How to culture neural organoids

Abstract

The present invention relates to methods for culturing neural cells and neural organoids for pharmaceutical or diagnostic uses.

Description

[Technical Field]

[0001] The present invention relates to methods for culturing neural cells and neural organoids for pharmaceutical or diagnostic uses. [Background technology]

[0002] Traditionally, research into human brain physiology and disease has relied primarily on animal models and postmortem tissue specimens due to ethical concerns surrounding the collection of human brain tissue. Animal brains, including those in widely used rodent models, differ significantly in size and structure from the human brain, posing significant challenges for studying human central nervous system development and disease. Due in part to these ultrastructural and cytoarchitectural differences between species, preclinical findings from these models have not been successfully translated into clinical practice.

[0003] However, stem cell technology based on the utilization of human induced pluripotent stem cells (hiPSCs) and the generation of pre-patterned 3D-like forebrain organoids in free-floating culture has opened new avenues toward personalized modeling of neurological diseases. Cerebral organoids can simulate the structure and function of the human brain and are now widely accepted as suitable models for investigating mechanisms of brain development and disease. For example, three-dimensional (3D) cerebral organoid ("mini-brain") systems generated from human pluripotent stem cells (hPSCs) have demonstrated great potential in recapitulating key features of the pathophysiology of brain cancer and neurological diseases (i.e., Alzheimer's disease).

[0004] Current brain organoid models and their production methods are promising for biological and medical research, but they are not without limitations. Current challenges and a lack of standardization in manufacturing procedures result in heterogeneity in brain organoid production, requiring users of organoid methodologies to implement quality control, growth planning, and "cherry-picking" of organoids for use in downstream applications. These can lead to heterogeneity between experiments and ultimately affect results. Further limitations include the variability in the time required for organoid maturation, the challenges of transporting organoids from the organoid production site to the end user (transporting biomaterials often involves freeze / thaw cycles, which, in the case of brain organoids, would lead to cell viability issues unfavorable for clinical use), and suitability for high-throughput approaches. Furthermore, organoid production routinely requires the use of natural soluble basement membrane matrices (e.g., Matrigel® droplets) to promote 3D organoid maturation. Because it is animal derived, there is batch-to-batch variability, which can result in the end user not being able to control the exact composition, including unknown or uncontrollable amounts of growth factors.

[0005] The present invention aims to overcome at least some of the limitations associated with the prior art. Summary of the Invention

[0006] According to the present invention, i) seeding induced pluripotent stem cells (iPSCs) onto a cell culture substrate in the presence of a culture medium that maintains pluripotency; ii) culturing the seeded iPSCs of i) in the presence of neural induction medium for a time sufficient to form neural stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) isolating the neural stem cells (NSCs) and / or early neural progenitor cells (eNPCs) of ii) to obtain a cell suspension containing single cells; iv) cryopreserving the cell suspension; v) aggregating the cryopreserved cell suspension of iv) in the presence of an aggregate formation medium for a time sufficient to form neural organoids; The present invention provides an in vitro method for producing neural organoids, comprising:

[0007] In some embodiments, the cell culture substrate comprises an extracellular matrix component, hi other embodiments, the cell culture substrate does not comprise an extracellular matrix component.

[0008] In some embodiments, the cell culture substrate is feeder cell-free.

[0009] In some embodiments, the culture medium that maintains pluripotency is a feeder-free medium.

[0010] In some embodiments, the culture medium that maintains pluripotency is a feeder-free medium selected from Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, stabilized Essential 8 medium, and StemFit medium.

[0011] In some embodiments, the culture medium that maintains pluripotency is supplemented with a Rho-associated coiled-coil kinase (ROCK) inhibitor.

[0012] In some embodiments, the neural induction medium is Dulbecco's Modified Eagle's Medium (DMEM). Preferably, the DMEM is DMEM medium containing nutrient mixture F-12 (DMEM / F12).

[0013] In some embodiments, the neural induction medium is supplemented with N2.

[0014] In some embodiments, the neural induction medium is supplemented with a TACC3 inhibitor. Preferably, the TACC3 inhibitor is KHS101.

[0015] In some embodiments, the neural induction medium is supplemented with a c-MYC inhibitor. Preferably, the c-MYC inhibitor is stauprimide.

[0016] In some embodiments, the neural induction medium is supplemented with heparin or heparin sulfate.

[0017] In some embodiments, the neural induction medium is serum-free.

[0018] In some embodiments, the neural induction medium is supplemented with glutamine or GlutaMAX™.

[0019] In some embodiments, the neural induction medium is supplemented with essential amino acids or MEM non-essential amino acids.

[0020] In some embodiments, the neural induction medium comprises DMEM / F12 medium supplemented with N2 supplement, heparin, GlutaMAX™, MEM non-essential amino acids, a TACC3 inhibitor, and a c-MYC inhibitor, optionally, the c-MYC inhibitor is stauprimide and the TACC3 inhibitor is KHS101.

[0021] In some embodiments, the culture of iPSCs seeded in the presence of neural induction medium is a 2D culture.

[0022] In some embodiments, the culture of the plated iPSCs in the presence of 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.

[0023] In some embodiments, the separation comprises enzymatic and / or mechanical separation.

[0024] In some embodiments, the separated cells are suspended in a cryopreservation medium to form a cell suspension.

[0025] In some embodiments, the cell suspension is stored at cryopreservation temperatures.

[0026] In some embodiments, the cryopreserved cell suspension is thawed prior to aggregation.

[0027] In some embodiments, the aggregates are formed in an aggregate formation medium that does not contain neural lineage inducers.

[0028] In some embodiments, the aggregate-forming medium is Dulbecco's Modified Eagle's Medium (DMEM). Preferably, the DMEM is DMEM medium containing nutrient mixture F-12 (DMEM / F12).

[0029] In some embodiments, the aggregate formation medium is supplemented with N2.

[0030] In some embodiments, the aggregate formation medium is supplemented with heparin or heparin sulfate.

[0031] In some embodiments, the aggregate formation medium is serum-free.

[0032] In some embodiments, the aggregate formation medium is supplemented with glutamine or GlutaMAX™.

[0033] In some embodiments, the aggregate formation medium is supplemented with non-essential amino acids or MEM non-essential amino acids.

[0034] In a further aspect, the present invention provides neural organoids obtainable or obtained by the methods described herein.

[0035] In a further aspect, the present invention provides neural organoids obtainable or obtained by the methods described herein for use in medicine or diagnosis.

[0036] In a further aspect, the present invention provides neural organoids obtainable or obtained by the methods described herein for use in screening the potential effects of substances on neural cells in vivo, optionally for use in screening for therapeutic or toxic effects.

[0037] Embodiments of the present invention are further described below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 is a schematic diagram of the differentiation procedure used in the method of the present invention. [Figure 2] FIG. 1 is a schematic diagram of the neural organoid aggregation method used in the methods of the present invention. [Figure 3]A. Neural organoids are characterized by low intrabatch heterogeneity and can mature with or without extracellular matrix (gel) support. Left: The intrabatch heterogeneity of "roundness" across various neural organoid batches (n=12) is significantly reduced compared to batches obtained with standard cerebral organoid (embryoid body-based) culture (n=12). Right: The intrabatch heterogeneity of "size" (n=12) is significantly reduced across neural organoid batches compared to standard cerebral organoid (embryoid body-based) culture. Points / boxes represent the calculated diameter range for each batch of neural organoids and standard cerebral organoids. Median and SD are shown. Parametric and nonparametric statistical tests indicate p<0.0001. B. Neural organoids are characterized by low intrabatch heterogeneity and can mature with or without extracellular matrix (gel) support. (Left) Schematic of image-based quantification of neural marker penetration scores for neural organoid sections and sections of standard brain organoids. (Right) Batch-to-batch neuronal (TuJ1-positive) marker penetration detected within tissue sections was significantly higher in neural organoids (n=8) compared to standard brain organoid (embryoid body-based) batches (n=9). Median and SD are shown. Parametric and nonparametric statistical tests indicate p<0.0001. (C) Neural organoids are characterized by low intrabatch heterogeneity and can be matured with or without extracellular matrix (gel) support. (D) Volcano plots show up- and down-regulated genes at the single-cell level in neural organoids compared to standard brain organoids (embryoid body-based) across batches (n=2) matured for 36 days under Matrigel / spinner flask conditions. (D) Neural organoids are characterized by low intrabatch heterogeneity and can be matured with or without extracellular matrix (gel) support. The 10 most up- and down-regulated genes (labeled C) are shown based on discrete log2 fold change values ​​(FC).E. Neural organoids are characterized by low intrabatch heterogeneity and can be matured with or without extracellular matrix (gel) support. Qualitative evaluation of neural organoid sections based on immunostaining for MAP2-positive neurons (arrows) and GFAP-positive astrocytes (arrowheads) after long-term maturation (180 days) in spinner flasks is shown. Note that the uniform distribution of neurons is consistent with the high TuJ1 expression penetration score (shown in B) across the batch of early maturation stage neural organoids. F. Neural organoids are characterized by low intrabatch heterogeneity and can be matured with or without extracellular matrix (gel) support. Qualitative evaluation of standard cerebral organoid (embryoid body-based) sections based on immunostaining for MAP2-positive neurons and GFAP-positive astrocytes after long-term maturation (180 days) in spinner flasks is shown. Scale bar, 200 μm. G. Neural organoids are characterized by low intrabatch heterogeneity and can be matured with or without extracellular matrix (gel) support. Marker penetration scores (as shown in Figure B) for the indicated markers within biologically distinct neural organoids (n = 3) and standard cerebral organoids (embryoid body-based) (n = 3) during long-term (180-day) maturation are shown. Data demonstrate that uniform marker distribution is clearly maintained in neural organoids. Median and SD are shown. Parametric and nonparametric statistical tests indicate p<0.001. H. Neural organoids are characterized by low intrabatch heterogeneity and can be matured with or without extracellular matrix (gel) support. Neural organoid maturation can be achieved without the use of gel (e.g., Matrigel), as demonstrated by sustained single-cell marker (NES, TUBB3, MAP2, DCX, NEFL) expression in "on-plate / no-gel" maturation compared to spinner flask / Matrigel culture conditions (shown in single-cell RNA-seq datasets fully visualized by UMAP). I. Neural organoids are characterized by low intra-batch heterogeneity and can mature with or without extracellular matrix (gel) support.Volcano plots showing up- and down-regulated genes at the single-cell level in neural organoids compared to standard cerebral organoids (embryoid body-based) across batches (n=2) matured "on a plate" for 36 days without Matrigel. J Neural organoids are characterized by low intrabatch heterogeneity and can be matured with or without extracellular matrix (gel) support. The 10 most up- and down-regulated genes (labeled I) are shown based on discrete log2 fold-change values ​​(FC). [Figure 4] This figure shows that neural organoids maintained their morphological characteristics after storage at -80°C for 1, 6, and 12 weeks. (A) Quantification of neural organoid diameter (µm). Error bars represent standard deviation (n = 5–12). (B) Representative images of neural organoids 96 hours after seeding. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention provides a simplified method for providing neural organoids that are useful for drug screening and neurological disease modeling.The method of the present disclosure can produce a uniform group of neural organoids, each of which has almost identical characteristics.In addition, the method of the present invention is advantageous because it simplifies the production of neural organoids, thereby achieving faster and cheaper production.Therefore, the method of the present invention provides an improved, robust and consistent method for providing neural organoids for use in neurological disease modeling and as a drug discovery tool.

[0040] The present invention provides an in vitro method for producing neural organoids, comprising: i) seeding induced pluripotent stem cells (iPCs) onto a cell culture substrate in the presence of a culture medium that maintains pluripotency; ii) culturing the seeded iPCs of i) in the presence of an initial neural induction medium for a time sufficient to form neural stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) dissociating the neural stem cells and / or early neural progenitor cells of ii) to obtain a cell suspension comprising single cells; iv) cryopreserving the cell suspension; and v) aggregating the cryopreserved cell suspension of iv) in the presence of an aggregate formation medium for a time sufficient to form neural organoids.

[0041] Step 1 of the culture method of the present invention involves seeding induced pluripotent stem cells (iPSCs) onto a cell culture substrate in the presence of a culture medium that maintains pluripotency. In this step, iPSCs are seeded onto a cell culture substrate.

[0042] The term "induced pluripotent stem cells" (iPSCs) refers to a type of pluripotent cell that has been created by reprogramming somatic cells 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 can be derived from cell types such as fibroblasts harvested from the skin, lungs, or veins of apparently healthy or diseased subjects.

[0043] Specific examples include cells obtained by reprogramming differentiated somatic cells, such as fibroblasts or peripheral blood mononuclear cells, to induce pluripotency by expressing any combination of multiple genes selected from a group of reprogramming genes including OCT3 / 4, SOX2, KLF4, MYC (c-MYC, N-MYC, L-MYC), GLIS1, NANOG, SALL4, LIN28, and ESRRB. Preferred examples of combinations 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;31:458-466), and (3) OCT3 / 4, SOX2, NANOG, and LIN28 (Science 2007;318:1917-1920).

[0044] In some embodiments, iPSCs are obtained from the Coriell Institute for Medical Research (e.g., catalog IDs gm25256(wtc-11), gm25430, gm23392, gm23396, gm24666, gm27177, gm24683), the California Institute for Regenerative Medicine: California's Stem Cell Cell lines from the American Type Culture Collection (ATCC®) (e.g., ATCC-DYR0530 human induced pluripotent stem (IPS) cells (ATCC® ACS-1012™, ATCC® ACS-1011™, ... The DNA may be obtained from a repository such as 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™.

[0045] The iPSCs used in the present invention are mammalian pluripotent stem cells, and preferably the iPSCs are human cells.

[0046] As used herein, the term "substrate" refers to any material that is a solid support that is free or substantially free of cytotoxins. In some embodiments, the solid substrate comprises one or a combination of silica, plastic, and metal.

[0047] In one embodiment, the cell culture substrate comprises an extracellular matrix component. Alternatively, or additionally, the cell culture substrate comprises matrigel, gelatin, vitronectin, laminin, fibronectin, collagen, and / or hydrogel. In another embodiment, the cell culture substrate does not comprise an extracellular matrix.

[0048] iPSCs may be seeded onto a substrate at an appropriate distribution in the presence of a medium that maintains pluripotency. The appropriate seeding distribution can be easily determined by those skilled in the art. For example, iPSCs may be seeded at a density of 50,000 to 300,000 cells / cm as single cells. 2 , 100,000~250,000 cells / cm 2 , 150,000~220,000 cells / cm 2 , 160,000~200,000 cells / cm 2 The cells can be inoculated at a density of 0.1 to 1.0 μg / ml.

[0049] The culture medium for maintaining pluripotency used in the culture method of the present invention has the advantage that it does not require the presence of feeder cells, which is undesirable because it complicates cell passaging and may also lead to contamination of the desired cells with the feeder cells.

[0050] In some embodiments, the culture medium that maintains pluripotency is a feeder-free medium 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 mTeSRplus medium.

[0051] Culture media that maintain pluripotency is preferably supplemented with a rho-associated kinase ("ROCK") inhibitor.

[0052] ROCK inhibitors are compounds that decrease 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 number Y0503, Sigma-Aldrich, St. Louis, MO), fasudil (HA-1077), hydroxyfasudil (HA1100 hydrochloride), thiazovivin, GSK429286A, narciclasine, and / or (+)-(R)-trans4-(1-aminoethyl)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)cyclohexanecarboxamide dihydrochloride (Y-30141).

[0053] Preferably, the ROCK inhibitor is Y-27632.

[0054] In some embodiments, the ROCK inhibitor is added on day 1 of seeding. If added on day 1, it may be removed later or left in place. For example, it may be removed by refreshing the medium two days after cell attachment.

[0055] In an embodiment, the culture medium for maintaining pluripotency is a feeder-free medium supplemented with a ROCK inhibitor, preferably supplemented with a ROCK inhibitor at 10 μM, more preferably supplemented with Y-27632 at 10 μM. In a preferred embodiment, the culture medium for maintaining pluripotency is mTeSRplus supplemented with a ROCK inhibitor, preferably supplemented with a ROCK inhibitor at 10 μM, more preferably supplemented with Y-27632 at 10 μM.

[0056] In a preferred embodiment, iPSCs are seeded as single cells onto extracellular matrix-coated plates in the presence of pluripotency-maintaining culture medium containing mTeSRplus supplemented with a rock inhibitor at 10 μM, preferably Y-27632 at 10 μM.

[0057] 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.

[0058] As used herein, "passaging," "passaged," or "passage" refers to the subculture of cells by harvesting individual cells from a colony and reseeding each individual cell into a new "daughter" culture. The number associated with the term "passage" refers to the consecutive number of times cells from a previous passage have been used to generate a new subculture. However, any suitable passaging method may be used. For example, hiPSCs need to be passaged to avoid overgrowth and maintain them in an undifferentiated state.

[0059] Step 2 of the culture method of the present invention involves culturing the seeded iPSCs of step 1 in the presence of an initial neural induction medium for a time sufficient to form neural stem cells (NSCs) and / or early neural progenitor cells (ENPCs), wherein the neural induction medium is

[0060] As used herein, the terms "neural stem cells" (NSCs) and "early neural progenitor cells" (ENPCs) refer to cells of the neural lineage. In certain cases, the cells express SOX2, PAX6, and NES, DCX. In certain cases, the cells are tripotent and can differentiate into neurons, astrocytes, or oligodendrocytes.

[0061] In some embodiments, the pluripotency-maintaining medium is removed before contacting the plated cells with neural induction medium. In such embodiments, the plated cells may be washed, for example with PBS, before adding neural induction medium.

[0062] As used herein, "neural induction media" and "neural induction medium" refer to a basal medium suitable for culturing neural progenitor cells or cells derived therefrom. Basal media suitable for culturing neural progenitor cells include BME medium, BGJB medium, CMRL1066 medium, Glasgow MEM medium, improved MEM zinc option medium, IMDM medium, Medium 199 medium, Eagle's MEM medium, αMEM medium, DMEM medium, HAM's medium, HAM's f-12 medium, RPMI1640 medium, Fischer's medium, neurobasal medium, and mixtures thereof. Preferably, the basal medium is Dulbecco's Modified Eagle's Medium (DMEM), preferably DMEM medium containing nutrient mixture f-12 (DMEM / f12).

[0063] In certain embodiments, the basal medium is supplemented with an N2 supplement. Preferably, the basal medium is DMEM-f12 medium containing an N2 supplement. The supplement may be present at a concentration of about 0.1-5%, about 0.5-2.0%, or about 1%.

[0064] As used herein, "N2" refers to a "hormone mix" containing transferrin, insulin, putrescine, selenium, and progesterone. For example, N2 contains 10 mg / ml transferrin, 2.5 mg / ml insulin, 1 mg / ml putrescine, 1 μl / ml selenium, and 1 μl / ml progesterone. N2 is commercially available from Gibco (Invitrogen / Thermoscientific), Sigma, and others, and can also be prepared.

[0065] In certain embodiments, the neural induction 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. The non-essential amino acids, or mem-non-essential amino acids, may be present at a concentration of about 0.1-5%, about 0.5-2.0%, or about 1%.

[0066] In certain embodiments, the neural induction medium further comprises glutamine or a glutamine product such as GlutaMAX™ (1:100, Thermo Fisher Scientific, 35050079). Glutamine may be present at a concentration of about 0.1-5%, about 0.5-2.0%, or about 1%.

[0067] In certain embodiments, the neural induction medium further comprises heparin or heparin sulfate, which may be present at a concentration of about 0.1-5 μg / mL, about 0.5-2.0 μg / mL, or about 1 μg / mL.

[0068] In certain embodiments, the neural induction medium further comprises a c-MYC inhibitor. Preferably, the c-MYC inhibitor is stauprimide. Suitable concentrations of stauprimide for use in these methods include 0.1 nm to about 10 μm, alternatively about 0.1 nm to about 1.5 μm, or alternatively 0.1 nm to about 1 μm.

[0069] In certain embodiments, the neural induction medium further comprises a TACC3 inhibitor. Preferably, the TACC3 inhibitor is KHS101, a selective inducer of neural differentiation. Suitable concentrations of stauprimide for use in these methods include 0.1 nm to about 10 μm, alternatively about 0.1 nm to about 3 μm, or alternatively 0.1 nm to about 2.5 μm.

[0070] In some embodiments, the neural induction medium comprises basal medium supplemented with a c-myc inhibitor and a TACC3 inhibitor.

[0071] In some embodiments, the neural induction medium comprises DMEM-F12 medium with N2 supplement supplemented with a c-myc inhibitor and a tacc3 inhibitor.

[0072] In some embodiments, the neural induction medium comprises DMEM-f12 medium with N2 supplement supplemented with stauprimide and KHS101.

[0073] In some embodiments, the neural induction medium comprises: mem non-essential amino acids; Glutamine or GlutaMAX™; Heparin or heparin sulfate; stauprimide; and Contains DMEM-f12 medium with N2 supplement supplemented with KHS101.

[0074] In some embodiments, the neural induction medium comprises: about 1% mem non-essential amino acids; approximately 1% GlutaMAX™; heparin approximately 1 μg / ml; Stauprimide about 0.1 nm to about 1 μm; and Contains DMEM-f12 medium containing N2 supplement supplemented with KHS101 approximately 0.1 nm to approximately 2 µm.

[0075] In certain embodiments, neural induction medium is initially supplied to the plated iPSCs, and additional medium is supplied to the cultures continuously or in discontinuous increments during culture prior to termination of the culture.

[0076] In certain embodiments, a time sufficient to form neural stem cells and / or early neural progenitor cells requires culturing the cells for about 1-10 days, about 2-8 days, or about 3-5 days, hi some embodiments, a time sufficient to form neural stem cells and / or early neural progenitor cells requires culturing the cells for 2, 3, 4, 5, 6, or 7 days.

[0077] Preferably, the cells are cultured for a sufficient time such that neural stem cells and / or early neural progenitor cells account for at least 50%, at least 75%, at least 85%, at least 95%, at least 99%, or about 100% of the cells in the culture.

[0078] In some embodiments, the culture is a two-dimensional culture. As used herein, the term "two-dimensional culture" refers to the culture of cells on a planar cell culture substrate disposed in a culture vessel.

[0079] Step 3 of the culture method of the present invention involves isolating the neural stem cells and / or early neural progenitor cells of ii) to obtain a cell suspension containing essentially single cells.

[0080] Suitable separation methods are known in the art. The separation step may involve enzymatic separation, mechanical separation, or a combination of both.

[0081] Cells can be detached from the culture substrate using enzymes, for example, enzymatic cell detachment solutions such as Accutase™, Dispase, ReLeSR, or TrypLE. Non-enzymatic solutions such as EDTA solutions can also be used.

[0082] After separation, the separated cells are suspended in a cryopreservation solution.

[0083] Cryopreservation solution refers to a medium containing a cryoprotectant. Cryoprotectants have a high affinity for water molecules and inhibit the growth of ice crystals in the cryopreservation solution. Examples of cryoprotectants include 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.

[0084] Cryopreservation media include Medium 3 10% (CS10), Medium 2 5% (CS5) and Medium 1 2% (CS2), Stem Cell Banker, Prime XV® Freezis, Hypothermasol®, CSB, Trehalose, etc.

[0085] In some embodiments, the separated cells are stored in a neutralizing solution and then suspended in a cryopreservation solution.

[0086] In some embodiments, the cells are stored in the cryopreservation medium for at least 1, 2, 3, 6, 12, 18, 24, or 48 hours prior to cryopreservation.

[0087] Step 4 of the culture method of the present invention involves cryopreserving the cell suspension. In some embodiments, cryopreservation requires maintaining the temperature of the cell suspension at or below -80°C. For example, the cell suspension 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.

[0088] Step 5 of the culture method of the present invention comprises aggregating the cryopreserved cell suspension of step 4 in the presence of an aggregate formation medium for a time sufficient to form neural organoids.

[0089] The cell suspension is placed in a culture vessel in the presence of aggregate formation medium.

[0090] The cryopreserved cell suspension is thawed. In some embodiments, thawing is performed at 30°C to 40°C, preferably 35°C to 38°C, and more preferably at about 37°C.

[0091] In embodiments, the culture vessel comprises a surface having topological features of various sizes, shapes, and depths, such as cavities or microwells. In embodiments, the surface of the culture vessel comprises one or more cavities or microwells.

[0092] Preferably, the culture vessel has a low- or non-adherent surface, which prevents cells from binding to the vessel surface, thereby promoting aggregation.

[0093] In some embodiments, aggregation requires forced spatial confinement of the cells, for example, centrifugation of the cells.

[0094] In one embodiment, the aggregate formation medium comprises a basal medium supplemented with a rho-associated kinase ("ROCK") inhibitor. Importantly, the aggregate formation medium does not contain neural lineage inducers. As used herein, neural lineage inducers refer to compounds that induce differentiation into neural lineages. Compounds that induce differentiation into neural lineages include TACC3 inhibitors and MY inhibitors. Suitably, the aggregate formation medium does not contain KHS101.

[0095] Preferably, the basal medium is supplemented with a rock inhibitor, preferably Y-27632, at a concentration of 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more.

[0096] Basal media suitable for aggregate formation include BME medium, BGJB medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM zinc option medium, IMDM medium, 199 medium, Eagle's MEM medium, αMEM medium, DMEM medium, HAM's medium, HAM's f-12 medium, RPMI1640 medium, Fisher's medium, neurobasal medium, and mixtures thereof. Preferably, the basal medium is Dulbecco's Modified Eagle's Medium (DMEM), preferably DMEM with nutrient mixture f-12 (DMEM / f12).

[0097] In certain embodiments, the basal medium is supplemented with an N2 supplement. Preferably, the basal medium is dmem-f12 medium containing an N2 supplement. The supplement may be present at a concentration of about 0.1-5%, about 0.5-2.0%, or about 1%.

[0098] 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. The non-essential amino acids or mem-non-essential amino acids may be present at a concentration of about 0.1-5%, about 0.5-2.0%, or about 1%.

[0099] In certain embodiments, the aggregate formation medium further comprises glutamine or a glutamine product such as GlutaMAX™ (1:100, Thermo Fisher Scientific, 35050079). Glutamine may be present at a concentration of about 0.1-5%, about 0.5-2.0%, or about 1%.

[0100] In certain embodiments, the aggregate formation medium further comprises heparin or heparin sulfate, which may be present at a concentration of about 0.1-5 μg / ml, about 0.5-2.0 μg / ml, or about 1 μg / ml.

[0101] In some embodiments, the aggregate formation medium comprises: mem non-essential amino acids; Glutamine or GlutaMAX; Heparin or heparin sulfate; and Contains DMEM-F12 medium with N2 supplemented with Y-27632.

[0102] In some embodiments, the aggregate formation medium comprises: about 1% mem non-essential amino acids; approximately 1% GlutaMAX; Heparin approximately 1 μg / ml; and Contains DMEM-F12 medium containing N2 supplemented with Y-27632 approximately 50 µM.

[0103] In embodiments, a time sufficient to form neural organoids requires at least 12 hours of cell culture, with culture of multiple cells containing one or more microparticles 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.

[0104] As used herein, "neural organoids" refer to aggregates of neural stem cells and / or early neural progenitor cells with a three-dimensional structure. Neural organoids are three-dimensional cell populations formed by cells adhering to each other through suspension culture or 3D culture. The shape of neural organoids is not particularly limited, and they may be spherical or non-spherical. There are no particular limitations on the size of neural organoids, but they typically have a spherical equivalent diameter of 150 μm to 1,000 μm, and in one embodiment, for example, 200 μm to 800 μm or 300 μm to 500 μm. Neural organoids typically contain 500 to 150,000 cells, and in one embodiment, for example, 1,000 to 100,000 cells, 1,000 to 70,000 cells, or 3,000 to 30,000 cells.

[0105] Neural organoid can also comprise other cells together with neural cell.Preferably, neural organoid is substantially homogeneous.In certain embodiments, neural organoid comprises at least 60% or more, 70% or more, 80% or more, more preferably 90% or more or 95% or more neural cell.

[0106] The neural organoid produced by the method of the present disclosure is useful as a neural disease model and a drug discovery tool.Neural organoid can be considered as a miniature model of the neural organ, including the brain.

[0107] Screening assays generally involve contacting neural organoids with a candidate agent and determining the phenotypic effect on the neural organoids.

[0108] Neural organoids can be used as disease models to investigate various diseases associated with nervous tissue, including stroke, brain inflammatory disorders, neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer's disease), neuroinflammatory diseases (e.g., multiple sclerosis), trauma (e.g., brain surgical injury), channelopathies (e.g., epilepsy), and psychiatric disorders (including autism and schizophrenia).

[0109] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to" and 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, it is to be understood that the specification contemplates the plural as well as the singular unless the context otherwise requires.

[0110] It is to be understood that any feature, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, unless inconsistent therewith. All features disclosed herein (including the accompanying claims, abstract, and drawings) and / or all steps of the disclosed methods or processes may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention extends to any novel or novel combination of features or steps of the disclosed methods or processes disclosed herein (including the accompanying claims, abstract, and drawings).

[0111] The reader is directed to all articles and documents related to this application that have been filed contemporaneously or previously hereto and that are publicly available herewith, the contents of all such articles and documents being incorporated herein by reference. [Example]

[0112] In this specification, particularly in the drawings, neural organoid is also referred to as neuroid or neuroid.In this specification, particularly in the drawings, cerebral organoid is also referred to as brainoid or brainoid.

[0113] The method of generating brain organoids is well known in the art and can include 3D differentiation of human pluripotent stem cells (hPSCs) into embryoid bodies (EBs), which are embedded in extracellular matrix and cultured in a rotating bioreactor.The present invention of neural organoids provides an in vitro approach based on the differentiation into chemically defined monolayer (2D) precursor neural organoid cells and the re-aggregation paradigm after freezing, as outlined in the examples.

[0114] [Example 1] 2D Differentiation of Stem Cells to Form Progenitor Neural Organoid Cell Suspensions iPSCs were cultured at 200,000 cells / cm in PSC culture medium supplemented with 10 μM of ROCK inhibitor (Y0503-5MG, reconstituted in HO) on coated (extracellular matrix-bearing) plates. 2After overnight adhesion, the medium is changed to standard neural induction medium. To induce / accelerate the differentiation state of postmitotic stem cells, standard neural induction medium is supplemented with the small molecule inhibitors stauprimide (e.g., S2951, supplied by Sigma, concentration range: 0.1 nM to 5 μM) and KHS101 (e.g., K4019, supplied by Sigma, concentration range: 0.1 nM to 5 μM). The compound-containing medium is replenished daily for four consecutive days. On day 5, the adherent cells are dissociated into a single-cell suspension and incubated with 0.5 mM EDTA at 37 °C for 4 min, followed by incubation with an enzyme mix (e.g., Accutase®) at 37 °C for 4 min. The single-cell suspension is cryopreserved in GMP-grade freezing medium (e.g., Stem Cell Banker, AMS-Biotechnology, 11890) and stored at -80 °C or in LN2.

[0115] The procedure is outlined in Figure 1.

[0116] [Example 2] Formation of 3D neural organoids To form one neural organoid from the cryopreserved progenitor neural organoid cell suspension, use 0.25 ml / cm of standard neural induction formulation medium supplemented with 50 µM of ROCK inhibitor (Y0503-5MG, reconstituted in HO). 2 Using a centrifuge tube, seed 40,000 neurally differentiated cells in 100 μl per microtiter well plate. Spin the plate at 400 g for 5 minutes at room temperature and allow the neural organoids to form a uniform structure for at least 16 hours (critical step). Remove half of the seeding medium (50 μl) and replace with 100 μl of fresh standard neural induction medium (without ROCK inhibitor, stauprimide, and KHS101). After a culture period of at least 16 hours, the neural organoids are ready for use in the desired assay (Figure 2).

[0117] The compositions of the various media used in the examples are shown in Table 1.

[0118] [Table 1]

[0119] The advantages of the method of the present invention compared to methods known in the art are outlined in Table 2.

[0120] [Table 2]

[0121] [Example 3] Characteristics of 3D neural organoids Neural organoids and standard cerebral organoids were formed as described in Examples 1 and 2 (paragraphs 112-119) or using a commercially available embryoid body-based organoid formation procedure (Stem Cell Technologies, Table 2). For morphological characterization at early time points, neural organoids were cultured for 96 h (medium changed at 48 h; Figure 3A). For longer-term maturation, neural organoids were embedded in Matrigel droplets on parafilm dimples formed in an empty P200 tip box. The embedded organoids were allowed to solidify at 37°C for 30 min before being transferred to a 10 cm dish containing organoid maturation medium (minus vitamin A). After 48 h, the majority of the medium was removed and replaced with fresh organoid maturation medium (containing vitamin A). After an additional 48 h, the organoids were transferred to a spinner flask containing organoid maturation medium (plus vitamin A) and allowed to mature until needed.

[0122] For immunofluorescence characterization, neural organoids or standard cerebral organoids (see Table 2) are transferred to a 24-well plate using a 1 mm-gauge pipette tip. The medium is removed and the organoids are washed with PBS. The PBS is then removed and a 4% paraformaldehyde solution (Thermo, 15670799) is added. The organoids are then incubated at 4 °C for 15 min (for early-stage organoids or overnight for organoids older than 36 days). The organoids are then washed with PBS and then incubated with a 30% sucrose solution (Sigma, S9378) overnight or until the organoids sink 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 min. The organoids are then transferred to a cryomold pre-coated with the gelatin / sucrose solution. The blocks are cryosectioned into 20 μm sections using a cryostat.

[0123] For immunofluorescence staining, slides were washed three times with PBS and incubated with blocking buffer (1% FBS, 0.3% Triton X-100 (Sigma, X100)) for 1 hour at room temperature. Next, the slides were incubated with primary antibodies diluted in blocking buffer for 1 hour at room temperature or overnight at 4°C. Next, the slides were washed three times with PBS and incubated with secondary antibodies diluted in blocking buffer for 1 hour at room temperature, protected from light. The slides were then washed three times with PBS and incubated with 2 μg / mL DAPI solution (Sigma, D9542) diluted in PBS for 10 minutes at room temperature, protected from light. Next, the slides were washed twice with PBS and then "mounted" onto polylysine-coated slides (VWR, 631-9483) using aqueous mounting medium (2B Scientific, H-5501-60). Slides were imaged using an inverted fluorescence microscope, and quantification was performed using the Cell Profiler pipeline.

[0124] Primary antibody

[0125] [Table 3]

[0126] Secondary antibody

[0127] [Table 4]

[0128] For single-cell sequencing analysis, organoids are incubated with Cell Recovery Solution (Thermo, 354253) at 4°C for 1 hour and then dissociated using a Neurosphere Dissociation Kit (Miltenyi Biotec, 130-095-943). Dead cells and cell debris are removed using a Dead Cell Removal Kit (Miltenyi Biotec, 130-090-101). Cells are counted and frozen in 500 µL of GMP-grade freezing medium (e.g., Stem Cell Banker) in aliquots of 1–2 million cells. Cells are then processed for single-cell sequencing according to the 10X3 gene expression protocol. The resulting data can then be mapped to the human GRCh38 reference genome using Cell Ranger (7.1.0). All subsequent analyses are performed using the online single-cell sequencing analysis tool, Bioturing.

[0129] [Example 4] Quantification of morphological changes after storage at -80°C for up to 12 weeks The culture medium was aspirated from the induced cells, and the wells were washed with 1 mL of PBS. The PBS was aspirated, 1 mL of 0.5 mM EDTA was added, and the cells were incubated at 37°C for 4 minutes. The EDTA was slowly removed with a P1000 pipette, and 1 mL of Accutase (e.g., Sigma-Aldrich A6964) was added. The cells were then incubated at 37°C for 4 minutes. Using a P1000 pipette, the cells were slowly resuspended in mTeSR medium and transferred to a 15 mL centrifuge tube. After centrifugation at 300 x g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of neural induction medium and 50 μM ROCK inhibitor for cell counting. 10 μL of cells were mixed with 10 mL of viability dye (e.g., trypan blue), and the cells were counted using a Countess automated cell counter. Next, the cells were frozen at 2 million cells per well in 500 μL of GMP-grade freezing medium (e.g., Stem Cell Banker). Cells were frozen to -80°C at a rate of 1°C per minute. Cells were maintained at -80°C for 1, 6, and 12 weeks. To revive cells, thaw them for approximately 3 minutes in a water bath set at 37°C and transfer them to a 15 mL tube. Next, at least 5 mL of medium was added dropwise to the cells, and the cell suspension was spun at 300 g for 5 minutes. The cell pellet was resuspended in 1 mL of neural induction medium containing 50 μM ROCK inhibitor. 10 μL of cells were mixed with 10 mL of trypan blue, and the cells were counted using a Countess automated cell counter. Cells were then seeded at 40,000 cells per well in 100 μL of neural induction medium containing 50 μM ROCK inhibitor in an ultra-low attachment 96-well plate. After 48 hours, 50 μL was removed from each well and 100 μL of neural induction medium was added. After another 48 hours, neural organoids were imaged using an inverted 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

1. i) seeding induced pluripotent stem cells (iPSCs) onto 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 neural induction medium for a time sufficient to form neural stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) isolating the neural stem cells (NSCs) and / or early neural progenitor cells (eNPCs) of ii) to obtain a cell suspension containing single cells; iv) cryopreserving the cell suspension; v) aggregating the cryopreserved cell suspension of iv) in the presence of an aggregate formation medium for a time sufficient to form neural organoids; A method for producing neural organoids in vitro, comprising:

2. The method of claim 1 , wherein the cell culture substrate comprises an extracellular matrix component.

3. 3. The method of claim 1 or claim 2, wherein the cell culture substrate is feeder cell-free.

4. The method of claim 1 , wherein the culture medium that maintains pluripotency is a feeder-free medium.

5. The method of claim 4, wherein the culture medium that maintains pluripotency is a feeder-free medium selected from Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, stabilized Essential 8 medium, and StemFit medium.

6. 6. The method of any one of claims 1 to 5, wherein the culture medium that maintains pluripotency is supplemented with a Rho-associated coiled-coil kinase (ROCK) inhibitor.

7. 7. The method of any one of claims 1 to 6, wherein the neural induction medium is Dulbecco's Modified Eagle's Medium (DMEM).

8. 8. The method of claim 7, wherein the DMEM is a DMEM medium with nutrient mixture F-12 (DMEM / F12).

9. 9. The method of claim 1, wherein the neural induction medium is supplemented with N2.

10. 10. The method of any one of claims 1 to 9, wherein the neural induction medium is supplemented with a TACC3 inhibitor.

11. 11. The method of claim 10, wherein the TACC3 inhibitor is KHS101.

12. The method of any one of claims 1 to 11, wherein the neural induction medium is supplemented with a c-MYC inhibitor.

13. 13. The method of claim 12, wherein the c-MYC inhibitor is stauprimide.

14. 14. The method of any one of claims 1 to 13, wherein the neural induction medium is supplemented with heparin or heparin sulfate.

15. 15. The method of any one of claims 1 to 14, wherein the neural induction medium is serum-free.

16. 16. The method of any one of claims 1 to 15, wherein the neural induction medium is supplemented with glutamine or GlutaMAX™.

17. 17. The method of any one of claims 1 to 16, wherein the neural induction medium is supplemented with non-essential amino acids or MEM non-essential amino acids.

18. 18. The method of any one of claims 1 to 17, wherein the neural induction medium comprises DMEM / F12 medium containing N2 supplement, heparin, GlutaMAX™, MEM non-essential amino acids, a TACC3 inhibitor and a c-MYC inhibitor, optionally wherein the c-MYC inhibitor is stauprimide and the TACC3 inhibitor is khs101.

19. 19. The method of any one of claims 1 to 18, wherein the culture of iPSCs seeded in the presence of neural induction medium is a 2D culture.

20. 20. The method of any one of claims 1 to 19, wherein the seeded iPSCs are cultured in the presence of neural induction medium for at least 2, 3, 4, or 5 days, preferably for 2 to 10 days, 4 to 9 days, or 3 to 5 days.

21. 21. The method of any one of claims 1 to 20, wherein the separation comprises enzymatic and / or mechanical separation.

22. 22. The method of any one of claims 1 to 21, wherein the separated cells are suspended in a cryopreservation solution to form a cell suspension.

23. 23. The method of any one of claims 1 to 22, wherein the cell suspension is stored at cryopreservation temperatures.

24. 24. The method of any one of claims 1 to 23, wherein the cell suspension is stored at frozen storage temperatures 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 more than 5% loss in product diameter upon aggregation.

25. 25. The method of any one of claims 1 to 24, wherein the cell suspension is stored at cryopreservation temperatures for at least 12 weeks without more than 5% loss in product diameter upon aggregation.

26. 26. The method of any one of claims 1 to 25, wherein the cryopreserved cell suspension is thawed prior to aggregation.

27. 27. The method of any one of claims 1 to 26, wherein the aggregates are formed in an aggregate formation medium that does not contain neural lineage inducers.

28. 28. The method of any one of claims 1 to 27, wherein the aggregate-forming medium is Dulbecco's Modified Eagle's Medium (DMEM).

29. 29. The method of claim 28, wherein the DMEM is a DMEM medium containing nutrient mixture F-12 (DMEM / F12).

30. 30. The method of any one of claims 1 to 29, wherein the aggregate formation medium is supplemented with N2.

31. 31. The method of any one of claims 1 to 30, wherein the aggregate formation medium is supplemented with heparin or heparin sulfate.

32. 32. The method of any one of claims 1 to 31, wherein the aggregate formation medium is serum-free.

33. 33. The method of any one of claims 1 to 32, wherein the aggregate formation medium is supplemented with glutamine or GlutaMAX™.

34. 34. The method of any one of claims 1 to 33, wherein the aggregate formation medium is supplemented with non-essential amino acids or MEM non-essential amino acids.

35. 35. A neural organoid obtainable or obtained by a method according to any one of claims 1 to 34.

36. 36. The neural organoid of claim 35 for use in medicine or diagnosis.

37. 36. The neural organoid of claim 35, for use in screening the potential effects of substances on neural cells in vivo, optionally for use in screening for therapeutic or toxic effects.

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