Hemorrhagic stroke organ-like model and construction method thereof
By injecting whole blood of hypertensive patients with hypertensive organoids into brain organoids, the hemorrhagic stroke organoid model was constructed, which solved the problem of lack of effective models in the existing technology, and achieved the simulation of the pathological mechanism of cerebral hemorrhage and the study on neural function recovery.
Patent Information
- Application Number
- CN202510178939.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-18
- Publication Date
- 2025-05-23
AI Technical Summary
The lack of effective hemorrhagic stroke organoid model in the prior art limits the in-depth study on the recovery of neurological function after cerebral hemorrhage.
By injecting whole blood of hypertensive patients into brain organoids, the pathophysiological characteristics of hemorrhagic stroke were simulated and the organoid model of hemorrhagic stroke was constructed.
The organoid model of hemorrhagic stroke was successfully constructed, which can show similar pathophysiological characteristics to patients with cerebral hemorrhage, and is used to explore the mechanism of injury formation and nerve repair after stroke, drug efficacy evaluation and mechanism of action.
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Figure CN120025977A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology and relates to a hemorrhagic stroke organoid model and a construction method thereof. Background Art
[0002] Intracerebral hemorrhage (ICH) is the most lethal subtype of stroke, with an incidence rate of about 23.4% of the total incidence of stroke, and is characterized by high mortality and disability rates. The injury mechanisms of ICH mainly include primary injury and secondary injury. Primary injury is the direct mechanical injury to the brain caused by hematoma formation and rupture into the ventricle. Secondary injury refers to the generation of a large amount of thrombin after blood coagulation, and the gradual degradation of hematoma in the later stage, releasing a variety of degradation products, which cause inflammatory reactions, perifocal tissue edema, neuronal death, etc., and ultimately lead to neurological deficits. Patients usually have long-term functional impairments, which seriously affect the quality of life and bring heavy economic burdens to families and society.
[0003] Currently, human brain tissue available for research is very scarce. Traditional animal models have certain limitations in in-depth research on neurological function recovery after cerebral hemorrhage due to the inconsistency between the brain anatomical structure and vascular network of the human brain. With the development of stem cell regenerative medicine technology, scholars have developed a three-dimensional (3D) culture system derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that is similar to the structure of the human brain, namely brain organoids. Organoid models can self-renew and self-organize, and exhibit spatial structures and physiological functions similar to the original organs. They are high-quality tools for studying disease formation mechanisms and testing drug efficacy.
[0004] Although brain organoid models have made great progress in recent years, the simulation of diseases is still in its early stages, and there are many disease brain organoid models that need technical breakthroughs. Among them, there are no reports of hemorrhagic stroke organoid models at home and abroad. Summary of the invention
[0005] The object of the present invention is to provide a hemorrhagic stroke organoid model and a construction method thereof to solve at least one of the above-mentioned technical problems.
[0006] According to one aspect of the present invention, a method for constructing a hemorrhagic stroke organoid model is provided, comprising the following steps:
[0007] Building brain organoids;
[0008] Whole blood from patients with hypertension was injected into the brain organoids at a volume occupancy of 8-15% and a depth of 0.8-1.2 mm below the surface of the cerebral cortex. The needle was retained for 4-7 minutes after injection before being withdrawn.
[0009] After the needle is removed, add brain organoid maturation culture medium and culture.
[0010] Based on brain organoids, the present invention chooses to use the method of injecting whole blood of hypertensive patients into brain organoids to construct a hemorrhagic stroke organoid model, and through research and optimization of parameters such as injection volume and injection depth, finally successfully constructs a hemorrhagic stroke organoid model that can exhibit pathological and physiological characteristics similar to those of patients with cerebral hemorrhage.
[0011] The method for constructing a hemorrhagic stroke organoid model provided by the present invention is simple to operate, has a high construction success rate, is easy to replicate, has good repeatability, and has small differences between batches.
[0012] In some embodiments, any method for constructing mature brain organoids disclosed in the prior art can be referred to, or any kit for constructing mature brain organoids disclosed in the prior art can be used to construct mature brain organoids containing basic cell types such as cortical neurons, astrocytes and mature neurons.
[0013] In some embodiments, brain organoids can be differentiated from human induced pluripotent stem cells (hiPSCs).
[0014] In some embodiments, whole blood from a hypertensive patient can be injected into the brain organoid at a volume percentage of about 10%. That is, in terms of volume percentage, the injected volume of whole blood from a hypertensive patient is about 10% of the volume of the brain organoid; taking a brain organoid with a diameter of 2 mm as an example, the injected dose of whole blood from a hypertensive patient is about 0.3 μL.
[0015] In some embodiments, whole blood from a hypertensive patient can be injected into the brain organoid using a single-point, single-injection injection method.
[0016] In some embodiments, the injection point can be in the central area of the brain organoid, for example, the injection can be performed within a range of 0 to 0.5 mm in diameter with the central point of the brain organoid as a circle, thereby preventing blood from leaking into the culture medium and causing unnecessary interference to the model.
[0017] In some embodiments, the inner diameter of the needle tip opening of the injection needle can be 25 to 35 μm. The needle tip of a conventional syringe needle is relatively thick, which can easily cause large mechanical damage to the organoids. In addition, under normal circumstances, the diameter of platelets in the blood is 2 to 3 μm, the diameter of red blood cells is 6 to 9 μm, and the diameter of white blood cells is 7 to 20 μm. Therefore, the present invention uses an injection needle with an inner diameter of 25 to 35 μm in the needle tip opening to inject whole blood from hypertensive patients into brain organoids, which can prevent blood cell rupture and hemolysis caused by the small needle tip aperture during the injection process, and can also effectively avoid large mechanical losses to the brain organoids.
[0018] In some embodiments, the inner diameter of the needle tip opening of the injection needle may be 30 μm.
[0019] In some embodiments, the bevel angle of the needle tip of the injection needle may be 25 to 40 degrees, thereby facilitating needle insertion.
[0020] In some embodiments, the bevel angle of the needle tip of the injection needle can be 30°.
[0021] In some embodiments, the injection needle can be a glass needle.
[0022] In some embodiments, the systolic blood pressure of the hypertensive patient is not less than 140 mmHg and / or the diastolic blood pressure is not less than 90 mmHg. Selecting whole blood of a hypertensive patient for injection can better simulate the occurrence of hemorrhagic stroke.
[0023] The hemorrhagic stroke organoid model constructed by the present invention can exhibit pathological and physiological characteristics similar to those of patients with cerebral hemorrhage, simulate the pathological mechanism of hemorrhagic stroke, bridge the gap between in vitro cell culture models and in vivo functional analysis, simulate the pathological process in the human body, and can be used to explore the formation of post-stroke damage and the mechanism of neural repair, the efficacy evaluation and mechanism of action research of therapeutic drugs, and observe the effects of inflammatory factors, drugs or other stimuli on neural regeneration after brain injury. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Representative images of brain organoids under a microscope at different time points during the induction and culture process of brain organoids;
[0025] Figure 2 This is a picture of the identification results of mature brain organoids;
[0026] Figure 3 This is a schematic diagram of the needle mouth structure of the oblique-mouth microinjection glass needle used in the present invention;
[0027] Figure 4 The results of hematoxylin-eosin staining of brain organoids 30 minutes and 1 hour after blood injection;
[0028] Figure 5 The results of Tunel staining of brain organoids 30 minutes and 1 hour after blood injection;
[0029] Figure 6 The results of immunofluorescence identification of Caspase-3 expression in brain organoids 30 minutes and 1 hour after blood injection;
[0030] Figure 7 These are the Fluoro jade staining results of brain organoids 30 minutes and 1 hour after blood injection. DETAILED DESCRIPTION
[0031] The present invention is further described in detail below in conjunction with the embodiments. The examples are only for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods in the examples where specific conditions are not specified are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer.
[0032] Example 1
[0033] 1Construction of brain organoids derived from human induced pluripotent stem cells (hiPSCs)
[0034] 1.1 Recovery and culture of human induced pluripotent stem cells (hiPSCs)
[0035] Human induced pluripotent stem cells (hiPSCs) were thawed and expanded using six-well Falcon culture dishes (BD Biosciences, San Jose, CA). Matrix (20×) was coated on the culture plate and incubated in a cell culture incubator for more than 0.5 to 2 hours. HiPSCs were taken out of the liquid nitrogen tank, placed in a 37°C water bath until completely thawed, and slowly added to a 15 mL sterile centrifuge tube containing 4 mL mTeSR1 medium (mTeSR1 medium, stemcell, 85850), centrifuged at 1000 rpm for 5 minutes, and 1 mL mTeSR1 medium and 10 μM Y-27632dihydrochloride (Tocris Bioscience, 1254 / 1) were added after aspirating the supernatant. The plate was inoculated into a well plate coated with Matrigel Matrix (20×) and placed in a cell culture incubator for culture. The medium was changed every day and the cell status was observed under an inverted optical microscope. When the hiPSCs clones were too large or the clones were fused, they were subcultured about every 4 days.
[0036] 1.2 Cryopreservation and passaging of human induced pluripotent stem cells (hiPSCs)
[0037] Place the hiPSCs in a clean bench, discard the old culture medium in the well plate, and add 1 mL (cell dissociation agent, Gibco, A1110501), digested in a cell culture incubator for 2 min, added 1 mL of mTeSR1 medium to terminate digestion, transferred to a 15 mL sterile centrifuge tube, centrifuged at 1000 rpm for 5 min, and seeded in a 1:6 ratio. Matrix (20×) coated cell culture plates were incubated at 37°C and CO 2 After cell digestion and centrifugation, follow the Knock Out TM The cell freezing solution was prepared in a volume ratio of SR-Multi-Species:DMSO=9:1 (compared with the cell freezing solution prepared in a volume ratio of culture medium: serum:DMSO=5:4:1, the cell survival rate was higher), the cells were resuspended in the prepared freezing solution, and the suspension was transferred to a cell freezing tube, and stored in liquid nitrogen after gradient cooling.
[0038] 1.3 Brain organoid induction and characterization
[0039] Cerebral organoids were constructed using the stemcell cerebral organoid kit (STEMdiff Cerebral Organoid Kit, 08570, stemcell). The specific operation was performed according to the kit instructions, including the following steps:
[0040] When the hiPSCs reached about 70% to 80% confluency in the well plate, the culture medium was aspirated and replaced with 1 mL of sterile D-PBS (without Ca2+). 2+ and Mg 2+ ) Rinse once, aspirate D-PBS, add 1 mL of cell dissociation reagent Accutase, and incubate at 37°C for 2 to 3 minutes. Resuspend with formation medium and plant at a density of 9000 cells / well in an ultra-low adsorption 96-well plate. Add 100 μL of formation medium and Y-27632 at a final concentration of 10 μM to each well and incubate at 37°C, CO 2 The day of the experiment was recorded as Day 0.
[0041] 100 μL of formation medium was added on day 2 and day 4. On day 5, the organoids were transferred to ultra-low attachment 24-well plates with 500 μL of induction medium. After 48 h, the organoids were transferred to matrix gel drops using the hanging drop method and cultured in ultra-low attachment six-well plates with 3 mL of expansion medium for 3 days. On day 10, the organoids were transferred to maturation medium and placed on an orbital shaker for long-term culture. The medium was changed every 3 to 4 days.
[0042] Representative images of brain organoids under a microscope at different time points during the induction and culture of brain organoids are shown below. Figure 1 shown.
[0043] Organoids were collected for identification on the 60th day, fixed with 4% paraformaldehyde overnight, dehydrated with 30% sucrose overnight, and then embedded in OCT. They were cut into 20 μm slices using a freezing microtome, and the markers TBR1, BRN2, SATB2, TUJ1, MAP2, NEUN, and GFAP were characterized by immunofluorescence and observed using a laser confocal microscope.
[0044] The results are as follows Figure 2 shown. Figure 2 In the figure, DAPI is a DNA dye, BRN2, SATB2, and TBR1 are markers of neurons in different cerebral cortex, respectively; GFAP is a marker of mature astrocytes; MAP2 and NEUN are markers of mature neurons; and TUJ1 is a marker of immature neurons. Figure 2 The results showed that mature brain organoids have been successfully constructed, containing basic cell types such as different cortical neurons, astrocytes and mature neurons.
[0045] 2 Construction of hemorrhagic stroke organoid model
[0046] 2.1 Microinjection needle preparation
[0047] The present invention uses an oblique microinjection glass needle, the needle structure is as follows Figure 3 As shown, the inner diameter d of the needle tip opening of the injection needle can be 25 to 35 μm, and the needle tip bevel angle ɑ is 25 to 40°.
[0048] The beveled microinjection glass needle selected in this embodiment has an inner diameter d of the needle tip opening of 30 μm and a bevel angle ɑ of 30°. The preparation method thereof comprises the following steps:
[0049] A 1mm outer diameter / 0.75mm inner diameter glass tube (WPI) was drawn using a P97 needle puller, with the specific parameters of pressure 200, heat 500, pull 60, velocity 120, and delay 90. Then, the needle forging instrument (NARISHIGE / MF-900) broke the needle at 30μm from the needle tip, and then the needle grinding instrument (NARISHIGE / EG-4001) was adjusted to an angle of 30° to grind the tip of the glass needle into an oblique mouth. Finally, hydrofluoric acid was used to clean the glass debris, and alcohol was rinsed several times until the debris was washed away, and then rinsed with pure water and dried for use.
[0050] 2.2 Preparation of whole blood from patients with hypertension
[0051] Whole blood from patients with hypertension (systolic blood pressure ≥ 140 mmHg and / or diastolic blood pressure ≥ 90 mmHg) was selected. The whole blood was obtained from the remaining whole blood samples from routine blood tests in the laboratory department. The blood samples were obtained after the patients signed the informed consent form voluntarily after communicating with the patients. The blood samples were mixed in sodium heparin blood collection tubes before use.
[0052] 2.3 Whole blood injection to simulate hemorrhagic stroke
[0053] Take brain organoids on the 65th day and fresh whole blood from hypertensive patients, use an RN Hamilton syringe to connect the oblique microinjection glass needle prepared in the above step "2.1 Preparation of microinjection needles", and fix the injection needle with a stereotaxic instrument (World Precision Instruments LLC, WPI) under a stereo microscope. In order to effectively prevent the movement of the organoids caused by the injection needle squeezing, use a brain slice press to fix the organoids. The pressure ring of the brain slice press is a C-shaped metal pressure ring with a diameter of 1.5 mm; the pressing wire is a nylon filament with a diameter of 100 microns. Use a brain slice press to fix the brain organoids for microinjection, and inject fresh blood into the brain organoids with a volume occupancy of 10%.
[0054] One injection point was selected for each brain organoid, and the injection point was in the central area of the organoid. Fresh blood was injected into the brain organoid at 10% volume occupancy, and the dose at each injection point was about 0.3 μL (the diameter of the organoid was about 2 mm). When the depth from the surface of the cerebral cortex was 1 mm, 0.3 μL of fresh whole blood from a hypertensive patient was slowly injected at one time within 1 minute. After each injection, the needle was retained for 5 minutes and then slowly withdrawn. After the end, 500 μL of fresh brain organoid maturation culture medium was added, and routine culture was continued, and samples were collected at different time points according to experimental needs.
[0055] 3 Pathophysiological testing of hemorrhagic stroke organoid models
[0056] 3.1 Hemorrhagic stroke organoid model slices
[0057] Brain organoids were collected 30 minutes and 1 hour after blood injection, fixed with 4% paraformaldehyde at 4°C overnight, and then washed with PBS 3 times, 5 minutes each time. Brain organoids were then embedded in OCT (Sakura, 4583). Slices were made in a freezing microtome with a thickness of approximately 20 μm. 15-20 slides were attached to each tissue, and 3-4 slices of tissue were attached to each slide. The slices were dried in a 37°C oven for 30 minutes and then transferred to a -80°C refrigerator for long-term storage.
[0058] 3.2 Observation of morphological changes of brain organoids by hematoxylin-eosin staining
[0059] (1) Staining: Take frozen sections and bake them in a 37°C oven for 30 minutes, then wash them with PBS three times, 5 minutes each time. Fix them with 95% alcohol for 1 minute. Stain them with hematoxylin for 30 seconds to 2 minutes (observe the specific time under the microscope), and rinse them with running water for 1 minute; observe the nuclear staining under the microscope, differentiate them with 1% hydrochloric acid ethanol for 1 second, and then rinse them with running water for 3 minutes. Soak them in 80% ethanol for 30 seconds, and then rinse them with running water for 1 minute. Stain them with eosin for 1 minute, and rinse them with running water for 1 minute.
[0060] (2) Dehydration and transparentization: Soak the stained sections in 75% ethanol for 30 seconds, 80% ethanol for 20 seconds, 95% ethanol for 20 seconds, anhydrous ethanol for 1 minute, xylene I for 10 minutes, and xylene II for 10 minutes.
[0061] (3) Sealing: Take the slices out of xylene, add a drop of neutral gum, and cover with a coverslip. Store at room temperature.
[0062] HE staining results of brain organoids 30 minutes and 1 hour after blood injection are as follows Figure 4 As shown, red-stained blood cells can be seen in the middle of the brain organoid.
[0063] 3.3 Tunel staining
[0064] The one-step TUNEL apoptosis detection kit (C1088, Bio-Tech) was used for the assay. The relevant operation steps were carried out according to the instructions of the kit. The results are shown in Figure 5 shown.
[0065] Figure 5 The results showed that there was Tunel-positive cell death at the injection site, indicating that the hemorrhagic stroke organoid model provided by the present invention can simulate the key pathological characteristics of hemorrhagic stroke.
[0066] 3.4 Immunofluorescence detection of Caspase-3 expression
[0067] Take the frozen sections and bake them in a 37℃ oven for 30min, then wash them with PBS 3 times, 5min each time, and wipe off excess water with a paper towel after the last wash. Incubate them in a 37℃ oven for 0.5h with 10% goat serum (prepared with 0.3% PBST, 50mL 0.3% PBST = 50mL PBS + 150μL Triton X-100). After blocking, do not wash, and incubate with the primary antibody caspase-3 (1:500, abcam) in a humidified box overnight at 4℃ refrigerator. Wash with PBS 3 times, 5min each time, and wipe off excess water with a paper towel. Incubate with the secondary antibody at 37℃ for 2h. Wash with PBS 3 times, 5min each time. DAPI staining nuclei and incubate at room temperature for 5min. Wash with PBS 3 times, 5min each time. The sections are sealed with anti-fluorescence quenching sealing agent for photography. Observe the fluorescence under a fluorescence microscope.
[0068] The results are as follows Figure 6 The results showed that cell apoptosis occurred at the injection site.
[0069] 3.5 Fluoro jade dyeing
[0070] The FJC staining kit (TR-100-FJ, biosensis) was used for detection. The relevant operation steps were carried out according to the instructions of the kit. The results are shown in Figure 7 shown.
[0071] Figure 7 The results showed that Fluorojade-positive neurons degenerated and died at the injection site.
[0072] In summary, after whole blood injection from hypertensive patients, HE staining can observe the presence of blood cells in brain organoids, Tunel staining around the injection area can observe apoptotic cells, and Fluoro jade staining can observe degenerated neurons, indicating that the hemorrhagic stroke organoid model provided by the present invention can exhibit pathological and physiological characteristics similar to those of patients with cerebral hemorrhage.
[0073] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.
Claims
1. A method for constructing a hemorrhagic stroke organoid model, characterized in that: The steps include: Building brain organoids; Whole blood from patients with hypertension was injected into the brain organoids at a volume occupancy of 8-15% and a depth of 0.8-1.2 mm below the surface of the cerebral cortex. The needle was retained for 4-7 minutes after injection before being withdrawn. After the needle is removed, add brain organoid maturation culture medium and culture.
2. The construction method according to claim 1, characterized in that: Construction of brain organoids using human induced pluripotent stem cells.
3. The construction method according to claim 1 or 2, characterized in that: Injections were performed in the central region of the brain organoids.
4. The construction method according to claim 3, characterized in that: The inner diameter of the needle tip opening of the injection needle is 25 to 35 μm.
5. The construction method according to claim 4, characterized in that: The inner diameter of the needle tip opening of the injection needle is 30 μm.
6. The construction method according to claim 4 or 5, characterized in that: The bevel angle of the needle tip of the injection needle is 25-40 degrees.
7. The construction method according to claim 6, characterized in that: The needle tip bevel angle of the injection needle is 30°.
8. The construction method according to claim 7, characterized in that: The injection needle is a glass needle.
9. The construction method according to claim 1, characterized in that: The hypertensive patient has a systolic blood pressure of not less than 140 mmHg and / or a diastolic blood pressure of not less than 90 mmHg.
10. The hemorrhagic stroke organoid model constructed according to the construction method according to any one of claims 1 to 8.