Whole-brain organoid based on human induced pluripotent stem cells as well as construction method and application of whole-brain organoid

Abstract

The invention provides a whole-brain organoid based on human induced pluripotent stem cells as well as a construction method and application thereof, and belongs to the technical field of organoid construction. The method comprises the following steps: culturing human induced pluripotent stem cells in a complete culture medium to obtain a cell aggregate; culturing in an embryoid body forming culture medium to obtain a spherical embryoid body; culturing in a nerve induction culture medium tissue to form a nerve epithelium structure; embedding the single embryoid body with the nerve epithelium structure in a Matrigel gel drop, culturing in a nerve expansion culture medium to form a nerve epithelium-like structure, and replacing a mature culture medium for culturing to obtain the whole-brain organ. According to the method, by dynamically and accurately regulating and controlling culture parameters, the control ability of a cell differentiation area is improved, and interference of non-target cell types is reduced, so that the efficiency and consistency of embryoid formation and neural differentiation are directly improved, and finally, the obtained mature organoid has a more complete epithelial structure and cell polarity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organoid construction, and particularly relates to a whole-brain organoid based on human induced pluripotent stem cells and a construction method and application thereof. BACKGROUND

[0002] The complexity of the human brain makes its related scientific research extremely dependent on reliable model systems. Traditional ischemic stroke models include: (1) two-dimensional cell models: single-layer culture derived from tumorized neural cell lines or primary cells. To construct a two-dimensional cell OGD / R model, the target cells are first cultured under normal conditions until they adhere to the wall and fuse, then the normal culture medium is removed and replaced with a sugar-free culture medium saturated with anoxic gas, and the cells are placed in an anoxic chamber or a three-gas incubator for a specific period of anoxic / sugar-free culture to simulate the ischemic stage; thereafter, the normal culture medium containing glucose is replaced again, and the cells are moved back to the regular oxygen-containing culture box for further culture for a specific period to simulate the reperfusion injury, thereby successfully establishing an OGD / R model in vitro for mechanism research and drug screening. (2) Animal models: to construct an animal ischemia / reperfusion model, the core is to temporarily block the main blood vessels of a specific organ (such as the brain, heart or kidney) of an animal through surgical means, causing severe ischemia and energy metabolism disorder in the local tissue of the organ to simulate ischemic injury; after a predetermined ischemic time, the blood vessel obstruction is removed to restore blood flow, thereby simulating the "reperfusion" process and causing typical reperfusion injury. Specifically, taking the construction of the most classic middle cerebral artery occlusion model as an example, the animal needs to be anesthetized first, then the carotid artery and its branches are exposed through a midline incision of the neck, a suture line with a coating is inserted into the internal carotid artery through the external carotid artery, and then pushed forward until the starting part of the middle cerebral artery is blocked, at which time the blood flow in the brain is suddenly reduced; after a certain time (for example, 60 or 90 minutes) of occlusion, the suture line is slowly withdrawn to restore blood flow, thereby achieving reperfusion, and the infarct volume, neural cell death and inflammatory response, and other reperfusion injury indicators are evaluated through neurobehavioral scoring, histological staining and molecular biology techniques. In recent years, breakthrough progress has been made in brain organoid technology based on human pluripotent stem cells (iPSC). This technology is mainly achieved through the following main steps: iPSCs are aggregated to form embryoid bodies (EBs), then neural induction is performed in a three-dimensional matrix using specific factors, and brain organoids with specific neuroepithelial structures are spontaneously differentiated. Such organs contain a variety of neural cell types and can partially simulate the development and structure of the brain.

[0003] However, all of the above models have certain problems. Two-dimensional cell models have significant limitations because they cannot simulate the three-dimensional microenvironment and complex cell-cell interactions in vivo. In traditional two-dimensional culture, cells grow on a rigid, planar plastic surface, altering their morphology, polarity, and movement, losing the spatial structure and mechanical sensation they should possess in three-dimensional tissues. More importantly, this simplified system severely lacks the complex cell communication networks in vivo; it cannot reproduce the dynamic regulation between different cell types (such as neurons and glial cells, cardiomyocytes and fibroblasts) through direct contact and paracrine signals, nor can it simulate the profound influence of extracellular matrix biochemical and physical signals on cell fate. Therefore, data obtained based on two-dimensional models often deviate significantly from the actual in vivo situation when explaining physiological and pathological processes such as cell differentiation, migration, metabolism, and drug response, greatly limiting our understanding of the essence of complex life phenomena such as ischemia-reperfusion injury. Although animal models can provide a holistic physiological environment integrating the nervous, vascular, and immune systems, and have irreplaceable value in understanding disease mechanisms, their application still faces multiple inherent limitations. Besides the translational bottlenecks caused by fundamental differences in brain structure, gene expression, and neural circuit function among species, the construction of animal disease models is often cumbersome and technically challenging. Furthermore, the models are prone to unpredictability due to individual differences and environmental interference during experiments. These factors collectively limit their reliability and applicability in simulating the pathological characteristics of complex human brain diseases, especially mental illnesses. While existing neural organoid culture techniques can simulate human brain development, they have certain limitations. On the one hand, poor control over cell differentiation regions easily leads to non-ideal differentiated cell types, affecting embryoid formation efficiency and cell aggregation, thus impacting organoid culture and the success rate of differentiation of various neural cells. On the other hand, the culture process (such as the temperature-sensitive Matrigel manipulation) is cumbersome and lacks stability, with demanding experimental conditions resulting in low reproducibility and hindering large-scale application. Moreover, the success of the technology highly depends on the high quality and homogeneity of the initial iPSC cell line. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a whole-brain organoid based on human induced pluripotent stem cells, its construction method and application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing whole-brain organoids based on human induced pluripotent stem cells, comprising the following steps: (1) Human induced pluripotent stem cells were cultured in a complete culture medium to obtain a cell suspension, and the cell suspension was cultured to obtain cell aggregates; (2) culturing the cell aggregate in a embryoid body formation medium to obtain a spherical embryoid body; (3) culturing the spherical embryoid body in a neural induction medium to form a neuroepithelial structure; (4) embedding the single embryoid body with the formed neuroepithelial structure in a Matrigel droplet, culturing in a neural expansion medium to form a neuroepithelial-like structure, and culturing in a maturation medium to obtain a whole brain organoid.

[0006] Preferably, the complete medium in step (1) is a DMEM / F12-based medium, further comprising the following components at the following concentrations: 60-70 mg / L L-ascorbic acid-2-phosphate magnesium, 10-20 µg / L sodium selenite, 80-120 µg / L FGF2, 15-25 mg / L insulin, 500-600 mg / L NaHCO3, 5-15 mg / L transferrin, 1-3 µg / L TGFβ1, 5-15 mg / mL BSA, 0.03-0.08 mM β-mercaptoethanol.

[0007] Preferably, the seeding density of the cell aggregate in step (2) is 6000 cells / well, the embryoid body formation medium comprises DMEM / F12, fibroblast growth factor and Y27632, the concentration of the fibroblast growth factor is 2-6 ng / mL, the concentration of the Y27632 is 5-15 mM, the volume ratio of the DMEM / F12, fibroblast growth factor and Y27632 is 2-6 mL:0.5-2 mL:3-8 µL, and the culturing time is 4-8 days.

[0008] Preferably, the neural induction medium in step (3) is prepared by mixing brain organoid basal medium 1 and brain organoid supplement A at a volume ratio of 99:1.

[0009] Preferably, the concentration of the Matrigel droplet in step (4) is 3.5-4 mg / mL, the embedding is followed by standing, the standing temperature is 35-40 °C, and the standing time is 20 min.

[0010] Preferably, the neural expansion medium in step (4) is prepared by mixing brain organoid basal medium 2, brain organoid supplement B and brain organoid supplement C at a volume ratio of 98:2:1.

[0011] Preferably, the maturation medium in step (4) is prepared by mixing brain organoid basal medium 2 and brain organoid supplement D at a volume ratio of 49:1.

[0012] Preferably, the frequency of the replacement in step (4) is once every 2 days, and after the 20th day of replacement of the mature culture medium, Matrigel matrix gel is added to the mature culture medium at a ratio of 1:100 at the same time of replacement of the culture medium, and the concentration of the Matrigel matrix gel is 3.5-4 mg / mL.

[0013] The application also provides the whole brain organoids constructed by the construction method.

[0014] The application also provides application of the whole brain organoids in research of neurological brain diseases or drug screening.

[0015] Compared with the prior art, the application has the following beneficial effects: The application improves the control ability of the cell differentiation area, reduces the interference of non-target cell types, and thus directly improves the efficiency and consistency of the embryoid body formation and neural differentiation by realizing dynamic and accurate regulation of the culture parameters. On the other hand, by standardizing the operation of the key materials such as Matrigel, the stability problem of the original process which is complicated and sensitive to temperature is overcome. Finally, these improvements reduce the harsh requirements for the starting cell lines, so that the technology as a whole presents better repeatability.

[0016] The application optimizes the number of starting induced cells to 6000 / well according to the specific state and proliferation rate of the iPSCs used; the treatment time of the four key differentiation stages is finely regulated according to the morphology and growth of the embryoid bodies at different stages, for example, the time of EBs formation stage is appropriately prolonged; the suitable gel setting time of the matrix gel after wrapping is determined to be 20 minutes through pre-experiments, which effectively guarantees the uniformity of the subsequent organoid structure and the experimental repeatability; the expression dynamics of the identity markers from the pluripotent state to different key brain regions are systematically verified at the molecular level, and the neural differentiation efficiency and regionalization characteristics of the organoids are comprehensively evaluated; according to the growth state of the organoids at the mature stage, the frequency of medium replacement is adjusted to once every two days, and the finally obtained organoids have more complete epithelial structure and cell polarity, which are more suitable for subsequent construction of hypoxia / reperfusion injury models. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a microscope image (4x) of human iPSCs in good condition and capable of continuous passage; Figure 2 is a microscope image of iPSC cells that can be induced; Figure 3 embryoid bodies formed after 24h of induction; Figure 4 is an embryoid body after 48h of induction; Figure 5 is an embryoid body grown to the fourth day; Figure 6 is a blastula grown to the sixth day; Figure 7 is a blastula grown to the eighth day; Figure 8 is a blastula grown to the eleventh day of budding; Figure 9 is a mature organoid; Figure 10 is the result of 16 marker qRT-PCR verification (blue represents the iPSC group; red is the whole brain organoid group); Figure 11 is the result of immunofluorescence identification; Figure 12 is the result of optimization of the initial cell density for induction; Figure 13 is the influence of the processing time of key differentiation stages on the morphology of organoids; Figure 14 is the influence of different matrix gel setting times on the morphology of blastulas; Figure 15 is the influence of key culture parameters on the mature morphology of organoids; Figure 16 is the result of immunofluorescence identification of the hypoxia-reperfusion (OGD / R) model (morphological changes of brain organoids after OGD / R modeling); Figure 17 is the result of CCK-8 detection of the hypoxia-reperfusion (OGD / R) model. DETAILED DESCRIPTION

[0018] The present application provides a method for constructing a whole brain organoid based on human induced pluripotent stem cells, comprising the following steps: (1) culturing human induced pluripotent stem cells in complete culture medium to obtain a cell suspension, and culturing the cell suspension to obtain a cell aggregate; (2) culturing the cell aggregate in blastula formation culture medium to obtain a spherical blastula; (3) culturing the spherical blastula in a neural induction culture medium to form a neural epithelial structure; (4) embedding a single blastula with a formed neural epithelial structure in a Matrigel gel droplet, culturing in a neural expansion culture medium to form a neural epithelial-like structure, and replacing the culture medium with a mature culture medium to obtain a whole brain organoid.

[0019] In the present application, the human induced pluripotent stem cells are cultured in a complete culture medium to obtain a cell suspension, and the cell suspension is cultured to obtain a cell aggregate. The complete culture medium is a DMEM / F12-based culture medium, and further comprises the following components at the following concentrations: 60-70 mg / L L-ascorbic acid-2-phosphorus magnesium, 10-20 μg / L sodium selenite, 80-120 μg / L FGF2, 15-25 mg / L insulin, 500-600 mg / L NaHCO3, 5-15 mg / L transferrin, 1-3 μg / L TGFβ1, 5-15 mg / mL BSA, 0.03-0.08 mM β-mercaptoethanol; the concentration of the L-ascorbic acid-2-phosphorus magnesium is preferably 62-68 mg / L, and further preferably 64 mg / L; the concentration of the sodium selenite is preferably 12-18 μg / L, and further preferably 14 μg / L; the concentration of the FGF2 is preferably 90-110 μg / L, and further preferably 100 μg / L; the concentration of the insulin is preferably 17-22 mg / L, and further preferably 19.4 mg / L; the concentration of the NaHCO3 is preferably 520-580 mg / L, and further preferably 543 mg / L; the concentration of the transferrin is preferably 8-12 mg / L, and further preferably 10.7 mg / L; the concentration of the TGFβ1 is preferably 1.5-2.5 μg / L, and further preferably 2 μg / L; the concentration of the BSA is preferably 8-12 mg / mL, and further preferably 10 mg / mL; the concentration of the β-mercaptoethanol is preferably 0.04-0.06 mM, and further preferably 0.05 mM.

[0020] In the present application, the cell aggregate is cultured in a blastocyst formation culture medium to obtain a spherical blastocyst. The seeding density of the cell aggregate is 6000 cells / well, and the blastocyst formation culture medium comprises DMEM / F12, fibroblast growth factor, and Y27632, the concentration of the fibroblast growth factor is preferably 2-6 ng / mL, further preferably 3-5 ng / mL, and more further preferably 4 ng / mL; the concentration of the Y27632 is preferably 5-15 mM, further preferably 8-12 mM, and more further preferably 10 mM; the volume ratio of the DMEM / F12, the fibroblast growth factor, and the Y27632 is preferably 2-6 mL:0.5-2 mL:3-8 μL, further preferably 3-5 mL:0.8-1.5 mL:4-7 μL, and more further preferably 4 mL:1 mL:5 μL; the culture time is preferably 4-8 d, and further preferably 6 d.

[0021] In the present application, the spheroid-like embryo is cultured in a neural induction medium to form a neuroepithelial structure. The neural induction medium is prepared by mixing brain organoid basal medium 1 and brain organoid supplement A at a volume ratio of 99:1.

[0022] In the present application, the single spheroid-like embryo forming a neuroepithelial structure is embedded in a Matrigel droplet, cultured in a neural expansion medium to form a neuroepithelial-like structure, and then cultured in a mature medium to obtain a whole brain organoid. The concentration of the Matrigel droplet is 3.5-4 mg / mL; the embedding is followed by a resting period, and the temperature of the resting period is preferably 35-40℃, further preferably 36-39℃, and more preferably 37℃; the resting period lasts for 20 min; the neural expansion medium is prepared by mixing brain organoid basal medium 2, brain organoid supplement B and brain organoid supplement C at a volume ratio of 98:2:1; the mature medium is prepared by mixing brain organoid basal medium 2 and brain organoid supplement D at a volume ratio of 49:1; the frequency of medium replacement is once every 2 days, and after the 20th day of replacement of the mature medium, Matrigel matrix gel is added to the mature medium at a ratio of 1:100 at the time of medium replacement, and the concentration of the Matrigel matrix gel is 3.5-4 mg / mL.

[0023] The present application also provides a whole brain organoid constructed by the construction method.

[0024] The present application also provides an application of the whole brain organoid in research of neurological and brain diseases or drug screening.

[0025] In the present application, the application of the whole brain organoid specifically includes: (1) exploring the human brain neural development mechanism in combination with the differentiation and maturation process of the whole brain organoid in the field of neuroscience; (2) constructing different brain disease models to explore the disease occurrence and development mechanism in the field of brain major disease research; (3) performing high-throughput screening and pharmacodynamic evaluation of central nervous system drugs in the field of neuropharmacological research; (4) providing an organoid model for the neural toxicity evaluation of environmental exposure factors and toxic and harmful substances in the field of neurotoxicity research; (5) constructing patient tissue-derived whole brain organoids to carry out drug screening of brain major diseases and promote the development of personalized medicine in the field of precision medicine research; (6) reducing the use of animal experiments and alleviating the death of a large number of animals in the aspect of medical ethics.

[0026] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0027] Example 1

[0028] I. Method of constructing whole brain organoids based on human iPSCs, the steps are as follows: 1. Culture and preparation of human iPSCs Human iPSC cell line (cell source: purchased from ATCC; ATCC number: ACS-1011TM) was used.

[0029] (1) Reagents and materials for culture

[0030] iPS cell complete medium kit (purchased from HAKATA); Matrigel matrix glue (5 mL purchased from HAKATA); iPS cell digestion solution (0.5 mM EDTA).

[0031] (2) Culture process

[0032] a. Preparation and coating of Matrigel working solution: Matrigel was placed in a 4°C refrigerator overnight to fully melt, and an appropriate volume of PBS diluent was pre-cooled to 4°C. After melting, quickly take out the Matrigel and pre-cooled PBS, open the tube cap, take a small amount of PBS pre-rinse the tube wall with a pre-cooled pipette, then add it to the Matrigel original tube and mix gently. Then transfer the Matrigel to the PBS diluent and mix gently. (Because Matrigel is prone to gelation above 15°C, it will adhere to the tube wall or the surface of the pipette, so all operations must be completed quickly in a low temperature environment, and the pipette must be pre-cooled to avoid gelation). After mixing, immediately use the diluted Matrigel (0.035~0.04 mg / mL) to coat the culture vessels. For a 6-well plate, add 1 mL per well; for a T25 culture bottle, add 3 mL per bottle. Gently shake the culture plate / bottle to evenly cover the bottom surface. Place the coated culture vessels in a 37°C incubator for 1 h, and they are ready for use.

[0033] b. Cell thawing: Prior to thawing procedure, sterile test tubes, pre-warmed complete medium and coated culture vessels are prepared to ensure a smooth and continuous process. After removing the cryo-vial from liquid nitrogen, it is immediately placed in a cell thawing apparatus for thawing until only a small amount of ice crystals remain in the cryo-preserved solution. The cryo-vial surface is disinfected by wiping with 70% ethanol. The cryo-preserved solution containing iPCS cells is gently transferred into a 50 mL centrifuge tube containing 2 mL of pre-warmed complete medium using a pipette, taking care to avoid vigorous pipetting to prevent cell clumps from being dispersed. The cell suspension is centrifuged at 300 x g for 5 minutes at room temperature. The supernatant is carefully aspirated, taking care not to disturb the intact cell pellet. One mL of complete medium [DMEM / F12 (Gibco, Cat# 11330057), 64 mg / L L-Ascorbic acid-2-phosphate magnesium, 14 µg / L Sodium selenite, 100 µg / L FGF2, 19.4 mg / L Insulin, 543 mg / L NaHCO3, and 10.7 mg / L Transferrin, 2 µg / L TGFβ1, Albumin (BSA, 10 mg / mL), 0.05 mM β-mercaptoethanol] is slowly added to the centrifuge tube, and the cell pellet is gently detached and evenly dispersed by flicking the tube bottom with a finger. Subsequently, the 1 mL cell suspension is gently transferred into the coated culture dish / plate / flask. The culture vessel is moved into a 37 °C incubator for incubation, and the medium is changed the next day.

[0034] c. Cell passaging: When the cell colonies significantly increase in size, the central region becomes dense and bright (clearly contrasted with the edges), and the adjacent colonies begin to contact and fuse, the cells can be passaged (as shown in Figure 1 ). Prior to passaging, 37 °C pre-warmed calcium- and magnesium-free PBS containing 0.5 mM EDTA is prepared. After aspirating the original medium, the cells are gently washed twice with pre-warmed PBS. Then, an appropriate amount of pre-warmed 0.5 mM EDTA (reference amount: 1 mL per well for a 6-well plate, and 2 mL for a T25 flask) is added, and the cells are incubated at 37 °C for 1-2 minutes. When most of the colony edges are rolled up and the internal cells are detached in clumps under a microscope, an equal volume of complete medium is immediately added to terminate the digestion. The cell suspension after digestion is transferred to a centrifuge tube and centrifuged at 300 x g for 5 minutes. The supernatant is discarded, and the cell pellet is resuspended in an appropriate amount of complete medium and passaged at a ratio of 1:3. The passaged culture vessel is placed in a 37 °C incubator for further incubation, and the medium is changed the next day.

[0035] 2. Standardized embryoid body (EB) formation

[0036] (1) When most of the cell colonies significantly increase in size, are closely arranged, and exhibit a typical multi-layered dense structure in the central region, while the cell confluence reaches about 70% and the differentiation ratio is less than 10% (as shown inFigure 2 iPSCs in logarithmic growth phase were collected with gentle cell dissociation reagent (GCDR) (1 ml per well of 6-well plate) as shown in FIG. 1. The collected cells were resuspended and accurately counted for subsequent passage or experimental use.

[0037] (2) Cells were seeded in ultra-low attachment 96-well plates at a density of 6000 cells per well and cultured with embryoid body formation medium [DMEM / F12 DMEM / F12 (Gibco, Cat# 11330057) 4 mL, Basic Fibroblast Growth Factor (4 ng / mL) 1 mL, 10 mM Y27632 (HAKATA, Cat# H-A005) 5 μL]. After 24 hours, regular and uniform spherical embryoid bodies (EBs) were formed (as shown in FIG. 2). Figure 3

[0038] 3. Temporal neural induction and patterning

[0039] (1) On day 2, the embryoid bodies were more regular in shape and about 350 μm in diameter (as shown in FIG. 3). Figure 4

[0040] (2) On day 4, the diameter of the embryoid bodies increased to about 440 μm, and the shape was round with smooth edges (as shown in FIG. 4). At this time, 100 μL of embryoid body formation medium (EB Formation Medium) was added to each well of the 96-well plate. Figure 5

[0041] (3) On day 6, the embryoid bodies were observed under a microscope. When the diameter reached about 510 μm, the shape was regular and the edges were smooth (as shown in FIG. 5), the second stage operation was started. Figure 6

[0042] Single embryoid bodies were transferred to low-attachment 24-well plates using wide-bore pipettes (inner diameter ≥ 1.5 mm), and 0.5 mL of neural induction medium [Brain Organoid Base Medium 1 (purchased from STEMdiff™, Cat# 08572) and Brain Organoid Supplement A (purchased from STEMdiff™, Cat# 08575) were prepared in a ratio of 99:1] was added to each well. This stage was continued until day 8.

[0043] 4. Three-dimensional maturation culture and vascularization strategy

[0044] On day 8, the formed embryoid bodies (EBs) were visible to the naked eye, showing the typical morphology of smooth edges and translucency (as shown in FIG. 6).​​​​Figure 7 At this point, the neural epithelial structures are formed and the third stage of culture can be initiated.

[0045] The single EBs were embedded in 15 pL Matrigel® droplets and incubated at 37 °C for 20 min to allow the construction of a three-dimensional culture environment. Subsequently, the gel pieces were gently washed with neural expansion medium [prepared from STEMdiff™ Brain Organoid Basal Medium 2 (purchased from STEMdiff™, Cat. No. 08573), STEMdiff™ Brain Organoid Supplement B (purchased from STEMdiff™, Cat. No. 08576) and STEMdiff™ Brain Organoid Supplement C (purchased from STEMdiff™, Cat. No. 08577) in a ratio of 98:2:1] and transferred to a low-attachment 6-well plate (6 gel pieces per well). This stage was continued until day 11.

[0046] By day 11, the embedded organoids further developed and increased in volume, and formed typical neuroepithelial-like structures with visible spore-like protrusions on their surface (as shown in Figure 8 ).

[0047] 5. Organoid maturation stage

[0048] From day 11, the original medium in the 6-well plate was aspirated using a 5 mL Pasteur pipette and replaced with 3 mL maturation medium [STEMdiff™ Brain Organoid Basal Medium 2 (purchased from STEMdiff™, Cat. No. 08573) and STEMdiff™ Brain Organoid Supplement D (purchased from STEMdiff™, Cat. No. 08578) in a ratio of 49:1] per well. The culture plates were then placed on a horizontal shaker (65 rpm) for continued culture. The maturation medium was replaced every 2 days thereafter, and at the fourth stage on day 30, 3.5-4 mg / mL Matrigel® matrix gel was added to the medium at a ratio of 1:100 to promote the stability of the organoid structure until the organoids were matured on day 40 (as shown in Figure 9 ).

[0049] 6. Identification of mature organoids

[0050] (1) qRT-PCR validation of 16 key markers

[0051] To verify the expression of 16 key markers, mature organoid samples and control iPSC samples were collected separately, and total RNA was extracted using TRIzol™ reagent. After detection, the A260 / A280 ratio of all RNA samples was between 1.8 and 2.0, meeting the quality requirements. Then, equal amounts of RNA were reverse transcribed into cDNA using a PrimeScript™ RT kit. The obtained cDNA was stored at -80°C for later use. The qPCR reaction was performed using cross-exon primers and TB Green Premix Ex Taq II on a QuantStudio™ 5 system, and the reaction program included: 95°C pre-denaturation for 30 seconds; followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds; finally, a melting curve analysis was performed. TBP was used as an internal reference gene, and each sample was set with technical repeats, and the Ct average value was taken, and the Log2 Fold Change of each target gene relative to the control group was calculated using the 2^(-ΔΔCt) method.

[0052] Experimental results: as shown in Figure 10 Compared with the parental iPSCs, the expression of pluripotency markers (OCT4, NANOG) in the organoids was significantly down-regulated, while the neural lineage-related markers (including SOX1, PAX6, etc. 14) were generally up-regulated. The down-regulation of pluripotency markers indicates that the stem cells in the organoids have successfully initiated the neural differentiation program. Further analysis showed that the markers involved in multiple key stages such as neural identity recognition, brain region development (such as ventricular region, forebrain and hindbrain), intermediate progenitor cells and cortical plate / neurons were significantly up-regulated, and the expression pattern covered multiple main divisions in the brain development process. These results collectively suggest that the organoids constructed in the present application have the potential to simulate the early brain regional development process in terms of molecular characteristics.

[0053] (2) Immunofluorescence identification

[0054] First, the organoid samples were fixed in 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS for 3 times, and temporarily stored in 4°C PBS. After paraffin sections were deparaffinated with environmentally friendly deparaffinizing solution and gradient ethanol, hydrated, antigen repair was performed and naturally cooled to room temperature. Then, the tissue area was circled using an immunohistochemical pen, and 3% BSA was used to block non-specific binding sites for 1 hour at room temperature. After washing with PBS for 3 times, the primary antibody was added and incubated overnight at 4°C. After washing with PBS for 3 times, the secondary antibody was added and incubated for 1 hour at room temperature in the dark. After washing with PBS for 3 times, the nucleus was stained with DAPI for 10 minutes, and the sections were mounted with a mounting medium. Finally, the sections were observed and photographed under a fluorescence microscope. Endogenous peroxidase was blocked and non-specific sites were blocked with BSA or rabbit serum. Subsequent multiple labeling was performed using tyramide signal amplification: the first primary antibody was added dropwise and incubated overnight at 4°C; after PBS washing, the corresponding HRP-labeled secondary antibody was added and incubated at room temperature for 50 minutes; signal amplification was performed using the first TSA fluorescent dye for 10 minutes in the dark, followed by TBST washing. Subsequently, the same antigen repair solution was used for microwave treatment to strip the antibody complex. The above incubation process was repeated to label the second target: the second primary antibody was added dropwise and incubated overnight at 4°C, and the HRP-labeled secondary antibody and the second TSA fluorescent dye were sequentially reacted. Finally, DAPI was added dropwise to stain the cell nucleus for 10 minutes, and after PBS washing, the optional step of spontaneous fluorescence quenching was performed to reduce the background. Anti-fluorescence quenching mounting agent was used for mounting, and images were collected under the DAPI, 488, CY3 and CY5 corresponding fluorescence channels, respectively. This process is used to detect the following markers: ventricular zone marker SOX2, neural precursor identity marker PAX6, intermediate progenitor cell marker TBR2, and cortical plate / neuronal precursor marker MAP2, CTIP2 and TBR1.

[0055] Experimental results: The above series of key neural development markers are highly expressed in organoids, as shown in Figure 11 The immunofluorescence analysis of the present application not only verifies the successful neural induction and regionalized specialization of organoids from the spatial distribution of proteins, but more importantly reveals the great potential of simulating the core process of human early brain development. This model provides a platform for studying specific human neural development events and disease mechanisms in vitro.

[0056] II. Optimization of conditions during the construction of whole brain organoids

[0057] 1. When the starting induction cell density is 6000 cells / well, regular-shaped blastocysts can be formed; while deviating from this density (such as 9000 cells / well), it cannot guarantee the normal morphology of the blastocyst, and problems such as unclear edges and irregular structures may occur Figure 12 ).

[0058] 2. According to the morphology and growth characteristics of blastocysts at different stages, the treatment time of key differentiation stages is finely adjusted, especially the culture time is appropriately prolonged at the blastocyst formation stage (extended by 1 day, which takes 6 days at this stage), to ensure that it is fully buffered before entering the induction and differentiation stage; without extending the treatment time at this stage (5 days), the epithelialization process will be affected, specifically manifested as unclear budding phenomenon Figure 13 ).

[0059] 3、Determine the appropriate gelation time of Matrigel after wrapping is 20 minutes, which helps to ensure the uniformity of organoid structure and experimental repeatability; when the gelation time exceeds 20 minutes, the blastula appears to have a large central necrotic area (indicated by a red circle), and the epithelial growth is stagnant; when the gelation time is less than 20 minutes, the blastula structure is loose, and the epithelial expansion is hindered. When the gelation time deviates from 20 minutes (more than 20 min, such as 28 min; too short: less than 20 min, such as 12 min), the integrity of the blastula structure is damaged, showing central necrosis or epithelial expansion, and the budding phenomenon is not significant Figure 14 ).

[0060] 4、In the maturation stage of the organoid (about the 30th day into the fourth stage), add 3.5~4mg / mL Matrigel to the culture medium at a ratio of 1:100 during the medium replacement process to promote the stability of the organoid structure; failure to do so will result in irregular organoid morphology, loose structure, and poor epithelial development Figure 15 ).

[0061] 5、According to the growth needs of the organoid in the maturation stage, the medium replacement frequency is optimized to once every two days; if it is extended to once every three days, it will result in insufficient nutrition supply and poor epithelial development, manifested as unclear epithelial tissue or long bubble formation Figure 15 ). After the above optimization, the obtained organoid has a more complete epidermal structure and cell polarity, and is more suitable for subsequent construction of hypoxia / reperfusion injury models.

[0062] III. Establishing a disease model using mature organoids (taking the hypoxia-reperfusion (OGD / R) model as an example)

[0063] (1) Model establishment: inoculate a single mature organoid into a low-adsorption 24-well plate, and add 1 mL of hypoxic medium (DMEM / F12, Gibco #11330057) to each well. Then, place the culture plate in a hypoxic chamber and introduce 95% / 5% C mixed gas (about 5 minutes) to fully displace the air in the chamber. After sealing, transfer to a 37°C incubator for continuous culture for 24 hours.

[0064] (2) Model verification: To verify the hypoxic model, we compared the morphological changes and cell viability of the organoids before and after treatment.

[0065] Morphological observation showed that compared with before treatment, the epithelial structure of the organoids after hypoxia was unclear, and a large number of cell fragments appeared around it Figure 16 ). CCK-8 cell viability detection further confirmed that after 8 hours of hypoxic treatment, the viability of the organoids decreased to 57.52% (. Figure 17 ).

[0066] From the above examples, by realizing the dynamic and accurate regulation of the culture parameters, the control ability of the cell differentiation area is improved, the interference of the non-target cell types is reduced, thereby directly improving the efficiency and consistency of the embryoid body formation and neural differentiation, and the mature organoids finally obtained have more complete epithelial structure and cell polarity.

[0067] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for constructing whole-brain organoids based on human induced pluripotent stem cells, characterized in that, Includes the following steps: (1) Human induced pluripotent stem cells were cultured in a complete culture medium to obtain a cell suspension, and the cell suspension was cultured to obtain cell aggregates; (2) Cell aggregates were cultured in embryoid formation medium to obtain globular embryoids; (3) Globular embryoids were cultured in a neural induction medium to form neuroepithelial structures; (4) The single embryoid body that has formed a neuroepithelial structure is embedded in Matrigel droplets, cultured in a neuro-expansion medium to form a neuroepithelial-like structure, and then cultured in a mature medium to obtain a whole brain organoid.

2. The construction method according to claim 1, characterized in that, The complete culture medium in step (1) is based on DMEM / F12 and also includes the following components at the following concentrations: 60~70 mg / L L-ascorbic acid-2-phosphate magnesium, 10~20 µg / L sodium selenide, 80~120 µg / L FGF2, 15~25 mg / L insulin, 500~600 mg / L NaHCO3, 5~15 mg / L transferrin, 1~3 µg / L TGFβ1, 5~15 mg / mL BSA, and 0.03~0.08 mM β-mercaptoethanol.

3. The construction method according to claim 1, characterized in that, In step (2), the seeding density of the cell aggregates is 6000 cells / well. The embryo-like body formation medium includes DMEM / F12, fibroblast growth factor and Y27632. The concentration of fibroblast growth factor is 2~6 ng / mL, the concentration of Y27632 is 5~15 mM, the volume ratio of DMEM / F12, fibroblast growth factor and Y27632 is 2~6 mL: 0.5~2 mL: 3~8 μL, and the culture time is 4~8 days.

4. The construction method according to claim 1, characterized in that, The neural induction culture medium in step (3) is prepared by mixing brain organoid basal culture medium 1 and brain organoid additive A at a volume ratio of 99:

1.

5. The construction method according to claim 1, characterized in that, In step (4), the concentration of the Matrigel droplets is 3.5~4 mg / mL. After embedding, the droplets are allowed to stand at a temperature of 35~40℃ for 20 min.

6. The construction method according to claim 1, characterized in that, The neuro-expansion culture medium in step (4) is prepared by mixing brain organoid basal culture medium 2, brain organoid additive B and brain organoid additive C in a volume ratio of 98:2:

1.

7. The construction method according to claim 1, characterized in that, The mature culture medium (4) described in step (4) is prepared by mixing brain organoid basal culture medium 2 with brain organoid additive D at a volume ratio of 49:

1.

8. The construction method according to claim 1, characterized in that, The replacement frequency in step (4) is once every 2 days. On the 20th day after the replacement of the mature culture medium, Matrigel is added to the mature culture medium at a ratio of 1:100 at the same time as the replacement of the culture medium. The concentration of Matrigel is 3.5~4 mg / mL.

9. The whole-brain organoid constructed by the construction method according to any one of claims 1 to 8.

10. The application of the whole-brain organoids as described in claim 9 in research on neuro-brain diseases or drug screening.

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