Method for culturing neural organoids

Abstract

The present invention relates to a method for culturing neural cells and to a neural organoid for use in medical or diagnostic treatment.

Description

Technical Field

[0001] This invention relates to methods for culturing nerve cells and to neural organoids for use in medical or diagnostic purposes. Background Technology

[0002] Traditionally, research on human brain physiology and disease has been based on animal models and post-mortem tissue samples, primarily due to ethical concerns regarding the collection of human brain tissue. Animal brains (including those from widely used rodent models) differ significantly in size and structure from the human brain, posing considerable challenges to the study of the development and diseases of the human central nervous system. These subtle structural and cellular differences between species are partly responsible for the failure to successfully translate preclinical findings from these models into clinical practice.

[0003] However, the availability of human induced pluripotent stem cells (hiPSCs) and stem cell technologies for generating pre-modeled 3D-like prebrain organoids in free-floating cultures have opened new avenues for personalized modeling of neurological diseases. Brain organoids, capable of mimicking the structure and function of the human brain, are now widely accepted as suitable models for studying brain development and disease mechanisms. For example, three-dimensional (3D) brain organoid (“mini-brain”) systems generated from human pluripotent stem cells (hPSCs) have demonstrated considerable potential in reproducing key features of the pathophysiology of brain cancer and neurological diseases, namely, Alzheimer's disease.

[0004] While current brain organoid models and their production methods hold promise for biological and medical research, they are not without limitations. The complexity and lack of standardization in their production protocols currently lead to heterogeneity in brain organoid production, thus requiring users of organoid methods to implement quality control, growth logistic management, and organoid "cherry-picking" for downstream applications. These factors contribute to heterogeneity between experiments, consequently affecting results. Further limitations include variability in organoid maturation time, organoid transportation issues from the production site to the end user (transporting biological materials often involves freeze-thaw cycles, which can cause cell viability problems in brain organoids, making them unacceptable for clinical use), and compliance with high-throughput methods. Furthermore, organoid production typically requires the use of naturally dissolved basement membrane matrices (such as Matrigel). (Droplets) to facilitate the maturation of 3D organoids. Due to their animal origin, batch-to-batch variability may exist, which could result in end-users having no control over their exact composition, such as unknown and uncontrollable amounts of growth factors.

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

[0006] According to the present invention, an in vitro method for producing neural organoids is provided, comprising:

[0007] i) In the presence of a culture medium that maintains pluripotency, induced pluripotent stem cells (iPSCs) are plated (seeded) on a cell culture substrate.

[0008] ii) In the presence of neural induction medium, the iPSCs plated in i) are cultured for a sufficient amount of time to form neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs).

[0009] iii) Dissociate the neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs) from ii) to obtain a cell suspension containing single cells;

[0010] iv) Cryopreservation of cell suspensions;

[0011] v) In the presence of an aggregate-forming medium, the cryopreserved cell suspension from iv) was aggregated for a sufficient amount of time to form neural organoids.

[0012] In some embodiments, the cell culture substrate includes an extracellular matrix component. In another embodiment, the cell culture substrate does not include an extracellular matrix component.

[0013] In some implementations, the cell culture substrate does not contain feeder cells.

[0014] In some implementations, the culture medium that maintains pluripotency is a feeder-free medium.

[0015] In some embodiments, the culture medium for maintaining pluripotency is selected from the following feeder-free culture media: Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, stabilized Essential 8 medium, and StemFit medium.

[0016] In some implementations, the culture medium used to maintain pluripotency is supplemented with a Rho-associated coiled-coil kinase (ROCK) inhibitor.

[0017] In some embodiments, the neural induction medium is Dulbecco modified Eagle medium (DMEM). Preferably, the DMEM is DMEM medium with nutrient mixture F-12 (DMEM / F12).

[0018] In some implementations, the neural induction medium is supplemented with N2.

[0019] In some embodiments, the neural induction medium is supplemented with a TACC3 inhibitor. Preferably, the TACC3 inhibitor is KHS101.

[0020] In some embodiments, the neural induction medium is supplemented with a c-MYC inhibitor. Preferably, the c-MYC inhibitor is stauprimide.

[0021] In some embodiments, the neural induction medium is supplemented with heparin or heparin sulfate (heparin sulfate).

[0022] In some implementations, the neural induction medium does not contain serum.

[0023] In some embodiments, the neural induction medium is supplemented with glutamine or GlutaMAX. TM .

[0024] In some implementations, the neural induction medium is supplemented with non-essential amino acids or MEM non-essential amino acids.

[0025] In some embodiments, the neural induction medium contains N2 supplement, heparin, and GlutaMAX. TM DMEM / F12 medium containing MEM non-essential amino acids, TACC3 inhibitor and c-MYC inhibitor, optionally wherein the c-MYC inhibitor is stataprolamine and wherein the TACC3 inhibitor is KHS101.

[0026] In some implementations, the iPSCs plated in the presence of a neural induction medium are cultured in 2D.

[0027] In some embodiments, the plated iPSCs are cultured in the presence of a neural induction medium for a period of at least 2, 3, 4 or 5 days, preferably 2-10 days, 4-9 days or 3-5 days.

[0028] In some implementations, dissociation includes enzymatic dissociation and / or mechanical dissociation.

[0029] In some implementations, the dissociated cells are suspended in a cryopreservation medium to form a cell suspension.

[0030] In some implementations, the cell suspension is stored at a cryopreservation temperature.

[0031] In some implementations, the frozen cell suspension is thawed before aggregation.

[0032] In some embodiments, aggregates are formed in an aggregate-forming medium that does not contain a neural lineage inducing factor.

[0033] In some embodiments, the aggregate-forming medium is Dulbecco-modified Eagle medium (DMEM). Preferably, the DMEM is DMEM medium with nutrient mixture F-12 (DMEM / F12).

[0034] In some implementations, the aggregate-forming culture medium is supplemented with N2.

[0035] In some implementations, the aggregate-forming culture medium is supplemented with heparin or heparin sulfate.

[0036] In some implementations, the aggregate-forming medium does not contain serum.

[0037] In some embodiments, the aggregate-forming medium is supplemented with glutamine or GlutaMAX. TM .

[0038] In some implementations, the aggregate forming medium is supplemented with non-essential amino acids or MEM non-essential amino acids.

[0039] In a further aspect of the invention, a neural organoid that can be obtained or acquired by the methods described herein is provided.

[0040] In a further aspect of the invention, a neural organoid that can be obtained or acquired by the methods described herein is provided for use in medicine or diagnosis.

[0041] In a further aspect of the invention, a neural organoid, obtainable or acquireable by the methods described herein, is provided for screening substances for potential effects on nerve cells in vivo, optionally for use in screening for therapeutic or toxic effects. Attached Figure Description

[0042] Embodiments of the present invention will be further described below with reference to the accompanying drawings, wherein:

[0043] Figure 1This is an overview of the differentiation process used in the method of the present invention;

[0044] Figure 2 This is an overview of the organ aggregation process of neural organoids used in the method of the present invention;

[0045] Figure 3 - Neural organoids are characterized by low intra-batch heterogeneity and the ability to mature in both cases with and without extracellular matrix (gel) support;

[0046] A) Left: Compared to batches (n=12) obtained from standard brain organoid (embryoid body-based) culture, intra-batch heterogeneity in "roundness" of neural organoids was significantly reduced. Right: Compared to standard brain organoid (embryoid body-based) culture, intra-batch heterogeneity in "size" of neural organoids (n=12) was significantly reduced. Dots / squares represent the calculated diameter range for each neural organoid and standard brain organoid batch. Median and SD are shown. Parametric and nonparametric statistical tests showed p<0.0001.

[0047] B) Left: Image-based quantitative illustration of neuronal marker penetration scores in sections of neural organoids and standard brain organoids. Right: Inter-batch neuronal (TuJ1-positive) marker penetration detected in tissue sections was significantly higher in neural organoids (n=8) compared to standard brain organoid (embryoid-based) batches (n=9). Median and SD are shown. Parametric and nonparametric statistical tests showed p<0.0001.

[0048] C) Volcano plots show the genes upregulated and downregulated at the single-cell level in neural organoids compared to standard brain organoids (based on embryoids) in each batch (n=2) matured for 36 days under Matrigel / spinner flask conditions.

[0049] D) Based on discrete log2 fold change (FC) values, the 10 most upregulated and 10 most downregulated genes are shown (as shown in C).

[0050] E) Qualitative assessment of neural organoid sections after long-term maturation (180 days) in roller bottles, based on immunostaining of MAP2-positive neurons (arrows) and GFAP-positive astrocytes (arrowheads). Note that in the early maturation stage, the uniform distribution of neurons throughout the neural organoid batch was consistent with a high TuJ1 expression penetration score (as shown in B).

[0051] F) Qualitative assessment of standard brain organoid (embryoid-based) sections after long-term maturation (180 days) in roller bottles, based on immunostaining of MAP2-positive neurons and GFAP-positive astrocytes, at a scale bar of 200 μm.

[0052] G) Biomarker penetration scores (as shown in B) of biomarkers in biologically distinct neural organoids (n=3) and standard brain organoids (based on embryoids) (n=3) after long-term (180-day) maturation. The data show a significant preservation of the uniform distribution of biomarkers in neural organoids. Median and SD are shown. Parametric and nonparametric statistical tests show p<0.001.

[0053] H) In “plate / gel-free” maturation, compared with roller bottle / Matrigel culture conditions, the expression of continuous single-cell markers (NES, TUBB3, MAP2, DCX, NEFL) (shown in the complete UMAP-visualized single-cell RNA sequencing (RNAseq) dataset) indicates that neural organoid maturation can be achieved without the use of gels (e.g., Matrigel).

[0054] I) Volcano plot, showing genes upregulated and downregulated at the single-cell level in neural organoids compared to standard brain organoids (based on embryoids) in batches (n=2) that matured for 36 days on Matrigel-free 'boards'.

[0055] J) Based on discrete log2 fold change (FC) values, the 10 most upregulated and 10 most downregulated genes are shown (as shown in I).

[0056] Figure 4 shows the retention of morphological features of neural organoids after storage at -80°C for 1, 6, and 12 weeks.

[0057] (A) Quantification of the diameter (μm) of neural organoids, with error bars representing the standard deviation n = 5-12.

[0058] (B) Representative images of neural organoids 96 hours after plating. Detailed Implementation

[0059] This invention provides a simplified method for providing neural organoids for drug screening and modeling of neurological diseases. The method disclosed herein can generate multiple sets of homogeneous neural organoids, each in the set having substantially the same characteristics. Furthermore, the method of this invention advantageously simplifies the production of neural organoids, thereby providing faster and less expensive production. Therefore, the method of this invention provides an improved, robust, and consistent way of providing neural organoids for use in models of neurological diseases and as a drug discovery tool.

[0060] This invention provides an in vitro method for producing neural organoids, comprising: i) plated induced pluripotent stem cells (iPCS) on a cell culture substrate in the presence of a pluripotency-maintaining medium; ii) culturing the plated iPCS from i) for a sufficient amount of time in the presence of a first neural induction medium to form neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) dissociating the neuronal stem cells and / or early neural progenitor cells from ii) to obtain a cell suspension containing single cells; iv) cryopreserving the cell suspension; and v) aggregating the cryopreserved cell suspension from iv) for a sufficient amount of time in the presence of an aggregate-forming medium to form neural organoids.

[0061] The first stage of the culture method of the present invention includes: plate-laying induced pluripotent stem cells (iPCS) on a cell culture substrate in the presence of a pluripotency-maintaining medium. In this stage, iPCS are seeded on the cell culture substrate.

[0062] The term "induced pluripotent stem cells" (iPSCs) refers to a type of pluripotent cell that is reprogrammed into somatic cells to possess the same characteristics 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 extracted from the skin, lungs, or veins of ostensibly healthy or diseased subjects.

[0063] Specific examples include: cells obtained by reprogramming differentiated somatic cells (such as fibroblasts or peripheral blood mononuclear cells) to induce pluripotency through the expression of any combination of genes selected from a reprogrammed genome group 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 (c-MYC or L-MYC). Stem Cells, 2013; 31: 458-466 ), and (3) OCT3 / 4, SOX2, NANOOG and LIN28 ( Science 2007;318:1917–1920 ).

[0064] In some implementations, iPSCs are obtained from repositories (cell banks), such as 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 StemCell Agency (e.g., cw60261, cw60354, cw60359, cw60480, cw60335, cw60280, cw60594, cw60083, cw60086, cw60087, cw60167, cw60186), and the American Type Culture Collection. Collection)(ATCC (For example, atcc-dyr0530 human induced pluripotent stem (ips) cells (atcc) acs-1012 TM ,atcc acs-1011 TM ,atcc Number: acs-1024 TM ,atcc Number: acs-1028 TM ,atcc Number: acs-1031 TM ,atcc Number: acs-1004 TM ,atcc Number: acs-1029 TM ,atcc Number: acs-1020 TM ,atcc Number: acs-1007 TM ,atcc Number: acs-1030 TM ).

[0065] The iPSCs used in this invention are mammalian pluripotent stem cells, and preferably, iPSCs are human cells.

[0066] As used herein, the term "substrate" refers to any substance that serves as a solid support that is free of or substantially free of cytotoxins. In some embodiments, the solid substrate comprises one or a combination of silica, plastic, and metal.

[0067] In one embodiment, the cell culture substrate comprises an extracellular matrix component. Alternatively or additionally, the cell culture substrate comprises matrigel, gelatin, fibronectin, laminin, fibronectin, collagen, and / or hydrogel. In another embodiment, the cell culture substrate does not contain an extracellular matrix.

[0068] iPSCs can be seeded onto substrates in a suitable distribution and in the presence of a medium that maintains pluripotency. Those skilled in the art can readily determine the appropriate seeding distribution. For example, iPSCs can be seeded as single cells at 50,000-300,000 cells / cm². 2 100,000-250,000 cells / cm 2 150,000-220,000 cells / cm 2 160,000-200,000 cells / cm 2 Density of vaccination.

[0069] The pluripotency-maintaining culture medium used in the culture method of the present invention advantageously does not require the presence of feeder cells. The use of feeder cells is undesirable because it complicates cell passage and can also lead to contamination of the desired cells by feeder cells.

[0070] In some embodiments, the culture medium for maintaining 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 for maintaining pluripotency is mTeSR plus medium.

[0071] Culture media that maintain pluripotency are preferably supplemented with rho-associated kinase (“ROCK”) inhibitors.

[0072] ROCK inhibitors are compounds that reduce rho kinase activity. In some embodiments, rho kinase inhibitors are n-[(3-hydroxyphenyl)methyl]-n'-[4-(4-pyridyl)-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 (ha-1077). 1100 hydrochloride), thiazovivin, gsk429286a, narciclasine and / or (+)-(r)-trans-4-(1-aminoethyl)-n-(1h-pyrrolo[2,3-b]pyridin-4-yl)cyclohexanecarboxamide dihydrochloride (y-30141).

[0073] Preferably, the ROCK inhibitor is Y-27632.

[0074] In some implementations, a ROCK inhibitor is added on day 1 of plating. If added on day 1, it can be removed or retained over subsequent days. For example, it can be removed by refreshing the culture medium on day 2 after cell adhesion (cell attachment).

[0075] In several embodiments, the culture medium for maintaining pluripotency is a feederless culture medium supplemented with a ROCK inhibitor, preferably supplemented with 10 μm of ROCK inhibitor, more preferably supplemented with 10 μm of Y-27632. In a preferred embodiment, the culture medium for maintaining pluripotency is mTeSR plus supplemented with a ROCK inhibitor, preferably supplemented with 10 μm of ROCK inhibitor, more preferably supplemented with 10 μm of Y-27632.

[0076] In a preferred embodiment, iPSCs are seeded as single cells on an extracellular matrix-coated culture plate in the presence of a culture medium that maintains pluripotency. This culture medium contains mTeSR plus supplemented with 10 μm of rock inhibitor (preferably 10 μm Y-27632).

[0077] In several embodiments, the inoculated iPSCs are amplified 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 several embodiments, the inoculated iPSCs are amplified in culture for at least 6, 12, 18, 24, 30, 36, or 42 hours.

[0078] As used herein, “passaging,” “passaged,” or “passage” refers to the process of subculturing cells by harvesting individual cells from a colony and reseeding those cells into new “daughter” cultures. The number associated with the term “passing” refers to the number of consecutive passages from which cells from a previous passage have been used to generate new subcultures. However, any suitable passaging method can be used. For example, hiPSCs should be passaged to avoid overgrowth and to maintain their undifferentiated state.

[0079] The second stage of the culture method of the present invention includes: culturing the plated ipscs of the first stage for a sufficient amount of time in the presence of a first neural induction medium to form neuronal stem cells (NSCs) and / or early neural progenitor cells (ENPCs), wherein the neural induction medium is present.

[0080] As used herein, the terms “neuronal stem cells” (NSCs) and “early neural progenitor cells” (ENPCs) refer to cells of the neural lineage. In some cases, these cells express SOX2, PAX6, and NES, DCX. In others, these cells possess tripotent differentiation potential and can differentiate into neurons, astrocytes, or oligodendrocytes.

[0081] In some embodiments, the pluripotency-maintaining medium is removed before contacting the plated cells with the neural induction medium. In such embodiments, the plated cells may be washed, for example, with PBS, before adding the neural induction medium.

[0082] As used herein, "neural induction media" and "neural induction medium" refer to basal media suitable for culturing neural progenitor cells or cells derived therefrom. Suitable basal media for culturing neural progenitor cells include: BME medium, BGJB medium, CMRL 1066 medium, Glasgow MEM medium, modified MEM zinc-selective medium, IMDM medium, Medium 199 medium, EagleMEM medium, αMEM medium, DMEM medium, HAM medium, such as HAM's f-12 medium, RPMI 1640 medium, Fischer's medium, neural basal media, and mixtures thereof. Preferably, the basal medium is Dulbecco modified Eagle medium DMEM medium, and more preferably DMEM medium containing the nutrient mixture f-12 (DMEM / f12).

[0083] In some embodiments, the basal medium is supplemented with an N2 supplement. Preferably, the basal medium is DMEM-f12 medium with an N2 supplement. The concentration of the supplement may be from about 0.1% to 5%, from about 0.5% to 2.0%, or from about 1%.

[0084] As used herein, "N2" refers to a "hormone mixture," which is a mixture of hormones containing transferrin, insulin, putrescine, selenium, and progesterone. For example, N2 may contain 10 mg / ml transferrin, 2.5 mg / ml insulin, 1 mg / ml putrescine, 1 μl / ml 15% selenium, and 1 μl / ml progesterone. N2 can be purchased from Gibco (Invitrogen / Thermoscientific), Sigma, and other companies, or can be prepared.

[0085] In some 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 concentration of non-essential amino acids or mem non-essential amino acids may be from about 0.1% to 5%, from about 0.5% to 2.0%, or from about 1%.

[0086] In some embodiments, the neural induction medium further comprises a glutamine product, such as glutamine or GlutaMAX. TM (1:100, Thermo Fisher Scientific, 35050079). The concentration of glutamine present can be approximately 0.1% to 5%, approximately 0.5% to 2.0%, or approximately 1%.

[0087] In some embodiments, the neural induction medium also contains heparin or heparin sulfate. The concentration of heparin or heparin sulfate may be from about 0.1 to 5 μg / mL, from about 0.5 to 2.0 μg / mL, or from about 1 μg / mL.

[0088] In some embodiments, the neural induction medium further comprises a c-MYC inhibitor. Preferably, the c-MYC inhibitor is stapoxetine. Suitable stapoxetine concentrations for this method include 0.1 nm to about 10 μm, optionally about 0.1 nm to about 1.5 μm or 0.1 nm to about 1 μm.

[0089] In some embodiments, the neural induction medium further comprises a TACC3 inhibitor. Preferably, the TACC3 inhibitor is KHS101, a selective inducer of neuronal differentiation. Suitable concentrations of stapillamine for this method include 0.1 nm to about 10 μm, optionally about 0.1 nm to about 3 μm or 0.1 nm to about 2.5 μm.

[0090] In some embodiments, the neural induction medium comprises a basal medium supplemented with c-myc inhibitors and TACC3 inhibitors.

[0091] In some embodiments, the neural induction medium comprises DMEM-f12 medium supplemented with c-myc inhibitors and tacc3 inhibitors and N2 supplementation.

[0092] In some embodiments, the neural induction medium comprises DMEM-f12 medium supplemented with stataprolol and KHS101 and N2 supplement.

[0093] In some embodiments, the neural induction medium comprises DMEM-f12 medium containing N2, supplemented with:

[0094] -mem non-essential amino acids;

[0095] - glutamine or GlutaMAX TM ;

[0096] -Heparin or heparin sulfate;

[0097] - Stapoxetine; and

[0098] -KHS101.

[0099] In some embodiments, the neural induction medium comprises DMEM-f12 medium containing N2, supplemented with:

[0100] - Approximately 1% of non-essential amino acids;

[0101] - Approximately 1% GlutaMAX TM ;

[0102] - Approximately 1 μg / ml heparin;

[0103] - Stapulamine, approximately 0.1 nm to approximately 1 μm; and

[0104] - Approximately 0.1 nm to approximately 2 μm KHS101.

[0105] In some implementations, neural induction medium is first supplied to the plated iPSCs, and additional medium is continuously or in discrete increments supplied to the culture during the culture period and before the culture is terminated.

[0106] In some embodiments, sufficient time for the formation of neuronal stem cells and / or early neural progenitor cells requires culturing the cells for approximately 1-10 days, approximately 2-8 days, or approximately 3-5 days. In some embodiments, sufficient time for the formation of neuronal stem cells and / or early neural progenitor cells requires culturing the cells for two, three, four, five, six, or seven days.

[0107] Preferably, the cells are cultured for a sufficient amount of time such that neuronal stem cells and / or early neural progenitor cells may constitute at least 50%, at least 75%, at least 85%, at least 95%, at least 99%, or about 100% of the cells in the culture.

[0108] In some implementations, the culture is a two-dimensional culture. As used herein, the term "two-dimensional culture" refers to cell culture on a flat cell culture substrate placed in a culture vessel.

[0109] The third stage of the culture method of the present invention includes: dissociating the neuronal stem cells and / or early neural progenitor cells of ii) to obtain a cell suspension that substantially contains single cells.

[0110] Suitable dissociation methods are known in the art. Dissociation steps may include enzymatic dissociation, mechanical dissociation, or a combination of both.

[0111] For example, enzymes can be used to detach cells from culture substrates, such as using enzyme cell separation solutions, like Accutase. TM Enzyme-based cell separation solutions such as Dispase, ReLeSR, or TrypLE can be used. Non-enzyme solutions, such as EDTA solutions, can also be used.

[0112] After dissociation, the dissociated cells were suspended in cryopreservation medium.

[0113] Cryopreservation medium refers to a culture medium containing cryoprotectants. A cryoprotectant is a substance with a high affinity for water molecules that inhibits the growth of ice crystals in the cryopreservation medium. 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), isopentyl glycol (IPG), dipropylene glycol (DPG), and glycerol.

[0114] The cryopreservation media include 10% medium 3 (CS10), 5% medium 2 (CS5), and 2% medium 1 (CS2), stem cell storage solution (stem cell banker), and Prime XV. freezis、hypothermasol CSB, trehalose, etc.

[0115] In some implementations, the dissociated cells are stored in a neutralization medium and then suspended in a cryopreservation medium.

[0116] In some implementations, the cells are stored in cryopreservation medium for at least 1, 2, 3, 6, 12, 18, 24, or 48 hours prior to cryopreservation.

[0117] The fourth stage of the culture method of the present invention includes: cryopreserving the cell suspension. In some embodiments, cryopreservation requires maintaining the temperature of the cell suspension at -80°C or lower. 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.

[0118] The fifth stage of the culture method of the present invention includes: allowing the cryopreserved cell suspension from the fourth stage to aggregate for a sufficient amount of time in the presence of an aggregate-forming culture medium to form neural organoids.

[0119] The cell suspension was placed in a culture vessel in the presence of an aggregate-forming medium.

[0120] Thawing of the frozen cell suspension. In some embodiments, thawing is carried out at 30°C to 40°C, preferably at 35°C to 38°C, and more preferably at about 37°C.

[0121] In several embodiments, the culture vessel includes a surface with topological features of various sizes, shapes, and depths, such as cavities or micropores. In several embodiments, the surface of the culture vessel includes one or more cavities or micropores.

[0122] Preferably, the culture container has a low-adhesion or non-adhesion surface. This low-adhesion surface prevents cells from binding to the container surface, thereby promoting aggregation.

[0123] In some implementations, aggregation involves forced spatial confinement of cells, such as cell centrifugation.

[0124] In one embodiment, the aggregate-forming medium comprises a basal medium supplemented with an inhibitor of rho-associated kinase (“ROCK”). Crucially, the aggregate-forming medium does not contain lineage-inducing factors. As used herein, lineage-inducing factors refer to compounds that induce differentiation into a neuronal lineage. Compounds that induce differentiation into a neuronal lineage include TACC3 inhibitors and MYC inhibitors. Appropriately, the aggregate-forming medium does not contain KHS101.

[0125] Preferably, the basal culture medium is supplemented with 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more of rock inhibitor, and the rock inhibitor is preferably Y-27632.

[0126] Suitable basal media for aggregate formation include BME medium, BGJB medium, CMRL 1066 medium, Glasgow MEM medium, modified MEM zinc selective medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, HAM medium, such as HAM's f-12 medium, RPMI 1640 medium, Fischer's medium, neural basal medium, and mixtures thereof. Preferably, the basal medium is Dulbecco modified Eagle MEM medium, and more preferably, DMEM medium with a nutrient mixture f-12 (DMEM / f12).

[0127] In some embodiments, the basal medium is supplemented with an N2 supplement. Preferably, the basal medium is a dmem-f12 medium with an N2 supplement. The concentration of the supplement may be from about 0.1% to 5%, from about 0.5% to 2.0%, or from about 1%.

[0128] In some embodiments, the aggregate-forming culture 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 concentration of non-essential amino acids or mem non-essential amino acids may be from about 0.1% to 5%, from about 0.5% to 2.0%, or from about 1%.

[0129] In some embodiments, the aggregate-forming medium also contains a glutamine product, such as glutamine or GlutaMAX. TM (1:100, Thermo Fisher Scientific, 35050079). The concentration of glutamine present can be approximately 0.1% to 5%, approximately 0.5% to 2.0%, or approximately 1%.

[0130] In some embodiments, the aggregate-forming culture medium further comprises heparin or heparin sulfate. The concentration of heparin or heparin sulfate present may be from about 0.1 to 5 μg / ml, from about 0.5 to 2.0 μg / ml, or from about 1 μg / ml.

[0131] In some embodiments, the aggregate formation medium comprises DMEM-f12 medium with N2, supplemented with:

[0132] -mem non-essential amino acids;

[0133] -Glutamine or GlutaMAX;

[0134] -Heparin or heparin sulfate; and

[0135] -Y-27632.

[0136] In some embodiments, the aggregate formation medium comprises DMEM-f12 medium with N2, supplemented with:

[0137] - Approximately 1% of non-essential amino acids;

[0138] - Approximately 1% GlutaMAX;

[0139] - Approximately 1 μg / ml heparin; and

[0140] - Approximately 50 μm y-27632.

[0141] In several embodiments, sufficient time is required to form neural organoids to culture cells for at least 12 hours; or to culture multiple cells together with 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.

[0142] As used herein, a “neural organoid” is an aggregate of neuronal stem cells and / or early neural progenitor cells with a three-dimensional structure. A neural organoid is a three-dimensional cell population formed by allowing cells to adhere to each other through suspension culture or 3D culture. The shape of a neural organoid is not particularly limited and can be spherical or non-spherical. The size of a neural organoid is not particularly limited, but it typically has an equivalent spherical diameter of 150 μm to 1000 μm, and for example, in one embodiment, the equivalent spherical diameter is 200 μm to 800 μm or 300 μm to 500 μm. A neural organoid typically comprises 500 to 150,000 cells, and for example, in one embodiment, it comprises 1,000 to 100,000 cells, 1,000 to 70,000 cells, or 3,000 to 30,000 cells.

[0143] Neural organoids may contain other cells and nerve cells. Preferably, neural organoids are substantially homogeneous. In some embodiments, neural organoids contain at least 60% or more, 70% or more, 80% or more, and more preferably 90% or more, or 95% or more nerve cells.

[0144] The neural organoids produced by the inventive method of this disclosure are useful as models of nervous system diseases and as tools for drug discovery. Neural organoids can be viewed as miniature models of neural organs, including the brain.

[0145] Screening trials typically involve exposing neural organoids to candidate drugs and determining the phenotypic effects on the neural organoids.

[0146] Neural organoids can be used as disease models for studying a variety of diseases related to neural tissue, including stroke, inflammatory brain disorders, neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), neuroinflammatory diseases (such as multiple sclerosis), traumatic injuries (such as injuries caused by brain surgery), channelopathy (such as epilepsy), and mental illnesses (including autism and schizophrenia).

[0147] Throughout the description and claims, the words “comprising” and “containing” and their variations mean “including, but not limited to,” and they are not intended (and do not) exclude other parts, additives, components, integers, or steps. Throughout the description and claims, the singular encompasses the plural unless the context requires otherwise. In particular, when the indefinite article is used, the description should be understood to consider both the plural and the singular unless the context requires otherwise.

[0148] Features, integers, properties, compounds, chemical parts, or groups described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel step or any novel combination of steps in any method or process so disclosed.

[0149] Readers are advised to note all papers and documents related to this application, submitted concurrently with or prior to this specification, and made publicly available together with this specification, the contents of which are incorporated herein by reference.

[0150] Example

[0151] Neural organoids are also referred to as Neuroyds or Neurooids in this paper, especially in the accompanying figures. Brain organoids are also referred to as Brainoyds or Brainoids in this paper, especially in the accompanying figures.

[0152] Methods for generating brain organoids are well known in the art and may include 3D differentiation of human pluripotent stem cells (hPSCs) into embryoid bodies (EBs), which are embedded in an extracellular matrix and cultured in a rotating bioreactor. The neural organoids of the present invention provide an in vitro method based on a chemically defined monolayer (2D) differentiation into pre-neural organoid cells and a cryopreservation and reassembly paradigm, as outlined in the examples.

[0153] Example 1 - Stem cell 2D differentiation to form a preneural organoid cell suspension

[0154] iPSCs were set at 200,000 cells / cm². 2 The cells were densely seeded on coated (extracellular matrix-laden) plates and cultured in PSC medium supplemented with 10 μM ROCK inhibitor (Y0503-5MG, reconstituted in H2O). After overnight adhesion, the medium was replaced with standard neural induction formulation medium. To achieve induced / accelerated postmitotic stem cell differentiation, the standard neural induction formulation medium was supplemented with the small molecule inhibitors stataprolamine (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 medium was supplemented daily for 4 consecutive days containing these compounds. On day 5, the adherent cells were dissociated into a single-cell suspension, incubated with 0.5 mM EDTA at 37°C for 4 minutes, and subsequently treated with an enzyme mixture (e.g., Accutase). Incubate at 37°C for 4 minutes. Cryopreserve the single-cell suspension in GMP-grade cryopreservation medium (e.g., StemCell Banker, AMS-Biotechnology, 11890) and store at -80°C or in LN2.

[0155] This plan is summarized as follows: Figure 1 As shown.

[0156] Example 2 - Formation of 3D Neural Organoids

[0157] To form a neural organoid from a cryopreserved suspension of preneurial organoid cells, 40,000 differentiated neural cells were seeded at 100 μl per well in a microtiter plate and dispensed at a rate of 0.25 ml / cm². 2Use a standard neural induction formulation supplemented with 50 μM ROCK inhibitor (Y0503-5 mg, reconstituted in H2O). Rotate the plate at 400 g for 5 minutes at room temperature and incubate the neural organoids for at least 16 hours to form a homogeneous structure (critical step). Remove half of the plate medium (50 μl) and replace it with 100 μl of fresh standard neural induction formulation (minus the ROCK inhibitor, statoxamine, and KHS101). After a culture period of at least 16 hours, the neural organoids are ready for the desired assays. Figure 2 ).

[0158] Table 1 shows the composition of the various culture media used in the examples.

[0159] Table 1

[0160]

[0161]

[0162] The advantages of the method of the present invention compared with known methods in the art are summarized in Table 2.

[0163] Table 2

[0164]

[0165]

[0166] Example 3 - Characterization of 3D Neural Organoids

[0167] The formation of neural organoids and standard brains was as described in Examples 1 and 2 (paragraphs 82-89), or using commercially available embryoid-based organoid formation protocols (Stem Cell Technologies, Table 2). For early time-point morphological characterization, neural organoids were cultured for 96 hours (with medium changed at 48 hours; Figure 3A). For long-term maturation, neural organoids were embedded in Matrigel droplets on parafilm dips prepared in empty P200 tip boxes. The embedded organoids were cured at 37°C for 30 minutes and then transferred to 10 cm culture dishes containing organoid maturation medium (-vitamin A). After 48 hours, most of the medium was removed and replaced with fresh organoid maturation medium (-vitamin A). After another 48 hours, the organoids were transferred to roller bottles containing organoid maturation medium (+vitamin A) and matured until desired.

[0168] For immunofluorescence characterization, neural organoids or standard brain organoids (see Table 2) were transferred to 24-well plates using 1 mm pipette tips. The culture medium was removed, and the organoids were washed with PBS. The PBS was then removed, and 4% paraformaldehyde solution (Thermo, 15670799) was added, and the organoids were incubated at 4°C for 15 minutes (15 minutes for early-stage organoids, or overnight for mature organoids 36+ days old). The organoids were then washed with PBS and incubated overnight with 30% sucrose solution (Sigma, S9378) or until they settled to the bottom of the wells. The sucrose solution was then removed and replaced with a warm 7.5% gelatin (Sigma, G2500) 10% sucrose solution and incubated at 37°C for at least 30 minutes. The organoids were then transferred to cryoforms pre-coated with gelatin / sucrose solution. The blocks were cryosectioned into 20 μm sections using a cryostat.

[0169] For immunofluorescence staining, slides were washed three times with PBS and incubated for 1 hour at room temperature with blocking buffer (1% FBS, 0.3% Triton X-100 (Sigma, X100)). The slides were then incubated with primary antibody diluted in blocking buffer for 1 hour at room temperature or overnight at 4°C. Afterwards, the slides were washed three times with PBS and incubated for 1 hour at room temperature in the dark with secondary antibody diluted in blocking buffer. The slides were then washed three times with PBS and incubated for 10 minutes at room temperature in the dark with 2 μg / mL DAPI solution diluted in PBS (Sigma, D9542). As a next step, the slides were washed twice with PBS and then mounted onto polylysine-coated slides (VWR, 631-9483) using an aqueous mounting agent (2B Scientific, H-5501-60). The slides were imaged using an inverted fluorescence microscope and quantified using the CellProfiler analysis pipeline.

[0170] Primary resistance

[0171] target protein host species Manufacturer Catalog Number TUJ1 mice Biolegend 801202 MAP2 chicken Abcam ab5392 GFAP rabbit Agilent Z033429-2

[0172] Secondary antibody

[0173] target host host species Coupled Manufacturer Catalog Number mice goat Alexa Fluor 488 Invitrogen 10696113 rabbit goat Alexa Fluor 546 Invitrogen 10789154 chicken rabbit Alexa Fluor 647 Agilent Z033429-2

[0174] For single-cell sequencing analysis, organoids were incubated with Cell Recovery Solution (Thermo, 354253) at 4°C for 1 hour and dissociated using the Neurosphere Dissociation Kit (Miltenyi Biotec, 130-095-943). Dead cells and cell debris were removed using a dead cell removal kit (Miltenyi Biotec, 130-090-101). Cells were counted and frozen in aliquots of 1 million to 2 million in 500 μL of GMP-grade cryopreservation medium (e.g., Stem Cell Banker). Cells were then processed according to a 10×3' gene expression protocol for single-cell sequencing. The resulting data were then mapped to the human GRCh38 reference genome using Cell Ranger (7.1.0). All subsequent analyses were performed using the online single-cell sequencing analysis tool Bioturing.

[0175] Example 4 - at -80 ℃ Quantification of morphological changes after storage for up to 12 weeks

[0176] Aspirate the culture medium from the induced cells and wash the wells with 1 mL of PBS. Discard the PBS and add 1 mL of 0.5 mM EDTA, then incubate the cells at 37°C for 4 minutes. Slowly remove the EDTA using a P1000 pipette and add 1 mL of Accutase (e.g., Sigma Aldrich A6964), then incubate the cells at 37°C for 4 minutes. Gently resuspend the cells in mTeSR medium using a P1000 pipette and transfer them to 15 mL centrifuge tubes. Centrifuge at 300 x g for 5 minutes, discard the supernatant, and resuspend the cells in 1 mL of neural induction medium supplemented with 50 μM ROCK inhibitor for cell counting. Mix 10 μL of cells with 10 mL of a live-dead cell distinguishing dye (e.g., trypan blue) and count the cells using a Counterss automated cell counter. Then, freeze the cells in 500 μL of GMP-grade freezing medium (e.g., Stem Cell Banker) in a 2 million cell aliquot. Cells were frozen at a rate of 1 °C per minute until reaching -80 °C. Cells were stored at -80 °C for 1, 6, and 12 weeks. Upon resuscitation, cells were thawed in a water bath set to 37 °C for approximately 3 minutes and transferred to 15 mL tubes. At least 5 mL of culture medium was then added dropwise to the cells, and the cell suspension was rotated at 300 g for 5 minutes. The cell pellets were 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 Counterss automated cell counter. Cells were then seeded at 100 μL per well in 96-well ultra-low adhesion neural induction medium containing 50 μM ROCK inhibitor in ultra-low adhesion plates at a rate of 40,000 cells per well. 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). A combination of the machine learning image analysis tool ilastik and the image analysis tool Cell Profiler was used to measure morphological parameters.

Claims

1. An in vitro method for producing neural organoids, comprising: i) In the presence of a culture medium that maintains pluripotency, induced pluripotent stem cells (iPSCs) are plated on a cell culture substrate; ii) In the presence of neural induction medium, the iPSCs plated in i) are cultured for a sufficient amount of time to form neuronal stem cells (NSCs) and / or early neural progenitor cells (eNPCs); iii) Dissociate the neural stem cells (NSCs) and / or early neural progenitor cells (eNPCs) of ii) to obtain a cell suspension containing single cells; iv) Cryopreservation of the cell suspension; v) In the presence of an aggregate-forming medium, the cryopreserved cell suspension from iv) was aggregated for a sufficient amount of time to form neural organoids.

2. The method according to claim 1, wherein, The cell culture substrate contains extracellular matrix components.

3. The method according to claim 1 or claim 2, wherein, The cell culture substrate does not contain feeder cells.

4. The method according to any one of claims 1 to 3, wherein, The culture medium that maintains pluripotency is a feeder-free culture medium.

5. The method according to claim 4, wherein, The culture medium for maintaining pluripotency is selected from the following feeder-free culture media: Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, stabilized Essential 8 medium, and StemFit medium.

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

7. The method according to any one of claims 1 to 6, wherein, The neural induction medium is a modified Eagle medium (DMEM) from Dulbecco.

8. The method according to claim 7, wherein, The DMEM is a DMEM culture medium (DMEM / F12) containing a nutrient mixture F-12.

9. The method according to any one of claims 1 to 8, wherein, The neural induction medium is supplemented with N2.

10. The method according to any one of the preceding claims, wherein, The neural induction medium was supplemented with a TACC3 inhibitor.

11. The method according to claim 10, wherein, The TACC3 inhibitor is KHS101.

12. The method according to any one of the preceding claims, wherein, The neural induction medium was supplemented with a c-MYC inhibitor.

13. The method according to claim 12, wherein, The c-MYC inhibitor is stapoxetine.

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

15. The method according to any one of the preceding claims, wherein, The neural induction medium does not contain serum.

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

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

18. The method according to any one of the preceding claims, wherein, The neural induction culture medium contains N2 supplement, heparin, and GlutaMAX. TM DMEM / F12 medium containing MEM non-essential amino acids, TACC3 inhibitors and c-MYC inhibitors, wherein the c-MYC inhibitor is optionally stataprolamine and the TACC3 inhibitor is khs101.

19. The method according to any one of the preceding claims, wherein, The iPSCs cultured in the presence of neural induction medium are 2D cultures.

20. The method according to any one of the preceding claims, wherein, The iPSCs plated in the presence of a neural induction medium are cultured for a period of at least 2, 3, 4 or 5 days, preferably 2-10 days, 4-9 days or 3-5 days.

21. The method according to any one of the preceding claims, wherein, Dissociation includes enzymatic dissociation and / or mechanical dissociation.

22. The method according to any one of the preceding claims, wherein, The dissociated cells were suspended in cryopreservation medium to form a cell suspension.

23. The method according to any one of the preceding claims, wherein, The cell suspension was stored at a cryopreservation temperature.

24. The method according to any one of the preceding claims, wherein, The cell suspension was stored at a cryopreservation temperature for at least 12, 13, 14, 15, 16, 17, 20, 30, 40 or 50 weeks, and the loss of the product diameter after aggregation was no more than 5%.

25. The method according to any one of the preceding claims, wherein, The cell suspension is stored at a cryopreservation temperature for at least 12 weeks, and the loss of the product diameter after aggregation is no more than 5%.

26. The method according to any one of the preceding claims, wherein, The frozen cell suspension was thawed before aggregation.

27. The method according to any one of the preceding claims, wherein, Aggregates were formed in aggregate-forming media that did not contain neural lineage inducing factors.

28. The method according to any one of the preceding claims, wherein, The aggregate formation medium is a modified Eagle medium (DMEM) from Dulbecco.

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

30. The method according to any one of the preceding claims, wherein, The aggregate-forming culture medium is supplemented with N2.

31. The method according to any one of the preceding claims, wherein, The aggregate-forming culture medium is supplemented with heparin or heparin sulfate.

32. The method according to any one of the preceding claims, wherein, The aggregate-forming culture medium does not contain serum.

33. The method according to any one of the preceding claims, wherein, The aggregate-forming culture medium is supplemented with glutamine or GlutaMAX. TM .

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

35. A neural organoid that can be obtained or acquired by the method of any one of claims 1-34.

36. The neural organoid of claim 335, for use in medical or diagnostic purposes.

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

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