Methods for producing brain organoids, multiwell plates containing said organoids, and screening methods

Abstract

The invention provides inter alia a method for the production of an adherent cortical organoid, comprising the steps of: (a) providing a cell culture comprising a population of neural progenitor cells (NPCs); (b) seeding neural progenitor cells (NPCs) of said population in a well of a multiwell plate; wherein said well comprises a bottom surface (106) and an upright wall (104), and wherein a distance (x) between a centre (112) of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm; and (c) culturing said seeded NPCs in a culture medium for neural differentiation for a period of time, to thereby allow formation of an adherent cortical organoid.

Description

[0001] Title: Methods for producing brain organoids, multiwell plates containing said organoids, and screening methods.

[0002] FIELD OF THE INVENTION

[0003] The invention is in the field of cortical brain organoid production. In particular, the present invention relates to methods for producing adherent cortical brain organoids which can be used in multiple applications, such as fundamental research that investigates brain development, as well as in screening for compounds of interest, such as food ingredients, that may modulate brain development. The invention also relates to the produced adherent cortical organoids as such, as well as to multiwell plates comprising wells containing said produced adherent cortical organoids. The invention also relates to methods for screening agents (compounds) of interest that may modulate formation of cortical brain organoids.

[0004] BACKGROUND OF THE INVENTION

[0005] Using human embryonic or induced pluripotent stem cell (hiPSC)- derived models to investigate the developing brain in health and disease has yielded considerable success (Marton and Pasca, Trends Cell Biol. 2020;30:133-143). The approaches have been varied, including single cell hiPSC-derived models grown in a monolayer (Sarkar et al., Cell Stem Cell. 2018;22:684-697.e9; Zhang et al., Neuron. 2013;78:785-798), multiple neural cell types in 2D neural networks (Astick and Vanderhaeghen, Curr Top Dev Biol. 2018;129:67-98; Bardy et al., Proc Natl Acad Sci U.S.A. 2015;112:E2725-34; Gunhanlar et al., Mol Psychiatry. 2018;23:1336-1344; Shi et al., Nat Neurosci. 2012;15:477-486), 3D free-floating regionalized neural organoids (Pa§ca et al., Nat Methods. 2015;12:671-8; Qian et al., Cell. 2016;165:1238-1254; Xiang et al., Cell Stem Cell. 2019;24:487-497; Zhang et al., 2023, Biol Psychiatry 93:594-605), and hiPSC-derived free-floating unguided neural organoids (Lancaster et al., Nature. 2013;501:373-379; Renner et al., EMBO J. 2017;36:1316-1329; Sawada et al., Mol Psychiatry. 2020;25:2695-2711; Gomes et al., Front Cell Dev Biol. 2020;8:1-18;

[0006] Pellegrini et al., Science. 2020;369:eaaz5626).

[0007] However, among the consistent findings across models of different types of brain organoids is that increasing cellular and topographical complexity has appeared to come at the cost of increased variability (Eichmuller et al., Nat Rev Neurol 18:661-680 (2022); Kelava et al., Cell Stem Cell 18:736-748 (2016)).

[0008] Therefore, a major current technical challenge is to identify hiPSC- derived models that recapitulate higher-order neural complexity with reduced heterogeneity.

[0009] Currently, most existing brain organoids are free-floating structures, which suffer from further variability due to the complex and heterogeneous nature of free-floating structures. A further challenge, in particular with free-floating organoids, is the necrotic core that emerges when tissue volumes exceed the limits of oxygen and nutrient diffusion beyond a radius of ~300-400pm. Although recent progress has been made with slicing organoids prior to the emergence of necrosis followed by organotypic air -liquid interface culture, even sliced organoids have to be repeatedly re-cut to prevent necrosis (Qian et al., Cell Stem Cell 26:766- 781.e9 (2020)), which is both laborious and risks introducing another potential source of variability.

[0010] Hence, there is a need for a simple method for generating cortical brain organoids in a reproducible manner, with low inter-organoid variability, preferably whereby the cortical brain organoids still exhibit higher-order neural complexity.

[0011] SUMMARY OF THE INVENTION The inventors have unexpectedly discovered a highly reproducible and simple method to produce a homogenous population of adherent cortical brain organoids, whereby the organoids can retain higher-order neural complexity. These cortical organoids are not free-floating but instead adhere to the bottom surface of a well of a multiwefl plate as a single adherent cortical organoid, with radial structure, per well. The inventors also established that this method can be employed using neural progenitor cells (NPCs) that are obtained from varying sources and using various cell culture conditions.

[0012] Therefore, the invention provides a method for the production of an adherent cortical organoid, comprising the steps of: (a) providing a cell culture comprising a population of neural progenitor cells (NPCs); (b) seeding neural progenitor cells (NPCs) of said population in a well of a multiwell plate; wherein said well comprises a bottom surface and an upright wall, and wherein a distance x between a centre of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm; and (c) culturing said seeded NPCs in a culture medium for neural differentiation for a period of time, to thereby allow formation of an adherent cortical organoid to occur.

[0013] Surprisingly, it was found that the confined space of a well of a standard flat (F) bottom multiwell plate, more specifically its bottom dimension, dictates that neural progenitor cells (NPCs) self-organize into a single cortical brain organoid, with a radial structure, per well. Moreover, it was discovered that the formation of a single cortical brain organoid with a radial structure, per well, was highly reproducible with low inter-organoid variability between seeded wells. In fact, single cortical organoids with a (single) radial structure, per well, were observed in about 80% of the wells seeded with NPCs after 60 days of differentiation. Even after about one year in culture, the number of single structure organoids per well was still about 50%. More specifically, the inventors have found that within the ranges of distance x specified herein, neural progenitor cells (NPCs) self-organize into a single cortical brain organoid, with a radial structure, per well. For instance, it was found that, when the distance % of a custom-made well was set at 0.5 mm or 1.0 mm, formation of a single cortical brain organoid per well was severely impacted (data not shown). The same was observed when the distance % of a custom-made well was set at 2.0 mm or when wells of a standard 96 well plate were used, the latter having a distance % of 3.5 mm (data not shown). The inventors also established that both square and circular bottom surfaces performed equally well (data not shown).

[0014] An adherent cortical brain organoid as disclosed herein can be maintained in long-term cell culture for more than 10 months, and can contain neurons that exhibit mature neuronal features including dendritic spines, as well as several classes of glial cells including oligodendrocyte precursor cells, myelinating oligodendrocytes, and morphologically distinct sub-types of astrocytes. Moreover, the adherent cortical organoids of the invention can exhibit robust electrophysiological activity measured by fluorescence-based calcium imaging.

[0015] In a preferred embodiment of said method for the production of an adherent cortical organoid, said well of said multiwell plate has a flat (F) bottom surface and / or said multiwell plate is a flat (F) bottom multiwell plate. In a highly preferred embodiment, the bottom surface of the well is a flat bottom surface.

[0016] In another preferred embodiment of said method for the production of an adherent cortical organoid; said distance x between a centre of the bottom surface and the upright wall is between 1.2 mm and 1.9 mm, preferably between 1.3 mm and 1.8 mm, preferably between 1.4 mm and 1.8 mm, more preferably between 1.5 mm and 1.8 mm such as between 1.6 and 1.7 mm. As an example, the distance x can be about 1.65 mm, which preferably corresponds to a well width or well diameter of about 3.3 mm. In another preferred embodiment of said method for the production of an adherent cortical organoid, said bottom surface of the well is a flat bottom surface; and said distance x between a centre of the bottom surface and the upright wall is between 1.5 mm and 1.8 mm.

[0017] In another preferred embodiment of said method for the production of an adherent cortical organoid, a height h of the well is between 5 and 20 mm, preferably between 5 and 15 mm, more preferably between 8 and 13 mm, more preferably between 10 and 13 mm, even more preferably between 11 and 12 mm. One example of the height h of the well is about 11.5 mm.

[0018] In another preferred embodiment of said method for the production of an adherent cortical organoid, said well of said multiwell plate is a well of a 384-well plate, preferably a flat (F) bottom 384-well plate.

[0019] In another preferred embodiment of said method for the production of an adherent cortical organoid, said multi-well plate is a flat (F) bottom 384-well plate.

[0020] In another preferred embodiment of said method for the production of an adherent cortical organoid, said well of said multiwell plate is coated with a coating for adherent cell culture, preferably wherein said coating for adherent cell culture is a laminin and / or poly-L-ornithine coating. Surprisingly, it was established that, especially, a laminin coating outperformed a standard Matrigel cell culture substrate (coating) by further improving reproducibility in the production of adherent cortical organoids (data not shown).

[0021] In another preferred embodiment of said method for the production of an adherent cortical organoid, in step (b), NPCs of said population are seeded in, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or in 100%, of the wells, calculated on the basis of the total number of wells, of said multiwell plate. In another preferred embodiment of said method for the production of an adherent cortical organoid, said NPCs are positive for one or more of the markers selected from the group formed by SOX2, Nestin, Vimentin and FOXG1.

[0022] In another preferred embodiment of said method for the production of an adherent cortical organoid, said NPCs are frontal cortical NPCs. Preferably, they are characterized by the presence of at least marker FOXG1.

[0023] In another preferred embodiment of said method for the production of an adherent cortical organoid, said NPCs are derived (or obtained) from induced pluripotent stem cells (iPSCs), preferably human iPSCs.

[0024] In another preferred embodiment of said method for the production of an adherent cortical organoid, wherein said NPCs are derived (or obtained) from induced pluripotent stem cells (iPSCs), said iPSCs are derived from somatic cells of a healthy or diseased subject. Hence, the NPCs may resemble a healthy or a diseased state, which may impact the structural and / or functional characteristics of an adherent cortical organoid when it forms. Preferably, if the NPC is derived from a diseased subject, the disease or disorder is a genetic disease, more preferably a genetic neurodevelopmental or neuropsychiatric disorder.

[0025] In another preferred embodiment of said method for the production of an adherent cortical organoid, said population of NPCs provided in step (a) is obtainable by a method comprising the steps of: (al) providing a cell culture comprising pluripotent stem cells (PSCs) and feeder cells; (a2) dissociating PSCs from said feeder cells; (a3) generating embryonic bodies from said PSCs, preferably by culturing said dissociated PSCs in a culture medium for maintaining PSCs on a non-adherent plate; preferably wherein the embryonic bodies are cultured for about 2 days in said culture medium for maintaining PSCs; (a4) culturing the embryonic bodies in a neural induction culture medium in suspension, preferably wherein the embryonic bodies are in suspension cell culture for about 4 days; (a5) optionally, at least partially dissociating said embryonic bodies, for example by trituration; (a6) plating the embryonic bodies, which are optionally at least partially dissociated, on a coated substrate, preferably a laminin-coated substrate such as a laminin-coated dish; and (a7) culturing the plated embryonic bodies, which are optionally at least partially dissociated, in a neural induction culture medium, followed by a step of culturing in an NPC culture medium so as to provide a population of NPCs; preferably wherein the optionally at least partially dissociated embryonic bodies are cultured for about 8 days in said neural induction culture medium and preferably wherein said optionally at least partially dissociated embryonic bodies are cultured for 1-10 days in said NPC culture medium; and optionally (a8) subjecting said population of NPCs to a step of antibody-based cell sorting; wherein said step of antibody-based cell sorting is performed on the basis of (i) the presence of cell surface markers CD 184 (CD 184+) and / or CD24 (CD24+) and (ii) the absence of cell surface markers CD44 (CD44-) and / or CD271 (CD271-) in order to provide a population of sorted NPCs.

[0026] In another preferred embodiment of said method for the production of an adherent cortical organoid, said step (a) is preceded by a step of: - subjecting neural progenitor cells (NPCs) to a step of antibody-based cell sorting; wherein said step of antibody-based cell sorting is performed on the basis of (i) the presence of cell surface marker CD 184 (CD 184+) and / or CD24 (CD24+) and (ii) the absence of cell surface marker CD44 (CD44-) and / or CD271 (CD271-), to thereby provide a population of sorted (also referred to as purified) NPCs. Such a purification step is optional, and allows for obtaining a more homogenous population of NPCs prior to seeding.

[0027] In another preferred embodiment of said method for the production of an adherent cortical organoid, in step (a), said cell culture comprising a population of neural progenitor cells (NPCs) is in an NPC culture medium (also referred to as NPC expansion medium) as disclosed herein. Preferably, said NPC culture medium is a culture medium comprising a basal medium and a basic fibroblast growth factor. More preferably, said NPC culture medium comprises DMEM / F12, an N2 supplement, a B-27 supplement without vitamin A (B-27-RA), and a basic fibroblast growth factor, and may optionally further comprise a laminin, a penicillin and / or a streptomycin.

[0028] In another preferred embodiment of said method for the production of an adherent cortical organoid, said culture medium for neural differentiation comprises a brain-derived neurotrophic factor, a glial cell- derived neurotrophic factor, dibutyryl cyclic adenosine monophosphate, and / or ascorbic acid. Preferably, the NPCs are seeded in a well of a multiwell plate as disclosed herein while they are in a culture medium for neural differentiation as disclosed herein.

[0029] In another preferred embodiment of said method for the production of an adherent cortical organoid, said culture medium for neural differentiation is a serum-free culture medium. Preferably, all culture media disclosed herein are serum -free culture media.

[0030] In another preferred embodiment of said method for the production of an adherent cortical organoid, said culture medium for neural differentiation further comprises a basal medium for neuronal cell culture.

[0031] In another preferred embodiment of said method for the production of an adherent cortical organoid, said culture medium for neural differentiation further comprises an N2 supplement and / or a B27 supplement without vitamin A (B27-RA).

[0032] In another preferred embodiment of said method for the production of an adherent cortical organoid, said culture medium for neural differentiation further comprises (i) a minimum essential medium and / or a non-essential amino acid supplement, (ii) a laminin and / or (iii) penicillin and / or streptomycin.

[0033] In another preferred embodiment of said method for the production of an adherent cortical organoid, said period of time for culturing said seeded NPCs in a culture medium for neural differentiation is at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months or at least 1 year.

[0034] In another preferred embodiment of said method for the production of an adherent cortical organoid, said culture medium for neural differentiation is refreshed once every 1-5 days, preferably once every 2-3 days.

[0035] In another preferred embodiment of said method for the production of an adherent cortical organoid, in at least 20%, in at least 30%, in at least 40%, in at least 50%, in at least 60%, in at least 70%, in at least 75%, in at least 80%, in at least 90%, or in at least 95%, of the seeded wells of said multiwell plate, a single adherent cortical organoid (preferably with a radial structure) is present. Preferably, after about 60 days from the start of step (c), in at least 60%, in at least 70%, in at least 75%, in at least 80%, in at least 90%, or in at least 95%, of the seeded wells of said multiwell plate, a single adherent cortical organoid (preferably with a radial structure) is present. Preferably, after about 1 year from the start of step (c), in at least 40%, or in at least 50%, of the seeded wells of said multiwell plate, a single adherent cortical organoid (preferably with a radial structure) is present.

[0036] In another preferred embodiment of said method, culturing of said seeded NPCs occurs in the presence of a further cell type, for example a microglial cell and / or a vascular cell.

[0037] In another preferred embodiment of said method for the production of an adherent cortical organoid, said adherent cortical organoid comprises neurons, astrocytes and (myelinating) oligodendrocytes, and optionally microglia or vascular cells. Examples of vascular cells are endothelial cells, pericytes, smooth muscle cells and fibroblasts.

[0038] In another preferred embodiment of said method, culturing of said seeded NPCs in said culture medium for neural differentiation in a well of said multiwell plate occurs in the presence of a test agent, and optionally, wherein, in a different well of said multiwell plate, culturing of said seeded NPCs in said culture medium for neural differentiation occurs in the absence of said test agent.

[0039] In another preferred embodiment of said method for the production of an adherent cortical organoid, said cortical organoid displays neuronal activity such as (i) synchronous network bursting and / or (ii) desynchronized activity such as desynchronized activity during time periods outside of synchronous network bursting. Preferably, said neuronal activity is measured by fluorescent calcium imaging.

[0040] In another preferred embodiment of said method for the production of an adherent cortical organoid, said method for the production of an adherent cortical organoid is a method for the production of a multi-well plate comprising a well that contains a single adherent cortical organoid (preferably with a radial structure).

[0041] In another preferred embodiment of said method for the production of an adherent cortical organoid, said method for the production of an adherent cortical organoid is a method for screening test agents for modulation, or modulation of formation, of an adherent cortical organoid. In such embodiments, in step (c), culturing of said seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation in a well (a test well) of said multiwell plate occurs in the presence of a test agent. In such embodiments it is investigated whether said test agent modulates or does not modulate an adherent cortical organoid or its formation or development.

[0042] In another aspect, the invention provides for a multiwell plate comprising a well; wherein said well comprises a bottom surface and an upright wall, and wherein a distance x between a centre of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm; wherein said well contains a single adherent cortical organoid (preferably with a radial structure); and wherein said adherent cortical organoid comprises neurons, astrocytes and (myelinating) oligodendrocytes, and optionally microglia or vascular cells. Embodiments disclosed in relation to a method for the production of an adherent cortical organoid also apply to this aspect and any other aspect where appropriate.

[0043] In a preferred embodiment of said multiwell plate; said distance x between a centre of the bottom surface and the upright wall is between 1.2 mm and 1.8 mm, preferably between 1.3 mm and 1.8 mm, more preferably between 1.4 mm and 1.8 mm, more preferably between 1.5 mm and 1.8 mm such as between 1.6 and 1.7 mm. As an example, the distance x can be about 1.65 mm, which preferably corresponds to a well width or well diameter of about 3.3 mm.

[0044] In another preferred embodiment of said multiwell plate, a height h of the well is between 5 and 20 mm, preferably between 5 and 15 mm, more preferably between 8 and 13 mm, more preferably between 10 and 13 mm, even more preferably between 11 and 12 mm. One example of the height h of the well is about 11.5 mm.

[0045] In another preferred embodiment of said multiwell plate, said bottom surface of the well is a flat bottom surface.

[0046] In another preferred embodiment of said multiwell plate, said bottom surface of the well is a flat bottom surface; and said distance x between a centre of the bottom surface and the upright wall is between 1.5 mm and 1.8 mm.

[0047] In another preferred embodiment of said multiwell plate, said well of said multiwell plate is a well of a 384-well plate, preferably a flat (F) bottom 384-well plate.

[0048] In another preferred embodiment of said multiwell plate, said multi-well plate is a flat (F) bottom 384-well plate.

[0049] In another preferred embodiment of said multiwell plate, said well of said multiwell plate is coated with a coating for adherent cell culture, preferably wherein said coating for adherent cell culture is a laminin and / or poly-L-ornithine coating.

[0050] In another preferred embodiment of said multiwell plate, in at least 20%, in at least 30%, in at least 40%, in at least 50%, in at least 60%, in at least 70%, in at least 75%, in at least 80%, in at least 90%, or in at least 95%, of the (seeded) wells of said multiwell plate, a single adherent cortical organoid (preferably with a radial structure) is present. Preferably, after about 60 days from the start of step (c), in at least 60%, in at least 70%, in at least 75%, in at least 80%, in at least 90%, or in at least 95%, of the seeded wells of said multiwell plate, a single adherent cortical organoid (preferably with a radial structure) is present. Preferably, after about 1 year from the start of step (c), in at least 40%, or in at least 50%, of the seeded wells of said multiwell plate, a single adherent cortical organoid (preferably with a radial structure) is present.

[0051] In another preferred embodiment of said multiwell plate, said cortical organoid is obtainable by a method for the production of an adherent cortical organoid of the invention.

[0052] In another aspect, the invention provides an adherent cortical organoid as disclosed herein, preferably wherein said adherent cortical organoid is obtainable by a method for the production of an adherent cortical organoid of the invention.

[0053] In another aspect, the invention provides a use of a multiwell plate or adherent cortical organoid of the invention, in screening a test agent for modulating an adherent cortical organoid, such as modulation of its formation or development. Such modulation may affect a structural and / or functional characteristic of said organoid. An example is the modulation of formation or development of an adherent cortical organoid as disclosed herein, for instance expressed by a structural or a functional change compared to the situation where the test agent is absent. In another aspect, the invention provides a method of screening for an agent, preferably a pharmacological agent or a food ingredient, that modulates an adherent cortical organoid or its formation (or development), comprising the steps of: - performing a method for the production of an adherent cortical organoid of the invention; whereby culturing of said seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation in a well (e.g. a test well) of said multiwell plate occurs in the presence of a test agent; -optionally, wherein, in a different well (e.g. a control well) of said multiwell plate, culturing of seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation occurs in the absence of said test agent; -comparing the cortical organoid produced in the presence of said test agent with a cortical organoid produced in the absence of said agent; - identifying said test agent as an agent that modulates an adherent cortical organoid, e.g. modulates the formation or development thereof, if the cortical organoid that is produced in the presence of said test agent is different from said cortical organoid that is produced in the absence of said agent. Differences may exist in structural and / or functional features of said adherent cortical organoid.

[0054] In the same manner, the invention provides a method of screening for an agent, preferably a pharmacological agent or a food ingredient, that modulates an adherent cortical organoid or its formation, comprising the steps of: (a) providing a cell culture comprising a population of neural progenitor cells (NPCs); (b) seeding neural progenitor cells (NPCs) of said population in a well of a multiwell plate; wherein said well comprises a bottom surface (106) and an upright wall (104), and wherein a distance (x) between a centre (112) of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm; (c) culturing said seeded NPCs in a culture medium for neural differentiation for a period of time; wherein said culturing occurs in the presence of a test agent; (d) optionally, wherein, in a different well (e.g. a control well) of said multiwell plate, culturing of seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation occurs in the absence of said test agent; and (e) identifying said test agent as an agent that modulates an adherent cortical organoid or its formation, if the cortical organoid that is produced in the presence of said test agent is different from said cortical organoid that is produced in the absence of said agent. Differences may exist in structural and / or functional features of said adherent cortical organoid.

[0055] Any embodiments disclosed in relation to a method for the production of an adherent cortical organoid of the invention also apply in relation to this aspect.

[0056] In another aspect, the invention provides a method for the production of an adherent cortical organoid, comprising the steps of: (a) providing a cell culture comprising a population of neural progenitor cells (NPCs); (b) seeding neural progenitor cells (NPCs) of said population in a well of a multiwell plate; wherein said well of said multi-well plate is a well of a flat (F) bottom 384-well plate; and (c) culturing said seeded NPCs in a culture medium for neural differentiation for a period of time, to thereby allow formation of an adherent cortical organoid.

[0057] DESCRIPTION OF THE DRAWINGS

[0058] Figure 1. Well of a multiwell plate.

[0059] Fig. 1A shows a well 100 as part of a multiwell plate, which well plate may in general comprise any number of wells. For example, the well plate can be a 384-well plate. Typically, all wells of a multiwell plate have essentially corresponding dimensions and shape. Fig. 1A in particular shows a schematic section view of the well 100. Figs. IB and 1C show examples of a top view of the well 100 of Fig. 1A.

[0060] The well 100 is formed by a base part 102 and an upright wall 104 protruding away from the base part 102. The base part 102 forms a bottom surface 106 of the well, which bottom surface 106 preferably is a (substantially) flat surface. In use, the flat surface typically is oriented essentially parallel to the horizon. A well with a flat bottom surface is typically indicated with “F -bottom”, “F / Bottom”, “F-type” or “F-well”, or generally with the indication “F”. A top opening 108 allows access into an inner volume 107 of the well 100. The inner volume 107 is typically delimited by an inner surface 105 of the upright wall 104, and the bottom surface 106. In general, the base part and upright wall may be integrally formed, in particular together with other wells of the multiwell plate. In the example depicted in Fig. 1A, the upright wall 104 is tapered towards the bottom surface 106. It will be appreciated that for any well of the present disclosure, the upright wall 104 may be tapered towards the bottom surface 106 at any angle, or the upright wall 104 may be oriented essentially perpendicular to the bottom surface 106.

[0061] In the example of the well 100 of which a top view is depicted in Fig. IB, the bottom surface 106 is formed as a rectangle with rounded corners. In the example of the well 100 of which a top view is depicted in Fig. 1C, the bottom surface 106 is formed as a circle. It will be appreciated that for any well of the present disclosure, the bottom surface 106 may have any shape, such as square or rectangular, in combination with straight or rounded corners, circular, oval, or any other shape. Preferably, the shape of the bottom surface 106 is square, with or without rounded corners, or circular. A centreline 110 of the well 100 is indicated in Figs. 1A-1C, which centreline 110 intersects the bottom surface 106 at a centre 112 of the bottom surface 106. A distance x is indicated in Figs. 1A-1C, which distance x is the distance between the centre 112 of the bottom surface 106 and the upright wall 104. The distance x may generally be determined in any direction, from the centre 112 of the bottom surface 106 to where the upright wall 104 meets the bottom surface 106.

[0062] As an option depicted in Fig. 1A, as a preference for any well disclosed herein, the upright wall 104 and the bottom surface 106 meet at an obtuse angle or a straight angle, essentially without the intersection of the upright wall 104 and the bottom surface 106 having a smoothed transition, for example with a radius, fillet, or chamfer.

[0063] For the example of Fig. IB with the quasi-rectangular bottom surface 106, the distance x corresponds to half of the width w of the bottom surface 106. For the example of Fig. 1C with the circular bottom surface 106, the distance x corresponds to the radius of the bottom surface 106.

[0064] For any well disclosed herein, the distance x is between 1.1 mm and 1.9 mm, for instance between 1.3 mm and 1.8 mm or between 1.4 mm and 1.8 mm. Preferably, the distance x is between 1.5 mm and 1.8 mm such as between 1.6 and 1.7 mm. As an example, the distance x can be about 1.65 mm, which preferably corresponds to a width or diameter of 3.3 mm. A height h of the well 100, indicated in Fig. 1A, may be between 5 and 15 mm, preferably between 10 and 13 mm, even more preferably between 11 and 12 mm. Any height h may be combined with any distance x for any well of the present disclosure. For example, the well plate can be a 384-well plate. Typically, all wells of a multiwell plate have essentially corresponding dimensions and shape.

[0065] For any well disclosed herein, the well may be coated with a coating for adherent cell culture. In particular, the bottom surface 106 or at least part thereof may be coated.

[0066] Figure 2: Adherent cortical organoid model.

[0067] A Schematic representation of the protocol used to obtain NPC line 1, and subsequent differentiation thereof. NPC lines 2 and 3 were obtained via a different protocol and not FACS sorted prior to seeding in the 384 well plate. A commercially available NPC line (NPC line 2), and a NPC line obtained via a different protocol (NPC line 3), were used to confirm that the claimed invention is not limited to specific NPC lines, and the specific cell culture conditions used to obtain them. B / C / D Representative NPCs from three different iPSC lines with markers S0X2, Nestin and F0XG1 (scale bar B, 50 jim; C / D, 20 jim).E Representative time course showing self-organization during differentiation, starting with radial organization between day 28 and day 42, seeding density 1500 NPCs per well (green is live-stain from Viability / Cytotoxicity kit; scale bars left to right 100 jim, 150 jim, 100 jim, 100 jim). F Full well showing radial organization at day 42 in culture (live- dead stain, 500 jim). Only very few dead cells are visible in red in the dense centre of the structure.

[0068] Figure 3 Adherent cortical organoids show an organized network of neurons and astrocyte subtypes. A MAP2+ somas and dendrites alongside Tau+ / MAP2- axons show segregation of dendritic and axonal compartments, with SOX2+ progenitors concentrated in the center of the well (Day 75, 200jim). B MAP2+ and NeuN+ cells indicate mature neurons (Day 72, 50 jim). C Deep-layer cortical marker CTIP2 and upper layer marker CUX1 show rudimentary segregation of cortical layers in expected inside-out pattern (Day 64, 50 jim). D A subset of the MAP2-positive neurons are GAD67+, indicating the presence of an interneuron population in the cortical organoids (Day 60, 50 jim). E Astrocyte markers GFAP and S100B show the general radial pattern of astrocyte outgrowth (Day 66, 500 jim). F / G / H GFAP staining reveals the morphologies of different astrocyte subtypes, including fibrous astrocytes (F, day 65, 100 jim), protoplasmic astrocytes (G, day 65, 50 jim) and interlaminar astrocytes (H, day 65, 100 jim), I Co-localization of astrocyte marker GFAP and PAX6 marks radial glia (day 65, 50 jim).

[0069] Figure 4 Adherent cortical organoids form oligodendrocyte lineage cells. A Adherent cortical organoids show OPCs as early as 44 days, indicated by OPC marker NG2 (Day 44, 20 jim). B / C The NG2+ OPCs are still present in the cortical organoids after 4 months (Day 119, B 50 jim, C 20 jim). D Young oligodendrocytes start to emerge after 4 months indicated by rudimentary MBP staining (Day 119 100 jim and 20 jim). E / F After 5 months, MBP-positive oligodendrocytes show more mature morphology and initial wrapping of NF200+ axons (Day 148; E, 50 jim and 20 jim, F 5 jim). G / H / I Oligodendrocyte distribution at organoid Day 161 where the MBP+ oligodendrocytes sit between axons and axon bundles and co-localize with NF200+ axons. (G / H / I Day 161, G 500 jim, H lOOjim, I 10 jim)

[0070] Figure 5 Adherent cortical organoids show synaptic connectivity and network bursts. A Synapsin staining shows synapse formation along MAP2+ dendrites (Day 70, 20 jim). B Co-localization of pre-synaptic marker Synapsin and post-synaptic marker PSD-95 (Day 205, 20 jim) C MAP2+ dendrites and soma are decorated with Synapsin+ synapses (Day 251, 100 jim) D Overview of entire well, showing alignment of Synapsin staining with MAP2 (Day 251, 500 jim). E Sparse labeling of neurons with AAV9.0amKII.eGFP allows detailed imaging of glutamatergic dendritic spines with mature mushroom morphology, contacting pre-synaptic Synapsin puncta (Day 310, 5 jim). F Snapshot of radially organized neurons transduced with AAVl.Syn.GCaMP6s.WPRE.SV40 (Day 60, 100 jim). G Calcium events per minute from 36 cells from 7 recordings (each showing a cluster of neurons) from 2 different cortical organoids at day 61. H Network bursts per minute from 37 cells from 6 recordings from 2 different cortical organoids. I Percentage of events that are part of network bursts indicates that the majority of cells are involved in network burst activity. J Representative calcium traces from 2 different clusters in which cells are showing individual activity as well as network bursts where multiple cells are active simultaneously.

[0071] Figure 6 Reproducibility of Neural Progenitor Cells and Adherent

[0072] Cortical Organoids. A NPCs are positive for SOX2, Nestin, FOXG1, PAX6 and TBR2 (scale bars 20 m). B LIVE / DEAD staining of adherent organoids (N=4) showing reproducibility of the structure formation. Seeding density line 1: 1250 NPCs, line 2: 750 NPCs, line 3: 1000 NPCs (scale bars 500 pm). C Function of the necessary NPC seeding density to form adherent cortical organoids in relation to the doubling time as a measure of the proliferation rate of the NPCs. The doubling time of the NPCs explains more than half the variation (r2= 0.67) of the required seeding density showing that fewer cells need to be seeded for NPC lines with a higher proliferation rate. D Proportion of successful, single structure adherent cortical organoids. Each dot represents a different batch of adherent cortical organoids where the size of the batch ranges from 10-40 organoids.

[0073] Figure 7 Neuronal maturation in cortical organoids is shown by a temporal shift in NPC and neuronal markers. A Representative images exhibiting change in expression of NPC and neuronal markers over time. Number of cells positive for NPC markers SOX2 and PAX6 decreases over time. Deep-layer CTIP2+ neurons start appearing at day 28, while upper-layer CUX1+ positive neurons appear in higher numbers around day 56. (Scale bars 20 pm). B The percentages of DAPI+ cells expressing each of the markers were quantified at four time points. Significant differences between week 2 and week 8 expression were seen for SOX2+ (down by 39.6 %), CTIP2+ (up by 13.5 %) and CUX1+ cells (up by 23.1 %). *** p < 0.0001, ANOVA one-way followed by Tukey- Kramer’s multiple correction test. Error bars indicate SEM, n=3-6 images taken over two wells, for each time point.

[0074] Figure 8 Distribution of axons and dendrites in adherent cortical organoids. Radially organized MAP2+ / Tau+ dendrites along with both radially and circumferentially organized NF200+ / Tau+ axons in duplicate for NPC cell lines 1-3 (Day 63, scale bars 500 pm) Figure 9 Cortical layering in the adherent cortical organoids A Rudimentary separation of SOX2+ NPCs, CTIP2+ deep layer neurons and CUX1+ upper layer neurons (Day 67, scale bar 100 pm) B-E Separation of CUX1 and CUX2 expression (B day 70, scale bar 100 pm; C day 70, scale bar 500 pm; D day 67, scale bar 500 jim; E day 67, scale bar 100 jim).

[0075] Figure 10 GAD67+ interneurons make up a small proportion of NeuN+ neurons in the adherent cortical organoids A Example image of GAD67+ interneuron proportion of NeuN+ neurons (Day 67, 50 jim). B Quantification of GAD67+ proportion of NeuN+ neurons. Every point is the percentage of GAD67+ interneurons out of all NeuN+ neurons in a spatially randomized selected image with on average 170 NeuN+ nuclei per frame. (Line 1: N= 17 images of 3 different cortical organoids at day 67; Line 2 / 3: 6 images of 2 different organoids at day 65)

[0076] Figure 11 Astrocyte distribution within adherent cortical organoids The abundantly present GFAP+ and S100B+ astrocytes have a comparable distribution with the MAP2+ neurons in the adherent cortical organoids. The astrocytes show radial patterns with many somas located in the centre as well as a large presence outside the center (Day 63, scale bars 500 pm).

[0077] Figure 12 Incorporation of microglia in adherent cortical organoids A Representative images of IBA1+ iPSC-derived microglia in all the models. Scale bar 20 pm. B Circularity analysis using individual cells. Monoculture: n= 142, NGN2: n= 43, Organoids: n= 122. C-E Skeleton analysis results. Violin plots show the average value per cell determined across all images: Monoculture: n= 10, NGN2: n= 9, Organoid: n= 12. *: p < 0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.0001. Figure 13 Microglia distribution and migration in adherent cortical organoids

[0078] A IBA1+ Microglia distribute widely (7 days post seeding) in adherent cortical organoids (Days in Vitro (DIV) 56) with neurons (MAP2+). B Microglia migrate inwards when cultured for longer (>50d post seeding) in organoids (DIV 150) Scale bar 300 pm. C-E Ramified microglia are observed in the core of the organoid while circular morphology is seen in periphery (circularity of microglia quantified in two organoids; n=210 IBA+ cells). F Somas of eGFP-labelled microglia become less motile over time (d = time in days in organoids); however, the processes of these cells remain highly motile.

[0079] DETAILED DESCRIPTION OF THE INVENTION Definitions

[0080] The term ‘organoid’, as used herein, includes reference to an in vitro produced three-dimensional miniaturized version of an organ, or part thereof. Under facilitating conditions, stem cells, such as embryonic stem cells or induced pluripotent stem cells, may self-organize into organoids. Organoids comprise more than one organ-specific cell type. The structure of the organoid generally resembles the structure of the organ or part thereof which it represents. It is also capable of mimicking some or all functions of the organ, or part thereof, that it represents. The term ‘cortical organoid’, as used herein, includes reference to an organoid representing the cerebral cortex, which is the outer layer of the brain. Preferably, a cortical organoid represents the mammalian cerebral cortex, more in particular the human cerebral cortex. A cortical organoid preferably comprises at least two cell types that are typically found in the cerebral cortex, such as neurons (e.g. excitatory glutamatergic neurons and inhibitory GABAergic neurons) and glial cells (e.g. astrocytes, oligodendrocyte precursor cells, myelinating oligodendrocytes and radial glia). Functionalities of a cortical organoid may include synaptic activity such as excitatory activity, inhibitory activity, network bursting and desynchronized activity. The term ‘cortical organoid’ and ‘cortical brain organoid’ are used interchangeably herein. Preferably, in an organoid as disclosed herein, different cell types co-develop together from a common NPC. Also, preferably, in an organoid as disclosed herein, a continuous population of (self-renewing) NPCs remains present.

[0081] The term ‘adherent’, as used herein, includes reference to cells of a cortical organoid being attached to a substrate, such as a coating for adherent cell culture that is introduced in a well of a multiwell plate prior to NPC seeding. Free floating cortical organoids such as spheroids are not adherent cortical organoids.

[0082] The term ‘pluripotent stem cell (PSC)’, as used herein, includes reference to inter alia embryonic stem cells and induced pluripotent stem cells (iPSC), and to other cells that have the capacity to self-renew by dividing and to develop into the three primary germ cell layers of the early embryo, and which can be differentiated into neural progenitor cells (NPCs).

[0083] The term induced pluripotent stem cell’, abbreviated as ‘iPSC’, as used herein, includes reference to a pluripotent stem cell that is derived from somatic cells or cell lines thereof. Said somatic cells or cell lines thereof can be genetically reprogrammed to iPSCs using well-established techniques known in the art (e.g. Takahashi et al., Cell, vol. 131, no. 5, pp. 861-72 (2007)). Such techniques generally involve introducing reprogramming factors, such as Oct3 / 4, Sox2, Klf4 and / or c-Myc, to a given somatic cell. The term ‘somatic cell’, as used herein, includes reference to a differentiated cell that is committed to a particular function and / or type. The iPSCs, which result from reprogramming, can be differentiated in vitro into any cell type, including a progenitor cell type such as NPCs, of the body, using directed differentiation with specific growth factors. These procedures are commonly known. The option to derive such iPSC lines from somatic cells of humans allows research using human cells of interest with the genetic make-up of a subject, which can be a subject having a disease (such as a neurological disease affecting the brain, e.g. a genetic neurological disease affecting the brain) or a subject not having a disease. Preferably, the iPSC is a human iPSCS. iPSCs can be produced from different somatic cell types, such as epithelial cells (e.g. IPSC0028 (Sigma-Aldrich)) or from skin fibroblasts (WTC11, Coriell Institute, #GM25256).

[0084] The term ‘progenitor cell’, as used herein, includes reference to an undifferentiated or partially differentiated cell that has the ability for (limited) self-renewal and is able to differentiate into different cell types. Progenitor cells as disclosed herein may have any potency, but are typically oligopotent. An example of a progenitor cell is a neural progenitor cell (NPC), which may also be referred to as a neural stem cell (NSC). The term ‘neural progenitor cell’, abbreviated as NPC, as used herein, includes reference to a progenitor cell of cells of the central nervous system. An NPC may terminally differentiate into for instance glial cells or neurons. NPCs are a well-characterized progenitor cell type, and can be obtained by different methods and means such as by isolation, by various PSC (e.g. iPSCs) differentiation protocols or can be directly acquired from commercial providers (e.g. human NPCs from Axol Biosciences (ax0015)). NPCs preferably express one or more markers selected from the group formed by SOX2, Nestin, Vimentin and FOXG1. Preferably, the NPC is a frontal cortical NPC. Frontal cortical NPCs are preferably characterized by the presence of marker FOXG1 and / or SOX2. It is shown in the Examples that the invention can be practiced with NPCs of varying origin, obtained via varying cell culture protocols. Preferably the NPC is an NPC derived from (or obtained from) a iPSC. Preferably, the NPC is a human NPC.

[0085] It is envisaged herein that the NPC (or the PSC from which the NPC is derived, or the somatic cell from which the iPSC is derived that was used to produce the NPC) may originate from a subject having a disorder such as a neurodevelopmental or neuropsychiatric disorder. In embodiments, the disorder is a genetic neurodevelopmental or neuropsychiatric disorder. Examples of disorders are Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Lafora disease, microcephaly, schizencephaly, lissencephaly, microgyria, poly microgyria and pachygyria. Using a method of producing adherent cortical organoids as disclosed herein, in which NPCs are used that originate from a subject having a disorder, it is possible to conduct mechanistic pathophysiological studies of said disorder by comparing adherent cortical organoid formation with adherent cortical organoid formation obtained with NPCs that originate from a subject not having a disease (healthy control). Hence, alternatively, the NPC (or the PSC from which the NPC is derived, or the somatic cell from which the iPSC is derived that was used to produce the NPC) may originate from a subject not having a disease (healthy subject).

[0086] The terms ‘expansion’ and ‘expanding’, as used herein, include reference to increasing the population size of progenitor cells, such as neural progenitor cells, in cell culture, i.e. progenitor cells are generating other progenitor cells by cell expansion. Such an expanded progenitor cell population can subsequently be cultured in differentiation media for differentiating said progenitor cells into specialized cell types, such as neurons and glial cells. The term ‘expansion’ can be used interchangeably with the term ‘proliferation’.

[0087] The term ‘population’, as used herein, includes reference to a plurality of cells, more particularly a plurality of cells in culture. The term ‘expanded population’, as used herein, includes reference to an unspecified number of cells that has previously been subject of expansion.

[0088] The term ‘sorting’, as used herein, includes reference to a process wherein cells of differential phenotypes are separated. A differential phenotype may for example be embodied by cells of different cell types and / or by cells expressing different cell surface makers. Most commonly, cells are sorted using antibody -based sorting such as fluorescence activated cell sorting (FACS), magnetic activated cell sorting, microfluidic cell sorting, and buoyancy-activated cell sorting. Cell sorting may require attachment of a foreign object or molecule to the cell or incorporation of such an object or molecule into the cell. Preferably, said object or molecule contacts a phenotypical feature relevant to the sorting of the cell, e.g. a cell surface marker.

[0089] The term ‘fluorescence activated cell sorting’, abbreviated as FACS, as used herein, includes reference to a protocol in which cells or cell populations are analyzed and separated using a fluorescent signal of one or more reporters and involves flow cytometry. In FACS, cellular components, preferably cell surface markers, attach to labeled reporters. Often, the reporters are antibodies that are labeled with fluorophores or quantum dots. Preferably, the cells are sorted based on their attachment to one or more fluorescent reporter, which is also referred to as cell purification. Cells are alive during and directly following FACS.

[0090] The term ‘well’, as used herein, includes reference to a cavity in a multiwell plate that functions as a small test tube.

[0091] The term ‘multiwell plate’, as used herein, includes reference to a plate that comprises a plurality of wells. The term can be used interchangeably with the terms ‘microtiterplate’ or ‘microplate’. The multiwell plate may comprise 384 wells arranged in a 2:3 rectangular matrix. Preferably, when the multiwell plate is a 384 well plate, the 384 well plate matches the prescribed dimensions of a standard 384-well plate as defined by ANSI SLAS 4-2004 (R2012) (formerly recognized as ANSI / SBS 4-2004). An example of such a 384-well plate is the flat (F) bottom 384-well plate of Greiner Bio (M1937-32EA), which was used in the Examples. Wells of this 384-well plate are characterized by having a distance x of about 1.65 mm, which corresponds with a width of about 3.3 mm. Furthermore, the wells of this 384-well plate have a height h of 11.5 mm. Furthermore, the shape of the wells of this plate are square with rounded corners. Wells of this plate have a working volume up to 131 pL, with an optimal working volume of 15-110 pL. Multiwell plates can be made from various materials such as polystyrene or glass. When reference is made to “well of a multiwell plate”, such as “well of a 384-well plate”, it also includes reference to multiwell plates, such as 384-well plates, that are processed in the sense that the plate is e.g. halved. In such a case, the well is still to be considered a well of said multiwell plate, such as a well of said 384-well plate, even though the plate has been processed thereby discarding a number of wells.

[0092] In the field of multiwell plates, the term “flat (F) bottom” is used to indicate that the bottom surface of the well is substantially flat, to distinguish over other multiwell types such as V-bottom or U-bottom multiwell types.

[0093] The terms ‘application of cells’ and ‘applying cells’, as used herein, include reference to putting cells on a designated surface, such as the bottom surface of a well. Said terms can be used interchangeably with the term ‘seeding’.

[0094] The term ‘seeding density’, as used herein, includes reference to the number of cells that are applied to a well. Seeding density is expressed in absolute numbers of cells per well. The person skilled in the art can routinely identify suitable seeding densities for a given NPC on the basis of the present disclosure.

[0095] The term ‘agent’ as used herein, includes reference to any compound or substance of interest, such as small molecule or a proteinbased molecule of interest. Preferably, the agent is a biologically or pharmacologically active compound or substance, and may modulate brain organoids, such as their formation. Such agents can be used for testing or screening for therapeutic effects, toxic effects, etc.

[0096] The term ‘cell surface marker’, as used herein, includes reference to proteins that are present on the surface of a cell and that are used to identify cell types. Cell surface markers have a variety of functionalities. Cell surface markers are often indicated by a CD number in the cluster of differentiation (CD) protocol. FACS can be used for the identification of cells by employing cell surface markers. Non-limiting examples of cell surface markers are CD 184, CD24, CD44 and CD271. Presence of a cell surface marker in or on a cell is generally indicated with a superscript ‘+’, for example CD184+; absence of a cell surface marker in or on a cell is generally indicated with a superscript for example CD271-. The term ‘CD 184’, as used herein, includes reference to a protein encoded by the human CXCR4 gene. CD 184 may also be referred to as C-X-C chemokine receptor type 4 (CXCR-4) and fusin. CD 184 is an alpha-chemokine receptor specific for stromal-derived-factor- 1 (SDF-1). The term ‘CD24’, as used herein, includes reference to a protein encoded by the human CD24 gene. CD24 is a sialoglycoprotein that plays a role in neural development. The term ‘CD44’, as used herein, includes reference to a protein encoded by human CD44 gene. CD44 may also be referred to as HCAM, Pgp-1, Hermes antigen, lymphocyte homing receptor, ECM-III and Hutch- 1. The term ‘CD271’, as used herein, includes reference to a protein encoded by the human NGFR gene. CD271 may also be referred to as p75 neurotrophin receptor (p75NTR) and low-affinity nerve growth factor receptor (LNGFR). CD271 is a member of the tumor necrosis factor receptor superfamily, and binds to Neurotrophins including Nerve Growth Factor, Neurotrophin- 3, Brain- derived neurotrophic factor and Neurotrophin-4. For instance, an NPC as disclosed herein can be CD 184+ and / or CD24+. For instance, an NPC as disclosed herein can be CD44- and / or CD271-. More specifically, an NPC as disclosed herein can be CD184+, CD24+, CD44- and CD271-.

[0097] The terms ‘coated’ and ‘coating’, as used herein, include reference to the application and binding of molecules to, amongst others, a bottom surface of a well of a multiwell plate, so that at least part of said bottom surface is covered by said molecules. A coating serves as a substrate for NPCs. Non-limiting examples of molecules that may be used for coating as disclosed herein, are laminin, poly-L-ornithine, poly-L-lysine, poly-D-lysine, collagen, streptavidin, fibronectin, vitronectin or PEG, or combinations thereof.

[0098] The term ‘neurotrophic factor’, as used herein, includes reference to a family of biomolecules involved in the differentiation of neural progenitor cells (NPCs). Examples of neurotrophic factors are brain-derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (also known as glial cell-derived neurotrophic factor; both abbreviated as GDNF). Preferably, a brain-derived neurotrophic factor as disclosed herein is a (recombinant) human brain-derived neurotrophic factor. Preferably, a glial cell-derived neurotrophic factor as disclosed herein is a (recombinant) human glial cell-derived neurotrophic factor.

[0099] The term ‘microglial cell’ or ‘ microglia’, as used herein, refers to a type of glial cell that acts as the primary immune cell of the central nervous system (CNS), including the brain and spinal cord. In nature, microglia originate from the mesoderm lineage. Preferably, the microglial cell is a human microglial cell. Preferably, the microglial cell is generated from an iPSC, for instance from a WTC11 hiPSC. Standard microglia differentiation kits are commercially available, e.g. cat# 05310 and cat# 100-0019 from Stemcell Technologies. Preferably, the microglia cell is IBA1+.

[0100] The term “vascular cell”, as used herein, includes reference to a cell that forms part of the vascular system, which includes blood vessels such as arteries, veins, and capillaries. Preferably, the vascular cell is a human vascular cell. The vascular cell can be generated from a iPSC but can also be non-iPSC derived. Examples of vascular cells are endothelial cells, pericytes, smooth muscle cells and fibroblasts. For instance, the vascular cell is an endothelial cell and / or a pericyte.

[0101] Methods for producing adherent cortical brain organoids The present inventors have discovered a method that allows for simple and highly reproducible production of single adherent cortical brain organoids per well.

[0102] In step (a) of this method, a cell culture comprising a population of neural progenitor cells (NPCs) is provided. NPCs can be obtained in different manners, such through isolation from human brain tissue, by differentiating PSCs, or from commercial providers. The Examples show that the method for producing adherent cortical organoids as disclosed herein can be practiced with a variety of NPCs derived from different origins and subjected to various cell culture conditions.

[0103] Preferably, the NPCs that are used in the methods of the invention are frontal cortical NPCs. Frontal cortical NPCs are a well-characterized NPC subtype. FOXG1 is a marker that is enriched in frontal cortical NPCs. Preferably, NPCs are positive for one or more of the markers selected from the group formed by SOX2, Nestin, Vimentin and FOXG1. More preferably, NPCs are positive for at least markers FOXG1 and SOX2, or FOXG1 and Nestin, or FOXG1 and Vimentin, or FOXG1, SOX2 and Nestin, or FOXG1, SOX2 and Vimentin. NPCs may also be positive for all four markers. As an example, NPC can be kept in a cell culture using a NPC culture medium (also referred to as NPC expansion) medium as described below.

[0104] As an example, human NPCs that are suitable for use in methods of invention are commercially available at Axol Biosciences (ax0015). Alternatively, well-established protocols exist to obtain NPCs from PSCs. One non-limiting example is described in Shi et al., Nat Neurosci 15:477-86, Si (2012)). Another non-limiting example of such a method is described in Gunhanlar et al., Mol Psychiatry 23:1336-1344 (2018)), which is described in more detail below. In some embodiments, step (a) of a method of the invention, in which a cell culture comprising a population of neural progenitor cells (NPCs) is provided, is preceded by steps that result in the generation of a population of neural progenitor cells from at least one pluripotent stem cell.

[0105] Steps that result in the generation of a population of NPCs from at least one pluripotent stem cell may include a step (al): providing a cell culture comprising pluripotent stem cells and feeder cells. Alternatively, said feeder cells may be substituted with a structure or composition suitable of serving as a substitute for feeder cells, such as decellularized extracellular matrix.

[0106] In such a step (al), any PSC can be used such as an iPSC obtained from any somatic cell type, but preferably from a cell type selected from the group formed by epithelial cells, hematopoietic progenitor cells and peripheral blood mononuclear cells. In some embodiments, commercially available iPSCs may be used, of which the product IPSC0028 from Sigma- Aldrich is an example.

[0107] In said step (al), said PSCs can be suspended in a (cell) culture medium. Said medium can be any suitable medium for maintaining PSCs, such as a medium that is referred to herein as ‘Human embryonic stem cell medium’. Human embryonic stem cell medium comprises at least one component selected from the group consisting of a basal medium; a formulation that replaces serum; L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-glycine; L-proline; L-serine; L-glutamine; 6- mercaptoethanol; penicillin; and streptomycin. More preferably, human embryonic stem cell medium as described herein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the components selected from the group consisting of a basal medium; a formulation that replaces serum; L-alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-glycine; L-proline; L- serine; L-glutamine; B-mercaptoethanol; penicillin; and streptomycin. Still more preferably, Human embryonic stem cell medium as described herein comprises at least a basal medium; a formulation that replaces serum; L- alanine; L-asparagine; L-aspartic acid; L-glutamic acid; L-glycine; L-proline; L-serine; L-glutamine; B-mercaptoethanol; penicillin; and streptomycin. Preferably, a basal medium present in Human embryonic stem cell medium as described herein is Dulbecco’s modified Eagle medium (DMEM); Ham’s F12 medium; or a combination thereof. More preferably, a basal medium present in Human embryonic stem cell medium as described herein is a mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12 medium; preferably mixed in a 1:1 volume ratio, such as commercially available DMEM / F12 (21331020; Thermo Fisher Scientific). Preferably, a formulation that replaces serum is a commercially available serum replacement, such as KnockOut™ Serum Replacement (10828028, Thermo Fisher Scientific). Preferably, a formulation that replaces serum is present in Human embryonic stem cell medium as described herein in a concentration of 0.2-80 %v / v; more preferably in a concentration of 2-40 %v / v; even more preferably in a concentration of 10-30 %v / v; still more preferably in a concentration of 15-25 %v / v; most preferably in a concentration of approximately 20 %v / v. Preferably, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline and L- serine in Human embryonic stem cell medium as described herein are provided as a mixture, such as MEM Non-Essential Amino Acid Solution (#07600, Stem Cell Technologies). Preferably, when MEM Non-Essential Amino Acid Solution is used, Human embryonic stem cell medium as described herein comprises approximately 1 %v / v MEM Non-Essential Amino Acid Solution. Preferably, B-mercaptoethanol in Human embryonic stem cell medium as described herein is present in a concentration of 0.07- 700 nl / ml; more preferably in a concentration of 0.7-70 nl / ml; most preferably in a concentration of approximately 7 nl / ml. Preferably, L- glutamine in Human embryonic stem cell medium as described herein is present in a concentration of 0.02-200 mM; more preferably in a concentration of 0.2-20 mM; most preferably in a concentration of approximately 2 mM. Alternatively, L-alanyl-L-glutamine, such as commercially available under the name GlutaMAX, may be used as a source of L-glutamine instead of L-glutamine. Preferably, penicillin and streptomycin in Human embryonic stem cell medium as described herein are provided as a mixture, such as Gibco™ penicillin-streptomycin (15140122, Thermo Fisher Scientific). Preferably, when Gibco™ penicillinstreptomycin is used, Neural Differentiation Medium as described herein comprises approximately 1 %v / v Gibco™ penicillin-streptomycin. More specifically, said Human embryonic stem cell medium may comprise Dulbecco’s modified Eagle’s medium (DMEM) / F12 (Thermo Fisher Scientific), 20% knockout serum (Thermo Fisher Scientific), 1% minimum essential medium / non-essential amino acid (Sigma-Aldrich, St Louis, MO, USA), 7 nlml-1B-mercaptoethanol (Sigma-Aldrich), 1% L-glutamine (Thermo Fisher Scientific) and 1% penicillin / streptomycin (Thermo Fisher Scientific).

[0108] In step (al) as described herein, said PSCs as described herein may be washed with said Human embryonic stem cell medium as described herein. Said washing preferably comprises resuspending said PCSs as described herein in a Human embryonic stem cell medium, subsequent centrifugation at 160g for 2 minutes at room temperature, and subsequent removing of supernatant. Preferably, the volume wherein PSCs are resuspended, is 10 times the cellular volume.

[0109] In step (al) as described herein, PSCs are applied while in suspension in Human embryonic stem cell medium to a well or culture dish coated with mouse embryonic fibroblasts (MEFs). MEFs function as feeder cells. The person skilled in the art knows how to coat a well or culture dish with MEFs. Preferably, a MEF coating comprises a single layer of mouse embryonic fibroblasts; more preferably a single layer of irradiated mouse embryonic fibroblasts, for example CF1 irradiated mouse embryonic fibroblasts (GSC-6001G, MTI-GlobalStem).

[0110] Further, steps that result in the generation of a population of NPCs from pluripotent stem cells may include a step (a2): dissociating PSCs from the feeder cells. This includes dissociation of PSCs from substitutes of feeder cells, if such substitute was used. Step (a2) follows step (al).

[0111] Step (a2) as described herein may be performed with any agent of which the person skilled in the art knows that it can be used for dissociation of PSCs. Examples of such agents include collagenase, dispase, trypsin and liberase. Preferably, collagenase is used; more preferably in a concentration of 100 U / ml. Preferably, the dissociation agent is allowed to incubate with the PSCs and feeder cells under suitable conditions, such as at 37 °C / 5% CO2 for 7 minutes.

[0112] Steps that result in the generation of a population of NPCs from pluripotent stem cells may include a step (a3): generating embryonic bodies from the PSCs obtained in step (a2). Step (a3) follows step (a2).

[0113] Step (a3) is preferably performed by culturing the dissociated PSCs from step (a2) in a culture medium for maintaining PSCs, preferably by culturing said dissociated PSCs in a culture medium for maintaining PSCs on a non-adherent plate; preferably wherein the embryonic bodies are cultured for about 2 days in said culture medium for maintaining PSCs. Preferably, said culturing is performed on a non-adherent plate.

[0114] The culture medium for maintaining PSCs that is preferably used in step (a3) is human embryonic stem cell medium as described hereinabove. Preferably, incubation is performed while on a shaker. Preferably, incubation takes place at conditions that facilitate generation of embryonic bodies, such as at 37°C and 5% CO2. PSCs are allowed to develop into embryonic bodies; preferably for 2 days. Said 2 days is preferably counted from the start of step (a3). During said 2 days, PSCs develop into embryonic bodies, and therefore the terms PSCs and embryonic bodies may be used for the developing embryonic bodies during this period.

[0115] Steps that result in the generation of a population of NPCs from pluripotent stem cells may include a step (a4): culturing the embryonic bodies in a neural induction culture medium in suspension, preferably wherein the embryonic bodies are in suspension cell culture for about 4 days. Step (a4) follows step (a3).

[0116] Said neural induction culture medium preferably comprises at least one component selected from the group consisting of a basal medium, transferrin, insulin, progesterone, putrescine, selenite, heparin, penicillin and streptomycin. More preferably, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 of the components from said list are present in said neural induction culture medium. Most preferably, said neural induction culture medium comprises basal medium, transferrin, insulin, progesterone, putrescine, selenite, heparin, penicillin and streptomycin. Preferably, a basal medium present in neural induction culture medium as described herein is Dulbecco’s modified Eagle medium (DMEM); Ham’s F12 medium; or a combination thereof. More preferably, a basal medium present in neural induction culture medium as described herein is a mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12 medium; preferably mixed in a 1:1 volume ratio, such as commercially available DMEM / F12 (21331020; Thermo Fisher Scientific). Preferably, transferrin, insulin, progesterone, putrescine, and selenite are provided as a mixture, such as Gibco™ N2 supplement (17502048, Thermo Fisher Scientific). Preferably, when Gibco™ N2 supplement is used, neural induction culture medium as described herein comprises approximately 1 %v / v Gibco™ N2 supplement. Preferably, heparin is heparin sodium (9041- 08-1, Sigma Aldrich), and is present in a concentration of 2 pg / ml in neural induction culture medium as described herein. Preferably, penicillin and streptomycin in neural induction culture medium as described herein are provided as a mixture, such as Gibco™ penicillin-streptomycin (15140122, Thermo Fisher Scientific). Preferably, when Gibco™ penicilhn-streptomycin is used, neural induction culture medium as described herein comprises approximately 1 %v / v Gibco™ penicilhn-streptomycin. More specifically, the neural induction medium comprises (DMEM / F12, 1% N2 supplement (Thermo Fisher Scientific), 2 pg ml-1heparin (Sigma- Aldrich) and 1% penicillin / streptomycin).

[0117] Preferably, in step (a4) as described herein, embryonic bodies are in suspension in neural induction culture medium for 3-5 days; more preferably for 4 days.

[0118] Steps that result in the generation of a population of NPCs from pluripotent stem cells may optionally include a step (a5): at least partially dissociating embryonic bodies obtained in step (a4). Step (a5) follows step (a4).

[0119] Dissociating, as described in step (a5), may for example be done by trituration. Trituration may for example comprise pipetting up and down, shaking or vortexing.

[0120] Steps that result in the generation of a population of NPCs from at pluripotent stem cells may include a step (a6): plating the embryonic bodies of step (a4) or step (a5) on a coated substrate. Step (a6) follows step (a4) or step (a5).

[0121] Preferably, said coated substrate is a laminin-coated substrate, such as a laminin-coated dish. Said laminin-coated substrate may for example be prepared by incubating a substrate with a basal medium, such as DMEM, wherein laminin is dissolved, for example in a concentration of 20 jig / ml. Laminin may for example be laminin from human fibroblasts (L4544, Sigma Aldrich). Said incubation of a substrate with laminin is preferably performed at 37°C for approximately 30 minutes.

[0122] Steps that result in the generation of a population of NPCs from pluripotent stem cells may include a step (a7): culturing the plated embryonic bodies of step (a6) in a neural induction culture medium, followed by a step of culturing in an NPC culture medium so as to provide a population of NPCs. Step (a7) follows step (a6).

[0123] Said neural induction medium is preferably a neural induction medium as described herein above.

[0124] Said NPC culture medium as described herein preferably comprises at least one component selected from the group consisting of a basal medium, transferrin, insulin, progesterone, putrescine, selenite, biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA, catalase, insulin, transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HC1, glutathione, L-carnitine HC1, linoleic acid, linolenic acid, progesterone, putrescine, selenite triodo-l-thyronine, laminin, basic fibroblast growth factor, penicillin and streptomycin. More preferably, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 or at least 27 of the components from said list are present in said NPC culture medium. Most preferably, said NPC culture medium comprises a basal medium, transferrin, insulin, progesterone, putrescine, selenite, biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA, catalase, insulin, transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HC1, glutathione, L-carnitine HC1, linoleic acid, linolenic acid, progesterone, putrescine, selenite triodo-l- thyronine, laminin, basic fibroblast growth factor, penicillin and streptomycin. Preferably, a basal medium present in NPC culture medium as described herein is Dulbecco’s modified Eagle medium (DMEM); Ham’s F12 medium; or a combination thereof. More preferably, a basal medium present in NPC culture medium as described herein is a mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12 medium; preferably mixed in a 1:1 volume ratio, such as commercially available DMEM / F12 (21331020; Thermo Fisher Scientific). Preferably, transferrin, insulin, progesterone, putrescine, and selenite are provided as a mixture, such as Gibco™ N2 supplement (17502048, Thermo Fisher Scientific). Preferably, when Gibco™ N2 supplement is used, NPC culture medium as described herein comprises approximately 1 %v / v Gibco™ N2 supplement. Preferably, biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA, catalase, insulin, transferrin, superoxide dismutase, corticosterone, D- galactose, ethanolamine HOI, glutathione, L-carnitine HOI, linoleic acid, linolenic acid, progesterone, putrescine, selenite and triodo-l-thyronine are provided as a mixture, such as Gibco™ B27-RA supplement (12587010, Thermo Fisher Scientific). Preferably, when Gibco™ B27-RA supplement is used, NPC culture medium as described herein comprises approximately 2 %v / v Gibco™ B27-RA supplement. Preferably, laminin is laminin from human fibroblasts (L4544, Sigma Aldrich), and is for example present in a concentration of 1 pg / ml in NPC culture medium as described herein. Preferably, basic fibroblast growth factor is human recombinant basic fibroblast growth factor (Merck Millipore), and is for example present in a concentration of 20 ng / ml in NPC culture medium as described herein. Preferably, penicillin and streptomycin in neural induction culture medium as described herein are provided as a mixture, such as Gibco™ penicillinstreptomycin (15140122, Thermo Fisher Scientific). Preferably, when Gibco™ penicillin-streptomycin is used, NPC culture medium as described herein comprises approximately 1 %v / v Gibco™ penicillin-streptomycin. More specifically, said NPC culture medium may comprise DMEM / F12, 1% N2 supplement, 2% B27-RA supplement (Thermo Fisher Scientific), 1 pg ml-1laminin, 20 ng ml-1basic fibroblast growth factor (Merck-Millipore, Darmstadt, Germany) and 1% penicilhn / streptomycin). The NPC culture medium can also be referred to as an NPC expansion medium. Preferably, in step (a7) as described herein, the optionally at least partially dissociated embryonic bodies are cultured for about 8 days in a neural induction culture medium.

[0125] Preferably, in step (a7) as described herein, the optionally at least partially dissociated embryonic bodies are subsequently cultured for 1-10 days in NPC culture medium.

[0126] Hence, this method provides a cell culture comprising a population of neural progenitor cells (NPCs).

[0127] Prior to step (a) of a method of the invention, or as a subsequent step (a8) in view of the method described above, the population of NPCs can be sorted so as to purify the population of NPCs.

[0128] Hence, a method of the invention may comprise a step of: subjecting neural progenitor cells (NPCs) to a step of antibody-based cell sorting, preferably FACS; wherein said step of antibody -based cell sorting is performed on the basis of (i) the presence of cell surface marker CD 184 (CD 184+) and / or CD24 (CD24+) and (ii) the absence of cell surface marker CD44 (CD44-) and / or CD271 (CD27F), to thereby provide a population of sorted NPCs. As an example, these sorted NPC can be kept in a cell culture using a NPC culture medium (also referred to as NPC expansion) medium as described above.

[0129] Preferably, said step of antibody-based cell sorting is performed on the basis of (i) the presence of cell surface markers CD 184 (CD 184+) and CD24 (CD24+) and (ii) the absence of cell surface markers CD44 (CD44-) and CD271 (CD271-).

[0130] In step (b) of a method for producing organoids of the invention, neural progenitor cells (NPCs) of said population are seeded in a well of a multiwell plate, wherein said well comprises a bottom surface 106 and an upright wall 104, and wherein a distance x between a centre 112 of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm. As explained herein above, it was established that the confined space of a well of a standard flat (F) bottom multiwell plate, more specifically its bottom dimension, dictates that neural progenitor cells (NPCs) self-organize into a single cortical brain organoid, with a radial structure, per well.

[0131] An exemplary description of a well, of a multiwell plate, that is suitable for use in the invention is as follows. Reference is also made to Figure 1, and its description above.

[0132] The well 100 is formed by a base part 102 and an upright wall 104 protruding away from the base part 102. The base part 102 forms a bottom surface 106 of the well, which bottom surface 106 preferably is a (substantially) flat surface. In use, the flat surface typically is oriented essentially parallel to the horizon. A top opening 108 allows access into an inner volume 107 of the well 100. The inner volume 107 is typically delimited by an inner surface 105 of the upright wall 104, and the bottom surface 106. In general, the base part and upright wall may be integrally formed, in particular together with other wells of the multiwell plate.

[0133] In the example depicted in Fig. 1A, the upright wall 104 is tapered towards the bottom surface 106. It will be appreciated that for any well of the present disclosure, the upright wall 104 may be tapered towards the bottom surface 106 at any angle, or the upright wall 104 may be oriented essentially perpendicular to the bottom surface 106.

[0134] In the example of the well 100 of which a top view is depicted in Fig. IB, the bottom surface 106 is formed as a rectangle with rounded corners. In the example of the well 100 of which a top view is depicted in Fig. 1C, the bottom surface 106 is formed as a circle. It will be appreciated that for any well of the present disclosure, the bottom surface 106 may have any shape, such as square or rectangular, in combination with straight or rounded corners, circular, oval, or any other shape. Preferably, the shape of the bottom surface 106 is square, with or without rounded corners, or circular. A centreline 110 of the well 100 is indicated in Fig. 1A, which centreline 110 intersects the bottom surface 106 at a centre 112 of the bottom surface 106. A distance x is indicated in Fig. 1A, which distance x is the distance between the centre 112 of the bottom surface 106 and the upright wall 104. The distance x may generally be determined in any direction, from the centre 112 of the bottom surface 106 to where the upright wall 104 meets the bottom surface 106.

[0135] As an option depicted in Fig. 1A, as a preference for any well disclosed herein, the upright wall 104 and the bottom surface 106 meet at an obtuse angle or a straight angle, essentially without the intersection of the upright wall 104 and the bottom surface 106 having a smoothed transition, for example with a radius, fillet, or chamfer.

[0136] For the example of Fig. IB with the quasi-rectangular bottom surface 106, the distance x corresponds to half of the width w of the bottom surface 106. For the example of Fig. 1C with the circular bottom surface 106, the distance x corresponds to the radius of the bottom surface 106.

[0137] For any well disclosed herein, the distance x is between 1.1 mm and 1.9 mm, for instance between 1.3 mm and 1.8 mm, between 1.4 mm and 1.8 mm or between 1.5 mm and 1.8 mm. Preferably, the distance x is between 1.5 mm and 1.8 mm or between 1.5 mm and 1.7 mm, such as between 1.6 and 1.7 mm. As an example, the distance x can be about 1.65 mm, which preferably corresponds to a width or diameter of 3.3 mm. Preferably, the distance x is a distance that allows for reproducible production of a single adherent cortical organoid, preferably with a radial structure, per well of a multiwell plate. Preferably, such reproducible production refers to a situation wherein, in at least 50%, in at least 60%, in at least 70%, in at least 75%, in at least 80%, in at least 90%, or in at least 95%, of the seeded wells of said multiwell plate, a single adherent cortical organoid, preferably with a radial structure, has formed or is present. A height h of the well 100, indicated in Fig. 1A, may be between 5 and 15 mm, preferably between 10 and 13 mm, even more preferably between 11 and 12 mm. Any height h may be combined with any distance x for any well of the present disclosure. For example, the well plate can be a 384-well plate. Typically, all wells of a multiwell plate have essentially corresponding dimensions and shape.

[0138] The well of a multiwell plate as described can be a well of a 384- well plate, such as a well of (a standard) flat (F) bottom 384-well plate. In such a case, the multiwelll plate can also be a 384-well plate, such as (standard) flat (F) bottom 384-well plate. Such plates are encompassed in the well definition provided above.

[0139] Preferably, when the multiwell plate is a 384-well plate, the 384- well plate matches the prescribed dimensions (and / or geometry) of a standard 384-well plate as defined by ANSI SLAS 4-2004 (R2012) (formerly recognized as ANSI / SBS 4-2004). An example of such a 384-well plate is the (standard) flat (F) bottom 384-well plate of Greiner Bio (M1937-32EA), which was used in the Examples. Wells of this 384-well plate are characterized by having a distance x of about 1.65 mm, which corresponds with a width of about 3.3 mm. Furthermore, the wells of this 384-well plate have a height h of 11.5 mm. Furthermore, the shape of the wells of this plate are square with rounded corners. Wells of this plate have a working volume up to 131 pL, with an optimal working volume of 15-110 jiL. Multiwell plates can be made from various materials such as polystyrene or glass.

[0140] Preferably, prior to seeding (NPCs) of said population in a well of a multiwell plate, wells of said multi-well plate are coated with a coating for adherent cell culture. The skilled person is well aware of suitable coatings for adherent cell cultures that can be applied in wells of multiwell plates. Examples of suitable coatings for adherent cell culture are laminin and poly- L-ornithine coatings. Wells can also be double coated, for instance using both laminin and poly-L-ornithine coatings. Exemplary conditions for coating a well with laminin are well known, and may for instance involve applying laminin, e.g. at about 50 gg / ml in dFLO, to a well of a multiwell plate, and incubating the well at approximately 37°C and e.g. at 5% CO2 Exemplary conditions for coating a well with poly-L-ornithine are for instance applying poly-L-ornithine, e.g. at concentration of approximately 0.01 %w / v in water, to a well of a multiwell plate and incubating said well for approximately 1 hour at room temperature.

[0141] Surprisingly, it was established that the coatings described herein, especially a laminin coating, outperformed a standard Matrigel cell culture substrate (coating) by further improving reproducibility in the production of adherent cortical organoids (data not shown).

[0142] Preferably, seeding of neural progenitor cells (NPCs) of said population is performed in a coated well of a multi-well plate. Preferably, all wells of a multiwell plate in which NPCs are seeded, are coated as described herein.

[0143] It is routine practice for a person skilled in the art to determine an appropriate seeding density for a given NPC line, especially on the basis of the present disclosure. In general, appropriate seeding densities can be in the range of 250-10000 NPCs per well of a multi-well plate, such as 1000- 7500 NPCs per well. Figure 6C provides guidance in that a seeding density can be determined on the basis of NPC proliferation rate. As an example, when the NPC proliferation rate (in terms of doubling time (h)) is between 15 and 25 hours, an NPC seeding density of between 500 and 2500 NPCs per well can be used, such as an NPC seeding density of between 1000 and 1875 NPCs per well or an NPC seeding density of around 1250 NPCs per well. When the NPC proliferation rate, in terms of doubling time (h)), is between 25 and 30 hours, an NPC seeding density of between 1000 and 4000 NPCs per well can be used, such as an NPC seeding density of between 1250 and 3500 NPCs per well. When the NPC proliferation rate, in terms of doubling time (h)), is between 30 and 35 hours, an NPC seeding density of between 2500 and 10000 NPCs per well can be used, such as an NPC seeding density of between 3000 and 1000 NPCs per well. NPC proliferation rate can for instance be determined using (standard) cell proliferation assays such as CyQUANT™ Direct Cell Proliferation Assay, C35011 (Thermo Fisher Scientific).

[0144] Preferably, prior to seeding, NPCs of said population are brought in a culture medium for neural differentiation and are seeded in a well of a multi-well plate while in said culture medium for neural differentiation. An example of a suitable culture medium for neural differentiation is provided herein below.

[0145] Preferably, NPCs of said population are seeded in, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or in 100%, of the (preferably coated) wells, calculated on the basis of the total number of wells of said multi-well plate. Different NPC lines can be seeded in different wells of a multi-well plate. For instance, NPCs that resemble cells of a subject having a disease can be seeded in wells of a multiwell plate together with NPCs that resemble cells of a subject not having a disease (health subject) as long as both NPC lines are seeded in different wells of said multi-well plate.

[0146] In step (c) of a method for the production of an adherent cortical organoid of the invention, said seeded NPCs are cultured in a culture medium for neural differentiation for a period of time. This is to allow an adherent cortical organoid to form or develop by self-organization.

[0147] Said culture medium for neural differentiation comprises one or more differentiation factors that allow for differentiation of NPCs into differentiated cell types of the cerebral cortex. More specifically, said culture medium for neural differentiation comprises one or more differentiation factors selected from the group consisting of a brain-derived neurotrophic factor, a glial cell-derived neurotrophic factor, a dibutyryl cyclic adenosine monophosphate, and an ascorbic acid. Combinations are also foreseen, such as a brain-derived neurotrophic factor and a glial cell-derived neurotrophic factor, a brain-derived neurotrophic factor and a dibutyryl cyclic adenosine monophosphate, a brain-derived neurotrophic factor and an ascorbic acid, a glial cell-derived neurotrophic factor and a dibutyryl cyclic adenosine monophosphate, a glial cell-derived neurotrophic factor and an ascorbic acid, or a dibutyryl cyclic adenosine monophosphate and an ascorbic acid. Preferably, all four differentiation factors are present in a culture medium for neural differentiation.

[0148] Preferably, said culture medium for neural differentiation is serum-free.

[0149] A culture medium for neural differentiation may further comprise (i) an N2 supplement and / or a B27 supplement without vitamin A (B27-RA). Said culture medium for neural differentiation may in addition thereto further comprises (ii) a minimum essential medium and / or a non-essential amino acid supplement, (iii) a laminin and / or (iv) penicillin and / or streptomycin.

[0150] A culture medium for neural differentiation may further comprise a basal medium for neuronal cell culture.

[0151] Alternatively, a culture medium for neural differentiation can be defined as follows. A culture medium for neural differentiation may comprise at least one component selected from the group consisting of neurobasal medium, transferrin, insulin, progesterone, putrescine, selenite, biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA, catalase, superoxide dismutase, corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, triodo-l-thyronine, L- alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, L-serine, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, dibutyryl cyclic adenosine monophosphate, ascorbic acid, laminin, penicillin, and streptomycin. More preferably, a culture medium for neural differentiation comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33 or at least 34 of the components selected from the group consisting of neurobasal medium, transferrin, insulin, progesterone, putrescine, selenite, biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA, catalase, superoxide dismutase, corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, triodo-l-thyronine, L- alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, L-serine, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, dibutyryl cyclic adenosine monophosphate, ascorbic acid, laminin, penicillin, and streptomycin. Still more preferably, a culture medium for neural differentiation comprises at least at least one brain-derived neurotrophic factor. Still more preferably, a culture medium for neural differentiation comprises a basal medium for neuronal cell culture, a brain- derived neurotrophic factor, a glial cell-derived neurotrophic factor, dibutyryl cyclic adenosine monophosphate and ascorbic acid; preferably additionally an N2 supplement or components thereof; and a B-27 supplement without vitamin A or components thereof; more preferably additionally a minimum essential medium and / or a non-essential amino acid supplement; and even more preferably a laminin and / or penicillin and / or streptomycin. Most preferably, a culture medium for neural differentiation as described herein comprises at least neurobasal medium, transferrin, insulin, progesterone, putrescine, selenite, biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA, catalase, superoxide dismutase, corticosterone, D-galactose, ethanolamine, glutathione, L- carnitine, linoleic acid, linolenic acid, triodo-l-thyronine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, L-serine, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, dibutyryl cyclic adenosine monophosphate, ascorbic acid, laminin, penicillin, and streptomycin. Preferably, said basal medium is neurobasal medium. More preferably, said neurobasal medium is a commercially available neurobasal medium, such as Gibco™ Neurobasal Medium (21103049, Thermo Fisher Scientific). Preferably, transferrin, insulin, progesterone, putrescine, and selenite are provided as a mixture, such as Gibco™ N2 supplement (17502048, Thermo Fisher Scientific). Preferably, when Gibco™ N2 supplement is used, a culture medium for neural differentiation as described herein comprises approximately 1 %v / v Gibco™ N2 supplement. Preferably, biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA, catalase, insulin, transferrin, superoxide dismutase, corticosterone, D- galactose, ethanolamine HOI, glutathione, L-carnitine HOI, linoleic acid, linolenic acid, progesterone, putrescine, selenite and triodo-l-thyronine are provided as a mixture, such as Gibco™ B27-RA supplement (12587010, Thermo Fisher Scientific). Preferably, when Gibco™ B27-RA supplement is used, a culture medium for neural differentiation as described herein comprises approximately 2 %v / v Gibco™ B27-RA supplement. Preferably, L- alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline and L-serine are provided as a mixture, such as MEM Non-Essential Amino Acid Solution (#07600, Stem Cell Technologies). Preferably, when MEM Non-Essential Amino Acid Solution is used, a culture medium for neural differentiation as described herein comprises approximately 1 %v / v MEM Non-Essential Amino Acid Solution. Preferably, brain-derived neurotrophic factor as described herein is provided as commercially available brain- derived neurotrophic factor, such as Human BDNF (CYT-207, ProSpec Bio). Preferably, brain-derived neurotrophic factor is present at a concentration of 0.2-2000 ng / ml; more preferably 2-200 ng / ml; most preferably approximately 20 ng / ml. Preferably, glial cell-derived neurotrophic factor as described herein is provided as commercially available glial cell-derived neurotrophic factor, such as Human GDNF (CYT-305, ProSpec Bio). Preferably, glial cell- derived neurotrophic factor is present at a concentration of 0.2-2000 ng / ml; more preferably 2-200 ng / ml; most preferably approximately 20 ng / ml. Preferably, dibutyryl cyclic adenosine monophosphate as described herein is provided as commercially available dibutyryl cyclic adenosine monophosphate, such as D0627 (Sigma- Aldrich). Preferably, dibutyryl cyclic adenosine monophosphate is present at a concentration of 0.01-100 pM; more preferably 0.1-10 pM; most preferably approximately 1 pM. Preferably, ascorbic acid as described herein is provided as commercially available ascorbic acid, such as L-ascorbic acid (A92902, Sigma- Aldrich). Preferably, ascorbic acid is present at a concentration of 2-20,000 pM; more preferably 20-2000 pM; most preferably approximately 200 pM. Preferably, laminin as described herein is provided as commercially available laminin, such as laminin from human fibroblasts (L4544, Sigma-Aldrich). Preferably, laminin is present at a concentration of 0.02-200 gg / ml; more preferably 0.20-20 pg / ml; most preferably approximately 2 pg / ml. Preferably, penicillin and streptomycin are provided as a mixture, such as Gibco™ penicillinstreptomycin (15140122, Thermo Fisher Scientific). Preferably, when Gibco™ penicillin-streptomycin is used, a culture medium for neural differentiation as described herein comprises approximately 1 %v / v Gibco™ p enicillin- str ep tomy cin .

[0152] As an example, a culture medium for neural differentiation comprises a neurobasal medium (Thermo Fisher Scientific), 1% N2 supplement (Thermo Fisher Scientific), 2% B27-RA supplement (Thermo Fisher Scientific), 1% minimum essential medium / non-essential amino acid (Stem Cell Technologies), 20 ng / ml brain-derived neurotrophic factor (ProSpec Bio), 20 ng / ml glial cell-derived neurotrophic factor (ProSpec Bio), 1 pM dibutyryl cyclic adenosine monophosphate (Sigma-Aldrich), 200 pM ascorbic acid (Sigma-Aldrich), 2 pg / ml laminin (Sigma-Aldrich) and 1% penicilhn / streptomycin (Thermo Fisher Scientific).

[0153] Preferably, NPCs that have been seeded in a well in step (c) of a method of the invention are allowed to develop into an adherent cortical brain organoid (by self-organization), wherein the temperature is kept at approximately 37°C.

[0154] In some embodiments, CO2 is present in gaseous form during a method for the production of adherent cortical organoid of the invention. Preferably, CO2 is present in a concentration of 0.05-50 %; more preferably in a concentration of 0.5-20 %; even more preferably in a concentration of 1- 10 %; still more preferably in a concentration of 3-7%; still more preferably in a concentration of 4-6 %; most preferably in a concentration of approximately 5%.

[0155] When culturing said seeded NPCs in a culture medium for neural differentiation, said medium is periodically refreshed. For instance, said medium is refreshed every 1-5 days; preferably every 2-3 days.

[0156] In step (c) of a method of the invention, the process of NPCs selforganizing into organoids is preferably not disturbed by slicing, cutting, drilling, poking, piercing and / or crushing. Preferably, refraining from slicing, cutting, drilling, poking, piercing and / or crushing applies throughout a method of the invention.

[0157] In step (c) of a method of the invention, said period of time for culturing said seeded NPCs in a culture medium for neural differentiation is for instance at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months or at least a year. Preferably, in step (c) of a method of the invention, at about 30-90 days following the start of culturing seeded NPCs in a culture medium for neural differentiation, more preferably at about 60 days following the start of culturing seeded NPCs in a culture medium for neural differentiation, in at least 20%, in at least 30%, in at least 40%, in at least 50%, in at least 60%, in at least 70%, in at least 75%, in at least 80%, in at least 90%, in at least 95%, or in 100%, of the seeded wells of said multiwell plate, a singular (single) adherent cortical organoid with a radial structure forms (by selforganization). Preferably, at about 60 days following the start of culturing seeded NPCs in a culture medium for neural differentiation, in at least, or about, 80% of the seeded wells of said multiwell plate, a singular (single) adherent cortical organoid with a radial structure has formed (by selforganization). Preferably, after about 1 year following the start of culturing seeded NPCs in a culture medium for neural differentiation, in at least 40%, preferably in about 50%, of the seeded wells of said multiwell plate a singular (single) adherent cortical organoid with a radial structure has formed (or is present).

[0158] During step (c) of a method for the production of an adherent cortical brain organoids of the invention, seeded NPCs may be cultured in the presence of Triiodothyronine (T3) to allow for oligodendrocyte maturation. For instance, 2 ng / ml T3 (Sigma-Aldrich) can be used.

[0159] In methods of the invention, seeded NPCs preferably self-organize into adherent cortical organoids with singular radial structures. Said radial structures resemble early human cortical development. More preferably, said structures are layered and have robust functional synaptic connectivity. Said layered structure may for example be visualized by identifying individual layers using markers, such as CTIP2 and CUX1. Segregation of those markers in an organoid may be indicative of layer formation. Preferably, said cortical organoid has a layered structure of at least 2 layers, more preferably 3, 4, 5 or 6 layers.

[0160] In a preferred embodiment, during step (c) of a method for the production of adherent cortical brain organoids of the invention, culturing of said seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation in a well (a test well) of said multiwell plate occurs in the presence of a test agent.

[0161] Preferably, a test agent can be any agent of interest, such as a pharmaceutical agent of interest (e.g. a small molecule or biological molecule) or a food supplement. Such a test agent can be added to the culture medium as recited in step (c) to investigate whether, and to what extent, it modulates an adherent cortical organoid such as its formation. Hence, in a method for the production of an adherent cortical brain organoids of the invention, when employing test agents, formation of an adherent cortical organoid may be modulated in the sense that organoid formation is counteracted (e.g. in the sense that the organoid underdevelops, e.g. lacking certain structural and / or functional characteristics) or may be improved (in the sense that organoid has additional or improved structural and / or functional characteristics). Said modulation can for instance be reflected in the following parameters, which can be used in the screening process: cell death, cell-type, organoid functionality, organoid cell number, organoid morphology, differentiation potential, cortical layering, and / or maturity of neuronal network (e.g. synaptic connections).

[0162] Optionally, when a test agent is included in a method for the production of an adherent cortical brain organoids of the invention, in a different well (control well) of said multiwell plate, culturing of seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation occurs in the absence of said test agent. This allows for comparing the formation of an adherent cortical organoid in the presence of said test agent with the formation of an adherent cortical organoid in the absence of said test agent. Alternatively, instead of providing of control sample by (co) -culturing (in a different well) said seeded neural progenitor cells (NPCs) in the absence of said test agent, a control adherent cortical organoid, previously cultured in the absence of test agents, or photos or videos of its development, can be used for said comparison. Said test agent can be identified as an agent that modulates an adherent cortical organoid, or its formation, if the cortical organoid that is produced in the presence of said test agent is different from said cortical organoid that is produced in the absence of said test agent. In other words, formation of an adherent cortical organoid in the presence of a test agent may be - compared to a control -modulated in the sense that organoid formation is counteracted (e.g. in the sense that the organoid underdevelops, e.g. lacking certain structural and / or functional characteristics), or may be improved (in the sense that organoid has additional or improved structural and / or functional characteristics).

[0163] Further, a test agent as disclosed herein may be added at any time point during step (c) of a method of the invention, for instance at the start of culturing when the seeded NPC start to self-organize, or more towards the end of culturing when the adherent cortical organoids are formed.

[0164] As an example, a test agent as disclosed herein can be used in toxicological screening, in which it is assessed to what extent the test agent is toxic. For instance, screening for cell death, cell-type, organoid functionality, organoid cell number, organoid morphology, differentiation potential, cortical layering, and maturity of neuronal network (e.g. synaptic connections) may be performed.

[0165] To that extent, the invention also provides a method of screening for (test) agents, such as pharmacological agents or food ingredients, that modulate an adherent cortical organoid, such as its formation, comprising the steps of: - performing a method for the production of an adherent cortical organoid as disclosed herein; wherein culturing of said seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation in a (test) well of said multiwell plate occurs in the presence of a test agent; -optionally, wherein, in a different well (control well) of said multiwell plate, culturing of said seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation occurs in the absence of said test agent;

[0166] -comparing the adherent cortical organoid produced in the presence of said test agent with a cortical organoid produced in the absence of said test agent; - identifying said test agent as an agent that modulates formation of an adherent cortical organoid if the cortical organoid that is produced in the presence of said test agent is different from said cortical organoid that is produced in the absence of said agent.

[0167] For instance, said difference may be differences in the parameters of cell death, cell-type (e.g. cell type quantification by cell-type specific markers over for instance a common cell nucleus marker identifying all cells), organoid functionality, organoid cell number, organoid morphology, differentiation potential, cortical layering, and maturity of neuronal network (e.g. synaptic connections). Differences in organoid functionality, such as neuronal activity (e.g. synchronous network bursting and / or desynchronized activity such as desynchronized activity during time periods outside of synchronous network bursting), can be measured by fluorescencebased calcium imaging.

[0168] Multiwell plates, and adherent cortical brain organoids

[0169] The invention also provides a multiwell plate comprising a well; wherein said well comprises a bottom surface 106 and an upright wall 104, and wherein a distance x between a centre 112 of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm; wherein said well contains a single adherent cortical organoid with a radial structure; and wherein said adherent cortical organoid comprises neurons, astrocytes and (myelinating) oligodendrocytes, and optionally microglia or vascular cells.

[0170] Such a multiwell plate is an intermediate or end-product of a method for the production of an adherent cortical organoid of the invention. Previous disclosure in relation to wells and / or multiwell plates provided hereinabove, also applies to this aspect.

[0171] Furthermore, the invention also provides an adherent cortical organoid with a radial structure, as such, and preferably comprises neurons, astrocytes and (myelinating) oligodendrocytes, and optionally microglia or vascular cells. Such an adherent cortical organoid is obtainable by a method for the production of an adherent cortical organoid as disclosed herein.

[0172] Adherent cortical brain organoids as disclosed herein are formed by self-organization of seeded NPCs. A cortical organoid as disclosed herein preferably resembles the frontal cortex. Resemblance to the frontal cortex, such as in early human cortical development, may for example be recognized by the presence of neurons and astrocyte subtypes, preferably in an organized network. Said resemblance is generally characterized by a low number of dead cells in the center of the organoid. Typically, in cortical organoids resembling a frontal cortex, dendritic and axonal compartments are segregated. Preferably, an organoid as disclosed herein comprises an interneuron cell population, which may for example be identified by checking for the presence of both MAP2 and GAD67. Also, preferably, a cortical organoid as disclosed herein comprises different astrocyte subtypes, such as fibrous astrocytes, protoplasmic astrocytes and interlaminar astrocytes. Additionally, an organoid produced by a method of the invention preferably displays myelination.

[0173] Another feature that cortical organoids as disclosed herein preferably possess, is dense packing of dendrites and somatic cells with synapses wherein the pre-synaptic marker Synapsin is present. Preferably, Synapsin co-localizes with the post-synaptic marker PSD-95.

[0174] Preferably, a cortical organoid as disclosed herein comprises neurons comprising dendritic spines. Dendritic spines may for example be visualized using sparse labeling of neurons by AAV9.CamKII.eGFP. Preferably, a cortical organoid as disclosed herein shows structure formation, i.e. segregation of upper and deeper cortical layer markers and radial orientation of neurons and glia cells.

[0175] Adherent cortical organoids compare favorably to free-floating brain organoid models on the basis of robust reproducibility in obtaining singular radial cortical structures that conform to the dimensions of each well and circumvent the internal necrosis that is common in free-floating cortical organoids.

[0176] Preferably, adherent cortical organoids as disclosed herein display neuronal activity such as (i) synchronous network bursting and / or (ii) desynchronized activity such as desynchronized activity during time periods outside of synchronous network bursting. For instance, organoids as disclosed herein may exhibit synchronous network level bursting (NB), as measured by fluorescence-based calcium imaging, of at least 0.3, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4 or at least 1.5, network bursts per minute.

[0177] For the purpose of clarity and a concise description, features may be described herein as part of the same or separate embodiments, however, it will be appreciated that the disclosure includes embodiments having combinations of all or some of the features described.

[0178] The content of the documents referred to herein is incorporated by reference. EXAMPLES

[0179] Example 1. Production of cortical brain organoids.

[0180] Materials and Methods

[0181] Generation of Neural Progenitor Cells (NPCs)

[0182] NPCs from 3 different source cell lines were used. NPC-line 1: in house generated NPCs from human iPSC line WTC11 (Gladstone Institute, GM25256, Miyaoka et al. Nat Methods, 11(3):291-3 (2014)). NPC-line 2: commercially available hNPCs from Axol Biosciences (ax0015). NPC-line 3: NPCs derived using the protocol of Shi et al., (Shi et al., Nat Neurosci. 2012;15:477-486) from hiPSC line IPSC0028 (Sigma-Aldrich). Line 1 NPCs were generated as previously described (Gunhanlar et al., Mol Psychiatry. 2018;23:1336-1344). For line 1, after passage 3, NPC cultures were purified using fluorescence-activated cell sorting (FACS). NPCs were detached from the culture plate using Accutase (Stem Cell Technologies) and CD184+ / CD447CD2717CD24+cefls (Yuan et al., PLoS One. 2011;6:el7540) were collected on a FACSAria III Cell Sorter (BD Bioscience) and expanded in NPC medium consisting of: DMEM / F12, 1% N2 supplement, 2% B27-RA supplement (Thermo Fisher Scientific), 1 pg / ml laminin, 20 ng / ml basic fibroblast growth factor (Merck-Millipore, Darmstadt, Germany) and 1% penicillin / streptomycin. NPCs were differentiated to neural cultures between passage 3 and 7 after sorting.

[0183] Neural differentiation

[0184] Standard 384-well plates (M1937-32EA. Life Technologies; flat (F) bottom) were coated with 50 gg / ml laminin in dFLO (Sigma, L2020) for 30 minutes at 37 °C. The NPCs in NPC medium were dissociated with Accutase (Stem Cell Technologies), live cells were counted with Trypan Blue (Stem Cell Technologies) in a B rker counting chamber. The NPCs were seeded in the wells of a 384-well plate at defined densities for each cell line. Specifically, the optimal seeding density was determined by visual inspection of the organoids between 28 to 42 days after seeding a range of cell densities in the 384-well plate wells. NPCs were seeded and differentiated in Neural Differentiation Medium: Neurobasal medium (Thermo Fisher Scientific, catalog number 21103049), 1% N2 supplement (Thermo Fisher Scientific), 2% B27-RA supplement (Thermo Fisher Scientific), 1% minimum essential medium / non-essential amino acid (Stem Cell Technologies), 20 ng / ml brain- derived neurotrophic factor (ProSpec Bio), 20 ng / ml glial cell-derived neurotrophic factor (ProSpec Bio), 1 pM dibutyryl cyclic adenosine monophosphate (Sigma-Aldrich), 200 pM ascorbic acid (Sigma-Aldrich), 2 pg / ml laminin (Sigma-Aldrich) and 1% penicillin / streptomycin (Thermo Fisher Scientific). For oligodendrocyte maturation the cells were grown in the presence of 2 ng / ml T3 (Sigma-Aldrich). Cells were refreshed every 2-3 days.

[0185] Immunocytochemistry

[0186] For live-dead staining, living cultures were incubated with LIVE / DEAD™ Viability / Cytotoxicity Kit according to manufacturer’s instructions (Thermo Fisher Scientific). For immunocytochemistry adherent cortical organoids were fixed for 20-30 minutes using 4% formaldehyde in phosphate-buffered saline (PBS), washed with PBS and blocked for 1 hour by pre-incubation in staining buffer containing 0.05 M Tris, 0.9% NaCl, 0.25% gelatin and 0.5% Triton-X-100 (pH 7.4). Primary antibodies were incubated for 48-72h at 4 °C in staining buffer, washed with PBS and incubated with the secondary antibodies in staining buffer for 2h at room temperature. The cultures were embedded in Mowiol 4-88 (Sigma-Aldrich), after which confocal imaging was performed with a Zeiss LSM700 and Zeiss LSM800 confocal microscope using ZEN software (Zeiss, Oberkochen, Germany). The following primary antibodies were used: SOX2 (Merck-Millipore AB5603, 1:200); Nestin (Merck -Millip ore MAB5326, 1:200); MAP2 (Synaptic Systems 188004, 1:100); NeuN (Merck ABN78, 1:200); GFAP (Merck-Millipore AB5804, 1:300); FOXG1 (Abeam AB 18259, 1:200); CUX1 (Abeam AB54583, 1:200); CTIP2 (Abeam AB 18465, 1:100); Synapsin (Synaptic Systems 106103, 1:200); and PSD95 (Thermo Fisher Scientific MAI-046, 1:100); Tau (Cell Signaling Technology 4019, 1:200); S100B (Sigma-Aldrich S2532, 1:200); Pax6 (Santa Cruz sc-81649, 1:100); NG2 (Gift from W. Stallcup Lab, 1:100); NF200 (Sigma-Aldrich 083M4833 1:200); MBP (Abeam AB7349, 1:100); GFP (Abeam abl3970, 1:100); GAD67 (Merck-Millipore MAB5406, 1:100) DAPI (Thermo Fisher Scientific D1306). The following secondary antibodies were used 1:200: Alexa-488, Alexa-555, Alexa-647 (Jackson ImmunoResearch, West Grove, PA, USA).

[0187] Sparse labelling of excitatory neurons pENN.AAV9.CamKII.4.eGFP.WPRE.rBG (Addgene viral prep # 105541- AAV9) was added to the cortical organoids at day 278 (1.68xl08GC / well). The transduced cortical organoids were fixed and stained at day 310.

[0188] Calcium imaging

[0189] For calcium imaging the genetically encoded calcium indicator AAVl.Syn.GCaMP6s.WPRE.SV40 (Penn Vector Core, 100843-AAV1) was added to the organoids at day 42 of differentiation (1.5xl08GC / well). Recordings were performed at day 60 on a Zeiss LSM800 confocal microscope using ZEN software (Zeiss, Oberkochen, Germany). The recordings were made with a 20x / 0.8NA Ph2 Plan -Apochromat objective, with a field of view of 150 x 100 pm and a pixel size of 0,3 pm. The acquisition rates of the recordings were between 4-5 f.p.s. 24 hours before the recordings, the medium was switched to BrainPhys Neural Differentiation Medium. BrainPhys Neuronal Media (Stem Cell Technologies), 1% N2 supplement (Thermo Fisher Scientific), 2% B27-RA supplement (Thermo Fisher Scientific), 1% minimum essential medium / non- essential amino acid (Stem Cell Technologies), 1% penicillin / streptomycin (Thermo Fisher Scientific), 20 ng / ml brain-derived neurotrophic factor (ProSpec Bio), 20 ng / ml glial cell-derived neurotrophic factor (ProSpec Bio), 1 pM dibutyryl cyclic adenosine monophosphate (Sigma-Aldrich), 200 pM ascorbic acid (Sigma-Aldrich) and 2 pg / ml laminin (Sigma- Aldrich). The calcium imaging recordings were processed using CNMF-E (Pnevmatikakis et al., Neuron. 2016 Jan 20;89(2):285-99). Calcium traces were then analysed using a custom script for event and network burst detection using an algorithm written in Python (v3.8.2) (code accessibility can be requested via Github).

[0190] Cyquant Proliferation Assay CyQUANT™ Direct Cell Proliferation Assay, C35011 (Thermo Fisher Scientific) was used according to manufacturer specifications. For each time point and each of the 3 NPC lines 10-12 wells of a 96-well plate were seeded with NPCs (2500 NPCs per well). In addition, NPC lines generated from 2 different clones from IPS line MHO 159020 (Rutgers University Cell and DNA Repository) were added to increase the dynamic range of the proliferation curve. At 24h and 96h NPCs were frozen at -80°C. All NPC lines and timepoints were thawed, lysed and measured together. Doubling time was calculated between 24h and 96h. None of the wells was confluent at 96h.

[0191] Statistical analysis

[0192] All data represent mean ± SEM. When comparing developmental markers in Figure 6 we used one-way ANOVA followed by Tukey- Kramer’s multiple correction test. n=3-6 images taken over two wells, for each time point. Results

[0193] Summary

[0194] In the growing diversity of human iPSC-derived models of brain development, we present here a novel method that exhibits 3D cortical layer formation in a highly reproducible topography of minimal dimensions. The resulting adherent cortical organoids develop by self-organization after seeding frontal cortex patterned iPSC-derived neural progenitor cells in 384- well plates during eight weeks of differentiation. The organoids have stereotypical dimensions of 3 x 3 x 0.2 mm, contain multiple neuronal subtypes, astrocytes and oligodendrocyte lineage cells, and are amenable to extended culture for at least 10 months. Longitudinal imaging revealed morphologically mature dendritic spines, axonal myelination, and robust neuronal activity. Moreover, adherent cortical organoids compare favorably to existing brain organoid models on the basis of robust reproducibility in obtaining topographically-standardized singular radial cortical structures and circumvent the internal necrosis that is common in free-floating cortical organoids. The adherent human cortical organoid platform holds considerable potential for high-throughput drug discovery applications, neurotoxicological screening, and mechanistic pathophysiological studies of brain disorders.

[0195] Self -organized topography of iPSC-derived adherent cortical organoids Adherent cortical organoids reproducibly self-organized into layered radial structures in 384-wells plates within 8 weeks of seeding with hiPSC-derived forebrain-patterned neural progenitor cells (NPCs). Three different hiPSC source cell lines were used to generate NPCs in this study. NPC line 1 was obtained from hiPSCs using a different protocol than NPC line 3, whereas NPC line 2 was commercially obtained. Hence, a variety of NPC lines with different origin and obtained under varying cell culture conditions were used. The protocol that was used to generate NPC line 1 is depicted in Fig.2A. NPCs were capable of neural rosette formation and expressed SOX2, Nestin, and the frontal cortical NPC-marker FOXG1 (Figure 2B, C, D, Figure 6A). The initial 4 weeks after seeding the NPCs in Neural Differentiation Medium (ND) were characterized by proliferative expansion of NPCs and the emergence of early neural differentiation markers (Figure 2E, Figure 7). Between 4- and 8-weeks post-seeding, neurons and glial cells emerged with a consistent spatial organization (Figure 2E, 2F, Figure 6B, Figure 7), in which the central region was densely packed with cell bodies while the periphery contained circumferentially and radially organized processes originating from cells in the centre. Typically, cortical organoids defined as a single radial structure per well were observed in ~ 80% of the wells seeded with NPCs after 60 days of differentiation. The number of single structure organoids slowly diminished over time to about 50% after 1 year in culture (Figure 6D).

[0196] As a side-experiment, it was established that organoid structure formation was dependent on the proliferation rate of NPCs, which can differ substantially between differentiation batches and hiPSC clones. Plotting the interaction between proliferation rate and the amount of NPCs required to be seeded for the successful generation of adherent cortical organoids, showed a significant correlation (R=0.67) that can be used as a guideline for testing a range of NPC densities (Figure 6C). For each NPC line an optimal seeding density was estimated based on the proliferation rate of that NPC line measured using CyQUANT™ Direct Cell Proliferation Assay. Multiple densities were seeded around the estimated optimal density and after 6 weeks it was visually determined which NPC density enabled adherent cortical organoid generation. Typically, too low density seeding generated neural networks without structure and too high density seeding caused overgrowth and had reduced structural organisation and long-term survival.

[0197] An appropriate NPC seeding density can be routinely established.

[0198] Cell type distribution and layer formation

[0199] The spatial organisation that evolved over the first 8 weeks after seeding was paralleled by a shift in cell type distribution. Tau+ / MAP2- axons exhibited long extensions in a circular pattern, while MAP2+ dendrites exhibited orthogonally-oriented radial outgrowth (Figure 3A, Figure 8). Overall, a reduction in progenitor markers (SOX2 day 14: 58.4%, day 56: 18.8% P = <0.001, PAX6 day 14: 34.5%, day 56: 8.0% P = 0.20) and a significant increase in neuronal cortical layer markers (CTIP2 day 14: 0.5%, day 56: 14.0% P = <0.001, CUX1 day 14: 1.3%, day 56: 24.4% P = <0.001) were observed (Figure 7). Cortical layer markers exhibited an inside-out pattern of development in which expression of the deep layer excitatory neuronal marker CTIP2 emerged before the upper layer marker CUX1 (Figure 7, Figure 3C). After 6-8 weeks following seeding, a self-organised rudimentary segregation of deep- and upper layer neurons emerged as shown by a clear macroscopic separation of deep- and upper layer neurons, although some neurons were spatially intermixed and some neurons were double-positive for CTIP2 and CUX1. Segregation was also observed between CUX1 and CUX2 positive cells as CUX2 is typically expressed over a wider range of upper cortical layers than CUX1 and also marks intermediate progenitors (Molyneaux et al., Nat Rev Neurosci 8:427-37 (2007)) (Figure 3C, Figure 9). Analogous to the broad distribution of cortical cell subclasses, the majority of the neurons were glutamatergic, while GAD67+ interneurons were also present (Figure 3D), constituting ~10% of the NeuN-positive neuronal population (Figure 10).

[0200] Adherent cortical organoids contain multiple glial cell types Within 8 weeks of seeding, a population of GFAP+ / S100B+ astrocytes emerged. Many astrocytes had their soma located in the central region with process outgrowth radially (Figure 3E), while other astrocytes exhibited subtype-specific morphologies including fibrous astrocytes (Figure 3F), protoplasmic -like astrocytes (Figure 3G) and interlaminar astrocytes (Figure 3H). GFAP / PAX6 double-positive radial glia were present at the outskirts of the densely populated centre of the well with processes growing radially outwards (Figure 31). Similar to free-floating organoids, adherent cortical organoids survived for longer periods compared to monolayer neural cultures grown on larger surfaces. The longevity allows for the development of cell types not usually seen in a monolayer culture that can typically be cultured up to a maximum of 2 to 3 months. By 6 weeks after seeding NPCs we observed the emergence of oligodendrocyte precursor cells (OPC), as shown by the expression of NG2 (Figure 4A). NG2+ cells remained present until at least 4 months (Figure 4B / C). Staining for Myelin Basic Protein (MBP) revealed the emergence of MBP+ oligodendrocytes around 4 months after NPC seeding when the organoids were continuously grown in the presence of T3 (2ng / ml) (Figure 4D). At 5 months, the oligodendrocytes showed increasingly mature morphologies (Figure 4E-I) and exhibited MBP co-localization along NF200+ axons (Figure 4F / I).

[0201] Adherent cortical orgaiwids slww synaptic connectivity and functional activity

[0202] Neurons within adherent cortical organoids exhibited clear evidence of synaptogenesis (Figure 5A-D). Sparse labelling of excitatory neurons with AAV9.0amKII.eGFP revealed the presence of synapsin 1 / 2 positive (Syn+) mushroom-shaped dendritic spines (Figure 5E). To assess the functional activity of the cortical organoids, we used the genetically-encoded calcium indicator GCaMP6s under the control of the human Synapsin promoter (Figure 5F), allowing cell-type specific quantification of neuronal activity. Calcium imaging revealed robust synchronous network -level bursting (NB) (1.4 ± 0.07 NB / min) in which the vast majority of recorded neurons participated. In addition, substantial desynchronized activity was also observed (3.9 ± 0.5 events / min) during time periods outside of network-level bursting (Figure 5G-J).

[0203] Discussion

[0204] The study of early human brain development and related diseases has long been hampered by the inherent complexity of the human brain and the inaccessibility of living brain tissue at cellular resolution. Technological advances in induced pluripotent stem cell technology have now facilitated the opportunity to obtain living human neurons derived from specific individuals.

[0205] We describe here a platform to model early human frontal cortical development with high reproducibility and simplified organization. While 3D floating organoids, or sliced organoids, nicely recapitulate layered cortex formation, they are subject to variation in the relative contribution of cortical tissue within the organoid, forming multiple cortical patches along the edges and complicating structured analysis (Giandomenico et al., Nat Neurosci 22:669-679 (2019); Quadrato et al., Nature 545:48-53 (2017)). Moreover, 3D floating organoids suffer from necrosis in the core of the organoid due to lack of oxygen and nutrient diffusion. Recently, other protocols have been published starting from rosette formation with a focus on very early development and leading to single structure free floating cortical organoids (Tidball et al., Stem Cell Reports 18:2498-2514 (2023); Pagliaro et al., Nat Commun 14. doi:10.1038 / s41467-023-43141-l (2023)). Our platform predefines a rosette-forming iPSC-derived cortical NPC population that self-organizes into adherent singular radial structures in a standard 384-well format. Our platform yields individual layered cortical structures with robust functional synaptic connectivity and neuronal activity and including advanced glial cell types such as myelinating oligodendrocytes and subclasses of astrocytes. The small reproducible format of the organoids in a 384-well format has the distinct advantage of being able to image entire organoids without slicing or clearing and to perform spatiotemporal functional analysis by fluorescence-based calcium imaging. We confirmed the self-organizing potential of adherent cortical organoids across multiple hiPSC lines and using different sources of NPCs, controlling the seeding density for the proliferation rate of the specific NPC batch. Seeding NPCs with frontal cortical identity in the defined geometry of a 384-well plate enabled the development of long-term functional neuronal networks in a complex radial structure resembling early human cortical development. Future studies aimed at single cell gene expression analysis and advanced image-based analysis solutions in this platform will be interesting in the context of the increasing knowledge on single cell topographical, typological and temporal hierarchies in the developing human cortex.

[0206] These functional adherent cortical organoids in a multiwell format are amenable to high-throughput screening applications, mechanistic pathophysiological studies of neuro develop mental and neuropsychiatric disorders, and pharmacological and phenotypic screening of disease phenotypes during early cortical development. Moreover, toxicological studies for novel therapeutic compounds also show an increasing need for testing specific effects in human neuronal models, whereas studies using rodent models have often shown poor predictive power for drug safety and efficacy in human central nervous system disorders.

[0207] Taken together, we present a novel platform for cellular-level human brain modeling using adherent cortical organoids that exhibit high reproducibility and robust neuronal activity. The ability to reliably generate human cortical organoids in multiwell plates combined with neural network functionality offers a unique potential for brain disease modeling and therapeutic screening applications.

[0208] Example 2. Integration of additional cell types to the adherent cortical organoids.

[0209] This Example is an add-on to Example 1 and shows that further cell types such as microglia can be added to the developing adherent cortical organoids and successfully integrate therein.

[0210] Materials and Methods hiPSC-derived microglia were generated from WTC-11 iPSCs based on the HPC differentiation protocol starting from hiPSCs, spanning 40 days (Abud et al., Neuron 2017, PMID28426964), using Stem cell Technologies HPC and microglia differentiation kits (cat# 05310 and 100-0019, including Supplement 2 supplementation for mono- and 2D co-cultures). The resulting microglia were cultured as monoculture, or supplemented to NGN2 neuronastrocyte co-cultures (according to Lendemeijer et al eNeuro 2024, PMID: 39227152) or adherent cortical organoids (according to Example 1). 1000 WTC-11 iPSC-derived microglial cells were added to each developing organoid via controlled introduction. The microglia were added once to each developing organoid, and were added at 2 -week intervals during the first 8 weeks after NPC seeding to study the effect of differences in introduction time. After five days of NGN2 differentiation, about 10.000-20.000 microglial cells were added to said NGN2 cultures per well of a 12-well plate. Subsequently, NGN2 cultures were analyzed after 21 days.

[0211] Microglia were labeled by immunocytochemistry using an antibody against microglia-specific marker IBA1 (Abeam AB 178846). Imaging IBA1+ positive cells and live tracking of microglia from an eGFP-transduced hiPSC line was performed using an LSM800 confocal microscope (Zeiss). Skeleton analysis was performed using Image J software.

[0212] Results

[0213] Adherent cortical organoids demonstrate compatibility with multimodal cellular integration, enabling systematic co-culture with for example hiPSC- derived microglia and vasculogenic lineages such as endothelial cells or pericytes. Experimental validation of microglial integration kinetics revealed temporal dependency, with controlled introduction of isogenic microglia at 2-week intervals (0-8 weeks post-NPC seeding) showing maximal structural preservation and microglial engraftment efficiency when adding the microglia after 6-8 weeks of organoid differentiation. Quantitative morphological analysis comparing microglia in 2D NGN2- induced neuron-astrocyte co-cultures versus adherent organoids demonstrated enhanced ramification complexity and reduced circularity in organoid-embedded microglia (Figure 12).

[0214] The multi-well format of the adherent cortical organoids permits following the integration of microglia with live imaging over time in the same organoid and studying different conditions at a high throughput scale reproducibly. Longitudinal tracking revealed sustained microglial viability exceeding 9 months in organoid cultures without exogenous cytokine supplementation, contrasting with 2D monoculture or co-culture systems requiring biweekly growth factor reinforcement. Interestingly, in adherent cortical organoids microglia seem to reside preferentially in denser regions in the centre with more ramified morphology and reduced motility over time.

Claims

Claims1. A method for the production of an adherent cortical organoid, comprising the steps of:(a) providing a cell culture comprising a population of neural progenitor cells (NPCs);(b) seeding neural progenitor cells (NPCs) of said population in a well of a multiwell plate; wherein said well comprises a bottom surface (106) and an upright wall (104), and wherein a distance (x) between a centre (112) of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm; and(c) culturing said seeded NPCs in a culture medium for neural differentiation for a period of time, to thereby allow formation of an adherent cortical organoid.

2. The method according to claim 1, wherein the bottom surface (106) of the well is a flat bottom surface; and wherein a distance (x) between a centre (112) of the bottom surface and the upright wall is between 1.5 mm and 1.8 mm.

3. The method according to claim 1 or claim 2, wherein said well of said multiwell plate is a well of a flat-bottom 384-well plate.4 The method according to any one of the preceding claims, wherein said well of said multiwell plate is coated with a coating for adherent cell culture, preferably wherein said coating for adherent cell culture is a laminin and / or poly-L-ornithine coating.

5. The method according to any one of the preceding claims, wherein, in step (b), NPCs of said population are seeded in, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or in 100%, of the wells, calculated on the basis of the total number of wells, of said multiwell plate.

6. The method according to any one of the preceding claims, wherein said NPCs are positive for one or more of the markers selected from the group formed by SOX2, Nestin, Vimentin and FOXG1.

7. The method according to any one of the preceding claims, wherein step (a) is preceded by a step of:- subjecting neural progenitor cells (NPCs) to a step of antibody-based cell sorting; wherein said step of antibody-based cell sorting is performed on the basis of (i) the presence of cell surface marker CD 184 (CD 184+) and / or CD24 (CD24+) and (ii) the absence of cell surface marker CD44 (CD44-) and / or CD271 (CD271-), to thereby provide a population of sorted NPCs.

8. The method according to any one of the preceding claims, wherein said culture medium for neural differentiation comprises a brain-derived neurotrophic factor, a glial cell-derived neurotrophic factor, dibutyryl cyclic adenosine monophosphate and / or ascorbic acid.

9. The method according to any one of the preceding claims, wherein said period of time for culturing said seeded NPCs in a culture medium for neural differentiation is at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months or at least 10 months.

10. The method according to any one of the preceding claims, wherein, in at least 50%, in at least 60%, in at least 70%, in at least 75%, inat least 80%, in at least 90%, or in at least 95%, of the seeded wells of said multiwell plate, a single adherent cortical organoid with a radial structure is present.

11. The method according to any one of the preceding claims, wherein culturing of said seeded NPCs occurs in the presence of a further cell type, for example a microglial cell and / or a vascular cell.

12. The method according to any one of the preceding claims, wherein said adherent cortical organoid comprises neurons, astrocytes and (myelinating) oligodendrocytes, and optionally microglia and / or vascular cells.

13. The method according to any one of the preceding claims, wherein culturing of said seeded NPCs in said culture medium for neural differentiation in a well of said multiwell plate occurs in the presence of a test agent, and optionally, wherein, in a different well of said multiwell plate, culturing of said seeded NPCs in said culture medium for neural differentiation occurs in the absence of said test agent.

14. A multiwell plate comprising a well; wherein said well comprises a bottom surface (106) and an upright wall (104), and wherein a distance (x) between a centre (112) of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm; wherein said well contains a single adherent cortical organoid with a radial structure; and wherein said adherent cortical organoid comprises neurons, astrocytes and (myelinating) oligodendrocytes, and optionally microglia and / or vascular cells.

15. The multiwell plate according to claim 14, wherein the bottom surface (106) of the well is a flat bottom surface; and wherein a distance (x) between a centre (112) of the bottom surface and the upright wall is between 1.5 mm and 1.8 mm.

16. The multiwell plate according to claim 14 or claim 15, wherein in at least 50%, in at least 60%, in at least 70%, in at least 75%, in at least 80%, in at least 90%, or in at least 95%, of the seeded wells of said multiwell plate, a single adherent cortical organoid with a radial structure is present.

17. The multiwell plate according to any one claims 14-16, wherein said cortical organoid is obtainable by a method according to any one of claims 1-13.

18. A method of screening for an agent that modulates an adherent cortical organoid or its formation, comprising the steps of:(a) providing a cell culture comprising a population of neural progenitor cells (NPCs);(b) seeding neural progenitor cells (NPCs) of said population in a well of a multiwell plate; wherein said well comprises a bottom surface (106) and an upright wall (104), and wherein a distance (x) between a centre (112) of the bottom surface and the upright wall is between 1.1 mm and 1.9 mm;(c) culturing said seeded NPCs in a culture medium for neural differentiation for a period of time; wherein said culturing occurs in the presence of a test agent;(d) optionally, wherein, in a different well of said multiwell plate, culturing of seeded neural progenitor cells (NPCs) in said culture medium for neural differentiation occurs in the absence of said test agent; and(e) identifying said test agent as an agent that modulates an adherent cortical organoid or its formation, if the cortical organoid that is produced inthe presence of said test agent is different from said cortical organoid that is produced in the absence of said agent.

19. Use of a multiwell plate according to any one of claims 14-17 in screening a test agent for modulating an adherent cortical organoid.

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