell pellet was resuspended in fresh smNPC medium (N2B27 with SAG and CHIR) and plated on Matrigel-coated 6-well plates.

[0084] AMO production After digestion with Accutase, the inventors seeded 9000 smNPCs into each well of a conical 96-well plate (Thermo Fisher) in smNPC medium and allowed the smNPCs to aggregate for 2 days. To increase cell-cell adhesion, the inventors added 0.4% (w / v) polyvinyl alcohol (PVA). Starting on day 2, by continuously maintaining SAG and removing CHIR99021, the cells experience ventral patterning over 4 days in two feedings. The addition of 0.5 ng / mL brain-derived neurotrophic factor (BDNF, PeproTech) and 1 ng / mL glial cell line-derived neurotrophic factor (GDNF, PeproTech) increases maturation and cell survival during the remainder of neural maturation. After ventralization, the inventors removed SAG on day 6 and supported midbrain differentiation and maturation by the addition of 0.5 ng / mL transforming growth factor β3 (TGFβ-3) and 100 μM dibutyryl cyclic adenosine monophosphate (dbcAMP, Sigma-Aldrich). A single dose of 5 ng / mL activin A was added only on day 6. Depending on the desired degree of maturity, the duration of the maturation period can be extended up to 100 days or more.

[0085] Generation of NABO Similar to automated midbrain organoids, non-patterned automated brain organoids were generated, maintained, and analyzed in a fully automated manner. Only the medium preparation and the timing of the medium differed, and the principle workflow remained the same, demonstrating the flexibility of the methods of the invention for adapting to various different, preferably neural structure generations. Briefly, the method for generating NABO is as follows:

[0086] After digestion with Accutase, the inventors seeded 9000 smNPCs into each well of a conical 96-well plate (Thermo Fisher) in smNPC medium and allowed the cells to aggregate for 2 days. To increase cell-cell adhesion, the inventors added 0.4% (w / v) polyvinyl alcohol (PVA) to the seeding medium. The smNPC medium was based on N2B27 medium supplemented with small molecule smoothened agonist (0.5 μM, SAG, Cayman Chemical) and CHIR 99021 (3 μM, Axon MedChem). The N2B27 medium consisted of 1:400 diluted N2 supplement (Thermo Fisher), and 1:200 diluted B27 supplement without vitamin A (Thermo Fisher), 1% penicillin / streptomycin / glutamine (Thermo Fisher), and a 1:1 ratio of DMEM-F12 (Thermo Fisher) and Neurobasal medium (Thermo Fisher) enriched with 200 μM ascorbic acid (Sigma-Aldrich). Starting on day 2, the aggregates experience non-directed neural differentiation by withdrawing SAG and CHIR from the medium and adding 1 ng / mL brain-derived neurotrophic factor (BDNF, PeproTech) and 1 ng / mL glial cell line-derived neurotrophic factor (GDNF, PeproTech). These growth factors increase maturation and cell survival for the remainder of neural maturation. Depending on the desired degree of maturity, the duration of the maturation stage can be extended up to 100 days or more. For analysis, as disclosed herein, the organoids were whole mount stained and optically cleared.

[0087] Size measurement of organoids For the size measurements of both AMO and NABO, the inventors took bright-field images of randomly selected organoids using a stereomicroscope (Leica MZ10 F, camera: Leica DFC425 C). The images were processed by ImageJ / Fiji (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682 (2012)) using a custom-adjusted standardization workflow. The auto threshold function was used to identify the organoids from the background, and then the area of the organoids was measured by the analyze particles function. The measured area corresponds to the maximum cross-section of the organoid. The data was output to Microsoft Excel and GraphPad Prism v7.0 (Graphpad Software, Inc.) for further analysis.

[0088] Whole-mount staining and clearing To analyze protein expression in 3D in the HTS-compatible format, the inventors adapted a whole-mount staining protocol based on Lee et al. ACT-PRESTO: Rapid and consistent tissue 579 clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep 6, 18631 (2016) to organoids and optimized the protocol for use in an automated liquid handling system. After fixation with 4% PFA (VWR) for 10 - 15 minutes, the inventors stained the organoids with primary and secondary antibodies (Alexa Fluor secondary antibodies, Thermo Fisher) for 6 days each. The inventors diluted the antibodies in a blocking and permeabilization solution (6% BSA, 0.5% Triton-X 100 (Roth), 0.1% (w / v) sodium azide (Sigma-Aldrich) in PBS (Sigma-Aldrich)) and refreshed it every 2 days. During the incubation of the primary and secondary antibodies, as well as after the staining procedure, the inventors washed the organoids 5 times for 1 hour each with 0.1% Triton X-100 in PBS. Due to this extremely long staining procedure, the antibodies penetrate the organoids well, regardless of the large size and high density of the organoids. To enable sufficient transmission by microscope illumination, BABB-based tissue clearing, Dent, J.A., Polson, A.G. & Klymkowsky, M.W. A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105, 61 - 74 (1989) is continued after the whole-mount staining procedure. First, the organoids were dehydrated stepwise with a methanol (Roth) series (25%, 50%, 70%, 90%, 100%, 15 minutes each). The organoids were then transferred to an organic solvent-resistant cycloolefin 96-well plate (“Screenstar”, Greiner Bio-One).Samples were incubated for 30 minutes in 1:1 methanol / 389 BABB (1:1 benzyl benzoate (Sigma-Aldrich) and benzyl alcohol (Sigma-Aldrich)) and subsequently maintained in BABB for imaging. We used Imaris v8.4 (Bitplane, Oxford Instruments) for 3D rendering of confocal sections (Figure 1d).

[0089] Quantitative real-time PCR For quantitative real-time PCR (qPCR) analysis, we isolated RNA using the NucleoSpin RNA XS kit (Macherey-Nagel) according to the manufacturer's instructions. Depending on the age of the organoids, we pooled 32 (d6), 24 (d16), or 18 (d30) organoids from one batch to obtain sufficient RNA for downstream analysis. We determined RNA concentration and purity using a NanoDrop 8000 spectrophotometer (Thermo Fisher) and performed reverse transcription according to a standard protocol using 1000 ng RNA per reaction. For quantification of gene expression, we used the Biomark 48.48 integrated fluidic circuit (IFC) Delta Gene assay (Fluidigm) according to the manufacturer's instructions. Briefly, after 14 cycles of preamplification, samples were subjected to exonuclease I (New England Biolabs) treatment (37 °C, 30 minutes and 80 °C, 15 minutes) and diluted 20-fold with DNA suspension buffer (TEKnova). Samples (in duplicate) and assay mixtures were loaded onto a 48.48 microfluidic ICF chip and run on a BioMark real-time PCR reader (Fluidigm), where samples were amplified and measured according to the manufacturer's instructions. Data analysis was performed using the BioMark real-time PCR analysis software 4.3.1 (Fluidigm) standard settings. Data were transferred to Microsoft Excel for further processing and to GraphPad Prism v7.0 for plotting. GAPDH served as a housekeeping gene.

[0090] Calcium imaging For calcium imaging, the inventors added 10 μM cell-permeable Fluo-4 AM (Thermo Fisher) diluted in organoid medium to the organoids and incubated them at 37 °C for 60 minutes. Imaging was performed using a Dragonfly spinning disk confocal microscope (Andor, Oxford Instruments) at a frequency of 10 Hz for 4 minutes. Data analysis was performed using ImageJ / Fiji (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682 (2012)). First, different ROIs were defined as shown in Fig. 4. Then, the average fluorescence intensity in these ROIs was measured over time and plotted using GraphPad Prism v7.0. Videos were assembled by ImageJ / Fiji (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682 (2012)) and the frame rate was accelerated to compress the real-time of 4 minutes at 10 Hz to an operating time of 20 seconds.

[0091] iPSC culture Previously described healthy control strains 45 Together with, human iPSC culture without feeder was performed using modified FTDA medium (Frank, S., Zhang, M., Scholer, H.R. & Greber, B. Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions. PLoS One 7, e41958 (2012)) in a 0.0125% (v / v) Matrigel-coated 6-well plate. 45。The FTDA medium consisted of DMEM-F12 supplemented with 1% human serum albumin (Biological Industries), 1% chemically defined lipid concentrate (Life Technologies), 0.1% insulin-transferrin-selenium (BD), and 1% penicillin / streptomycin / glutamine. The inventors supplied nutrients to the iPSCs daily and added 10 ng / mL FGF2 (PeproTech GmbH), 0.2 ng / mL TGFβ3 (PeproTech GmbH), 50 nM Dorsomorphin (Santa Cruz), 5 ng / mL activin A (eBioscience), and 20 nM C59 (Tocris) before each medium change. The inventors dissociated the iPSCs into single cells every 3 to 5 days using Accutase at 37°C for approximately 10 minutes. The inventors transferred 600,000 cells per well of the 6-well plate to be seeded into DMEM-F12 with 0.1% BSA and centrifuged at 1200 g for 2 minutes. The inventors resuspended the cell pellet in fresh FTDA medium supplemented with 1:2000 ROCK inhibitor Y-27632 (tebu-bio) and plated the iPSCs on Matrigel-coated 6-well plates.

[0092] iPSC-derived organoid culture Regarding the generation of iPSC-derived organoids, the inventors followed the protocol of Lancaster et al. (Cerebral organoids model human brain development and microcephaly. Nature 501, 373-379 (2013)) with minor modifications. Briefly, the inventors dissociated iPSCs into single cells by Accutase treatment and plated 9000 cells per well in a conical 96-well plate in a low FGF stem cell medium (DMEM-F12 with KnockOut Serum Replacement (KOSR, Thermo Fisher) 1:5, fetal bovine serum (Biochrom) 1:33.3, 1% penicillin / streptomycin / glutamine, 1% non-essential amino acids (NEAA, Thermo Fisher), β-mercaptoethanol (Thermo Fisher) 1:143, 4 ng / μL FGF2, 50 μm ROCK inhibitor Y-27632, and 0.4% PVA) to facilitate aggregation on the seeding day. The inventors changed the medium every other day and withdrew FGF2 and Y-27632 on day 6. Neural induction was initiated on day 8 (neural induction medium: DMEM-F12 with KOSR 1:5, 1% penicillin / streptomycin / glutamine, 1% 439 non-essential amino acids, N2 supplement 1:100, and heparin (Sigma-Aldrich) 1 μg / mL), and the medium was changed every other day for 6 days. On day 13, the inventors embedded the aggregates in 30 μL Matrigel drops and transferred them to a 6 cm2 suspension tissue culture dish (Sarstedt) in a cerebral organoid differentiation medium (DMEM-F12 and Neurobasal 1:1 with 1% penicillin / streptomycin / glutamine, 1% NEAA, N2 supplement 1:200, vitamin A-free B27 supplement 1:100, insulin (Sigma-Aldrich) 1:4000, and β-mercaptoethanol 1:285714). The inventors placed the culture dish on a shaker at 37 °C and 5% CO2 and fed the organoids every other day. On day 20, the B27 supplement was replaced with B27 with vitamin A (Thermo Fisher), and the organoids were cultured until day 30 / 45.

[0093] RNA Sequencing To isolate the RNA of a single organoid, the inventors used the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. The inventors evaluated the RNA concentration and purity using a NanoDrop 8000 spectrophotometer and the RNA integrity using a Bioanalyzer (Agilent Technologies) according to standard protocols. Subsequently, mRNA was enriched using the NEBNext Poly(A) Magnetic Isolation Module (NEB) followed by strand-specific cDNA NGS library preparation (NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, NEB). The size of the resulting library was adjusted using a D1000 ScreenTape (Agilent 2200 TapeStation) and quantified using the NEBNext Library Quant Kit for Illumina (NEB). Equimolar pooled libraries were sequenced in single-read mode (75 cycles) on a NextSeq 500 System (Illumina) using v2 chemistry that yields an average Q-score distribution with a 95% >= Q30 score, then demultiplexed and converted to FASTQ files by bcl2fastq v2.20 conversion software (Illumina).

[0094] RNA Sequencing Analysis The inventors aligned the RNA sequencing reads to the human genome hg19 using the TopHat2 aligner (v2.1.1)46 with default input parameters. Gene annotation from Ensembl (version GRCh37.87) was used in the mapping process. The number of reads mapped to each gene was determined using the Python package HTSeq (v0.7.2) together with "htseq-count -m o 464 de union-stranded no" Counting was performed using (Anders, S., Pyl, P.T. & Huber, W. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166-169 (2015)). Principal component analysis and differential expression analysis were performed using the R package DESeq2 (v1.18.1) using raw counts. The variance within groups was calculated using the mean distance between data points and the centroid. Genes were considered deregulated when |log2FC| > 2 and FDR < 0.05 using Benjamini-Hochberge multiple testing adjustment (Benjamini, Y. & Hochberg, Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society. Series B (Methodological) 57, 289-300 (1995)). Enrichment of gene ontology (GO) terms was analyzed by the bioinformatics web server Gorilla (Eden, E., Navon, R., Steinfeld, I., Lipson, D. & Yakhini, Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10, 48 (2009)) and visualized by REViGO40. All RNA sequencing data were deposited in the NCBI GEO database.

[0095] Quantification of whole-mount staining and clearing To evaluate how quantitative the inventors' imaging workflow was, the inventors performed dilution experiments. The inventors mixed unlabeled smNPCs with CellTracker Deep Red Dye (Life technologies)-labeled cells (labeled according to a standard protocol, dye concentration 1:20,000) at different percentages (1.25%, 2.5%, 5%, 10%, 20%, 40%) and aggregated them in smNPC maintenance medium with 0.4% PVA. To examine the effect of aggregate size on quantification, the inventors generated aggregates with a total of 100,000 and 200,000 cells. One day after aggregation, the aggregates were fixed with 4% PFA, subjected to BABB-based tissue clearing, imaged, and analyzed as follows.

[0096] High-content imaging and analysis After staining and clearing, the inventors achieved a uniform aggregate arrangement within the wells by tilting the plates 60° off horizontal for 1 minute. Image acquisition was performed on an Operetta high-content imager (Perkin Elmer), and the images were analyzed with Harmony 4.1 software. The inventors acquired a total of 16 confocal planes at an inter-plane spacing of 36.6 μm for a total stack of 549 μm, covering the height of all organoids, in three channels (DAPI, Sox2-488, and MAP2-647). To define the organoid regions on each image plane, all three channels were summed, filtered with a median filter to remove small localized features, and bright regions were identified using the "Find Image Regions" function. After cleaning the edges of the organoid regions with respective dilation and erosion steps of 10 and 3 pixels, the inventors identified true organoids by selecting regions with a minimum of 300 arbitrary brightness units (abu) and a size of 4000 μm2. To better isolate Sox2+ nuclei from the general background, the inventors ran a sliding parabola algorithm with 2 curvature settings across each image plane in the 488 channel. Next, nuclei were identified within each organoid region by the "Find Nuclei" function, algorithm "M", and further selected as Sox2+ if the nuclei were larger than 10 um2 and brighter than 1200 abu. The inventors excluded image artifacts, small dust particles, and overlapping nuclei by omitting nuclei brighter than 6000 abu and larger than 70 μm2 from further quantification. For the final output, the number and total brightness of nuclei in 488 and organoid regions in 647 were summed for all planes and all fields of view in each well and transferred to Microsoft Excel and TIBCO Spotfire for further annotation, analysis, and plotting. The inventors excluded data from wells containing dust particles, organoids that were incompletely imaged due to improper placement, or organoids that may have been damaged or compromised during culture or downstream processing.Plates 1, 2, and 3 represent independent differentiations of separately thawed and cultured cells from the same frozen batch.

[0097] Electrophysiological analysis of single cells by patch clamp Due to the form of AMO (the fact that high optical density and most cell bodies are located at a depth of at least 10 - 20 μm), it was technically impossible to perform patch-clamp measurements on intact aggregates. Therefore, the organoids were treated with 1 mg / ml trypsin and then mechanically dispersed to obtain single cells. These were seeded onto PDL-coated cover glasses and cultured in AMO medium for 1 - 3 days (the inventors described the age of AMO at the time of dissociation). Transmembrane currents were recorded from the isolated cells using the whole-cell configuration of the patch-clamp technique (Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85 - 100 (1981)). Patch pipettes were fabricated from borosilicate glass on a Sutter P1000 (Sutter Instrument company) pipette puller. When filled with the pipette solution, the pipettes had a tip resistance of 4 - 6 MΩ. Recordings were made using an EPC-10 amplifier (HEKA Elektronik) and Patchmaster acquisition software (HEKA Elektronik). A series of resistances, liquid junction potentials, and pipette and whole-cell capacitances were electronically compensated. The bath solution contained (mM): NaCl 140, KCl 2.4, MgCl2 1.2, CaCl2 2.5, HEPES 10, D-glucose 10, pH 7.4, and the pipette solution contained (mM): K-aspartate 125, NaCl 10, EGTA 1, MgATP 4, HEPES 10, D-glucose 10, pH 7.4 (KOH). The inventors performed all experiments at room temperature. Recordings of the current-voltage relationship (I-V curve) were made in voltage-clamp mode at a holding potential of -70 mV. Recordings of the induced action potentials were made in current-clamp mode.Data was analyzed using the Patcher's Power Tool routine for IgorPro (WaveMetrics), SciDAVis (http: / / scidavis.sourceforge.net / ), and Origin Pro 2019 (Origin Lab). To clarify the shape of the I-V curve, a single trace was normalized against the peak amplitude and then averaged.

[0098] 3D Toxicology Assay On day 50, the inventors treated AMO with increasing concentrations (0, 5, 50, 100, 250, 500, 1000 μg / mL) of G418 added directly to the culture medium. Two days later, the inventors refreshed the medium (containing the same inhibitor concentration) and fixed the aggregates after a total of 4 days of treatment. Fixation, whole-mount immunostaining for cCasp3 and Sox2, and BABB-based clearing were performed as described above. Image analysis was continued after the steps outlined in the high-content analysis section with slight modifications to adapt to the individual brightness, morphology, and background features of the cCasp3 staining. Briefly, after identifying AMO and Sox2+ cells as described above, a sliding parabola algorithm with 10 curvature settings was run across each confocal slice of AMO to background correct the cCasp3 channel. The inventors identified apoptotic cells using the "Nuclear Discovery" function in the 647 channel and algorithm "M", and selected cells to be further cCasp3+ if they were larger than 11 μm2, smaller than 100 μm 2 and brighter than 2700 abu. The inventors considered cells to be Sox2 / cCasp3 double positive if they met the criteria for both filters simultaneously. Results were output to Microsoft Excel, reformatted, and then transferred to GraphPad Prism v8.0.2 for plotting, data analysis, and IC50 calculation.

[0099] Example 2 Results Automation enables high-throughput generation of homogeneous midbrain organoids Screening applications require biological systems that operate within predictable physiological parameters. To limit cellular heterogeneity during differentiation, the inventors started with small molecule neural progenitor cells (smNPCs) derived from pluripotent stem cells (PSCs) to generate human neural midbrain organoids (Reinhardt, P. et al. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One 8, e59252 (2013)). The neuro-restricted developmental capacity of these cells still allows for the self-organization required for organoid formation (Di Lullo, E. & Kriegstein, A.R. The use of brain organoids to investigate neural development and disease. Nat Rev Neurosci 18, 573-584 (2017); and Monzel, A.S. et al. Derivation of Human Midbrain-Specific Organoids from Neuroepithelial Stem Cells. Stem Cell Reports 8, 1144-1154 (2017)), but results in more homogeneous organoids compared to PSC-based protocols. Surprisingly, matrigel embedding was found to be dispensable, and the use of an automated liquid handling system (ALHS) reduces variability between batches of matrigel embedding as if the standardized mechanical stress is the same. The resulting automated midbrain organoids (AMOs) are not structurally more complex than PSC-derived aggregates, but the organoids show little within-batch and between-batch variability in size distribution (Figure 1b, mean coefficient of variation (CV) 3.56% within 1 batch; minimum 2.2%, maximum 5.6%), morphology (Figure 1c), as well as cell composition and organization (Figure 2), making the organoids ideal for HTS approaches.Furthermore, our workflow generates one organoid per well, maintained independently from other organoids, thus minimizing batch effects for paracrine signaling observed in bioreactor-based strategies (Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48-53 (2017)). If paracrine signaling is desired, our workflow can also accommodate several organoids per well.

[0100] Automated midbrain organoids express typical neural and midbrain markers and show structural organization. To characterize protein localization in our large-scale AMOs (>500 μm diameter) and to evaluate the efficiency of their neural / mesencephalic differentiation at cellular resolution and in an HTS-compatible format, we adapted an extended 3D staining protocol by Lee et al. ACT-PRESTO: Rapid and consistent tissue 579 clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep 6, 18631 (2016) for use in organoids and combined this with benzyl alcohol and benzyl benzoate (BABB)-based tissue clearing (Dent, J.A., Polson, A.G. & Klymkowsky, M.W. A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105, 61-74 (1989)). We found that BABB-based clearing was both the fastest and most effective method compared to different clearing protocols. The combination of whole-mount staining and clearing enabled 3D reconstruction of entire organoids via confocal imaging, allowing for more detailed 3D quantification and analysis, such as neurite tracing throughout the organoid that could not be performed using typical tissue sectioning procedures (see Fig. 1d).

[0101] The results of immunostaining are shown as either single confocal optical sections (Fig. 2a, b, c, d, f, g, h, j) or maximum intensity projections (MIP, Fig. 2e). Already on day 25, the AMO contains many neurons as shown by the expression of Map2 (Shafit-Zagardo, B. & Kalcheva, N. Making sense of the multiple MAP-2 transcripts and their role in the neuron. Mol Neurobiol 16, 149-162 (1998)) (Fig. 1d), β-tubulin III (TUBB3) (Leandro-Garcia, L.J. et al. Tumoral and tissue-specific expression of the major human beta tubulin isotypes. Cytoskeleton (Hoboken) 67, 214-223 (2010)) (Fig. 2e) and doublecortin (Gleeson, J.G., Lin, P.T., Flanagan, L.A. & Walsh, C.A. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 23, 257-271 (1999)) (DCX, Fig. 2c and 2d).The presence of tyrosine hydroxylase (TH, Figures 2a and 2b), the rate-limiting enzyme for dopamine synthesis (Nagatsu, T. Tyrosine hydroxylase: human isoforms, structure and regulation in physiology and pathology. Essays Biochem 30, 15-35 (1995)), and the expression of the transcription factors Nurr1 and Foxa2 (Hegarty, S.V., Sullivan, A.M. & O'Keeffe, G.W. Midbrain dopaminergic neurons: a review of the molecular circuitry that regulates their development. Dev Biol 379, 123-138 (2013)) (Figures 2f and 2g) are consistent with the dopaminergic midbrain differentiation of our organoids.As seen in common to all neural organoids, AMO retains a population of neural precursors identified by the expression of Sox2 (Ellis, P. et al. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev Neurosci 26, 148-165 (2004)) (FIGS. 1d, 2a and b), Brn2 (Dominguez, M.H., Ayoub, A.E. & Rakic, P. POU-III transcription factors (Brn1, Brn2, and Oct6) influence neurogenesis, molecular identity, and migratory destination of upper-layer cells of the cerebral cortex. Cereb Cortex 23, 2632-2643 (2013)) (FIGS. 2c and d) and the more general neural marker nestin (Hendrickson, M.L., Rao, A.J., Demerdash, O.N. & Kalil, R.E. Expression of nestin by neural cells in the adult rat and human brain. PLoS One 6, e18535 (2011)) (FIGS. 2a and 2b).

[0102] Over time, AMO further matures. On day 50, the expression of the presynaptic marker synaptophysin and the postsynaptic marker Homer, which frequently co-localize with each other on Map2-positive neurites (Tadokoro, S., Tachibana, T., Imanaka, T., Nishida, W. & Sobue, K. Involvement of unique leucine-zipper motif of PSD-Zip45 (Homer 1c / vesl-1L) in group 1 metabotropic glutamate receptor clustering. Proc Natl Acad Sci U S A 96, 13801-13806 (1999)) (Figure 2h), indicates the presence of synapses. Since gliogenesis follows neurogenesis in vivo (Miller, F.D. & Gauthier, A.S. Timing is everything: making neurons versus glia in the developing cortex. Neuron 54, 357-369 (2007)), the inventors predict the emergence of astrocytes after the initial formation of neurons. Consequently, AMO contains GFAP- and S100b-double positive astrocytes on day 75 (Gotz, M., Sirko, S., Beckers, J. & Irmler, M. Reactive astrocytes as neural stem or progenitor cells: In vivo lineage, In vitro potential, and Genome-wide expression analysis. Glia 63, 1452-1468 (2015)) (Figure 2j).

[0103] Generally, different cell types within the AMO (i.e., neurons, astrocytes, and neural precursors) do not form localized structures such as neural rosettes, but rather are organized in four concentric regions around the center of the AMO (Figs. 2a and 2c). The outermost region 4, which is a layer oriented circumferentially around the TH+ / nestin+ / DCX+ cell processes, contains few nuclei at high density. The cell orientation changes in the underlying region 3 closer to the core of the organoid, where TH+ dopamineergic and DCX+ neurons show a distinct radial organization (Figs. 2b and 2c). Region 2, which separates this radially organized neuronal region from the core, contains DCX+ neurons oriented circumferentially and few Brn2+ neural precursors (Figs. 2c and 2d). Region 1, which is the core itself, contains mainly neural precursors and few neurons. Within a given distance from the center, different cell types are uniformly distributed around the entire radius of the microtissue. In the context of HTS compatibility, this radial symmetry is an advantage over protocols that yield more complex and even more heterogeneous organoids with locally randomly branching subdomains, as it makes optical quantification independent of the orientation of the microtissue in the well.

[0104] Superstructural analysis of the AMO (Fig. S2) supports immunofluorescence data revealing a high-density 3D cell structure consistent with neuronal cell bodies surrounded by nerve fibers. Analysis of nerve fibers at high magnification reveals regularly spaced neurofilaments and microtubules that can be identified. Furthermore, vesicles with the characteristic size and localization of synaptic vesicles are frequently seen within these nerve fibers.

[0105] Further quantitative real-time PCR (qPCR) analysis showed increased expression levels of various neuronal (DCX, Map2, NEFL, NeuN, TBR2, TUBB3, Syt1), midbrain (EN1, GIRK2, MIXL1, NURR1, TH) and glia-specific (GLAST, MBP, S100b) markers, accompanied by simultaneous decreases in neural progenitor markers (Brn2, nestin, Pax6, Sox1, Sox2) at different developmental stages, confirming neural midbrain maturation over time (Figure 3).

[0106] Calcium imaging reveals spontaneous and synchronous activity across entire organoids To evaluate the functional connectivity of individual cells within the AMO, the inventors performed Fluo-4 acetoxymethyl ester (AM)-based calcium imaging that can be used as a readout for spiking neuronal activity (Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862-885 (2012)). In addition to the spontaneous activity of individual cells, the inventors observed simultaneous and periodic calcium spikes throughout the organoids in all analyzed organoids (n = 5). To further characterize this behavior, the inventors defined different regions of interest (ROIs) and evaluated the change in fluorescence intensity over time in each region (Figure 4). By measuring the entire organoid, two consecutive spikes are revealed in the brightness of Fluo-4 at a period of approximately 30 seconds (Figure 4a). When the region measured by the inventors is further divided into four quadrants, the inventors see simultaneous spike activity in all four resulting ROIs (Figure 4b). This similar activity pattern can be seen at many structural levels of the organoid, even for single cells (Figure 4c). By changing the time scale, in addition to the spikes of the entire organoid as a whole, a further level of simultaneity between selected single cells is revealed (Figure 4d). Considering the presence of synaptic vesicles at the ultrastructural level (Figure S2), synapses by immunostaining (Figure 2h), and synaptotagmin 1 (Syt1) by qPCR (Figure 3), the results of calcium imaging support the existence of functionally connected neurons within the AMO. The simultaneous spike pattern suggests that not only some neurons but actually the entire organoid is functionally connected.

[0107] RNA sequencing reveals lower variability within and between batches in automated midbrain organoids compared to established protocols To examine the variability of AMO at the gene expression level, the inventors performed RNA sequencing of single organoids from three different batches of AMO and one batch of iPSC-derived organoids manually generated according to an established protocol from Lancaster et al. (Cerebral organoids model human brain development and microcephaly. Nature 501, 373-379 (2013)) as a control. Since iPSC-derived organoids need to first experience a period of neural precursors before entering their neural maturation, the inventors performed sequencing of AMO on day 30 and of iPSC-derived organoids on days 30 and 45 for a more even comparison. This comparison revealed that the variance in the whole-genome expression levels measured by principal component analysis (PCA, Figure 5a) in AMO was approximately 4-fold lower than that in iPSC-derived organoids (Figure 5b), indicating that AMO has higher reproducibility within and between batches than current standard protocols.

[0108] Further gene ontology (GO) (Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25, 25-29 (2000); and Supek, F., Bosnjak, M., Skunca, N. & Smuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6, e21800 (2011)) analysis of genes significantly upregulated (padj. < 0.05) in AMO on day 30 compared to iPSC-derived organoids on day 45 (Figure 5c) yielded almost exclusively GO terms related to neuron maturation, particularly synaptic activity (Figure 5d, see Table 1 for the complete list of GO terms and further analysis), indicating that AMO matures earlier than established organoids.

[0109] In screening settings, wells at the edges of plates often show different readouts than those placed further towards the center of the plate ("edge effect") (Malo, N., Hanley, J.A., Cerquozzi, S., Pelletier, J. & Nadon, R. Statistical practice in high-throughput screening data analysis. Nat Biotechnol 24, 167-175 (2006)). Therefore, the inventors decided to sequence half of a 96-well plate for one AMO batch and test for differences arising from the position of the wells within the plate (group "1 inner" = central plate vs. "1 outer" = edge of FIGS. 5a / b). AMOs cluster independently of their positions on the plate, indicating that AMOs do not show a measurable edge effect at the gene expression level. Together, the results of RNA sequencing show higher homogeneity and earlier neuronal maturation of AMOs compared to standard iPSC-based protocols.

[0110] Automated whole-mount immunostaining is highly quantitative and reveals the homogeneity of automated midbrain organoids Immunofluorescence-based screening compatibility techniques for whole organoids have been reported, but the technique can only detect cells in the outer layer of large organoids (Vergara, M.N. et al. Three-dimensional automated reporter quantification (3D-ARQ) technology enables quantitative screening in retinal organoids. Development 144, 3698-3705 (2017)), or small aggregates with a diameter of about 100 μm can be used (Verissimo, C.S. et al. Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening. Elife 5 (2016)) or cystic organoids in which antibodies and fluorescence proofs can penetrate more easily (Czerniecki, S.M. et al. High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping. Cell Stem Cell 22, 929-940 e924 (2018)) can be used. In contrast, the inventors' workflow enables the quantification of high-density whole large organoids (>800 μm in diameter) with single-cell resolution and high sensitivity, as highlighted by a dose-response assay for 3D cell detection (Figure 6a). The inventors mixed cells tracked by a fluorescent dye and unlabeled cells at a known ratio, aggregated them, cleared them, and then analyzed them in a confocal high-content imaging system. The resulting relationship between the amount of tracked cells and the measured brightness is highly linear (R2>0.99), indicating the quantitative nature of the inventors' optical HTS 3D whole mount analysis workflow.

[0111] Next, the inventors demonstrated the homology of AMO at the protein level. A fully automated 96-well AMO whole mount optical assay (left in Fig. 6b) shows the ability to detect both abundant fibrous structures (neuronal marker Map2) and nuclear marker (Sox2) in a HTS-compatible format (right in Fig. 6b, single section from one organoid). Using nuclear markers such as Sox2, the inventors' technique enables quantification at single cell resolution by identifying, counting, and summing the brightness of Sox2+ nuclei for each imaged confocal plane (Fig. 6c / d / f / h). Fibrous abundant signals such as Map2 can be quantified in organoids by summing the overall average brightness for each confocal plane (Fig. 6e / g). Comparison of three 96-well plates from independent differentiations revealed a uniform cell composition of AMO within and between batches (Fig. 6d - g) (average CVSox2 = 5%, CVMap2 = 9%).

[0112] By positional analysis, an effect of plate position (edge effect) on Map2 levels rather than Sox2 levels was detected, and the Map2 brightness of organoids at the center of the plate was reduced by 10% compared to the wells at the edge (Fig. S3). Considering the absence of an edge effect in the RNA sequencing results, this may indicate that only a specific subset of proteins is altered by edge conditions, while most of the cellular processes are uniform across the plate (see Table 2 for a list of differential gene expression between organoids inside and at the edge of the plate).

[0113] Automated midbrain organoids enable toxicity assessment in specific cell subpopulations at the single cell level in a fully automated high-throughput screening format To evaluate the ability of our workflow to quantify drug effects, we treated AMOs with increasing concentrations of the known cytotoxic compound G418 and stained for the apoptosis marker cleaved caspase 3 (cCasp3) together with Sox2. By plotting the number of apoptotic cCasp3+ cells against the logarithmic drug concentration, a typical sigmoidal dose–response curve with an IC50 of 339 μg / mL was revealed (Figure 7a), which is in agreement with the published value (Delrue, I., Pan, Q., Baczmanska, A. K., Callens, B. W. & Verdoodt, L. L. M. Determination of the Selection Capacity of Antibiotics for Gene Selection. Biotechnol J 13, e1700747, doi:10.1002 / biot.201700747 (2018)). Co-localization analysis between cCasp3 and Sox2 (Figure 7b) shows that the workflow can also be used to evaluate cell-type-specific toxicity in 3D. This also indicates that G418 mainly does not affect Sox2+ neural precursors other than the other (more mature) cell types. A part of the precursors in apoptotic cells increases with higher inhibitor concentrations, but it remains at a low level of only up to 15%. Figure 7c shows an example of a high-content image showing the increase in cCasp3 signal with increasing inhibitor concentration. We stained, imaged, and analyzed all AMOs for this experiment, but we understood that by considering only a single confocal plane during the analysis, almost the same results are obtained. This can be used to significantly reduce the required imaging time and cost in large-scale drug screening campaigns. Finally, we also generated AMOs from a second independent smNPC line derived from patient-derived iPSCs and confirmed the applicability of our workflow for different cell lines with different genetic backgrounds.

[0114] Homogeneity - AMOs of the present invention, NABOs of the present invention, and prior art organoids The AMO is significantly more homogeneous than other published brain organoids in terms of overall shape and size. See Figure 8 and the description.

[0115] The internal organization / structure of the state-of-the-art brain organoids is highly variable and unpredictable compared to the AMO. See Figure 9 and the description.

[0116] Compared to only other midbrain organoids, the AMO still exhibits the highest level of homogeneity. See Figure 10 and the description.

[0117] Analysis of the cell composition reveals significant variability in the state-of-the-art organoids. See Figure 11 and the description.

[0118] The NABO of the present invention See Figure 12 and the description. Examples of aspects of the present invention include the following. Item 1 (a) A step of seeding a plurality of tissue-specific progenitor cells in a container; (b) (i) Aggregation of the cells; and (ii) Maturation of the aggregate formed in (i) into a single organoid A method for producing an organoid, comprising or consisting of the steps of A method that does not include embedding the cells or the aggregate in a gel. Item 2 (i) The organoid is a neural organoid, preferably a midbrain organoid or a homogeneous brain organoid without pattern formation, The tissue-specific progenitor cells are neuron tissue-specific progenitor cells, preferably small molecule neuron progenitor cells (smNPC); (ii) The organoid has a reproducible or homogeneous size and / or cell composition, and homogeneous preferably means a standard deviation of less than about 20% or less of the average; (iii) Step (b) includes (b-i) Culturing in an aggregation medium, preferably for about 2 days, where the aggregation medium preferably contains polyvinyl alcohol; (b-ii) Culturing in a maturation medium; and (b-iii) Preferably between (b-i) and (b-ii), culturing in a ventral patterning medium, preferably for about 4 days Including; (iv) The plurality of cells are about 100 to about 1,000,000, preferably about 10,000 cells; and / or (v) The container is a well of a multi-well plate, where preferably a plurality of the cells are seeded in a plurality of wells or each well of the multi-well plate, and a multi-well plate is obtained in which a plurality of wells or each well contains one single organoid. (v) The container is a well of a multi-well plate, where preferably a plurality of the cells are seeded in a plurality of wells or each well of the multi-well plate, and a multi-well plate is obtained in which a plurality of wells or each well contains one single organoid. The method according to Item 1. Item 3 An organoid or a plurality of organoids obtained by the method according to any of the preceding items. Item 4 (a) The organoid(s) is / are (a) neural organoid(s), (a) preferably midbrain organoid(s) or (a) non-patterned homogeneous brain organoid(s); (b) The organoid(s) is / are (i) showing a plurality of concentric regions, preferably at least three regions, where each region is different from any of the other regions with respect to cell composition and organization; and / or (ii) the organoid(s) showing tissue-specific cell activity, preferably electrical activity in neurons in the case of neural organoids; and / or (c) the plurality of organoids being homogeneous with respect to structure and / or size, an organoid or a plurality of organoids; the organoid or the plurality, preferably obtained by the method according to item 2, of an organoid or a plurality of organoids. Item 5 A multi-well plate in which each of a plurality of wells contains one single organoid or each well contains one single organoid, preferably the plurality of organoids or each organoid being obtained by the method defined in item 3 or 4 or according to item 1 or 2. Item 6 Use of tissue-specific progenitor cells for organoid production, wherein no gel is used for embedding cells or aggregates, preferably the tissue-specific progenitor cells being neuron tissue-specific progenitor cells, preferably small molecule neuron progenitor cells (smNPC). Item 7 A method for producing an organoid or spheroid for analysis, (a) a step of staining the organoid or spheroid; (b) a step of performing tissue clearing using the organoid or spheroid comprising or consisting of the step. Item 8 (a) The staining is (i) performed using an antibody, preferably a primary antibody and a secondary antibody, where the staining using the primary antibody and / or the secondary antibody is performed for about 5 to about 10 days, preferably about 6 days; (ii) a fluorescent label; (iii) a luminescent label; (iv) performed using a radioactive label; and / or (b) the clearing is benzyl alcohol and benzyl benzoate (BABB)-based clearing, preferably the clearing being performed in a cycloolefin container, more preferably a cycloolefin multi-well plate. The method according to item 7. Item 9 (a) The method does not include slicing of the organoid or spheroid, and / or the staining is whole-mount staining; and / or (b) The organoid is the organoid according to item 3 or 4 or obtained by the method according to item 1 or 2, The method according to item 7 or 8. Item 10 A method for analyzing an organoid or spheroid, the method being the method according to any of items 7 to 9; and (c) Analysis of the stained and cleared organoid or spheroid, preferably (c-i) Optical analysis, where the optical analysis preferably includes microscopy and / or image analysis; (c-ii) Genetic analysis such as RNA sequencing; and / or (c-iii) Protein analysis such as mass spectrometry or Western blot, comprising or consisting of such methods. Item 11 A method for preparing and analyzing an organoid, comprising or consisting of the method according to item 1 or 2 and the method according to item 10. Item 12 A method for identifying an organoid, an organoid-forming and / or an organoid-specific function regulator, (a) (i) A step of adding a test compound to an organoid preferably according to item 3 or 4 or an organoid obtained by the method according to item 1 or 2; (ii) A step of performing the method according to item 1 or 2 after adding the test compound to tissue-specific progenitor cells; or (iii) A step of performing the method according to item 1 or 2, where the test compound is added at one or more time points during the performance of the method according to item 1 or 2; (b) A step of performing the method according to item 10; (c) A step of comparing the result of the analysis in the presence of the test compound with the result of the analysis in the absence of the test compound, where the difference indicates a regulator, comprising or consisting of such steps. Item 13 (a) If the analysis shows an improvement in the function of the organoid, organoid formation and / or an organoid-specific function, the test compound is a lead compound, and the method optionally further comprises or consists of a step of developing the lead compound to produce a drug; or (b) If the analysis shows a decrease in the function of the organoid and / or a negative interference with organoid formation and / or an organoid-specific function, this indicates that the test compound is toxic. The method according to item 12. Item 14 (a) An automated format; and / or (b) A high-throughput format preferably using means for multiwell plates, pipetting robots, automated liquid handling, plate readers and / or plate transport The method according to any one of claims 1, 2 or 7 to 13, which is carried out in Claim 15 (a) Tissue-specific progenitor cells, preferably neuron tissue-specific progenitor cells, more preferably smNPCs; and (b) A culture medium A kit comprising or consisting of, wherein the culture medium is (b-i) An aggregation medium, wherein the aggregation medium preferably contains polyvinyl alcohol; (b-ii) A maturation medium; and (b-iii) Optionally, a ventral patterning medium A kit comprising or consisting of.

Claims

1. (a) A step of seeding a plurality of neuron tissue-specific progenitor cells into a container; (b) (b-i) A step of culturing in an aggregation medium to cause aggregation of the cells; and (b-ii) A step of culturing in a maturation medium to cause maturation of the aggregate formed in (b-i) into a single neural organoid ; A method for producing a plurality of neural organoids comprising or consisting of the steps; The method, wherein the cells or the aggregates are not embedded in a gel, and the plurality of neural organoids have a homogeneous size, and homogeneous means a standard deviation of less than 20% or less of the average.

2. (i) The neural organoid is a midbrain organoid or a homogeneous brain organoid that is not patterned; (ii) In (b-i), the culturing in the aggregation medium is for 2 days; (iii) The plurality of cells are from 100 to 1,000,000; and / or (iv) The container is a well of a multi-well plate. The method according to claim 1.

3. (i) The aggregation medium contains polyvinyl alcohol; and / or (ii) The method includes culturing in a ventral patterning medium between (b-i) and (b-ii); and / or (iii) The neuron tissue-specific progenitor cells are small molecule neuron progenitor cells (smNPCs). The method according to claim 1 or 2.

4. A multi-well plate in which a plurality of the cells are seeded into a plurality of wells or each well of the multi-well plate, and the plurality of wells or each well contains a single neural organoid. The method according to claim 2.

5. A plurality of neural organoids, (a) The plurality of neural organoids are a plurality of midbrain organoids or a plurality of homogeneous brain organoids that are not patterned; and / or (b) The plurality of neural organoids (i) A plurality of regions with the same center, where each region is different from any of the other regions in terms of cell composition and organization. ; and / or (ii) The plurality of neural organoids exhibit tissue-specific cell activity; and / or (c) The plurality of neural organoids are homogeneous in size and optionally homogeneous in structure. The plurality of neural organoids have a homogeneous size, and homogeneous means a standard deviation of less than 20% or less of the average. The plurality of neural organoids are those obtained by the method according to claim 2. Plurality of neural organoids.

6. (I) The plurality of regions of the same center include at least three regions; and / or (II) The tissue-specific cell activity is electrical activity in neurons. The plurality of neural organoids according to claim 5.

7. A multi-well plate in which each of the plurality of wells contains one single neural organoid or each well contains one single neural organoid, and the plurality of neural organoids or each neural organoid is as described in claim 5 or 6.

8. Use of neuron tissue-specific progenitor cells for the production of a plurality of neural organoids, wherein the use comprises (a) a step of seeding a plurality of neuron tissue-specific progenitor cells in a container; and (b) (b-i) culturing in an aggregation medium to cause aggregation of the cells; and (b-ii) culturing in a maturation medium to cause maturation of the aggregates formed in (b-i) into single neural organoids wherein no gel is used to embed the cells or their aggregates, and the plurality of neural organoids have a homogeneous size, where homogeneous means a standard deviation of less than 20% or less of the average.

9. The use according to claim 8, wherein the neuron tissue-specific progenitor cells are small molecule neuron progenitor cells (smNPC).

10. A method for producing neural organoids for analysis, comprising (a) a step of staining the plurality of neural organoids according to claim 5 or 6; (b) a step of performing tissue clearing using the plurality of neural organoids wherein the method comprises or consists of the steps.

11. (a) The staining is performed using (i) an antibody; (ii) a fluorescent label; (iii) a luminescent label; or (iv) a radioactive label; and / or (b) The clearing is benzyl alcohol and benzyl benzoate (BABB)-based clearing. The method according to claim 10.

12. (i) The staining in (a) is performed using a primary antibody and a secondary antibody, wherein the staining using the primary antibody and / or the secondary antibody is performed for 5 to 10 days; and / or (ii) The clearing in (b) is performed in a cycloolefin container. The method according to claim 11.

13. (a) The method does not include sectioning of the neural organoids, and / or (b) The staining is whole-mount staining, The method according to any one of claims 10 to 12.

14. A method for analyzing a neural organoid, the method comprising The method according to any one of claims 10 to 13; and (c) Analysis of the stained and cleared neural organoid, A method comprising or consisting of these.

15. The analysis is (c-i) Optical analysis; (c-ii) Genetic analysis; and / or (c-iii) Protein analysis, is, The method according to claim 14.

16. The method according to claim 15, wherein the optical analysis includes microscopy and / or image analysis.

17. The method according to claim 15, wherein the genetic analysis is RNA sequencing.

18. The method according to claim 15, wherein the protein analysis is mass spectrometry or Western blot.

19. The method according to claim 14 or 15, further comprising the step of producing the neural organoid by performing the method according to any one of claims 1 to 4.

20. A method for identifying a neural organoid, a neural organoid-forming and / or a regulator of a neural organoid-specific function, (a) (i) The step of adding a test compound to a plurality of neural organoids according to claim 5 or 6; (ii) After adding the test compound to the neuron tissue-specific progenitor cells, the step of performing the method according to any one of claims 1 to 4; or (iii) The step of performing the method according to any one of claims 1 to 4, wherein the test compound is added at one or more time points during the performance of the method according to any one of claims 1 to 4; (b) The step of performing the method according to claim 14 or 15; (c) The step of comparing the result of the analysis in the presence of the test compound with the result of the analysis in the absence of the test compound, wherein the difference indicates a regulator, A method comprising or consisting of the step.

21. (a) When the result of the analysis in the presence of the test compound shows an improvement in the function of the neural organoid, neural organoid formation and / or a neural organoid-specific function, the test compound is a lead compound, and the method according to claim 20 optionally further comprises or consists of the step of developing the lead compound to produce a drug; or (b) If the results of the analysis in the presence of the test compound show a decrease in the function of the neural organoid and / or a negative interference with neural organoid formation and / or neural organoid-specific functions, the test compound is toxic. The method according to claim 20. **Claim 22** (a) In an automated manner; and / or (b) In a high-throughput format The method according to any one of claims 1 to 4 or 10 to 21, which is carried out **Claim 23** The method according to claim 22, wherein the analysis in the high-throughput format involves the use of multi-well plates, pipetting robots, automated liquid handling, plate readers and / or means for plate transport.

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