A brain extracellular matrix, organoid and methods of making and uses thereof

Abstract

The application belongs to the technical field of organoid culture, and particularly relates to a brain extracellular matrix, an organoid and a preparation method and application thereof. The brain extracellular matrix is prepared according to the following method: step 1, brain tissue is treated with ultrapure water or trypsin; step 2, the sample is treated with a mixed solution containing 0.1-0.5 % v / v Triton, 0.05-0.15 % v / v ammonia water and 0.01-0.1 % w / v EDTA; step 3, the sample is treated with 0.1 % ‑ 1.0 % w / v SDS; step 4, the sample is treated with 0.5 M‑1.8 M sucrose permeation; step 5, the sample is treated with 5‑25 μg / ml DNA enzyme; and step 6, the sample is treated with ultrapure water, and the brain extracellular matrix is obtained. The brain extracellular matrix is suitable for vascularized neural organoid culture and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology, specifically relating to a brain cell extracellular matrix, organoids, their preparation methods and uses. Background Technology

[0002] Brain injury and neurodegenerative diseases are among the leading threats to human health. The central nervous system is highly complex, and once damaged, its self-repair and regeneration capabilities are extremely limited. Current clinical treatments struggle to fundamentally repair damaged neural networks or reverse neuronal death. To further explore the mechanisms of brain diseases, screen effective neuroprotective drugs, and develop neuroregeneration therapies, constructing in vitro models that highly simulate the brain's physiological environment, cellular composition, and neural network connections is particularly urgent.

[0003] In recent years, with the development of stem cell technology, brain organoids have attracted much attention as an emerging in vitro model. Compared with traditional two-dimensional (2D) cell culture, brain organoids can reproduce the layered structure and histological features similar to in vivo brain tissue through cell self-assembly, and simulate complex neurophysiological functions. Therefore, brain organoids show great application potential in the fields of neural development mechanism research, disease model construction, and drug screening.

[0004] Despite significant progress in brain organoid technology, the lack of vascularization remains a key bottleneck hindering its further development. Traditional culture systems rely solely on the passive diffusion of nutrients, which is insufficient to meet the growth requirements of organoids. This often leads to necrosis in the core region due to hypoxia and nutrient deficiency, thus impeding long-term survival and structural maturation. Furthermore, the absence of vascular-neuronal interactions severely limits the application of brain organoids in simulating complex brain physiological functions, evaluating drug transport across the blood-brain barrier, and in vivo transplantation therapy.

[0005] To overcome this bottleneck, researchers have gradually developed vascularized neural organoids technology. These organoids are created in vitro by co-culturing human vascular endothelial cells, pericytes, and other vascular-related cells, or by using transgenic strategies to induce the spontaneous formation of a blood vessel-like network within the organoid, thereby reconstructing a microvascular system with perfusion function. Vascularized neural organoids not only significantly improve the survival rate and maturity of organoids, but also provide a more physiologically accurate in vitro model for studying neurovascular interactions, cerebrovascular diseases, and drug transport mechanisms across the blood-brain barrier. This represents a key direction for advancing organoid technology towards higher complexity, longer culture periods, and wider applications.

[0006] Currently, Matrigel, the mainstream matrix used for organoid culture, is derived from mouse sarcoma tissue. Rich in laminin, type IV collagen, and various basement membrane-related components, it provides cells with excellent adhesion sites and basic three-dimensional physical support, playing a crucial role in organoid formation and early maintenance. However, as a non-brain-derived matrix, Matrigel is primarily composed of basement membrane proteins and lacks the brain-specific composition and spatial structure characteristics of the extracellular matrix (ECM). This makes it difficult to simulate the precise signal regulation within the central nervous system microenvironment, particularly limiting its ability to actively induce vascular network formation and neurovascular co-development. In contrast, brain extracellular matrix (BEM) retains the complex biochemical components and three-dimensional topology of natural brain tissue while being rich in endogenous signaling molecules closely related to neurogenesis, synapse formation, and angiogenesis. Theoretically, it could provide biologically induced cues that more closely resemble in vivo conditions for neural differentiation and vascularization.

[0007] However, previous studies have found that different BEM preparation methods result in differences in the components and structures retained by the BEM, and these differences have a significant impact on the vascularization of brain organoids. Therefore, there is an urgent need in this field to develop BEMs with appropriate composition and structure to achieve better vascularization culture of brain organoids. Summary of the Invention

[0008] To address the problems of existing technologies, this invention provides a brain extracellular matrix, organoids, their preparation methods, and applications.

[0009] A brain cell extracellular matrix was prepared according to the following method: Step 1: Treat the brain tissue with ultrapure water or 0.025%-0.1 w / v trypsin; Step 2: The sample treated in Step 1 is treated with a mixture containing 0.1-0.5% v / v Triton, 0.05-0.15% v / v ammonia and 0.01-0.1% w / v EDTA. Step 3: Treat the samples processed in Step 2 with 0.1% - 1.0% w / v SDS; Step 4: Permeate the sample treated in Step 3 with 0.5 M-1.8 M sucrose. Step 5: Treat the sample after step 4 with 5-25 μg / ml DNase; Step 6: Treat the sample processed in Step 5 with ultrapure water to obtain the final product.

[0010] Preferably, in step 1, the processing conditions include: temperature 2-8 °C, processing time 8-24 h; And / or, in step 2, the processing conditions include: temperature 2-8 °C, processing time 8-24 h; And / or, in step 3, the processing conditions include: temperature 2-8 °C, processing time 8-24 h; And / or, in step 4, the processing conditions include: temperature 2-8 °C, processing time 0.5-2 h; And / or, in step 5, the processing conditions include: temperature 25-37 °C, processing time 1-4 h; And / or, in step 6, the treatment conditions include: temperature 2-8 °C, liquid changes 3-8 times, with an interval of 4-8 hours between each change.

[0011] Preferably, the extracellular matrix of brain cells is in the form of a hydrogel, and the preparation method further includes the following steps: Step 7: Freeze-dry the sample processed in step 6 and grind it into a fine powder; Step 8: The fine powder is suspended in a pepsin solution for digestion to obtain a pre-gel solution; Step 9: Adjust the pH of the pregelation solution to neutral to form a gel, thus obtaining a hydrogel.

[0012] Preferably, in step 7, the particle size of the fine powder is selected from 10–200 μm; And / or, in step 8, the concentration of the pepsin solution is 0.5–3 mg / mL, the pH is 1–3, the suspension concentration of the fine powder in the pepsin solution is 5–20 mg / mL, and the digestion conditions are digestion at 20–37 °C for 12–72 h; And / or, in step 9, adjust the pH to 7.0–7.4 and incubate at 37 °C for 10–30 min to form a gel.

[0013] The present invention also provides a method for preparing the above-mentioned extracellular matrix of brain cells, comprising the following steps: Step 1: Treat the brain tissue with ultrapure water or 0.025%-0.1% w / v trypsin; Step 2: The sample treated in Step 1 is treated with a mixture containing 0.1-0.5% v / v Triton, 0.05-0.15% v / v ammonia and 0.01-0.1% w / v EDTA. Step 3: Treat the samples processed in Step 2 with 0.1% - 1.0% w / v SDS; Step 4: Permeate the sample treated in Step 3 with 0.5 M-1.8 M sucrose. Step 5: Treat the sample after step 4 with 5-25 μg / ml DNase; Step 6: Treat the sample processed in Step 5 with ultrapure water to obtain the final product.

[0014] The present invention also provides the above-mentioned extracellular matrix of brain cells as an independent culture medium, or as a component of a culture medium, for the culture of brain organoids, vascular organoids or vascularized neural organoids.

[0015] The present invention also provides an organoid, which is prepared according to the following method: Step A: Culture at least one of neural stem cells or human umbilical vein endothelial cells into spheres; Step B involves encapsulating the spheres obtained in Step A within the aforementioned brain cell extracellular matrix and continuing to culture them to obtain the final product.

[0016] Preferably, the organoid is a brain organoid, a blood vessel organoid, or a vascularized neural organoid.

[0017] The present invention also provides a method for preparing organoids, comprising the following steps: Step A: Culture at least one of neural stem cells or human umbilical vein endothelial cells into spheres; Step B involves encapsulating the spheres obtained in Step A within the aforementioned brain cell extracellular matrix and continuing to culture them to obtain the final product.

[0018] The present invention also provides the use of the above-mentioned organoids for research on neural development mechanisms, construction of disease models or drug screening.

[0019] This invention is the first to discover that a brain extracellular matrix prepared according to a specific preferred method can be used to culture vascularized neural organoids. Comparative experimental studies have shown that, based on differences in the brain extracellular matrix preparation process (e.g., variations in process settings, reagent selection, and process conditions), the prepared brain extracellular matrix has different effects on the vascularization of vascularized neural organoids. Therefore, this invention provides a brain extracellular matrix suitable for culturing vascularized neural organoids (or brain organoids containing blood vessels, vascular organoids, etc.), and organoids obtained by culturing using this brain extracellular matrix. This invention has promising applications in the fields of neural development mechanism research, disease model construction, drug screening, and in vivo implantation.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 A comparison of the macroscopic morphology of brain tissue before and after decellularization; Figure 2 The results of the histological evaluation of brain tissue before and after decellularization; Figure 3 Results of the experiment measuring DNA content before and after brain decellularization; Figure 4 Measurement of α-gal content before and after brain decellularization; Figure 5 The results show the changes in the content of bioactive factors before and after decellularization. A: Epidermal growth factor (EGF) test results; B: Fibroblast growth factor (FGF) test results; C: Vascular endothelial growth factor (VEGF). Figure 6 The results of the extracellular matrix cell tube formation experiment are as follows: A: Microscopic imaging of the HUVEC tube formation experiment; B: Quantitative statistical analysis of the total number of principal connection points. Figure 7 The results of the brain extracellular matrix cell scratch assay are as follows: A: Microscopic imaging of the HUVEC scratch assay; B: Quantitative statistical analysis of the healing rate. Figure 8 The results of the experiment evaluating the angiogenic capacity of the brain extracellular matrix are as follows: A: Microscopic imaging of brain extracellular matrix encapsulating HUVEC vascular organoids; B: Quantitative statistical analysis of the number and length of blood vessels. Figure 9 To evaluate the influence of brain extracellular matrix on the co-development of neurovascular organoids, A: Immunofluorescence staining of organoid cytoskeleton (F-actin / DAPI) in each group; B: Immunofluorescence staining of neural marker (Tuj-1, green) and vascular marker (CD31, red); C: qPCR relative expression analysis of neural differentiation-related gene (TUBB3) and vascular endothelial-related gene (PECAM1) in each group of organoids. Detailed Implementation

[0023] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0024] Example 1: Extracellular matrix of brain cells This embodiment provides a brain cell extracellular matrix, the preparation method of which is as follows: Fresh pig brains (taken from individuals approximately 6 months old and processed within 6 hours of death) were cut into small tissue pieces of approximately 1 cm³ after the pia mater was removed to promote initial exudation of cellular contents. Subsequently, a decellularization process combining physical and chemical methods was employed, with gentle stirring at 4°C for systematic processing.

[0025] The systematic processing includes the following steps: Step 1: Treat the brain tissue with ultrapure water at 4 °C for 24 h (in other embodiments, treatment with 0.05% w / v trypsin may also be used). Step 2: The sample treated in Step 1 was treated with a mixture containing 0.3% v / v Triton, 0.1% v / v ammonia and 0.05% w / v EDTA (4 °C, 24 h). Step 3: Treat the sample after Step 2 with 0.2% w / v SDS (4 °C, 24 h). Step 4: Permeate the sample treated in Step 3 with 1 M sucrose (4 °C, 1 h). Step 5: Treat the sample after step 4 with 5 μg / ml deoxyribonuclease I (DNase) (37 °C, 2 h). Step 6: Treat the sample after step 5 with ultrapure water (4 °C, 6 liquid changes, each time interval is 6 hours) to obtain the final product.

[0026] The extracellular matrix prepared through the above steps can be further prepared into a hydrogel, as follows: Step 7: Freeze-dry the sample processed in step 6, and grind it thoroughly in a ball mill to obtain a uniform fine powder. Then, sieve it to obtain particles with a particle size of about 10–200 μm (in this embodiment, particles smaller than 75 μm are preferred to obtain higher dissolution efficiency and gel uniformity).

[0027] Step 8: Subsequently, the screened particles are suspended in a pepsin solution at a mass concentration of 5–20 mg / mL (preferably 10 mg / mL in this embodiment). The pepsin working solution is prepared at a concentration of 0.5–3 mg / mL (preferably 1 mg / mL in this embodiment), and the pH is adjusted to 1–3 (preferably pH 2 in this embodiment) to simulate an acidic environment to promote the controlled degradation of ECM. The suspension is continuously and gently digested at 20–37 °C for 12–72 h (preferably 48 h at 25 °C in this embodiment) to fully enzymatically hydrolyze the ECM into a flowable pre-gel solution. This process effectively breaks down the collagen and polysaccharide network, transforming the ECM into a homogeneous solution suitable for subsequent gelation, while preserving the bioactive components of the matrix to the greatest extent.

[0028] Step 9: Slowly add 0.5 M NaOH / PBS solution (10 mM PBS concentration) to the pre-gel solution (4 °C) to adjust the pH to neutral (approximately 7.0–7.4) to neutralize the acidic environment and restore the native conformation of ECM proteins. Then incubate at 37 °C for 10 min; the solution rapidly forms a stable three-dimensional hydrogel network. During this process, BEM protein molecules self-assemble to form β-sheet fibrous structures, causing ECM fibers to cross-link and stabilize into a network, ultimately generating a cell-supporting, bioactive three-dimensional scaffold. If the system volume is large, the incubation time can be appropriately extended to 30 min to ensure uniform gelation.

[0029] Example 2: Extracellular matrix of brain cells The method for preparing the extracellular matrix of brain cells in this embodiment is the same as that in Example 1. The difference is that the actual dosage or processing conditions are adjusted to obtain an extracellular matrix of brain cells with similar performance, biosafety and angiogenesis promotion effect.

[0030] Specifically, the adjustable parameters and their ranges include: Step 1: Treat brain tissue with ultrapure water or 0.025%-0.1% w / v trypsin; treatment conditions include: temperature 2-8 °C, treatment time 8-24 h; Step 2: The sample treated in Step 1 is treated with a mixture containing 0.1-0.5% v / v Triton, 0.05-0.15% v / v ammonia and 0.01-0.1% w / v EDTA. The treatment conditions include: temperature 2-8 °C and treatment time 8-24 h. Step 3: Treat the samples processed in Step 2 with 0.1% - 1.0% w / v SDS; the treatment conditions include: temperature 2-8 °C, treatment time 8-24 h; Step 4: The samples treated in Step 3 are permeated with 0.5 M-1.8 M sucrose; the treatment conditions include: temperature 2-8 °C, treatment time 0.5-2 h. Step 5: Treat the samples processed in Step 4 with 5-25 μg / ml DNase; the treatment conditions include: temperature 25-37 °C, treatment time 1-4 h. Step 6: Treat the sample after step 5 with ultrapure water. The treatment conditions include: temperature 2-8 °C, liquid replacement 3-8 times, with an interval of 4-8 hours between each replacement. Step 7: Freeze-dry the sample processed in step 6 and grind it into a fine powder; the particle size of the fine powder is selected from 10–200 μm; Step 8: The fine powder is suspended in a pepsin solution for digestion to obtain a pre-gel solution; the concentration of the pepsin working solution is 0.5–3 mg / mL, the pH is 1–3, the suspension concentration of the fine powder in the pepsin solution is 5–20 mg / mL, and the digestion conditions are 20–37 °C for 12–72 h. Step 9: Adjust the pH of the pregelation solution to neutral to form a gel. Adjust the pH to 7.0–7.4 and incubate at 37°C for 10–30 min.

[0031] Example 3 Vascularized Neural Organoids This embodiment uses the hydrogel prepared in Example 1 or Example 2 as the culture medium for culturing vascularized neural organoids. The specific steps are as follows: Step A: Neural stem cells and human umbilical vein endothelial cells (at a ratio of 10:1) are co-cultured to form a neurovascular sphere; wherein, the neural stem cells can be selected from human pluripotent stem cells (hPSCs / hiPSCs) induced differentiation, fetal brain tissue isolation, or fetal mouse brain; The culture time for this step is 5-7 days. The culture medium composition is: DMEM / F12 medium with 20% v / v knockout serum substitute (KSR), 2 mM Glutamax, 0.1 mM non-essential amino acids (NEAA), 0.1 mM beta-mercaptoethanol, 3 μM endogenous IWR1, 0.1 μM LDN-193189 (small molecule inhibitor) and 10 μM SB431542 (cell permeability compound).

[0032] Step B: Encapsulate the neurovascular sphere in the extracellular matrix of the brain cells and continue culturing to obtain the final product.

[0033] The packaging process is as follows: 1) Use a pipette tip with the tip cut off to transfer the neurovascular bulb into a centrifuge tube. After settling, be sure to aspirate the liquid. 2) Add BEM hydrogel at a rate of 25 uL of BEM hydrogel per neurovascular bulb; 3) Place a small droplet of gel on the sealing film or petri dish, and quickly use the pipette tip to move the neurovascular bulb to the exact center of the droplet; 4) Place in a 37 °C incubator for 20-30 minutes to form a gel and seal; 5) The encapsulated organoids were then placed in a mature culture medium for further culture. The culture medium consisted of: DMEM / F12+Neurobasal (1:1), 1:50 B27, 1:100 N2 additive, 2 mM Glutamax, 0.1 mM NEAA, 55 μM 2-mercaptoethanol, 50 ng / mL VEGF, and 20 ng / mL FGF.

[0034] The encapsulated culture conditions were: temperature 37 °C, 5% CO2, and time 28 days.

[0035] Example 4: Method for culturing vascular organoids The culture method in this embodiment is the same as that in embodiment 3, except that only human umbilical vein endothelial cells are used for culture in step A.

[0036] To demonstrate the crucial role of the processing sequence in the brain extracellular matrix preparation method of the present invention, two comparative examples are provided below as control experimental samples. Comparative Example 1 and Comparative Example 2 are brain extracellular matrix preparation methods based on existing technologies (references: "Nature Biomedical Engineering, 2, 2018, 522-539" and "TISSUE ENGINEERING: Part A, 17, 2011, 2583-2592").

[0037] Comparative Example 1: Brain cell extracellular matrix comparison samples In this comparative method for preparing extracellular matrix of brain cells, steps 1-5 in the method of Example 1 are replaced by the following steps 1a-12a, while the other steps are the same as in Example 1: Step 1a: Treat the brain tissue with ultrapure water (24 h, 4 °C). Step 2a: Use 0.05% v / v Trypsin-EDTA solution and treat with constant temperature shaking at 37 °C for 90 min.

[0038] Step 3a: Treat with 3% v / v Triton X-100 (120 min, 120 rpm, 4 °C). Step 4a: Treat with 1 M sucrose solution (30 min, 120 rpm, 4 °C). Step 5a: Ultrapure water treatment (15 min, 120 rpm, 4 °C). Step 6a: Treat with 3% w / v SDS (60 min, 150 rpm, 4 °C). Step 7a: Treat with 4% v / v ethanol (120 min, 150 rpm, 4 °C). Step 8a: Treatment with 1% v / v Triton X-100 and 0.1% v / v ammonia (60 min, 150 rpm, 4 °C). Step 9a: Treat with PBS (15 min, 120 rpm, 4 °C). Step 10a: Treatment with 1% v / v penicillin / streptomycin (30 min, 60 rpm, 4 °C). Step 11a: Ultrapure water treatment (15 min, 90 rpm, 4 °C). Step 12a: PBS treatment (15 min, 90 rpm, 4 °C).

[0039] Comparative Example 2: Brain cell extracellular matrix comparison samples In this comparative method for preparing extracellular matrix of brain cells, steps 1-5 in the method of Example 1 are replaced by steps 1b-4b as follows, while the other steps are the same as in Example 1: Step 1b: Treat with 0.1% w / v SDS and 1% penicillin / streptomycin in PBS for 24 h, change the medium once, and continue for 3–4 days before taking out the supernatant; Step 2b: Transfer to a 50 mL centrifuge tube and centrifuge at 10,000 rpm for 5 min; Step 3b, cleaning BEM: Add deionized water to the centrifuge tube, shake well and centrifuge again, repeat 10–12 times; Step 4b: The cleaned BEM is cryo-embedded and sectioned.

[0040] The technical solution of the present invention will be further illustrated by the following experiments. In the following experimental examples, the brain extracellular matrix samples used were prepared according to the methods described in Example 1, Comparative Example 1 and Comparative Example 2, respectively.

[0041] Example 1: Characterization of brain extracellular matrix prepared by different methods and comparison of its effects on the culture of neurovascular organoids I. Experimental Methods 1.1 Histological evaluation (HE and DAPI staining) Brain tissue samples before and after decellularization were embedded in OCT embedding medium and then continuously sectioned using a cryostat.

[0042] HE staining: Hematoxylin-eosin staining was performed according to standard procedures to assess tissue microstructure and nuclear clearance.

[0043] DAPI staining: Incubate with DAPI solution at room temperature in the dark for 5-10 minutes, and observe the DNA residue using a fluorescence microscope.

[0044] 1.2 Quantitative determination of decellularized components DNA concentration determination: Take the freeze-dried samples from each group, extract total DNA using a DNA extraction kit, measure the DNA concentration using a NanoDrop micro spectrophotometer, and normalize it to the content per unit dry weight (ng / mg).

[0045] ELISA assay (α-gal, EGF, FGF, VEGF): Take equal volumes of dried tissue, pulverize it, and add protein extraction buffer to homogenize thoroughly. Collect the supernatant and strictly follow the instructions of the relevant ELISA kit to determine the concentrations of the heteroantigen (α-gal) and bioactive factors (EGF, FGF, VEGF).

[0046] 1.3 In vitro functional experiments of HUVEC Tube formation experiment: Different groups of brain cell extracellular matrix were pre-coated in 96-well plates. HUVECs were seeded in the wells and cultured for 6 hours. The formation of capillary-like tubular structures was observed and photographed.

[0047] Scratch assay: HUVECs were seeded in 6-well plates until complete confluence. Vertical scratches were made using a pipette tip. Culture medium containing different groups of brain cell extracellular matrix components was added. Photos were taken at 0 and 24 hours, and the scratch healing rate was calculated to evaluate cell migration ability.

[0048] 1.4 Construction and Encapsulation of Three-Dimensional Organoids Vascular organoids (HUVEC spheroidization): Using low-adhesion 96-well plates, an appropriate amount of HUVECs was seeded into each well and cultured for 48 hours to form uniform cell spheroids. The cell spheroids were then encapsulated into prepared decellularized hydrogels for further culture and observation of angiogenesis. The culture medium for this step consisted of Endothelial Cell Medium supplemented with 50 ng / mL VEGF, 10 ng / mL LFGF, and 100 ng / mL Ang-1.

[0049] Neurovascular organoids (NSC-HUVEC co-culture): NSCs (derived from fetal rat brains) and HUVECs were mixed at a ratio of 10:1 and co-formed into spheres in a low-adhesion plate.

[0050] The culture time for this step is 7 days. The culture medium composition is: DMEM / F12 medium with 20% v / v knockout serum substitute (KSR), 2 mM Glutamax, 0.1 mM non-essential amino acids (NEAA), 0.1 mM beta-mercaptoethanol, 3 μM endogenous IWR1, 0.1 μM LDN-193189 (small molecule inhibitor) and 10 μM SB431542 (cell permeability compound).

[0051] After forming co-culture spheres, they were encapsulated in extracellular matrix hydrogels of each group of brain cells to induce neurovascular development. The encapsulation process was as follows: 1) Use a pipette tip with the tip cut off to transfer the neurovascular bulb into a centrifuge tube. After settling, be sure to aspirate the liquid. 2) Add BEM hydrogel at a rate of 25 uL of BEM hydrogel per neurovascular bulb; 3) Place a small droplet of gel on the sealing film or petri dish, and quickly use the pipette tip to move the neurovascular bulb to the exact center of the droplet; 4) Place in a 37 °C incubator for 20-30 minutes to form a gel and seal; 5) The encapsulated organoids were then placed in a mature culture medium for further culture. The culture medium consisted of: DMEM / F12+Neurobasal (1:1), 1:50 B27, 1:100 N2 additive, 2 mM Glutamax, 0.1 mM NEAA, 55 μM 2-mercaptoethanol, 50 ng / mL VEGF, and 20 ng / mL FGF.

[0052] The encapsulated culture conditions were: temperature 37 °C, 5% CO2, and time 28 days.

[0053] 1.5 Molecular Biology and Statistical Analysis Immunofluorescence staining: After fixation, permeabilization and blocking, organoid samples were added with primary antibodies (Tuj-1, CD31), followed by fluorescent secondary antibodies and DAPI to restain the nuclei. Images were then acquired using a confocal microscope.

[0054] RT-qPCR (PCR): Total RNA was extracted from organoids using the Trizol method and reverse transcribed into cDNA. Amplification was performed using specific primers for TUBB3 (Tuj-1) and PECAM1 (CD31), with GAPDH as an internal control, and the relative gene expression levels were calculated using the 2-ΔΔCT method.

[0055] Quantitative statistics: Image analysis was performed using ImageJ software (e.g., scratch healing rate, number of tube nodes, etc.), and multiple comparisons (one-way ANOVA) and significance labeling were performed using GraphPad Prism software. All experiments were repeated at least 3 times.

[0056] II. Experimental Results 1. Safety Comparison Figure 1 This study presents a comparison of the macroscopic morphology of brain tissue treated by different methods. As shown in the figures, the original brain tissue is a pale pink and opaque. After treatment in Comparative Example 1, the outline color lightens, indicating the presence of residual cells and impurities. In Comparative Example 2, the tissue shows significant shrinkage and morphological changes. In contrast, the tissue treated in Example 1 is a uniform, translucent, milky white, and retains the macroscopic outline and volume of the original tissue well, suggesting that this method effectively removes cellular components from the brain tissue while maximally maintaining the physical structural integrity of the extracellular matrix scaffold.

[0057] Histological evaluation before and after decellularization was performed by HE and DAPI staining. Figure 2 The results showed that the sample prepared in Comparative Example 1 maintained its morphological structure intact, but had a small number of residual cell nuclei. Although some cells were removed from the sample prepared in Comparative Example 2, the matrix structure showed obvious breakage and collapse. No obvious residual cell nuclei were observed in the BEM prepared in Example 1, and the extracellular matrix structure was effectively preserved.

[0058] Quantitative analysis results show that ( Figure 3 The DNA content of the original brain tissue was as high as 257.6 ± 11 ng / mg. After different processing methods, the DNA content of each group decreased to varying degrees: the sample prepared in Comparative Example 1 decreased to 151.2 ± 15.3 ng / mg, and the sample prepared in Comparative Example 2 decreased to 40.8 ± 16.2 ng / mg. However, the DNA content of the sample prepared in Example 1 was significantly reduced to 21.3 ± 2.1 ng / mg, which is far below the internationally recognized safety threshold for decellularized matrix (<50 ng / mg), further confirming the high efficiency and safety of the decellularization process in Example 1 from a quantitative perspective.

[0059] Immunogenicity testing against the major heterologous antigen α-galactoside (α-gal) showed ( Figure 4The content of each component showed a significant stepwise decrease. The α-gal content of the original brain tissue was 88.63±8.3 pg / mg, which decreased to 74.13±4.6 pg / mg and 48.90±3.5 pg / mg in Comparative Example 1 and Comparative Example 2, respectively, after decellularization. Notably, the α-gal content in the sample prepared in Example 1 decreased significantly to 18.07±4.6 pg / mg, which is only about 20.4% of that in the natural tissue. This result indicates that the process in Example 1 can effectively remove the key antigenic component α-gal that causes immune rejection, greatly improving the biosafety of the decellularized matrix material.

[0060] The experimental results above show that the BEM prepared according to the method in Example 1 has higher biocompatibility compared to other methods.

[0061] 2. Comparison of angiogenesis-promoting effects Figure 5 The effects of the decellularization process on bioactive factors in brain tissue were further evaluated. Experimental results showed that Example 1 demonstrated a significant advantage in preserving key neurovascular factors. Quantitative analysis indicated that the levels of EGF (5.5 ± 0.2 pg / mg) and VEGF (2.4 ± 0.1 pg / mg) in the treated Example 1 tissue were significantly higher than those in the original brain tissue and the comparative group (P < 0.05); simultaneously, the FGF content (approximately 27.3 pg / mg) was also effectively preserved, and was significantly higher than that in Comparative Example 1 and Comparative Example 2. These data fully demonstrate that the decellularization process of Example 1 successfully enriched and preserved core bioactive factors in the extracellular matrix while removing cellular components, endowing the BEM material with an excellent neurovascular biochemical microenvironment.

[0062] The pro-angiogenic bioactivity of each group of materials was evaluated using an endothelial cell tube formation assay. Figure 6 Microscopic imaging observations show ( Figure 6 In Example 1 group, endothelial cells rapidly migrated and interconnected, forming a dense and complete tubular network structure; while in the control group, only scattered cell clusters were observed, failing to form an effective luminal structure. Quantitative statistical results ( Figure 6 B) shows that the total number of connection points in Example 1 was significantly higher than that in Comparative Example 1 and Comparative Example 2 (P<0.0001), with an increase of approximately 4-8 times. Combined with the aforementioned results of bioactive factor detection, Figure 6 It was confirmed that Example 1 not only enriched pro-angiogenic factors such as VEGF in terms of biochemical components, but also showed excellent pro-angiogenic ability at the biological function level.

[0063] Figure 7The regulatory effect of BEM on endothelial cell migration was further evaluated using a cell scratch assay. The results showed that cells in all groups exhibited a tendency to migrate towards the damaged area after scratching. Quantitative analysis indicated that the scratch healing rate of the Example 1 (BEM) treatment group was significantly higher than that of the control groups within the same time frame, reaching approximately 80%, while the healing rates of Comparative Examples 1 and 2 were only 50.1% and 43.7%, respectively. Microscopic examination revealed that the scratch gaps in the Example 1 group were essentially eliminated, and cell connections were dense. In conclusion, the decellularization process of Example 1 not only preserved the biochemical components that promote angiogenesis but also significantly enhanced the biological activity of extracellular matrix-induced endothelial cell migration. This highly efficient cell recruitment ability, combined with its excellent angiogenesis-promoting activity, provides solid in vitro experimental evidence for the rapid revascularization of damaged areas using BEM in vivo.

[0064] Figure 8 The ability of BEM to induce the construction of complex vascular networks was further evaluated using a three-dimensional vascular organoid model. Experimental results showed that HUVEC organoids encapsulated in the BEM prepared in Example 1 exhibited strong pro-angiogenic potential. Microscopic imaging revealed that the organoids in Example 1 group showed dense radial vascular buds growing at their edges, with significantly higher vessel numbers (104.3±9.5) and average vessel length (438.7±31.2 μm) compared to the control groups (P<0.0001). In contrast, the angiogenesis ability of Control Group 2 was severely limited. Therefore, the 3D culture results confirm that the BEM prepared in Example 1 can successfully simulate the microenvironment of the natural brain matrix, effectively preserving key pro-angiogenic biochemical signals on a three-dimensional scale and providing a suitable physical framework for the maturation of vascular organoids. This discovery lays a crucial technological foundation for its application in the construction of vascularized neural organoids and the regeneration of ischemic brain tissue.

[0065] Neurovascular spheres were formed by co-culturing NSCs and HUVECs, and then encapsulated in BEMs prepared in Comparative Example 1, Comparative Example 2, and Example 1, respectively, to evaluate their neurovascular synergistic induction effect. Results are as follows: Figure 9 As shown, in Example 1 group, the CD31 positive signal was significantly enhanced, and it showed obvious spatial proximity and even partial co-distribution with Tuj-1 positive neurons, suggesting that neurogenesis and angiogenesis have a synergistic characteristic. Quantitative PCR results further confirmed that the gene expression levels of the neural marker Tuj-1 and the vascular marker CD31 in Example 1 group were several times higher than those in the control group (P<0.001).

[0066] The above experimental data strongly demonstrate that the extracellular matrix of brain cells obtained by the treatment in Example 1 not only provides the necessary physical support, but also successfully initiates the synergistic effect of neurogenesis and angiogenesis by retaining key biochemical signals, thereby promoting the effective vascularization of neural organoids and brain organoids.

[0067] As can be seen from the above embodiments and experimental examples, by optimizing the preparation process and conditions of the brain cell extracellular matrix, the present invention has obtained a biosafety-enhancing agent that can promote the effective vascularization of neural organoids and brain organoids, and has good application prospects.

Claims

1. A brain cell extracellular matrix, characterized in that, It was prepared according to the following method: Step 1: Treat the brain tissue with ultrapure water or 0.025%-0.1 w / v trypsin; Step 2: The sample treated in Step 1 is treated with a mixture containing 0.1-0.5% v / v Triton, 0.05-0.15% v / v ammonia and 0.01-0.1% w / v EDTA. Step 3: Treat the samples processed in Step 2 with 0.1% - 1.0% w / v SDS; Step 4: Permeate the sample treated in Step 3 with 0.5 M-1.8 M sucrose. Step 5: Treat the sample after step 4 with 5-25 μg / ml DNase; Step 6: Treat the sample processed in Step 5 with ultrapure water to obtain the final product.

2. The extracellular matrix of brain cells according to claim 1, characterized in that, In step 1, the processing conditions include: temperature 2-8°C, processing time 8-24 h; And / or, in step 2, the processing conditions include: temperature 2-8 °C, processing time 8-24 h; And / or, in step 3, the processing conditions include: temperature 2-8 °C, processing time 8-24 h; And / or, in step 4, the processing conditions include: temperature 2-8 °C, processing time 0.5-2 h; And / or, in step 5, the processing conditions include: temperature 25-37 °C, processing time 1-4 h; And / or, in step 6, the treatment conditions include: temperature 2-8 °C, liquid changes 3-8 times, with an interval of 4-8 hours between each change.

3. The extracellular matrix of brain cells according to claim 1, characterized in that, The extracellular matrix of the brain cells is in the form of a hydrogel, and the preparation method further includes the following steps: Step 7: Freeze-dry the sample processed in step 6 and grind it into a fine powder; Step 8: The fine powder is suspended in a pepsin solution for digestion to obtain a pre-gel solution; Step 9: Adjust the pH of the pregelation solution to neutral to form a gel, thus obtaining a hydrogel.

4. The extracellular matrix of brain cells according to claim 3, characterized in that, In step 7, the particle size of the fine powder is selected from 10–200 μm; And / or, in step 8, the concentration of the pepsin solution is 0.5–3 mg / mL, the pH is 1–3, the suspension concentration of the fine powder in the pepsin solution is 5–20 mg / mL, and the digestion conditions are 20–37 °C for 12–72 h. And / or, in step 9, adjust the pH to 7.0–7.4 and incubate at 37 °C for 10–30 min to form a gel.

5. The method for preparing the extracellular matrix of brain cells according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Treat the brain tissue with ultrapure water or 0.025%-0.1% w / v trypsin; Step 2: The sample treated in Step 1 is treated with a mixture containing 0.1-0.5% v / v Triton, 0.05-0.15% v / v ammonia and 0.01-0.1% w / v EDTA. Step 3: Treat the samples processed in Step 2 with 0.1% - 1.0% w / v SDS; Step 4: Permeate the sample treated in Step 3 with 0.5 M-1.8 M sucrose. Step 5: Treat the sample after step 4 with 5-25 μg / ml DNase; Step 6: Treat the sample processed in Step 5 with ultrapure water to obtain the final product.

6. The extracellular matrix of brain cells as described in any one of claims 1-4, used independently as a culture medium or as a component of a culture medium, for the culture of brain organoids, vascular organoids or vascularized neural organoids.

7. An organoid, characterized in that, It was prepared according to the following method: Step A: Culture at least one of neural stem cells or human umbilical vein endothelial cells into spheres; Step B: Encapsulate the spheres obtained in Step A in the extracellular matrix of brain cells as described in any one of claims 1-4, and continue culturing to obtain the final product.

8. The organoid according to claim 7, characterized in that, The organoids are brain organoids, blood vessel organoids, or vascularized neural organoids.

9. The method for preparing the organoid according to claim 7 or 8, characterized in that, Includes the following steps: Step A: Culture at least one of neural stem cells or human umbilical vein endothelial cells into spheres; Step B: Encapsulate the spheres obtained in Step A in the extracellular matrix of the brain cells and continue culturing to obtain the final product.

10. The use of the organoids of claim 7 or 8 for research on neural development mechanisms, construction of disease models, or drug screening.

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