A method for vascularizing human brain organoids by preparing artificial blood vessels using 3D printing

Abstract

The present invention discloses a method for vascularizing human brain organoids by preparing artificial blood vessels using 3D printing. The method comprises the following steps: culturing human pluripotent stem cells (hPSCs) adherently, suspending and culturing them in a neural induction culture medium to begin differentiation, embedding them with matrix gel and then culturing them in suspension on the 7th day of differentiation, designing artificial blood vessels using 3D printing design software, and printing them using a bioprinter to obtain the artificial blood vessels; immersing and incubating the artificial blood vessels, and transferring a single human brain organoid to a separate culture dish on the 30th day of differentiation, passing the artificial blood vessel through the central region of the human brain organoid, and then transferring the human brain organoid to another culture dish for culture. After 60 days of differentiation, the artificially vascularized human brain organoids show significant differences, and can significantly improve the supply of oxygen and nutrients during the long-term culture of human brain organoids and increase the volume of human brain organoids.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical engineering and biotechnology, and specifically relates to a 3D printed micro-region gradient structure high entropy alloy / titanium and titanium alloy composite material, and a preparation method and application thereof. Background Art

[0002] The structural and functional complexity of the brain, particularly the unique functional compartmentalization of the human brain, poses significant challenges to brain science research. With the advent of induced pluripotent stem cell (iPSC) reprogramming technology, researchers can now derive iPSCs (iPSCs) directly from patients carrying disease-causing genes, which, under certain conditions, can be differentiated into any type of somatic cell carrying the gene. While cells derived from traditional 2D culture systems have established commercial and research applications for scientific research, drug screening, and precision medicine, these cells still struggle to replicate the realistic 3D internal environment of the human brain and fail to reflect the 3D intercellular interactions that are crucial for brain development and neuroscience research. In recent years, human brain organoids derived from hPSCs have been recognized as effective experimental models for studying human brain development and pathogenesis. However, brain maturation, especially in the late stages, is highly dependent on vascularization of the subventricular zone. The lack of vascularization limits the oxygen and nutrient supply to organoids, often leading to necrosis in the central region and disrupting neuronal migration. Therefore, achieving vascularization in human brain organoids has become a critical challenge in the field.

[0003] Currently, methods for vascularizing human brain organoids are still immature, primarily through co-culturing endothelial cells with human brain organoids or transplanting human brain organoids into mouse brains. However, while the first approach introduces interaction between endothelial cells and human brain organoids, it still fails to form a mature vascular network, and it remains unclear whether vascular structures contribute to oxygen and nutrient supply. The second approach, while vascularizing organoids with mature vascular networks, requires xenotransplantation, involving the introduction of non-homologous cells from different species. Current bioengineering approaches to enhance organoid culture primarily focus on whole-organ culture, such as using microbioreactors to improve nutrient supply for entire batches of organoids. Bioengineering approaches for individual human brain organoids primarily exploit the inherent properties of biomaterials, such as using spider silk fibers to promote the differentiation of dopamine neurons in midbrain organoids. Currently, no clear method has been proposed for using biomaterials to construct vascularized nutrient transport pathways to enhance oxygen and nutrient supply in human brain organoids. However, existing cultures fail to establish nutrient pathways, resulting in only nominal vascularization. Currently, there are no studies using 3D printing technology to fabricate artificial blood vessels from commercially available materials to achieve vascularization of brain organoids.

[0004] Therefore, it is particularly important to establish a method to use 3D printing to prepare artificial blood vessels to achieve vascularization of human brain organoids. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the purpose of the present invention is to provide a method for vascularizing human brain organoids by preparing artificial blood vessels using 3D printing, which can significantly improve the supply of oxygen and nutrients during the long-term culture of human brain organoids and increase the volume of human brain organoids.

[0006] The present invention also provides artificial blood vessels prepared by the method of using 3D printing to prepare artificial blood vessels to achieve vascularization of human brain organoids and vascularized human brain organoids.

[0007] Technical Solution: To achieve the above-mentioned purpose, the present invention provides a method for preparing artificial blood vessels using 3D printing to achieve vascularization of human brain organoids, characterized by comprising the following steps:

[0008] Step 1: hPSCs were cultured adherently, proliferated, and then digested to form embryoid bodies, and culture and differentiation began. This day was designated as day 0.

[0009] Step 2: The first day of differentiation is recorded as day 1, and the culture medium is changed halfway every day;

[0010] Step 3: On the 7th to 10th day of differentiation, the cells were embedded in Matrigel and cultured in suspension, with the medium changed halfway every day for human brain organoid culture.

[0011] Step 4: Design an artificial blood vessel using 3D printing design software and print it using a bioprinter to obtain an artificial blood vessel;

[0012] Step 5: The human brain organoid is obtained on the 30th to 35th day of differentiation in step 3, the artificial blood vessel obtained in step 4 is immersed in the cell culture medium for incubation, and then inserted into the single human brain organoid differentiated in step (3), so that the artificial blood vessel passes through the central area of ​​the human brain organoid, and then the artificially vascularized human brain organoid is transferred to another culture dish;

[0013] Step 6: After culturing the artificially vascularized human brain organoids for 60 days of differentiation, vascularized human brain organoids were obtained.

[0014] The adherent culture described in step 1 is carried out in a culture dish coated with matrigel Vitronectin the night before at 4°C. When the hPSCs proliferate to a density of 60-80% in the culture dish, they are digested and incubated for 1-7 minutes. When the edges of the clones are observed to be shiny and slightly curled under a microscope, the Dispase is aspirated, the cells are gently washed with DMEM / F12, and the clones are gently blown off with DMEM / F12. While blowing, the blown-off clones are aspirated from the liquid and transferred to a centrifuge tube. After centrifugation, the supernatant is aspirated and the cells at the bottom of the tube are transferred to a cell culture flask for suspension culture. The culture medium is replaced and the cells are made into EBs. This is day 0 of cell differentiation.

[0015] Preferably, in step 1, the adherent cells are cultured in a culture dish coated with matrigel Vitronectin the night before at 4°C. When the hPSCs proliferate to a density of 60-80% in the culture dish, they are digested and incubated in a 37°C, 5% CO2 incubator for 1-7 minutes. When the edges of the clones are observed to be shiny and slightly curled up under a microscope, the Dispase is aspirated, the cells are gently washed with DMEM / F12, and the clones are gently blown off with DMEM / F12. While blowing, the blown-off clones are aspirated from the liquid and transferred to a 15 ml centrifuge tube. The centrifuge tube is centrifuged at 800 rpm for 1 minute, and the supernatant is discarded. The cells at the bottom of the tube are transferred to a cell culture flask for suspension culture, and the neural induction medium (NIM) is replaced. The cells are made into EBs. This is day 0 of cell differentiation.

[0016] The culture medium in the culture dish coated the night before is Essential 8. TM Basal Medium DMEM / F12(Ham) and Essential 8 TM Prepared with Supplement; digested with Dispase.

[0017] Preferably, the culture medium in the culture dish coated the night before is Essential 8, which is composed of Essential 8 at a volume ratio of 50:1. TM Basal Medium DMEM / F12(Ham)(1:1) and Essential 8 TM Supplement (50X) was prepared; 1 U / ml Dispase was used for digestion; the neural induction culture medium NIM was prepared by DMEM / F12 culture medium, NEAA, and N2 at a volume ratio of 98:1:1.

[0018] The matrix gel described in step 3 is Matrigel, which is embedded in 1X stock solution.

[0019] The culture medium for differentiation culture in steps 1 to 6 is all neural induction culture medium.

[0020] Among them, the specific steps of preparing the artificial blood vessel in step 4 are: designing a hollow cylinder with a length of 2 to 3 mm and a diameter of 200 μm through design software, and designing a circular hole with a diameter of 20 μm on the side wall, saving the design information in .stl format, importing the design information in .stl format into the software for slicing, using photoresist to print the designed two-photon polymerization in a 3D bioprinter, soaking it in a developer overnight, washing off excess photoresist, and then soaking it in isopropyl alcohol overnight to wash off the developer, thereby obtaining an artificial blood vessel.

[0021] The design software is Nanoscribe's DeScribe, the 3D bioprinter is Nanoscribe, and the developer is Nanoscribe developer.

[0022] In step 4, the 3D printing design software is 3Ds MAX or AutoCAD, and the photoresist is IP-S, a product of Nanoscribe.

[0023] Preferably, the design is made in 3dMAX and imported into the Nanoscribe printer.

[0024] In step 5, DMEM / F12 containing Fibronectin is used for immersion incubation.

[0025] The artificial blood vessels prepared by the method of using 3D printing to prepare artificial blood vessels and realize vascularization of human brain organoids described in the present invention.

[0026] The vascularized human brain organoids are prepared by the method of the present invention for preparing artificial blood vessels using 3D printing to achieve vascularization of human brain organoids.

[0027] This invention pioneers a method for 3D printing artificial blood vessels and constructs vascularized human brain organoids by inserting tubes into the organoids to establish nutrient pathways. This method establishes nutrient transport pathways within the core regions of the brain organoids, ensuring better nutrition for the organoids. This effectively overcomes the inability of existing methods, such as co-culture of endothelial cells or re-aggregation methods and material combinations, to achieve the desired vascularization effect.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] The present invention uses photoresist for the first time to prepare a new type of artificial blood vessel, and co-cultures the artificial blood vessels with human brain organoids induced and differentiated from stem cells on the 30th day, thereby realizing the vascularization of human brain organoids. This can significantly improve the supply of oxygen and nutrients during the long-term culture of human brain organoids, increase the volume of human brain organoids, and is conducive to experimental simulation of human brain organoids at different stages. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the process of inducing hPSCs to differentiate into whole brain organoids according to the present invention;

[0031] Figure 2 This is a schematic diagram of the artificial blood vessel preparation process of the present invention;

[0032] Figure 3 is a schematic diagram of the design details of the artificial blood vessel of the present invention;

[0033] Figure 4 is the artificial vascularized state of the human brain organoid of the present invention;

[0034] In the figure: a is a schematic diagram of artificial vascularization of human brain organoids, b is an optical microscopic image of human brain organoids before co-culture with artificial blood vessels on day 30 of differentiation, and c is an optical microscopic image of human brain organoids after co-culture with artificial blood vessels on day 30 of differentiation. Scale bar = 250 μm;

[0035] Figure 5 This is a fluorescence diagram of the vascularized human brain organoids on day 60 of induced differentiation in the present invention, showing normal expression of the neural progenitor cell marker PAX6, neuronal markers DCX, TUJ1, NeuN and MAP2, forebrain marker FOXG1, and cortical markers TBR1, SATB2 and CTIP2. Scale bar = 50 μm.

[0036] Figure 6 Figure 1 is a size comparison of artificially vascularized and non-vascularized human brain organoids from an embodiment of the present invention, wherein: a is an optical microscope image of the area of ​​artificially vascularized and non-vascularized human brain organoids on days 60-90 of induced differentiation; b is an optical microscope image of artificially vascularized and non-vascularized human brain organoids on day 60 of induced differentiation. Scale bar = 250 μm.

[0037] Figure 7Figure 90 shows the oxygen supply of artificially vascularized and non-vascularized human brain organoids detected using the hypoxia probe on day 90 of differentiation induction in an embodiment of the present invention. Figure a is a schematic diagram of the fluorescence intensity of the hypoxia probe in artificially vascularized and non-vascularized human brain organoids on day 90 of differentiation induction, with a scale of 50 μm. Figure b is a statistical graph of the fluorescence intensity of the hypoxia probe in artificially vascularized and non-vascularized human brain organoids on day 90 of differentiation induction.

[0038] Figure 8 Figure 90 is a graph showing the fluorescence intensity of the apoptosis marker TUNNEL on the 90th day of induced differentiation in artificially vascularized and non-vascularized human brain organoids, with a scale of 50 μm. Figure 90 shows ... DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and examples.

[0040] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0041] hPSCs

[0042] The hPSCs cells used in the embodiments of the present invention are the commercial cell line H9, which are commercially available human embryonic stem cells (WiCell, an American stem cell company). It does not require the decomposition of human embryos or blastocysts to obtain pPS or embryonic stem cells as the starting material for producing oligodendrocytes. They can be obtained from established cell lines in public depositories and are mature, conventional, and well-known cell lines.

[0043] Essential 8 TM Basal Medium & Essential 8 TMSupplement (50X) (Gibco; Catalog No.: A1517001); DMEM / F12 medium, i.e. DMEM / F12 (Ham) (1:1) (Gibco; Catalog No.: 11320033); MEM NEAA, i.e. MEM non-essential amino acid solution (Gibco; Catalog No.: 11140050); N2, i.e. N-2 supplement (100X), (Gibco; Catalog No.: 17502048); liquid Matrigel (Corning; Catalog No.: 354234); Vitronectin (Gibco; Catalog No.: A14700); Fibronectin (Gibco; Catalog No.: 33016015).

[0044] Photoresist (Nanoscribe, IP-S), 3D bioprinter (Nanoscribe Photonic ProfessionalGT2).

[0045] Example 1

[0046] A method for vascularizing human brain organoids by preparing artificial blood vessels using 3D printing

[0047] like Figure 1 Figure 2 shows a schematic diagram of the process for inducing hPSCs to differentiate into whole brain organoids. The steps are as follows:

[0048] Step 1: hPSCs (H9 cells) were cultured in a culture dish coated with Vitronectin overnight at 4°C. The culture medium used for hPSCs was Essential 8, which was composed of Essential 8 at a volume ratio of 50:1. TM BasalMedium DMEM / F12(Ham)(1:1) and Essential 8 TM Supplement (50X); culture conditions: 37°C, 5% CO2, culture to 60-80% density.

[0049] When hPSCs proliferate to 60-80% density in a culture dish, they are digested with 1 mL of Dispase (1 U ml-1) and incubated in a 37°C, 5% CO2 incubator for 2 minutes. When the edges of the colonies are observed to be shiny and slightly curled up under a microscope, the Dispase is aspirated. The cells are gently washed with DMEM / F12, and the colonies (i.e., a piece of cells) are gently blown off with DMEM / F12. While blowing, the colonies are aspirated out of the liquid and transferred to a 15 mL centrifuge tube. The centrifuge tube is centrifuged at 800 rpm for 1 minute, the supernatant is discarded, and the cells at the bottom of the tube are transferred to a cell culture flask for suspension culture. Neural induction medium (NIM) is replaced with the EBs, and the cells are suspended in the EBs to begin differentiation culture (culture conditions are 37°C, 5% CO2). This day is recorded as day 0. NIM is DMEM / F12 medium, and MEM is 1% CO2. NEAA and N2 were prepared in a volume ratio of 98:1:1.

[0050] Step 2: Differentiation day 1 is designated as day 1. Culture cells were maintained at 37°C, 5% CO2, with half the medium changed daily. The replaced medium was neural induction medium (NIM), which consisted of DMEM / F12 medium, NEAA, and N2 at a volume ratio of 98:1:1.

[0051] Step 3: On day 7 of differentiation, EBs were embedded in Matrigel. Specifically:

[0052] On day 7 of differentiation, transfer approximately 20 differentiated EB spheres to a 5cm x 5cm parafilm. Aspirate the culture medium surrounding the EB spheres and embed each EB sphere with a drop of 1X Matrigel stock solution. Incubate the embedded EB spheres in a 37°C, 5% CO2 incubator for 10 minutes. Use a 1mL pipette to aspirate the neural induction culture medium and transfer the embedded EBs from the parafilm to a new culture dish to culture human brain organoids. Change the culture medium halfway daily, using the neural induction culture medium.

[0053] Step 4, use the 3D printing design software 3Ds Max to design a hollow cylinder with a length of 2-3mm and a diameter of 200μm, and design circular holes with a diameter of 20μm on the side wall. The holes should cover the entire tube wall as much as possible, and save the design information in .stl format. Import the design information in .stl format into Nanoscribe's official design software DeScribe for slicing, and use commercial photoresist IP-S in Nanoscribe's commercial 3D bioprinter Nanoscribe GT2 to print through two-photon polymerization. The formed product is soaked in Nanoscribe developer solution 1 (poly(ethylene glycol) methyl ether acrylate, 32171-39-4) overnight to wash away excess photoresist, and then soaked in developer solution 2 isopropyl alcohol overnight to wash away developer solution 1, thereby obtaining an artificial blood vessel. Figure 2 The schematic diagram of the artificial blood vessel preparation process of the present invention is shown as follows: Figure 3 A schematic diagram showing the design details of the artificial blood vessel of the present invention is shown.

[0054] Step 5: On the 30th day of differentiation in step 3, the prepared artificial blood vessels are co-cultured with the human brain organoids differentiated from EBs, specifically:

[0055] Before using artificial blood vessels to co-culture with human brain organoids, the blood vessels obtained in step 4 were incubated in DMEM / F12 containing 10 μg / mL Fibronectin in a clean bench for 1 hour. On day 30 of differentiation, individual human brain organoids were transferred to a culture dish pre-lined with 5 cm × 5 cm parafilm. Under an optical microscope, the 3D-printed artificial blood vessels were grasped with forceps and inserted into the individual organoids, ensuring that the artificial blood vessels passed through the center of the organoid (one printed blood vessel per brain organoid; all blood vessels were inserted. The blood vessels were longer than the brain organoids, with both ends transparent and the dimensions of the blood vessels corresponding to the dimensions printed in the previous step). The artificially vascularized human brain organoids are then transferred to other new culture dishes for continued differentiation and culture. The above process is repeated until all engineering operations of the artificial blood vessels and human brain organoids in this batch are completed (i.e., one brain organoid is taken each time and an artificial blood vessel is inserted. After completion, it is transferred to another culture dish for culture, and another organoid and artificial blood vessel are processed until the processing of the artificial blood vessels in this batch is completed); the culture medium is changed halfway every day, and the replaced culture medium is all neural induction culture medium;

[0056] Step 6: Continue to culture the artificially vascularized human brain organoids at 37°C and 5% CO2. After differentiation to day 60, vascularized human brain organoids are obtained. Change the culture medium halfway every day. The culture medium replaced is the neural induction culture medium. The state of the artificial vascularized human brain organoids (vascularized human brain organoids) is as follows: Figure 4 As shown, the human brain organoids of the present invention achieved vascularization by co-culturing with artificial blood vessels on the 30th day of differentiation induction (the 30th day of differentiation of the organoids, i.e., the first day of insertion into the blood vessels), and the scale bar = 250 μm. Step 7, in step 6, the cells were fixed with 4% paraformaldehyde for 2-4 hours on the 60th and 90th days of differentiation, rinsed 3 times with Phosphate Buffered Saline (PBS), dehydrated with 30% sucrose, and allowed to stand at 4°C until the organoids sank to the bottom of the centrifuge tube. Afterwards, the organoids were embedded in OCT and sliced. The slices were rinsed 3 times with PBS for 5 minutes each time, permeated with 0.2% Triton X-100, blocked with 10% donkey serum at room temperature for 1 hour, and then the primary antibody was diluted with 5% donkey serum and 0.1% Triton in PBS and refrigerated at 4°C overnight. The next day, the sections were washed with PBS three times for 10 minutes each time, incubated with secondary antibody diluted with 5% donkey serum at room temperature for 1 hour, then rinsed three more times with PBS and finally mounted with anti-fade immunofluorescence mounting medium. The sections were observed and photographed using a Nikon upright microscope or a Zeiss laser confocal microscope (Zeiss 700b). Figure 5 As shown, on the 60th day of induced differentiation in the embodiment of the present invention, co-culture with artificial blood vessels did not affect the normal growth of organoids, and the artificially vascularized human brain organoids still normally expressed the neural progenitor cell marker PAX6, neuronal markers DCX, TUJ1, NeuN and MAP2, forebrain marker FOXG1, and cortical markers TBR1, SATB2 and CTIP2. Scale bar = 50 μm.

[0057] like Figure 6 As shown, starting from the 60th day of differentiation induction, the human brain organoids in the embodiment of the present invention increased significantly in size after artificial vascularization compared with non-vascularized organs (i.e., cultured using the above method without inserting artificial blood vessels and cultured in the same way for 60 days), and the size increase was still significant after long-term culture until the 90th day. Scale bar = 250 μm.

[0058] like Figure 7 As shown, on the 90th day of differentiation induction in an embodiment of the present invention, detection using a hypoxia probe (Hypoxyprobe, lnc; Cat NO. HP1-100) revealed that the oxygen supply condition of artificially vascularized human brain organoids was significantly improved compared with that of non-vascularized organs (differentiation cultured under the same conditions for 90 days). Scale bar = 50 μm.

[0059] like Figure 8 As shown, on the 90th day of differentiation induced by the embodiment of the present invention, TUNNEL staining (Beyotime, CatNO.C1090) was used to detect that the level of cell apoptosis in human brain organoids after artificial vascularization was significantly reduced compared with non-vascularized organs (differentiation cultured under the same conditions for 90 days), scale bar = 50 μm.

[0060] Figure 6-8 Experimental results confirm that the method described in the present invention, which uses 3D printing technology to prepare artificial blood vessels to achieve vascularization of human brain organoids, uses 3D technology to simulate human brain organs, can significantly increase the volume of human brain organoids, and improve the supply of oxygen and nutrients during the long-term culture of human brain organoids, effectively avoiding hypoxia and reducing apoptosis; human brain organoids co-cultured with artificial blood vessels have significant differences in size and other effects compared with organoids that are not artificially vascularized.

Claims

1. A method for vascularizing human brain organoids by preparing artificial blood vessels using 3D printing, characterized in that: The steps include: Step 1: hPSCs were cultured adherently, proliferated, and then digested to form embryoid bodies, and culture and differentiation began. This day was designated as day 0. Step 2: The first day of differentiation is recorded as day 1, and the culture medium is changed halfway every day; Step 3: On the 7th to 10th day of differentiation, the cells were embedded in Matrigel and cultured in suspension, with the medium changed halfway every day for human brain organoid culture. Step 4: Design an artificial blood vessel using 3D printing design software and print it using a bioprinter to obtain an artificial blood vessel; Step 5: The human brain organoid is obtained on the 30th to 35th day of differentiation in step 3. The artificial blood vessel obtained in step 4 is immersed in the cell culture medium and incubated, and then inserted into the single human brain organoid differentiated in step (3), so that the artificial blood vessel passes through the central area of ​​the human brain organoid, and then the artificially vascularized human brain organoid is transferred to another culture dish; Step 6: After culturing the artificially vascularized human brain organoids for 60 days of differentiation, vascularized human brain organoids were obtained; The specific steps of preparing the artificial blood vessel in step 4 are: designing a hollow cylinder with a length of 2-3 mm and a diameter of 200 μm using 3Ds MAX or AutoCAD design software, and designing a circular hole with a diameter of 20 μm on the side wall, saving the design information in .stl format, importing the design information in .stl format into the software for slicing, and printing the designed two-photon polymerization using photoresist in a 3D bioprinter, soaking it in a developer overnight, washing away excess photoresist, and then soaking it in isopropyl alcohol overnight to wash away the developer, thereby obtaining an artificial blood vessel; wherein the design information in .stl format is imported into Nanoscribe's DeScribe software, the 3D bioprinter is Nanoscribe, the developer is Nanoscribe developer, and the photoresist is IP-S.

2. The method for preparing artificial blood vessels using 3D printing to achieve vascularization of human brain organoids according to claim 1, characterized in that: The adherent cells described in step 1 were cultured at 4°C in a culture dish coated with Vitronectin overnight. When the hPSCs proliferated to a density of 60-80% in the culture dish, they were digested and incubated for 1-7 minutes. When the edges of the clones were observed to be shiny and slightly curled under a microscope, the Dispase was aspirated and the cells were gently washed with DMEM / F12. The clones were then gently blown off with DMEM / F12, while being aspirated from the liquid and transferred to a centrifuge tube. After centrifugation, the supernatant was aspirated and the cells at the bottom of the tube were transferred to a cell culture flask for suspension culture. The culture medium was replaced and the cells were made into EBs. This was day 0 of cell differentiation.

3. The method for preparing artificial blood vessels using 3D printing to achieve vascularization of human brain organoids according to claim 2, characterized in that: The culture medium in the culture dish coated the night before was Essential 8. TM BasalMedium vs. Essential 8 TM Prepared with Supplement; digested with Dispase.

4. The method for preparing artificial blood vessels using 3D printing to achieve vascularization of human brain organoids according to claim 1, characterized in that: The matrix gel described in step 3 is Matrigel, which is embedded in 1X stock solution.

5. The method for preparing artificial blood vessels using 3D printing to achieve vascularization of human brain organoids according to claim 1, characterized in that: The culture medium used for differentiation culture in steps 1 to 6 is all neural induction culture medium.

6. The method for preparing artificial blood vessels using 3D printing to achieve vascularization of human brain organoids according to claim 1, characterized in that: In step 5, immerse and incubate in DMEM / F12 containing Fibronectin.

7. A vascularized human brain organoid prepared by the method for vascularizing human brain organoids by preparing artificial blood vessels using 3D printing as described in any one of claims 1 to 6.

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