EP4677072A1 - Culture method for neural organoids - Google Patents

Abstract

This invention relates to a methods of culturing neural cells and to neural organoids for use in medicine or diagnostics

Description

Culture Method [001] This invention relates to a methods of culturing neural cells and to neural organoids for use in medicine or diagnostics BACKGROUND [002] Traditionally, the study of human brain physiology and disease has been based on animal models and post-mortem tissue specimens mainly due to ethical concerns regarding the collection of human brain tissue. Animal brains (including the ones of widely used rodent models) overtly differ from human brain size and architecture, which imposes considerable challenges for studying the development and disease of the human central nervous system. The existence of these subtle structural and cellular compositional differences across species is partially responsible for failure of preclinical findings acquired from those models to be translated into clinical practice successfully. [003] However, stem cell technology based on the availability of human induced pluripotent stem cells (hiPSCs) as well as the generation of pre-patterned 3D-like forebrain organoids in free floating culture, have opened new avenues towards personalised modelling of neurological diseases. Brain organoids are able to simulate the architecture and functionality of the human brain, and in turn are now widely accepted as suitable models for investigating brain development and the mechanisms of diseases. For example, three-dimensional (3D) brain organoid (‘mini brain’) systems generated from human pluripotent stem cells (hPSCs) have demonstrated considerable potential in recapitulating key features of brain cancer and neurological disease (i.e., Alzheimer’s) pathophysiology. [004] Whilst current brain organoid models and methods for their production are promising for biological and medical research, they are not without their limitations. The current difficulty and lack of standardisation in their production protocols leads to heterogeneity in brain organoid production and as a result requires organoid methodology users having to carry out quality control, growth logistics and ‘cherry-picking’ of organoids for use in downstream applications. These can lead to heterogeneity between experiments, and in turn may affect results. Further limitations include variability in the time required for organoid maturation, issues with transporting organoids from the site of organoid generation and end-user (transportation of biomaterials often involves freeze/thaw cycles which for brain organoids would lead to cell viability issues that would be unacceptable for clinical use), and amenability to high throughput approaches. Additionally, organoid production routinely requires the use of a natural solubilised basement membrane matrices (such as Matrigel® droplets) to aid 3D organoid maturation. Owing to its animal original, there may be variability from batch to batch lack which can result in lack of end-user control over its exact composition, such as unknown and uncontrollable amount of growth factors. [005] The present invention aims to address at least some of the limitations associated with the prior art. BRIEF SUMMARY OF THE DISCLOSURE In accordance with the present inventions there is provided an in vitro method of producing a neural organoid comprising: i) plating induced pluripotent stem cells (iPSCs) on a cell culture substrate in the presence of a culture medium that maintains pluripotency; ii) culturing the plated iPSCs of i) in the presence of a neural induction medium for an amount of time sufficient to form neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) disassociating the neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs) of ii) to obtain a cell suspension comprising single cells; iv) cryopreserving the cell suspension; v) aggregating the cryopreserved cell suspension of iv) in the presence of an aggregate formation medium for an amount of time sufficient to form a neural organoid. In some embodiments the cell culture substrate comprises extracellular matrix components. In another embodiment the cell culture substrate does not comprise an extracellular matrix component. In some embodiments the cell-culture substrate feeder cell-free. In some embodiments the culture medium that maintains pluripotency is a feeder free media. In some embodiments the culture medium that maintains pluripotency is a feeder free media is selected from Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit medium. In some embodiments the culture medium that maintains pluripotency is supplemented with Rho-associated coiled-coil kinase (ROCK) inhibitor. In some embodiments the neural induction medium is a Dulbecco's Modified Eagle Medium (DMEM). Preferably the DMEM is a DMEM medium with nutrient mixture F-12 (DMEM/F12). In some embodiments the neural induction medium is supplemented with N2. In some embodiments the neural induction medium is supplemented with a TACC3 inhibitor. Preferably the TACC3 inhibitor is KHS101. In some embodiments the neural induction medium is supplemented with a c-MYC inhibitor. Preferably the c-MYC inhibitor Stauprimide In some embodiments the neural induction medium is supplemented with heparin or heparin sulfate. In some embodiments the neural induction medium is serum free. In some embodiments the neural induction medium is supplemented with glutamine or GlutaMAX™. In some embodiments the neural induction medium is supplemented with non-essential amino acids or MEM non-essential amino acids. In some embodiments the neural induction medium comprises DMEM/F12 medium with N2 supplement, heparin, GlutaMAX™, MEM non-essential amino acids, a TACC3 inhibitor and a c-MYC inhibitor, optionally wherein the -MYC inhibitor Stauprimide and wherein the TACC3 inhibitor is KHS101. In some embodiments culturing the plated iPSCs in the presence of a neural induction medium is 2D culturing. In some embodiments wherein culturing the plated iPSCs in the presence of a neural induction medium is for a period of at least 2, 3, 4, or 5 days, preferably for a period of 2 - 10 days, 4 - 9 days or 3 – 5 days. In some embodiments disassociating comprises enzymatic disassociation and / or mechanical disassociation. In some embodiments dissociated cells are suspended in a cryopreservation media to form a cell suspension. In some embodiments the cell suspension is stored at a cryopreservation temperature. In some embodiments the cryopreserved cell suspension is thawed prior to aggregation. In some embodiments the aggregates are formed in an aggregate formation medium free of neural lineage inducing factors. In some embodiments the aggregate formation medium is a Dulbecco's Modified Eagle Medium (DMEM). Preferably the DMEM is a DMEM medium with nutrient mixture F-12 (DMEM/F12). In some embodiments the aggregate formation medium is supplemented with N2 In some embodiments the aggregate formation medium is supplemented with heparin or heparin sulfate. In some embodiments the aggregate formation medium is serum free. In some embodiments the aggregate formation medium is supplemented with glutamine or GlutaMAX™. In some embodiments the aggregate formation medium is supplemented with non- essential amino acids or MEM non-essential amino acids. In a further aspect the invention provides a neural organoid obtainable or obtained by the method described herein. In a further aspect the invention provides a neural organoid obtainable or obtained by the method described herein for use in medicine or diagnostics. In a further aspect the invention provides a neural organoid obtainable or obtained by the method described herein for use screening the potential effect of a substance on neural cells in vivo, optionally for use in screening for a therapeutic or toxic effect. BRIEF DESCRIPTION OF THE DRAWINGS [006] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 is an overview of the differentiation procedure used in the method of the invention; Figure 2 is an overview of the neural organoid organ aggregation process used in the method of the invention; Figure 3 - Neural organoids are characterised by low intra-batch heterogeneity and can be matured both with and without extracellular matrix (gel) support; A) left, Intra-batch heterogeneity of ‘roundness’ is markedly reduced across different batches (n=12) of neural organoids compared to batches (n=12) obtained by Standard cerebral organoid (embryoid body-based) culture. Right, Intra-batch heterogeneity (n=12) of ‘size’ is markedly reduced in neural organoids compared to Standard cerebral organoid (embryoid body-based) cultures. Dots/squares represent the range of diameters calculated per neural organoid and Standard cerebral organoid batch. The median with SD is shown. Parametric and non-parametric statistical tests indicate p<0.0001. B) left, schematical illustration of image-based quantification of the neuronal marker penetration score across sections of neural organoids and Standard cerebral organoids. Right, Inter-batch neuronal (TuJ1-positive) marker penetration detected within the tissue sections is markedly higher in neural organoids (n=8) compared with Standard cerebral organoid (embryoid-body based) batches (n=9). The median with SD is shown. Parametric and non-parametric statistical tests indicate p<0.0001. C) Volcano plot showing up- and downregulated genes at the single cell level in neural organoids compared to Standard cerebral organoids (embryoid body-based) across batches (n=2) matured for 36 days in Matrigel/spinner flask conditions. D) The 10 most up- and downregulated genes (depicted in C) are shown based on discrete log2 fold change (FC) values. E) Qualitative assessment of neural organoid section based on immunostaining for MAP2-postive neurons (arrows) and GFAP-positive astrocytes (arrowheads) upon long term maturation in spinner flask (180 days). Note that the homogenous distribution of neurons is consistent with the high TuJ1 expression penetration score (as shown in B) throughout neural organoid batches at an earlier maturation stage. F) Qualitative assessment of Standard cerebral organoid (embryoid body-based) section based on immunostaining for MAP2-positive neurons and GFAP-positive astrocytes upon long term maturation in spinner flask (180 days), scale bar, 200 ^m. G) Marker penetration score (as depicted B) for the shown markers within biologically-different neural organoids (n=3) and Standard cerebral organoids (embryoid body-based) (n=3) undergoing long term (180-day) maturation. The data indicate a marked maintenance of homogenous marker distribution in neural organoids. The median with SD is shown. Parametric and non-parametric statistical tests indicate p<0.001. H) Neural organoids maturation can be achieved without the use of gels (e.g., Matrigel) as indicated by sustained single cell marker (NES, TUBB3, MAP2, DCX, NEFL) expression (shown within the full UMAP-visualised single cell RNAseq data set) in the ‘on plate/non-gel’ maturation versus spinner flask/Matrigel culture conditions. I) Volcano plot showing up- and downregulated genes at the single cell level in neural organoids compared to Standard cerebral organoids (embryoid body-based) across batches (n=2) matured for 36 days ‘on plate’ without Matrigel. J) The 10 most up- and downregulated genes (depicted in I) are shown based on discrete log2 fold change (FC) values. Figure 4 Maintenance of morphological features of neural organoids following 1, 6, and 12 weeks of storage at -80 °C. (A) Quantification of neural organoid diameter (µm) Error bars represent standard deviation n=5-12. (B) Representative images of neural organoids 96 hrs post plating. DETAILED DESCRIPTION [007] The present invention provides a simplified method for providing neural organoids useful in drug screening and neural disease modelling. The methods of the present disclosure can produce sets of homogeneous neural organoids in which each neural organoid in the set has nearly identical features. Moreover, the method of the invention advantageously simplifies the production of neural organoids thereby providing faster and cheaper production. Accordingly, the method of the invention provides an improved, robust, and consistent method of providing neural organoids for use in neurological disease models and as drug discovery tools. [008] The invention provides an in vitro method of producing a neural organoid comprising: i) plating induced pluripotent stem cells (iPCSs) on a cell culture substrate in the presence of a culture medium that maintains pluripotency; ii) culturing the plated iPCSs of i) in the presence of a first neural induction medium for an amount of time sufficient to form neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) disassociating the neuronal stem cells and / or early neural progenitor cells of ii) to obtain a cell suspensions comprising single cells; iv) cryopreserving the cell suspension; v) aggregating the cryopreserved cell suspension of iv) in the presence of an aggregate formation medium for an amount of time sufficient to form a neural organoid. [009] Stage 1 of the culture method of the invention comprises plating induced pluripotent stem cells (iPCSs) on a cell culture substrate in the presence of a culture medium that maintains pluripotency. In this stage, iPSCs are seeded on a cell culture substrate. [0010] The term “induced pluripotent stem cell” (iPSC) means a type of pluripotent cell made by reprogramming a somatic cell to have the same properties as embryonic stem cells, namely, the ability to self-renew and differentiate into the three primary germ layers. Induced pluripotent stem cells may be derived from cell types such as fibroblasts taken from the skin, lung, or vein of subjects that are apparently healthy or diseased. [0011] Specific examples thereof include cells obtained by reprogramming differentiated somatic cells such as fibroblasts or peripheral blood mononuclear cells by expression of any combination of a plurality of genes selected from a reprogramming gene group including OCT3/4, SOX2, KLF4, MYC (c-MYC, N-MYC, L-MYC), GLIS1, NANOG, SALL4, LIN28, ESRRB to induce pluripotency. Preferred examples of the combination of reprogramming factors include (1) OCT3/4, SOX2, KLF4, and MYC (c-MYC or L-MYC), (2) OCT3/4, SOX2, KLF4, LIN28, and L-MYC (Stem Cells, 2013; 31: 458 to 466), and (3) OCT3/4, SOX2, NANOG, and LIN28 (Science 2007; 318: 1917 to 1920). [0012] In some embodiments iPSCs are obtained from a repository, such as the Coriell Iinstitute for Medical Research (e.g., catalog id gm25256 (wtc-11), gm25430, gm23392, gm23396, gm24666, gm27177, gm24683), California Institute for Regenerative Medicine: California's Stem Cell Agency (e.g., cw60261, cw60354, cw60359, cw60480, cw60335, cw60280, cw60594, cw60083, cw60086, cw60087 cw60167, cw60186), and the American Type Culture Collection (ATCC®) (e.g., atcc-dyr0530 human induced pluripotent stem (ips) cells (atcc® acs-1012™, atcc® acs-1011™, atcc® number: acs-1024™, atcc® number: acs-1028™, atcc® number: acs-1031™, atcc® number: acs-1004™, atcc® number: acs- 1029™, atcc® number: acs-1020™, atcc® number: acs-1007™, atcc® number: acs- 1030™). [0013] iPSCs used in the present invention are mammalian pluripotent stem cells, preferably the iPSCs are human cells. [0014] As used herein the term “substrate” as used herein refers to any substance that is a solid support that is free of or substantially free of cellular toxins. In some embodiments, the solid substrate comprises one or a combination of silica, plastic, and metal. [0015] In one embodiment the cell culture substrate comprises extracellular matrix components. Alternatively, or additionally the cell culture substrate comprises matrigel, gelatin, vitronectin, laminin, fibronectin, collagen, and / or hydrogels. In another embodiment the cell culture substrate does not comprise an extracellular matrix. [0016] The iPSCs may be plated onto the substrate in a suitable distribution and in the presence of medium that maintains pluripotency. An appropriate seeding distribution can readily be determined by one of skill in the art. For example, iPSCs can be seeded as single cells at a density of from 50,000 – 300,000 cells/cm2, 100,000 – 250,000 cells/cm2, 150,000 – 220,000 cells/cm2, 160,000 – 200,000 cells/cm2. [0017] The culture medium that maintains pluripotency used in the culture methods of the invention advantageously does not require the presence of feeder cells. The use of feeder cells is undesirable because it complicates passaging of the cells and can also lead to contamination of the desired cells with the feeder cells. [0018] In some embodiments the culture medium that maintains pluripotency is a feeder free media is selected from Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit medium. Preferably, the culture medium that maintains pluripotency is mTeSR plus medium. [0019] The culture medium that maintains pluripotency is preferably supplemented with a rho associated kinase ("ROCK") inhibitor. [0020] A ROCK inhibitor is a compound that decreases the activity of rho kinase. In some embodiments, the rho kinase inhibitor is n-[(3-hydroxyphenyl)methyl]-n′-[4-(4- pyridinyl)-2-thiazolyl]urea dihydrochloride (rki-1447), (+)-(r)-trans-4-(1-aminoethyl)-n-(4- pyridyl)cyclohexanecarboxamide dihydrochloride (y-27632) (rock inhibitor, catalog no. Y0503, sigma-aldrich, st. Louis, mo), fasudil (ha-1077), hydroxyfasudil (ha 1100 hydrochloride), thiazovivin, gsk429286a, narciclasine, and/or (+)-(r)-trans4-(1-aminoethyl)- n-(1h-pyrrolo[2,3-b]pyridin-4-yl)cyclohexanecarboxamide dihydrochloride (y-30141). [0021] Preferably, the ROCK inhibitor is Y-27632. [0022] In some embodiments, the ROCK inhibitor is added on day 1 of plating. It can be removed or left in for subsequent days if added on day 1. It can be removed for example by refreshing the media on day 2 after cell adhesion. [0023] In embodiments the culture medium that maintains pluripotency is a feeder free medium supplemented with a ROCK inhibitor, preferably supplemented with 10 μm of a ROCK inhibitor, more preferably 10 μm of Y-27632. In preferred embodiments the culture medium that maintains pluripotency is mTeSR plus supplemented with a ROCK inhibitor, preferably supplemented with10 μm of a rock inhibitor, more preferably 10 μm of Y-27632. [0024] In preferred embodiments, iPSCs are seeded as single cells on extracellular matrix coated plates in the presence of culture medium that maintains pluripotency comprising mTeSR plus supplemented with 10 μm of a rock inhibitor, preferably 10 μm of Y-27632. [0025] In embodiments the seeded iPSCs are expanded in culture for at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 passages. In embodiments the seeded iPSCs are expanded in culture for at least 6, 12, 18, 24, 30, 36, or 42 hours. [0026] As used herein “passaging”, “passaged” or “passage” refers to the subculturing of cells by the harvesting of individual cells from colonies and reseeding individual cells into new “daughter” cultures. The number associated with the term “passage” refers to the sequential number of times that a cell from a previous passage has been used to generate a new subculture. Any suitable method of passaging however can be used. For example, hiPSCs should be passaged in order to avoid overgrowth and to maintain them in an undifferentiated state. [0027] Stage 2 of the culture method of the invention comprises culturing the plated ipscs of stage 1 in the presence of a first neural induction medium for an amount of time sufficient to form neuronal stem cells (NSCs) and / or early neural progenitor cells (ENPCs), wherein the neural induction medium. [0028] As used herein, the term “neuronal stem cells” (NSCs) and “early neural progenitor cells” (ENPCs) refer to cells of a neural linage. In certain instances, said cells express SOX2, PAX6 and NES, DCX. In certain instances, said cells are tripotent and differentiable to neurons, astrocytes, or oligodendrocytes. [0029] In some embodiment the medium that maintains pluripotency is removed prior to contacting the plated cells with the neural induction medium. In such embodiments the plated cells may be washed, e.g. washed with pbs, prior to addition of neural induction medium. [0030] As used herein “neural induction media” and “neural induction medium” refers to a base media suitable for culturing neural precursor cells or cells derived therefrom. Base media suitable for culturing neural progenitor include BME medium, BGJB medium, CMRL 1066 medium, Glasgow MEM medium, improved MEM zinc option medium, IMDM medium, medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, HAM medium, HAM's f-12 such as a medium, RPMI 1640 medium, Fischer's medium, neurobasal medium, and a mixed medium thereof. Preferably, the base media is a Dulbecco's modified eagle medium DMEM media, preferably a DMEM medium with nutrient mixture f-12 (DMEM/f12). [0031] In certain embodiments the base media is supplemented with N2 supplement. Preferably, the base media is a DMEM-f12 medium with N2 supplement. The supplement may be present in a concentration of about 0.1 to 5%, about 0.5 to 2.0%, or about 1%. [0032] As used herein “N2” refers toto as “hormone mix” refers to a hormone mix comprising transferrin, insulin, putrescine, selenium and progesterone. For example, N2 can comprise 10mg/ml transferrin, 2.5 mg/ml insulin, 1 mg/ml putrescine, 1 ul/ml 15 selenium, 1 ul/ml progesterone. N2 can be purchased commercially from gibco (invitrogen/thermoscientific), sigma and others or can be prepared. [0033] In certain embodiments the neural induction media further comprises non- essential amino acids or mem non-essential amino acids. Non-essential amino acids include glycine, l-alanine, l-asparagine, l-aspartic acid, l-glutamic acid, l-proline and l- serine. Non-essential amino acids or mem non-essential amino acids may be present in a concentration of about of about 0.1 to 5%, about 0.5 to 2.0%, or about 1%. [0034] In certain embodiments the neural induction media further comprises a glutamine product such as glutamine or GlutaMAX™ (1 :100, thermo fisher scientific, 35050079). The glutamine may be present in a concentration of about of about 0.1 to 5%, about 0.5 to 2.0%, or about 1%. [0035] In certain embodiments the neural induction media further comprises heparin or heparin sulfate. The heparin or heparin sulfate may be present in a concentration of about 0.1 to 5 µg/ML, about 0.5 to 2.0 µg/ML, or about 1 µg/ML. [0036] In certain embodiments the neural induction media further comprises a c-MYC inhibitor. Preferably, the c-MYC inhibitor is stauprimide. Suitable concentrations of stauprimide for use in the methods include 0.1 nm to about 10 μm, alternatively from about 0.1 nm to about 1.5 μm or 0.1 nm to about 1 μm. [0037] In certain embodiments the neural induction media further comprises a TACC3 inhibitor. Preferably, the TACC3 inhibitor is KHS101, a selective inducer of neuronal differentiation. Suitable concentrations of stauprimide for use in the methods include 0.1 nm to about 10 μm, alternatively from about 0.1 nm to about 3 μm or 0.1 nm to about 2.5 μm. [0038] In some embodiments the neural induction media comprises a base media supplemented with a c-myc inhibitor and a TACC3 inhibitor. [0039] In some embodiments the neural induction media comprises DMEM-f12 medium with N2 supplement supplemented with a c-myc inhibitor and a tacc3 inhibitor. [0040] In some embodiments the neural induction media comprises DMEM-f12 medium with N2 supplement supplemented with a stauprimide and KHS101. [0041] in some embodiments the neural induction media comprises DMEM-f12 medium with N2 supplemented with: - Mem non-essential amino acids; - Glutamine or GlutaMAX™.; - Heparin or heparin sulfate; - Stauprimide; and - KHS101. [0042] In some embodiments the neural induction media comprises DMEM-f12 medium with N2 supplemented with: - about 1%.mem non-essential amino acids; - about 1% GlutaMAX™; - about 1 µg/ml heparin; - about 0.1 nm to about 1 μm stauprimide; and - about 0.1 nm to about 2 μm KHS101. [0043] In certain embodiments the neural induction medium are supplied to the plated iPSCs initially and additional medium are supplied, continuously or in discrete increments, to the culture during culturing, before termination of culture. [0044] In certain embodiments the time sufficient to form neuronal stem cells and / or early neural progenitor cells requires that the cells be cultured for about 1-10 days, about 2-8 days, about 3-5 days. In some embodiments the time sufficient to form neuronal stem cells and / or early neural progenitor cells requires that the cells be cultured for two, three, four, five, six or seven days. [0045] Preferably, the cells are cultured for an amount of time sufficient that neuronal stem cells and / or early neural progenitor cells may comprise at least 50%, at least 75%, at least 85%, at least 95%, at least 99% or about 100% of the cells in said culture. [0046] In some embodiment, the culture is a two-dimensional culture. As used herein, the term “two-dimensional culture” refers to cultures of cells on flat cell culture substrates disposed in culture vessels. [0047] Stage 3 of the culture method of the invention comprises disassociating the neuronal stem cells and / or early neural progenitor cells of ii) to obtain a cell suspension comprising essentially single cells. [0048] Suitable disassociation methods are known in the art. The disassociating step may comprise enzymatic disassociation, mechanical disassociation, or a combination of both. [0049] For example, cells can be dislodged from the culture substrate using enzymes, for example using an enzyme cell detachment solution such as the enzyme cell detachment solution such as Accutase™, Dispase, ReLeSR or TrypLE. Non-enzymatic solutions, like EDTA solution, can also be used. [0050] Following disassociation, the dissociated cells are suspended in a cryopreservation medium. [0051] A cryopreservation medium refers to medium comprising a cryoprotectant. A cryoprotectant is a substance that has a high affinity with water molecules and inhibits the growth of ice crystals in the cryopreservation medium. Cryoprotectants include, for example, dimethyl sulfoxide (dmso), ethylene glycol (eg), propylene glycol (pg), 1,2- propanediol (1,2-pd), 1,3-propanediol (1,3-pd), butylene glycol (bg), isoprene glycol (ipg), dipropylene glycol (dpg), and glycerin. [0052] Cryopreservation media include media 310% (cs10), media 25% (cs5) and media 12% (cs2), stem cell banker, prime xv® freezis, hypothermasol®, csb, trehalose, etc. [0053] In some embodiments, the dissociated cells are stored in a neutralization medium followed by suspension in cryopreservation medium. [0054] In some embodiments the cells are stored in a cryopreservation medium for at least 1, 2, 3, 6, 12, 18, 24, or 48 hours prior to cryopreservation. [0055] Stage 4 of the culture method of the invention comprises cryopreserving the cell suspension. In some embodiments cryopreserving requires that the temperature of the cell suspension is maintained at −80° C or less. For example, it may be maintained at about - 90° C, about -100° C, about -110° C, about -120° C, about -130° C, about -140° C, about -150° C, about −160° C., about −170° C., about −180° C., or about −190° C. [0056] Stage 5 of the culture method of the invention comprises aggregating the cryopreserved cell suspension of stage 4 in the presence of an aggregate formation medium for an amount of time sufficient to form a neural organoid. [0057] Cell suspensions are placed in a culture vessel in the presence of aggregate formation medium. [0058] The cryopreserved cell suspension is thawed. In some embodiments the thawing is performed at 30° C to 40° C., preferably at 35° C to 38° C., and more preferably at about 37° C. [0059] In embodiments the culture vessel comprises a surface has topological features of various sizes, shapes, and depths, e.g., cavities, or microwells. In embodiments, a surface of the culture vessel comprises one or more cavities or microwells. [0060] Preferably the culture vessel has a low-adhesion or non-adhesive surfaces. Such a low-adhesion surface prevents binding of cells to the vessel surface, thereby promoting aggregation. [0061] In some embodiments aggregating involves forced spatial confinement of the cells, for example centrifugation of cells. [0062] In one embodiment, the aggregate formation medium comprises a base media supplemented with a rho associated kinase ("ROCK") inhibitor. Critically, the aggregate formation medium does not comprise neural lineage inducing factors. As used herein neural lineage inducing factors refers to compounds that induce differentiation into neuronal lineages. Compounds that induce differentiation into neuronal lineages include TACC3 inhibitors, MYC inhibitor. Suitably, the aggregate formation medium does not include KHS101. [0063] Preferably the base media is supplemented with 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more of a rock inhibitor, preferably Y-27632. [0064] Base media suitable for aggregate formation medium include BME medium, BGJB medium, CMRL 1066 medium, Glasgow MEM medium, improved MEM zinc option medium, IMDM medium, medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, HAM medium, HAM's f-12 such as a medium, RPMI 1640 medium, Fischer's medium, neurobasal medium, and a mixed medium thereof. Preferably, the base media is a Dulbecco's modified eagle medium DMEM media, preferably a DMEM medium with nutrient mixture f-12 (DMEM/f12). [0065] In certain embodiments the base media is supplemented with N2 supplement. Preferably, the base media is a dmem-f12 medium with N2 supplement. The supplement may be present in a concentration of about 0.1 to 5%, about 0.5 to 2.0%, or about 1%. [0066] In certain embodiments the aggregate formation medium further comprises non- essential amino acids or mem non-essential amino acids. Non-essential amino acids include glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L- serine. Non-essential amino acids or mem non-essential amino acids may be present in a concentration of about of about 0.1 to 5%, about 0.5 to 2.0%, or about 1%. [0067] In certain embodiments the aggregate formation medium further comprises a glutamine product such as glutamine or GlutaMAX™ (1 :100, thermo fisher scientific, 35050079). The glutamine may be present in a concentration of about of about 0.1 to 5%, about 0.5 to 2.0%, or about 1%. [0068] In certain embodiments the aggregate formation medium further comprises heparin or heparin sulfate. The heparin or heparin sulfate may be present in a concentration of about of about 0.1 to 5 µg/ml, about 0.5 to 2.0 µg/ml, or about 1 µg/ml. [0069] In some embodiments the aggregate formation medium comprises DMEM-f12 medium with N2 supplemented with: - Mem non-essential amino acids; - Glutamine or GlutaMAX; - Heparin or heparin sulfate; and - Y-27632. [0070] In some embodiments the aggregate formation medium comprises DMEM-f12 medium with N2 supplemented with: - About 1%.mem non-essential amino acids; - About 1% GlutaMAX.; - About 1 µg/ml heparin; and - About 50 μm y-27632. [0071] In embodiments, the time sufficient to form a neural organoid requires culture of cells for at least 12 hours; culturing the plurality of cells with the one or more microparticles occurs for up to 8 hours, up to 16 hours, up to 24 hours, up to 2 days, up to 3 days, up to 4 days, or up to 5 days. [0072] As used herein a “neural organoid” is an aggregate of neuronal stem cells and / or early neural progenitor cells having a three-dimensional structure. The neural organoid is a three-dimensional cell population is formed by cells adhering to each other through a suspension culture or a 3d culture. The shape of the neural organoid is not particularly limited and may be spherical or non-spherical. The neural organoid has no particular restrictions on its size but usually has an equivalent spherical diameter of 150 µm to 1000 µm, and for example, 200 µm to 800 µm or 300 µm to 500 µm in one embodiment. The neural organoid usually includes 500 to 150000 cells, and in one embodiment, for example, 1000 to 100000 cells, 1000 to 70000 cells, or 3000 to 30000 cells. [0073] The neural organoid may comprise other cells together with the neural cells. Preferably the neural organoids are substantially homogeneous. In certain embodiments the neural organoid comprises at least 60% or more, 70% or more, 80% or more, and more preferably 90% or more or 95% or more of neural cells. [0074] The neural organoids produced by the methods of the invention of the present disclosure are useful as neurological disease models and as drug discovery tools. The neural organoids may be regarded as miniaturized models of neural organs, including the brain. [0075] Screening assays generally involve contacting neural organoids with a candidate agent and determining a phenotypic effect on the neural organoids. [0076] The neural organoids can be used as disease models for investigating various diseases related to neural tissues including stroke, brain inflammation disorders, neurodegenerative diseases ( e.g.., Parkinson’s disease and Alzheimer’s disease), neuroinflammatory diseases ( e.g.., multiple sclerosis), traumatic injury (e.g., brain- surgery-induced injury), channelopathy (e.g., epilepsy), and psychiatric diseases (including autism and schizophrenia). [0077] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. [0078] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. [0079] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. [0080] EXAMPLES [0081] Neural organoid has also been referred to herein as Neuroyd or Neuroids, in particular in the figures. Brain organoid has also been referred to herein as Brainoyd or Brainoids, in particular in the figures. [0082] Methods for brain organoid generation are well known in the art, they can include the 3D differentiation of human pluripotent stem cells (hPSCs) into embryoid bodies (EB), which are embedded into an extracellular matrix and cultivated in a rotating bioreactor. The present invention of neural organoid provides an in vitro approach that is based on a chemically defined monolayer (2D) differentiation into pre-neural organoid cells and a post- freezing re-assembly paradigm as outlined in the examples. [0083] Example 1 – 2D differentiation of stem cells to form a pre-neural organoid cell suspension [0084] iPSCs are plated on coated (extracellular matrix-carrying) plates at a density of 200,000cells/cm² in PSC culturing medium supplemented with 10 μM ROCK inhibitor (Y0503-5MG, reconstituted in H₂O). Following adhesion overnight medium is changed to a standard neural induction formulation medium. To enable an induced/accelerated post mitotic stem cell differentiation state, the standard neural induction formulation medium is supplemented with the small molecule inhibitors Stauprimide e.g., supplied by Sigma, S2951; concentration range: 0.1 nM to 5 μM) and KHS101 e.g., supplied by Sigma, K4019; concentration range: 0.1 nM to 5 μM). The compound-containing medium is replenished daily for 4 consecutive days. On day 5 adherent cells are dissociated into a single cell suspension following a 4-minute incubation with 0.5 mM EDTA at 37°C, and a subsequent 4-minute incubation with an enzymatic mix (e.g., Accutase®) at 37°C. Single cell suspensions are cryopreserved in GMP grade freezing medium (e.g., Stem Cell Banker, AMS-Biotechnology, 11890) and stored at -80°C or in LN₂. [0085] The protocol is outline in figure 1. [0086] Example 2 – formation of the 3D neural organoid [0087] For the formation of one neural organoid from the cryopreserved pre-neural organoid cell suspension, 40,000 neural differentiated cells are plated per microtiter well plate in 100 μl using standard neural induction formulation at 0.25ml/cm² supplemented with 50 μM ROCK inhibitor (Y0503-5MG, reconstituted in H2O). The plate is spun at 400g for 5 minutes at room temperature and neural organoids are left to form homogenous structures for at least 16 hours (critical step). Half of the plating medium is removed, 50 μl, and replaced with 100 μl fresh standard neural induction formation (minus ROCK inhibitor, stauprimide and KHS101). Following at least 16 hours of culture period, neural organoids are ready to be used in desired assays (Figure 2). [0088] The composition of the various media used in the Examples is shown in Table 1. Table 1 Medium Composition PSC culturing medium 0.25ml/cm², mTeSR Plus, StemCell Technologies,100-0276 Human embryonic stem cell medium (hESC DMEM/F12 (Thermo, 31330-038), MEM-NEAA medium) 0.5% (Thermo, 11140-035), GlutaMAX™ 1% (Thermo,35050-038), KSOR 20% (Thermo, 10828028, FBS 3% (THermo, 10270106), β- Mercaptoethanol 100µM (Thermo, 21985023). Standard neural induction formulation DMEM/F12 (Thermo, 31330-038), N2 supplement 1% (Thermo, 17502048), GlutaMAX™ 1% (Thermo,35050-038), MEM- NEAA 1% (Thermo, 11140-035), and Heparin 1μg/ml (Sigma, H3149) at 0.25ml/cm². Organoid maturation medium (No vitamin A) DMEM/F12 (Thermo, 31330-038), Neural Basal Medium (Thermo, 21103049), MEM-NEAA 0.5% (Thermo, 11140-035), GlutaMAX 1% (Thermo, 35050038), B-27 supplement minus vitamin A 0.5x (Thermo, 12587010), N2 supplement 0.5x (Thermo, 17502048), β- Mercaptoethanol 50µM (Thermo, 21985023), Insulin 2.5 µg/mL (Sigma, I9278), Penicillin- Streptomycin x1 (Sigma, P0781). Organoid maturation medium (with vitamin A) DMEM/F12 (Thermo, 31330-038), Neural Basal Medium (Thermo, 21103049), MEM-NEAA 0.5% (Thermo, 11140-035), GlutaMAX 1% (Thermo, 35050038), B-27 supplement with vitamin A 0.5x (Thermo, 17504044), N2 supplement 0.5x (Thermo, 17502048), β- Mercaptoethanol 50µM (Thermo, 21985023), Insulin 2.5 µg/mL (Sigma, I9278), Penicillin- Streptomycin x1 (Sigma, P0781). [0089] The advantages of the method of the present invention as compared to the known methods of the art are outlined in Table 2. Table 2 Stem Cell technologies protocol Neural organoid protocol Neural differentiation via embryoid body Monolayer-based neural differentiation bias formation No differentiation priming with small molecule Use of small molecule inhibitors accelerating inhibitors postmitotic state/stem cell differentiation Matrigel droplets required to aid 3D organoid Currently xeno-free at neural organoid maturation (not xeno-free) formation stage (in the ‘end user’ hands). Around 40 days of differentiation required from Ready to use 2 days post plating plating day before able to use, no guarantee of exact date they will be ready for assay Manual checking required regularly to assess Predictable workflow with consistent timeline for whether correct differentiation stage has been the next stages reached and when to move onto next stage Weekend feeding (medium change) required No weekend feeding required Variability in resulting organoids Homogeneous neural organoids – opening an avenue towards customisable options to contain relevant and desired ratios of physiological or disease phenotype-carrying brain cell mixes in ‘Brainoyds’ (using the neural organoid as a tissue scaffold). [0090] Example 3 – Characterisation of the 3D neural organoid Neural organoids and standard cerebral are formed as described in examples 1 and 2 (paragraphs 82 to 89) or using a commercially-available embryoid body-based organoids formation protocol (Stem Cell Technologies, table 2). For early time point morphological characterisation, the neural organoids are cultured for 96 hrs (with a media change at 48 hrs; Figure 3A). For longer term maturation, neural organoids are embedded into Matrigel droplets on parafilm dimples made in an empty P200 tip box. The embedded organoids are solidified at 37 °C for 30 mins before being transferred into a 10 cm dish containing Organoid Maturation media (- vit A). After 48 hrs, the majority of the media is removed and replaced with fresh Organoid Maturation media (- vit A). After a further 48 hrs, the organoids are transferred to spinner flasks containing Organoid Maturation Media (+ vit A) and are matured until required. [0091] For immunofluorescent characterisation, neural organoids or standard cerebral organoids (see Table 2) are transferred to a 24 well plate using a 1mm gauge pipette tip. The medium is removed, and the organoids washed with PBS. The PBS is then removed, and a 4% Paraformaldehyde solution added (Thermo, 15670799), and the organoids incubated for 15 minutes (for early-stage organoids, or overnight for mature 36 days+ organoids) at 4 °C. The organoids are then washed in PBS before being incubated with a 30% sucrose solution (Sigma, S9378) overnight or until they have sunk to the bottom of the well. The sucrose solution is then removed and replaced with a warmed 7.5% gelatin (Sigma, G2500) 10% sucrose solution and incubated at 37 °C for at least 30 minutes. The organoids are then transferred to cryomolds precoated with gelatin/sucrose solution. Blocks are cryosectioned into 20 µm slices using a cryostat. [0092] For immunofluorescent staining, slides are washed three times with PBS and incubated with blocking buffer (1% FBS 0.3% Triton X-100 (Sigma, X100) for 1 hr at room temperature. The slides are then incubated with primary antibody diluted in blocking buffer at room temperature for 1 hr or overnight at 4 °C. The slides are then washed three times with PBS and incubated with the secondary antibody diluted in blocking buffer for 1 hr at room temperature, protected from light. The slides are subsequently washed three times with PBS and incubated with a 2µg/mL DAPI solution (Sigma, D9542) diluted in PBS for 10 minutes at room temperature, protected from light. As a next step, the slides are washed twice with PBS before being ‘mounted’ onto poly-lysine coated slides (VWR, 631-9483) with aqueous mounting medium (2B Scientific, H-5501-60). Slides are imaged using an invert fluorescent microscope and quantification is carried out using a Cell Profiler pipeline. [0093] Primary Antibodies Target Protein Host Species Manufacturer Catalogue No. TUJ1 Mouse Biolegend 801202 MAP2 Chicken Abcam ab5392 GFAP Rabbit Agilent Z033429-2 [0094] Secondary Antibodies Target Host Host Species Conjugate Manufacturer Catalogue No. Mouse Goat Alexa fluor 488 Invitrogen 10696113 Rabbit Goat Alexa fluor 546 Invitrogen 10789154 Chicken Rabbit Alexa fluor 647 Agilent Z033429-2 [0095] For single cell sequencing analysis, Organoids are incubated with Cell Recovery Solution (Thermo, 354253) for 1 hr at 4°C and dissociated using with the Neurosphere Dissociation Kit (Miltenyi Biotec, 130-095-943). Dead cells and cellular debris are removed using the Dead Cell Removal kit (Miltenyi Biotec, 130-090-101). Cells are counted and frozen down in aliquots of 1-2 million in 500 µL GMP grade freezing medium (e.g., Stem Cell Banker). Cells are then processed for single cell sequencing according to the 10X 3' Gene Expression protocol. The result data can then be mapped against the Human GRCh38 reference genome using Cell Ranger (7.1.0). All subsequent analysis is performed using the online single cell sequencing analysis tool Bioturing. [0096] Example 4 – Quantification of Morphological Changes Following up to 12 Weeks Storage at -80 °C The culture media was aspirated from inducted cells and the wells washed with 1 mL PBS. The PBS was aspirated and 1 mL of 0.5mM EDTA added, and the cells incubated at 37 °C for 4 minutes. The EDTA was slowly removed with P1000 pipette and 1 mL Accutase (e.g., Sigma Aldrich A6964) added, and cells incubated at 37 °C for 4 minutes. Using a P1000 pipette, cells were gently resuspended with mTeSR media and transferred to a 15 mL centrifuge tube. Following centrifugation at 300 x g for 5 minutes, the supernatant was discarded, and cells resuspended in 1 mL of neural induction media plus 50 µM ROCK inhibitor for cell counting.10 uL of cells were mixed with 10 mL of a life-dead distinguishing dye (e.g., Trypan Blue) and cells counted using the countess automated cell counter. The cells are then frozen in aliquots of 2 million cells in 500 µL GMP grade freezing medium (e.g., Stem Cell Banker). The cells are frozen at a rate of 1 °C a minute until at –80 °C. The cells are kept at –80 °C for 1, 6 and 12 weeks. When reviving the cells, they are thawed in a water bath set to 37 °C for approximately 3 minutes and transferred to a 15 mL tube. At least 5 mL of medium is then added to the cells dropwise and the cell suspension is spun at 300 g for 5 minutes. The cell pellet is resuspended in 1 mL of Neural Induction medium containing 50 µM ROCK inhibitor.10 uL of cells were mixed with 10 mL of Trypan Blue and cells counted using the countess automated cell counter. The cells were then plated at 40,000 cells in 100 μL per well in an ultra-low adherence 96 well plate in neural induction medium containing 50 μM ROCK inhibitor. After 48 hrs, 50 μL was removed from each well and 100 μL of Neural induction medium added. After a further 48 hrs, the neural organoids are imaged on an invert microscope (Evos). Morphological parameters are measured using a combination of the machine learning image analysis tool ilastik and the image analysis tool Cell Profiler.

Claims

CLAIMS An in vitro method of producing a neural organoid comprising: i) plating induced pluripotent stem cells (iPSCs) on a cell culture substrate in the presence of a culture medium that maintains pluripotency; ii) culturing the plated iPSCs of i) in the presence of a neural induction medium for an amount of time sufficient to form neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) disassociating the neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs) of ii) to obtain a cell suspension comprising single cells; iv) cryopreserving the cell suspension; v) aggregating the cryopreserved cell suspension of iv) in the presence of an aggregate formation medium for an amount of time sufficient to form a neural organoid. The method of claim 1, wherein the cell culture substrate comprises extracellular matrix components. The method of claim 1 or claim 2, wherein the cell-culture substrate feeder cell- free. The method of any one of claims 1 to 3, wherein the culture medium that maintains pluripotency is a feeder free media. The method of claim 4, wherein the culture medium that maintains pluripotency is a feeder free media is selected from Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit medium. The method of any one of claims 1 to 5, wherein the culture medium that maintains pluripotency is supplemented with Rho-associated coiled-coil kinase (ROCK) inhibitor. The method of any one of clams 1 to 6, wherein the neural induction medium is a Dulbecco's Modified Eagle Medium (DMEM). The method of claim 7, wherein the DMEM is a DMEM medium with nutrient mixture F-12 (DMEM/F12). The method of any one of claims 1 to 8 wherein the neural induction medium is supplemented with N2. The method of any one of the preceding claims wherein the neural induction medium is supplemented with a TACC3 inhibitor. The method of claims 10, wherein the TACC3 inhibitor is KHS101. The method of any one of the preceding claims wherein the neural induction medium is supplemented with a c-MYC inhibitor. The method of claims 12, wherein the c-MYC inhibitor Stauprimide The method of any one of the preceding claims, wherein the neural induction medium is supplemented with heparin or heparin sulfate. The method of any one of the preceding claims, wherein the neural induction medium is serum free. The method of any one of the preceding claims, wherein the neural induction medium is supplemented with glutamine or GlutaMAX™. The method of any one of the preceding claims, wherein the neural induction medium is supplemented with non-essential amino acids or MEM non-essential amino acids. The method of any one of the preceding claims wherein the neural induction medium comprises DMEM/F12 medium with N2 supplement, heparin, GlutaMAX™, MEM non-essential amino acids, a TACC3 inhibitor and a c-MYC inhibitor, optionally wherein the -MYC inhibitor Stauprimide and wherein the TACC3 inhibitor is khs101. The method according to any one of the preceding claims wherein culturing the plated iPSCs in the presence of a neural induction medium is 2D culturing. The method according to any one of the preceding claims wherein culturing the plated iPSCs in the presence of a neural induction medium is for a period of at least 2, 3, 4, or 5 days, preferably for a period of 2 - 10 days, 4 - 9 days or 3 – 5 days. The method of any one of the preceding claims, wherein disassociating comprises enzymatic disassociation and / or mechanical disassociation. The method of any one of the preceding claims wherein dissociated cells are suspended in a cryopreservation media to form a cell suspension. The method of any one of the preceding claims wherein the cell suspension is stored at a cryopreservation temperature. The method of any one of the preceding claims wherein the cell suspension is stored at a cryopreservation temperature for at least 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 20 weeks, 30 weeks, 40 weeks or 50 weeks without greater than 5% loss of product diameter upon aggregation. The method of any one of the preceding claims wherein the cell suspension is stored at a cryopreservation temperature for at least 12 weeks without greater than 5% loss of product diameter upon aggregation. The method of any one of the preceding claims wherein the cryopreserved cell suspension is thawed prior to aggregation. The method of any one of the preceding claims, wherein the aggregates are formed in an aggregate formation medium free of neural lineage inducing factors. The method of any one of the preceding claims, wherein the aggregate formation medium is a Dulbecco's Modified Eagle Medium (DMEM). The method of claim 28, wherein the DMEM is a DMEM medium with nutrient mixture F-12 (DMEM/F12). The method of any one of the preceding claims wherein the aggregate formation medium is supplemented with N2 The method of any one of the preceding claims, wherein the aggregate formation medium is supplemented with heparin or heparin sulfate The method of any one of the preceding claims, wherein the aggregate formation medium is serum free. The method of any one of the preceding claims, wherein the aggregate formation medium is supplemented with glutamine or GlutaMAX™. The method of any one of the preceding claims, wherein the aggregate formation medium is supplemented with non-essential amino acids or MEM non-essential amino acids. A neural organoid obtainable or obtained by the method of any one of claims 1 – 34. The neural organoid of claim 335 for use in medicine or diagnostics. The neural organoid of claim 35 for use screening the potential effect of a substance on neural cells in vivo, optionally for use in screening for a therapeutic or toxic effect.