A method for bioprinting preparation of human vascular organoids and a kit therefor

Abstract

The application discloses a method for preparing human vascular organoids by bioprinting and a special kit thereof. The kit contains culture solution I, culture solution II, culture solution III and printing solution IV; the culture solution I comprises serum albumin, transferrin, vitamin C, sodium selenite, VEGF and bFGF; the culture solution II comprises serum albumin, transferrin, vitamin C, sodium selenite, PDGF-BB and TGF-beta; the culture solution III comprises serum albumin, transferrin, vitamin C, sodium selenite, VEGF, bFGF, PDGF-BB and TGF-beta; and the printing solution IV comprises serum albumin, transferrin, vitamin C, sodium selenite, VEGF, bFGF, PDGF-BB, TGF-beta, GelMA and LAP. The method can obtain vascular organoids, and the method is very efficient, the obtained organoids are uniform, and the vascular structure is more complex.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing human vascular organoids by bioprinting and a dedicated reagent kit. Background Technology

[0002] Human pluripotent stem cells (including human induced pluripotent stem cells and human embryonic stem cells) can differentiate into functional cells of various tissues and organs. They are used to study human development, create disease models, and, through cell transplantation, replace damaged or diseased cells, promoting wound repair and treating diseases. Stem cell and regenerative medicine will revolutionize traditional treatments for necrotic and invasive diseases, bringing about revolutionary changes in disease mechanism research and clinical treatment.

[0003] Vascular organoids derived from human pluripotent stem cells are important models for studying de novo regeneration and angiogenesis of human blood vessels in vitro, and have significant application value in damage repair, vascular reconstruction, treatment of ischemic diseases, tissue engineering, drug development, and the establishment of disease models. However, it is currently difficult to efficiently prepare homogeneous vascular organoids, and the complexity of these organoids is limited, which greatly restricts their applications. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to use bioprinting technology to prepare human vascular organoids and the application of such organoids in biomedical research and treatment of vascular diseases.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a kit A for bioprinting human vascular organoids.

[0007] The kit A includes composition 1, composition 2, composition 3 and printing composition 4;

[0008] The composition 1 may include the following components: serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).

[0009] The composition 2 may include the following components: serum albumin, transferrin, vitamin C, sodium selenite, platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β);

[0010] The composition 3 may include the following components: serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β).

[0011] The printing composition 4 may include methacrylic hydrogel (GelMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), matrix gel, and serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β).

[0012] Further, the composition 1 may consist of the following components: serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).

[0013] Furthermore, in the composition 1, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) is (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (10-250) ng : (2-50) ng.

[0014] According to an embodiment of the present invention, in the composition 1, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) may be 0.1 mg: 1 μg: 0.04 mg: 2 ng: 10 ng: 2 ng.

[0015] Alternatively, in the composition 1, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) may be 0.5 mg: 5 μg: 0.2 mg: 10 ng: 50 ng: 10 ng.

[0016] Alternatively, in composition 1, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) may be 2.5 mg: 25 μg: 1 mg: 50 ng: 250 ng: 50 ng.

[0017] Further, the composition 2 may consist of the following components: serum albumin, transferrin, vitamin C, sodium selenite, platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β).

[0018] Furthermore, in the composition 2, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) is (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (5-10) ng : (1-2) ng.

[0019] According to an embodiment of the present invention, in the composition 2, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) is 0.5 mg: 5 μg: 0.2 mg: 10 ng: 10 ng: 2 ng.

[0020] Further, the composition 3 may consist of the following components: serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β).

[0021] Furthermore, in the composition 3, the mass ratios of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) are (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (10-50) ng : (2-20) ng : (5-10) ng : (1-2) ng.

[0022] According to an embodiment of the present invention, in the composition 3, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) is 0.5 mg: 5 μg: 0.2 mg: 10 ng: 50 ng: 20 ng: 5 ng: 1 ng.

[0023] Further, the printing composition 4 may consist of the following components: methacrylic hydrogel (GelMA), phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) solution, Matrigel, serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β).

[0024] Furthermore, in the printing composition 4, the proportions of methacrylic hydrogel (GelMA), phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) solution, Matrigel, serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) are (0.05-0.3) g : (50-100) μL : (50-100) μL : (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (10-50) ng : (2-20) ng : (5-10) ng : (1-2) ng.

[0025] According to an embodiment of the present invention, in the printing composition 4, the proportions of methacrylic hydrogel (GelMA), phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) solution, Matrigel, serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) are respectively 0.05g:100μL:100μL:0.5mg:5μg:0.2mg:10ng:50ng:20ng:5ng:1ng.

[0026] The concentration of the phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) solution can be 0.25%-0.9% (w / v), specifically such as 0.25%, 0.3%, 0.5%, 0.9%, etc.

[0027] Each of the above reagent kits A may also contain composition 5.

[0028] The composition 5 may include the following components: serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor.

[0029] Furthermore, the composition 5 may consist of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor.

[0030] Furthermore, in the composition 5, the ratio of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor is (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (2-50) ng : (1-10) mmol.

[0031] According to an embodiment of the present invention, in the composition 5, the ratio of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor is 0.1 mg: 1 μg: 0.04 mg: 2 ng: 50 ng: 10 mmol.

[0032] Alternatively, in composition 5, the ratio of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor is 0.5 mg: 5 μg: 0.2 mg: 10 ng: 50 ng: 10 mmol, respectively.

[0033] Alternatively, in the composition 5, the ratio of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor is 2.5 mg: 25 μg: 1 mg: 50 ng: 50 ng: 10 mmol, respectively.

[0034] Alternatively, in composition 5, the ratio of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor is 0.5 mg: 5 μg: 0.2 mg: 10 ng: 2 ng: 1 mmol, respectively.

[0035] Alternatively, in composition 5, the ratio of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor is 0.5 mg: 5 μg: 0.2 mg: 10 ng: 10 ng: 5 mmol, respectively.

[0036] In the composition 5, the GSK3 inhibitor may be a1), a2), a3), or a4): a1) CHIR-99021; a2) B216763; a3) BIO; a4) TWS119.

[0037] Any of the above kits A may also contain culture medium VI-1 and / or culture medium VI-2;

[0038] The culture medium VI-1 may be TeSR-E8 complete culture medium or other culture medium used for culturing human pluripotent stem cells;

[0039] The culture medium VI-2 can be a TeSR-E8 complete culture medium containing 2-10 μM (e.g., 2-5 μM, 5-10 μM, 2 μM, 5 μM or 10 μM) ROCK inhibitor.

[0040] The TeSR-E8 complete culture medium mentioned above can be a product of STEM CELL Technologies, catalog number 05940.

[0041] The TeSR-E8 complete culture medium (500 mL) described above can be specifically composed of 474 mL of TeSR-E8 basal culture medium, 25 mL of 20× supplement 1, and 1 mL of 500× supplement 2. The TeSR-E8 basal culture medium, 20× supplement 1, and 500× supplement 2 can be components of the TeSR-E8 complete culture medium.

[0042] In the culture medium VI-2, the Rho-associated protein kinase (ROCK) inhibitor may specifically be Y27632.

[0043] Secondly, the present invention provides a kit B for bioprinting human vascular organoids.

[0044] The kit B includes culture medium I, culture medium II, culture medium III, and printing solution IV;

[0045] The culture medium I includes composition 1 as described in the first aspect of the present invention and a serum-free basic culture medium.

[0046] Furthermore, the culture medium I consists of composition 1 and a serum-free basic culture medium.

[0047] Furthermore, each 1 mL of the culture medium I may include 0.1-2.5 mg serum albumin (e.g., 0.1-0.5 mg serum albumin, 0.5-2.5 mg serum albumin, 0.1 mg serum albumin, 0.5 mg serum albumin, or 2.5 mg serum albumin), 1-25 μg transferrin (1-5 μg transferrin, 5-25 μg transferrin, 1 μg transferrin, 5 μg transferrin, or 25 μg transferrin), 0.04-1 mg vitamin C (0.04-0.2 mg vitamin C, 0.2-1 mg vitamin C, 0.04 mg vitamin C, 0.2 mg vitamin C, or 1 mg vitamin C), 2-50 ng sodium selenite (2-10 ng sodium selenite, 10-50 ng sodium selenite, 2 ng sodium selenite, 10 ng sodium selenite, or 50 ng sodium selenite), 10-250 ng The culture medium contains VEGF (10-50 ng VEGF, 50-250 ng VEGF, 10 ng VEGF, 50 ng VEGF, or 250 ng VEGF) and 2-50 ng bFGF (2-10 ng bFGF, 10-50 ng bFGF, 2 ng bFGF, 10 ng bFGF, or 50 ng bFGF), with the remainder being the serum-free basal culture medium; the pH of the culture medium I can be 7.0-7.6 (e.g., 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).

[0048] The culture medium I can specifically be culture medium I-1, culture medium I-2 or culture medium I-3 as shown in Table 1.

[0049] Table 1

[0050]

[0051] The culture medium II is composed of composition 2 as described in the first aspect of the present invention and a serum-free basic culture medium.

[0052] Furthermore, the culture medium II consists of composition 2 and a serum-free basal culture medium.

[0053] Furthermore, each 1 mL of the culture medium II may include 0.1-2.5 mg serum albumin (e.g., 0.1-0.5 mg serum albumin, 0.5-2.5 mg serum albumin, 0.1 mg serum albumin, 0.5 mg serum albumin, or 2.5 mg serum albumin), 1-25 μg transferrin (1-5 μg transferrin, 5-25 μg transferrin, 1 μg transferrin, 5 μg transferrin, or 25 μg transferrin), 0.04-1 mg vitamin C (0.04-0.2 mg vitamin C, 0.2-1 mg vitamin C, 0.04 mg vitamin C, 0.2 mg vitamin C, or 1 mg vitamin C), 2-50 ng sodium selenite (2-10 ng sodium selenite, 10-50 ng sodium selenite, 2 ng sodium selenite, 10 ng sodium selenite, or 50 ng sodium selenite), and 5-10 ng PDGF-BB (5 ng The culture medium contains PDGF-BB (6 ng PDGF-BB, 7 ng PDGF-BB, 8 ng PDGF-BB, 9 ng PDGF-BB or 10 ng PDGF-BB) and 1-2 ng TGF-β (1 ng TGF-β or 2 ng TGF-β), with the remainder being the serum-free basal culture medium; the pH of the culture medium II can be 7.0-7.6 (e.g. 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6).

[0054] Each 1 mL of culture medium II may specifically contain 0.5 mg recombinant human serum albumin, 5 μg human transferrin, 0.2 mg vitamin C, 10 ng sodium selenite, 10 ng PDGF-BB and 2 ng TGF-β, with the remainder being RPMI 1640 culture medium; the pH value of culture medium II is 7.0 to 7.6.

[0055] The culture medium III includes composition 3 as described in the first aspect of the present invention and a serum-free basic culture medium.

[0056] Furthermore, the culture medium III is composed of composition 3 and a serum-free basic culture medium.

[0057] Furthermore, each 1 mL of the culture medium III may include 0.1-2.5 mg serum albumin (e.g., 0.1-0.5 mg serum albumin, 0.5-2.5 mg serum albumin, 0.1 mg serum albumin, 0.5 mg serum albumin, or 2.5 mg serum albumin), 1-25 μg transferrin (1-5 μg transferrin, 5-25 μg transferrin, 1 μg transferrin, 5 μg transferrin, or 25 μg transferrin), 0.04-1 mg vitamin C (0.04-0.2 mg vitamin C, 0.2-1 mg vitamin C, 0.04 mg vitamin C, 0.2 mg vitamin C, or 1 mg vitamin C), 2-50 ng sodium selenite (2-10 ng sodium selenite, 10-50 ng sodium selenite, 2 ng sodium selenite, 10 ng sodium selenite, or 50 ng sodium selenite), and 10-50 ng VEGF (30-50 ng...). The culture medium contains VEGF (10 ng VEGF, 30 ng VEGF, 40 ng VEGF or 50 ng VEGF) and 2-20 ng bFGF (10-20 ng bFGF, 2 ng bFGF, 10 ng bFGF, 15 ng bFGF or 20 ng bFGF), 5-10 ng PDGF-BB (5 ng PDGF-BB, 6 ng PDGF-BB, 7 ng PDGF-BB, 8 ng PDGF-BB, 9 ng PDGF-BB or 10 ng PDGF-BB) and 1-2 ng TGF-β (1 ng TGF-β or 2 ng TGF-β), with the remainder being the serum-free basal culture medium; the pH of the culture medium III can be 7.0-7.6 (e.g. 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6).

[0058] Each 1 mL of the culture medium III may specifically contain 0.5 mg recombinant human serum albumin, 5 μg human transferrin, 0.2 mg vitamin C, 10 ng sodium selenite, 50 ng VEGF-A, 20 ng bFGF, 5 ng PDGF-BB and 1 ng TGF-β, with the remainder being RPMI 1640 culture medium; the pH value of the culture medium III is 7.0 to 7.6.

[0059] The printing solution IV may include the printing composition 4 described in the first aspect of the present invention and a serum-free basic culture medium.

[0060] Furthermore, the printing solution IV consists of printing composition 4 and serum-free basal culture medium.

[0061] Furthermore, each 1 mL of the printing solution IV may include (0.05-0.3) g GelMA (e.g., 0.05-0.15 g GelMA, 0.05-0.10 g GelMA, 0.05 g GelMA, 0.15 g GelMA, or 0.3 g GelMA), (50-100) μL LAP solution (e.g., 80-100 μL LAP solution, 50 μL LAP solution, 80 μL LAP solution, or 100 μL LAP solution), and (50-100) μL Matrigel (e.g., 80-100 μL Matrigel, 50 μL Matrigel, 80 μL Matrigel, or 100 μL Matrigel). Matrigel), 0.1-2.5 mg serum albumin (e.g., 0.1-0.5 mg serum albumin, 0.5-2.5 mg serum albumin, 0.1 mg serum albumin, 0.5 mg serum albumin, or 2.5 mg serum albumin), 1-25 μg transferrin (1-5 μg transferrin, 5-25 μg transferrin, 1 μg transferrin, 5 μg transferrin, or 25 μg transferrin), 0.04-1 mg vitamin C (0.04-0.2 mg vitamin C, 0.2-1 mg vitamin C, 0.04 mg vitamin C, 0.2 mg vitamin C, or 1 mg vitamin C), 2-50 ng sodium selenite (2-10 ng sodium selenite, 10-50 ng sodium selenite, 2 ng sodium selenite, 10 ng sodium selenite, or 50 ng sodium selenite), 10-50 ng VEGF, 2-20 ng bFGF, 5-10 ng The solution contains PDGF-BB and 1-2 ng TGF-β, with the remainder being the serum-free basal culture medium; the pH of the printing solution IV can be 7.0-7.6 (e.g., 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6).

[0062] Each 1 mL of the printing solution IV may specifically contain 0.5 mg recombinant human serum albumin, 5 μg human transferrin, 0.2 mg vitamin C, 10 ng sodium selenite, 50 ng VEGF-A, 20 ng bFGF, 5 ng PDGF-BB, 1 ng TGF-β, 0.05 g GelMA, 100 μL LAP solution, and 100 μL Matrigel. The pH value of the printing solution IV is 7.0–7.6.

[0063] The concentration of the phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) solution can be 0.25%-0.9% (w / v), specifically such as 0.25%, 0.3%, 0.5%, 0.9%, etc.

[0064] Any of the above kits B may further include culture medium V, which includes the composition 5 and a serum-free basal culture medium.

[0065] Furthermore, the culture medium V consists of composition 5 and a serum-free basal culture medium.

[0066] Furthermore, each 1 mL of the culture medium V may include 0.1-2.5 mg serum albumin (e.g., 0.1-0.5 mg serum albumin, 0.5-2.5 mg serum albumin, 0.1 mg serum albumin, 0.5 mg serum albumin, or 2.5 mg serum albumin), 1-25 μg transferrin (1-5 μg transferrin, 5-25 μg transferrin, 1 μg transferrin, 5 μg transferrin, or 25 μg transferrin), 0.04-1 mg vitamin C (0.04-0.2 mg vitamin C, 0.2-1 mg vitamin C, 0.04 mg vitamin C, 0.2 mg vitamin C, or 1 mg vitamin C), 2-50 ng sodium selenite (2-10 ng sodium selenite, 10-50 ng sodium selenite, 2 ng sodium selenite, 10 ng sodium selenite, or 50 ng sodium selenite), and 2-50 ng BMP4 (2-10 ng BMP4). BMP4, 10-50 ng BMP4, 2 ng BMP4, 10 ng BMP4 or 50 ng BMP4) and 1-10 mmol GSK3 inhibitor (1-5 mmol GSK3 inhibitor, 5-10 mmol GSK3 inhibitor, 1 mmol GSK3 inhibitor, 5 mmol GSK3 inhibitor or 10 mmol GSK3 inhibitor), with the remainder being the serum-free basal culture medium; the pH of the culture medium I is 7.0-7.6 (e.g. 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6).

[0067] The culture medium V can specifically be culture medium V-1, culture medium V-2, culture medium V-3, culture medium V-4 or culture medium V-5 as shown in Table 2.

[0068] Table 2

[0069]

[0070] Any of the above kits B may also contain culture medium VI-1 and / or culture medium VI-2;

[0071] The culture medium VI-1 may be TeSR-E8 complete culture medium or other culture medium used for culturing human pluripotent stem cells;

[0072] The culture medium VI-2 can be a TeSR-E8 complete culture medium containing 2-10 μM (e.g., 2-5 μM, 5-10 μM, 2 μM, 5 μM or 10 μM) ROCK inhibitor.

[0073] The TeSR-E8 complete culture medium mentioned above can be a product of STEM CELL Technologies, catalog number 05940.

[0074] The TeSR-E8 complete culture medium (500 mL) described above can be specifically composed of 474 mL of TeSR-E8 basal culture medium, 25 mL of 20× supplement 1, and 1 mL of 500× supplement 2. The TeSR-E8 basal culture medium, 20× supplement 1, and 500× supplement 2 can be components of the TeSR-E8 complete culture medium.

[0075] In the culture medium VI-2, the Rho-associated protein kinase (ROCK) inhibitor may specifically be Y27632.

[0076] In the composition or culture medium of the present invention, the VEGF may specifically be VEGF-A or VEGF165.

[0077] In the composition or culture medium of the present invention, the serum albumin may be recombinant human serum albumin, bovine serum albumin, or other non-animal-derived recombinant human serum albumin, such as plant-derived recombinant human serum albumin.

[0078] In the composition or culture medium of the present invention, the transferrin may be human transferrin or other non-animal recombinant human transferrin, such as plant-derived recombinant human transferrin.

[0079] In the composition or culture medium of the present invention, the serum-free basic culture medium may be RPMI 1640 medium or other basic culture media commonly used for cell culture.

[0080] Thirdly, the application of reagent kit A as described in the first aspect and reagent kit B as described in the second aspect of the present invention is provided.

[0081] The application is the use of kit A or kit B in the preparation of vascularized organoids.

[0082] Fourthly, this invention provides a method for bioprinting human vascular organoids.

[0083] The method for preparing human vascular organoids by bioprinting provided by this invention includes the following steps:

[0084] (a) Using human pluripotent stem cells, culture medium V and culture medium I to prepare vascular endothelial cells; using human pluripotent stem cells, culture medium V and culture medium II to prepare vascular smooth muscle cells;

[0085] (b) Collect the vascular endothelial cells and vascular smooth muscle cells obtained in step (a), mix the two types of cells together and add the resulting printing solution IV into the printing syringe, and store at 4°C for 30 seconds to 5 minutes.

[0086] (c) Using a bio-3D printer, the mixture in the printing syringe is used as the bio-ink for 3D printing;

[0087] (d) Add culture medium III to the printed vascularized organoids and culture them, changing the culture medium III every 48 hours.

[0088] In step (a) of the above method, the method for preparing vascular endothelial cells includes the following steps:

[0089] (3) Human pluripotent stem cells were seeded into the culture medium V and cultured to obtain mesodermal cells;

[0090] (4-1) After completing step (3), mesodermal cells are seeded into culture medium I for culture; vascular endothelial cells are obtained.

[0091] In step (a) of the above method, the method for preparing vascular smooth muscle cells includes the following steps:

[0092] (3) Human pluripotent stem cells are seeded into the culture medium V described in claim 7 and cultured to obtain mesodermal cells;

[0093] (4-2) After completing step (3), mesodermal cells are seeded into culture medium II for culture; vascular smooth muscle cells are obtained.

[0094] The above method for preparing vascular endothelial cells or vascular smooth muscle cells may further include the following steps: performing steps (1) and (2) before performing step (3);

[0095] Step (1) can be: seeding human pluripotent stem cells into any of the above-mentioned culture medium VI-1 and culturing for 1 day ± 0.5 days;

[0096] Step (2) can be: seeding human pluripotent stem cells that have completed step (1) into any of the above-mentioned culture medium VI-2 and culturing for 1 day ± 0.5 days.

[0097] In step (1), the concentration of human pluripotent stem cells in the culture system can be 2.0 × 10⁻⁶. 4 Units per square centimeter (cm) 2)-5.0×10 4 Units per square centimeter (cm) 2 ), specifically 2.0×10 4 pcs / cm 2 -2.5×10 4 pcs / cm 2 2.5×10 4 pcs / cm 2 -5.0×10 4 pcs / cm 2 2.0×10 4 pcs / cm 2 5.0×10 4 pcs / cm 2 Or 2.5×10 4 pcs / cm 2 .

[0098] In step (4), the seeding density of the cells obtained from step (3) into any of the above-mentioned culture medium II can be 0.25 × 10⁻⁶. 5 pcs / cm 2 -1.0×10 5 pcs / cm 2 Specifically, such as 0.25×10 5 pcs / cm 2 -5×10 4 pcs / cm 2 5×10 4 pcs / cm 2 -1.0×10 5 pcs / cm 2 0.25×10 5 pcs / cm 2 5×10 4 pcs / cm 2 Or 1.0×10 5 pcs / cm 2 .

[0099] In step (3), the culture time can be 3 days ± 0.5 days.

[0100] In step (4), the culture time can be 3 to 10 days, specifically 3 days, 6 days or 10 days.

[0101] In any of the methods described above, the cultivation parameters may be 36℃-38℃ (e.g., 36℃, 37℃, or 38℃).

[0102] The above-described culture can be carried out using a CO2 incubator. The parameters set for the CO2 incubator can be: 5% CO2.

[0103] In any of the methods described above, the human pluripotent stem cells may be single cells digested with digestive fluid. Specifically, the digestive fluid may be Accutase. Accutase may be a product of Merk Millipore, catalog number SF006.

[0104] The human pluripotent stem cells mentioned above can be any one of b1)-b6):

[0105] b1) Human embryonic stem cell line;

[0106] b2) Human induced pluripotent stem cell lines;

[0107] b3) Human embryonic stem cell line H1;

[0108] b4) Human embryonic stem cell line H7;

[0109] b5) Human embryonic stem cell line H9;

[0110] b6) Human induced pluripotent stem cell line CD34-iPSC.

[0111] The human embryonic stem cell lines can be purchased commercially. Human embryonic stem cell lines H1, H7, and H9 can all be products of the WiCell Cell Bank in the United States.

[0112] In step (b) of the above method, the vascular endothelial cells and vascular smooth muscle cells need to be digested into single cells with a digestive solution before being mixed with the printing solution IV. Specifically, the digestive solution can be Accutase. Accutase can be a product of Merk Millipore, catalog number SF006.

[0113] In step (b) of the above method, the concentration of endothelial cell precursor cells in the mixture obtained by mixing is 1×10⁻⁶. 6 -2×10 6 The concentration of smooth muscle cell precursor cells was 5 × 10⁶ cells / mL. 5 -1×10 6 per mL.

[0114] In step (c) of the above method, the bio-3D printer can be a SunP BioMaker 4 bio-3D printer.

[0115] When using the SunP BioMaker 4 bio-3D printer, the printer parameters are set as follows: printhead temperature 18-20℃, plateau temperature 4-10℃, print speed 2.5-3mm / s, extrusion speed 0.7-1mm. 3 / s, retraction distance 0.2-0.3mm, 405nm ultraviolet light intensity 15%, each layer of printed structure photocuring time 5-10s, the last layer time 10-15s, the printer photocuring light distance 30mm, light mode 1.

[0116] The preferred printer settings are as follows: printhead temperature 19℃, platform temperature 10℃, printing speed 2.5mm / s, extrusion speed 0.7mm. 3 / s, pullback distance 0.3mm, 405nm ultraviolet light intensity 15%, each layer of printed structured light curing time 5s, the last layer time 10s, distance 30mm, light mode 1.

[0117] In step (d) of the above method, the parameters can be 36℃-38℃ (e.g., 36℃, 37℃, or 38℃). The culture can be carried out using a CO2 incubator. The parameters set for the CO2 incubator can be 5% CO2.

[0118] In step (d) of the above method, the culture time is 7-14 days.

[0119] As used herein, the term "bioprinting" refers to the printing of biological materials (including, but not limited to, cells such as cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates and multicellular bodies; subcellular structures such as organelles and cell membranes). As used herein, the term "printing" refers to the process of depositing material according to a predetermined pattern. In this invention, bioprinting is preferably achieved through a method compatible with an automated or semi-automated, computer-aided three-dimensional prototyping device (e.g., a bioprinter).

[0120] As used in this article, the term "organoid" refers to a three-dimensional (3D) cell culture containing some of the key characteristics of its representative organ, belonging to an in vitro culture system. Organoids comprise a self-renewing population of stem cells capable of differentiating into multiple organ-specific cell types, possessing similar spatial organization to the corresponding organ, and able to reproduce some of the functions of the corresponding organ, thus providing a highly physiologically relevant system.

[0121] Experiments have demonstrated that the preparation method provided by this invention can obtain vascular organoids. This method is simple, efficient, and produces uniform organoids. Furthermore, the corresponding culture medium has a defined chemical composition, contains no animal-derived proteins, and requires no added insulin. The preparation method provided by this invention allows for large-scale production of vascular organoids with stable quality and high safety, offering a new approach for tissue engineering, drug development, and cell therapy. This invention has significant application value. Attached Figure Description

[0122] Figure 1A clonal morphology diagram of undifferentiated human induced pluripotent stem cells (CD34-iPSC).

[0123] Figure 2 Morphological changes at different time points during the differentiation of human induced pluripotent stem cells (CD34-iPSCs) into endothelial cells.

[0124] Figure 3 Morphological changes at different time points during the differentiation of human induced pluripotent stem cells (CD34-iPSC) into smooth muscle cells.

[0125] Figure 4 The corresponding process for bioprinting human vascularized organoids.

[0126] Figure 5 Image of the appearance of a human vascularized organoid obtained by bioprinting.

[0127] Figure 6 The results are shown in bright-field imaging of human vascularized organoids at different time points after printing.

[0128] Figure 7 The results of confocal analysis of the expression of characteristic proteins CD31 and SM22a in human vascularized organoids were obtained 1 week and 2 weeks after printing. Detailed Implementation

[0129] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0130] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0131] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0132] The reagents and biological materials involved in the following embodiments are:

[0133] RPMI 1640 culture medium and recombinant human vitronectin are products of Thermo Fisher Scientific. VEGF-A is a product of SinoBiological, catalog number 11066-HNAB. CHIR-99021 is a product of Tocris Biosciences, catalog number 4423. Y27632 is a product of TargetMol, catalog number T1725. Vitamin C (catalog number A8960) and human transferrin (catalog number T1147) are both products of Sigma-Aldrich. Sodium selenite is a product of Sigma-Aldrich, catalog number S5261. VEGF165 is a product of Nearshore Proteins, catalog number C083. bFGF is a product of Nearshore Proteins, catalog number C779. PDGF-BB is a product of Nearshore Proteins, catalog number C119. TGF-β is a product of Nearshore Proteins, catalog number CA72. PBS buffer is a product of Wincent, catalog number 311-010-CL. Fetal bovine serum is a product of BI, catalog number 04-001-1ACS. Paraformaldehyde is a product of Sinopharm Chemical Reagent Co., Ltd. Digestive fluid Accutase is a product of Merk Millipore, catalog number SF006. TeSR-E8 complete culture medium is a product of STEMCELL, catalog number 05940. BMP4 is a product of Peprotech, catalog number 120-05. Recombinant human serum albumin is a product of Wuhan Heyuan Biotechnology Co., Ltd. The secondary antibody binding DyLight 488 and 549 is a product of Thermo. DAPI is a product of Solarbio, catalog number C0065. Anti-human CD31 antibody is a product of Santa Cruz, catalog number sc-13537. The SM22a antibody is a product of Abcam, catalog number ab14106. Matrigel is a product of BD Biosciences, catalog number 356231. GelMA is a product of Shanghai Pu Biotechnology Co., Ltd., catalog number SunP Gel G1. LAP is a product of Shanghai Pu Biotechnology Co., Ltd., catalog number SunP Gel LAP. The printer and printing consumables (such as printing syringes and needles) required for bioprinting are products of Shanghai Pu Biotechnology Co., Ltd., catalog number SunP BioMaker 4.

[0134] Human induced pluripotent stem cell line CD34-iPSC (CD34-iPSC cells for short): Obtained by reprogramming human umbilical cord blood hematopoietic stem cells (CD34-positive cells) using the Sendai virus reprogramming kit (Invitrogen, catalog number: A16517). In the following text, human induced pluripotent stem cell line CD34-iPSC will be referred to as CD34-iPSC cells.

[0135] 4% Paraformaldehyde: PBS buffer containing 4% (4g / 100mL) paraformaldehyde.

[0136] PBST buffer: PBS buffer containing 0.1% (v / v) Tween-20.

[0137] In the following examples, the pore size of the filter membrane is 0.22 μm.

[0138] Example 1: Preparation of Culture Medium

[0139] I. Preparation of the storage liquid

[0140] Recombinant human serum albumin was dissolved in sterile RPMI 1640 buffer and then filtered through a filter membrane to obtain a stock solution of recombinant human serum albumin with a concentration of 50 mg / mL.

[0141] Human transferrin was taken, dissolved in sterile PBS buffer, and then filtered through a filter membrane to obtain a human transferrin stock solution with a concentration of 1 mg / mL.

[0142] Vitamin C was dissolved in cell culture grade water and then filtered through a filter membrane to obtain a vitamin C stock solution with a concentration of 50 mg / mL.

[0143] Sodium selenite was dissolved in cell culture grade water and then filtered through a filter membrane to obtain a sodium selenite stock solution with a concentration of 100 μg / mL.

[0144] CHIR-99021 was dissolved in DMSO to obtain a CHIR-99021 stock solution with a concentration of 20 mM.

[0145] GelMA stock solution preparation: Weigh 0.0833g of GelMA powder using weighing paper for every 1mL of stock solution. Add 1mL of the corresponding culture medium to a clean bench and place in a 55℃ water bath to dissolve for 5-10 minutes, briefly shaking the solution a few times during this period to ensure complete dissolution. Immediately after dissolution, add the solution to a preheated disposable sterile syringe. Connect the syringe to a preheated 0.22μm filter membrane and filter the solution into a new sterile centrifuge tube. The stock solution preparation is now complete. Wrap the centrifuge tubes in aluminum foil to protect them from light and store them at 4℃ for later use.

[0146] Culture medium VI-1 is composed of 474 mL TeSR-E8 basal culture medium, 25 mL 20× supplement 1 and 1 mL 500× supplement 2.

[0147] Culture medium VI-2: Culture medium III-1 and Y27632 are mixed to obtain culture medium III-2; the concentration of Y27632 in culture medium III-2 is 5 μM.

[0148] Culture medium V, the composition of which is shown in Table 2 (culture medium V-1), consists of recombinant human serum albumin, human transferrin, vitamin C, sodium selenite, BMP4, CHIR-99021, and RPMI 1640 medium. Each 1 mL of culture medium V-1 contains 0.1 mg of recombinant human serum albumin, 1 μg of human transferrin, 0.04 mg of vitamin C, 2 ng of sodium selenite, 50 ng of BMP4, and 10 mmol of CHIR-99021.

[0149] Culture medium I has the following composition as shown in Table 1 (Culture Medium I-2). Culture medium II-2 consists of recombinant human serum albumin, human transferrin, vitamin C, sodium selenite, VEGF-A, bFGF, and RPMI 1640 medium. Each 1 mL of culture medium II-2 contains 0.5 mg of recombinant human serum albumin, 5 μg of human transferrin, 0.2 mg of vitamin C, 10 ng of sodium selenite, 50 ng of VEGF, and 20 ng of bFGF. The pH of culture medium I is 7.0–7.6.

[0150] Culture medium II consists of recombinant human serum albumin, human transferrin, vitamin C, sodium selenite, PDGF-BB, TGF-β, and RPMI 1640 medium. Each 1 mL of culture medium II contains 0.5 mg of recombinant human serum albumin, 5 μg of human transferrin, 0.2 mg of vitamin C, 10 ng of sodium selenite, 10 ng of PDGF-BB, and 2 ng of TGF-β. The pH of culture medium II is 7.0–7.6.

[0151] Culture medium III consists of recombinant human serum albumin, human transferrin, vitamin C, sodium selenite, VEGF-A, bFGF, PDGF-BB, TGF-β, and RPMI 1640 medium. Each 1 mL of culture medium III contains 0.5 mg of recombinant human serum albumin, 5 μg of human transferrin, 0.2 mg of vitamin C, 10 ng of sodium selenite, 50 ng of VEGF-A, 20 ng of bFGF, 5 ng of PDGF-BB, and 1 ng of TGF-β. The pH of culture medium III is 7.0–7.6.

[0152] Printing solution IV consists of recombinant human serum albumin, human transferrin, vitamin C, sodium selenite, VEGF-A, bFGF, PDGF-BB, TGF-β, GelMA, LAP, Matrigel, and RPMI 1640 culture medium. Each 1 mL of printing solution IV contains 0.5 mg of recombinant human serum albumin, 5 μg of human transferrin, 0.2 mg of vitamin C, 10 ng of sodium selenite, 50 ng of VEGF-A, 20 ng of bFGF, 5 ng of PDGF-BB, 1 ng of TGF-β, 0.05 g of GelMA, 100 μL of LAP solution (0.25% concentration), and 100 μL of Latrigel. The pH of printing solution IV is 7.0–7.6.

[0153] Example 2: Bioprinting of human vascular organoids

[0154] In this embodiment, CD34-iPSC cells were used as human pluripotent stem cells. The specific steps for differentiating and preparing vascular cells using human pluripotent stem cells are as follows:

[0155] I. Differentiation of human pluripotent stem cells into vascular endothelial cells and vascular smooth muscle cells

[0156] 1. Human pluripotent stem cells were seeded into 12-well plates (1.5 × 10⁶ cells per well). 5 Cells were cultured in TeSR-E8 complete medium (medium VI-1) at 37°C until the cell confluence reached 70%-80%.

[0157] 2. After completing step 1, remove the 12-well plate, aspirate the culture supernatant, and wash twice with RPMI 1640 medium preheated to 37°C. At this point, the morphology of CD34-iPSC cells is shown in the image. Figure 1 .

[0158] 3. After completing step 2, take the 12-well plate, add 1 ml of cell digestion solution Accutase to each well, incubate at 37°C for 3-5 minutes, then terminate the digestion with 1 ml of RPMI 1640 basal culture medium, and collect the cells by centrifugation.

[0159] 4. Seed the cells collected in step 3 into 12-well plates (the plates were coated with Matrigel at 37°C for 2 hours) at a seeding density of 1×10⁻⁶ cells / well. 5 pcs / cm 2 -1.5×10 5 pcs / cm 2 Add 1 mL of culture medium VI-2 to each well and incubate at 37°C and 5% CO2 for 1 day. Discard the original culture medium from each well and replace it with culture medium V-1, then incubate at 37°C and 5% CO2 for 3 days to obtain mesodermal cells.

[0160] 5. The mesodermal cells were prepared at a ratio of 0.35 × 10⁻⁶. 5 pcs / cm 2 -0.4×10 5 pcs / cm 2 The cells were seeded into 6-well plates (the plates were coated with Matrigel at 37°C for 2 hours), and culture medium I-2 was added. The cells were then cultured at 37°C in a 5% CO2 incubator for 3 days. The morphological changes of human induced pluripotent stem cells (CD34-iPSCs) at different time points during Day 6 differentiation into corresponding endothelial cells are shown below. Figure 2 As shown. By Figure 2 It can be seen that the corresponding stem cells have begun to transform into endothelial cells.

[0161] Mesodermal cells were divided into 0.35×10 5 pcs / cm 2 -0.4×10 5 pcs / cm 2 Inoculate into 6-well plates (the plates were coated with Matrigel at 37°C for 2 hours), add the culture medium II, and incubate at 37°C in a 5% CO2 incubator for 3 days.

[0162] Morphological changes in smooth muscle cells at different time points during Day 6 differentiation of human induced pluripotent stem cells (CD34-iPSCs) are as follows: Figure 3 As shown. By Figure 3 It can be seen that the corresponding stem cells have begun to transform into smooth muscle cells.

[0163] Note: The above differentiation to Day 6 refers to a total of six days of differentiation. Counting from the stem cells, it takes a total of six days. The first three days differentiate into mesodermal cells, and the next three days (using different culture media) the mesodermal cells differentiate into endothelial cells and smooth muscle cells, respectively, for a total of six days.

[0164] II. Bioprinting for Human Vascular Organoids

[0165] 6. After completing step 5, add the differentiated Day 6 endothelial cells and Day 6 smooth muscle cells to the cell digestion solution Accutase, incubate at 37°C for 3-4 min, then add RPMI 1640 medium for neutralization, centrifuge at 350xg for 3 min and 30 s-4 min to collect the cells.

[0166] 7. Resuspend the corresponding cell pellet in printing solution IV at 37°C, so that the endothelial cell concentration is 1×10⁻⁶. 6 The concentration of smooth muscle cells was 5 × 10⁶ cells / mL. 5 Mix the sample at a concentration of 1 / mL and add it to the printing syringe. Then place it in a 4°C refrigerator for 3 min 30 s.

[0167] 8. A SunP BioMaker 4 bio-3D printer was used, with the mixture in the printing syringe as the bio-ink for 3D printing. The printer parameters were set as follows: printhead temperature 19℃, platform temperature 10℃, printing speed 2.5mm / s, and extrusion speed 0.7mm. 3 / s, pullback distance 0.3mm, 405nm UV light intensity 15%, each layer's structure photocuring time 5s, the last layer's time 10s, the required photocuring distance is 30mm, and the illumination mode is 1. The corresponding printing process is as follows: Figure 4 As shown, the obtained human vascularized organoid structure is as follows: Figure 5 As shown, a layered structure can be observed.

[0168] 9. Add culture medium III to the printed human vascularized organoids and incubate them at 37°C in a 5% CO2 incubator for 7-14 days, changing the culture medium III every 48 hours. Perform bright-field microscopy imaging at different time points; the corresponding results are shown in the figures below. Figure 5 As shown, some tubular structures are visible on days 2 and 3 after printing and culture, but they become relatively less noticeable due to increased cell density. By day 7 after printing, relatively clearer and longer tubular structures reappear. On days 14 and 21, the tubular structures become more prominent and robust.

[0169] III. Detection of Human Vascular Organoids

[0170] 1. Take vascularized organoids from day 7 and day 14 of culture, remove the culture medium, wash once with PBS, add 4% paraformaldehyde and fix at room temperature for 30-45 minutes. Remove the 4% paraformaldehyde.

[0171] 2. Treat the fixed vascular organoids with perforation solution (PBS containing 0.2% Triton X-100) for 30 minutes, then add 5% BSA or 5% sheep serum for 1 hour for blocking.

[0172] 3. After blocking, the primary antibodies against human CD31 and SM22a were diluted with PBST containing 1% BSA (PBS + 0.1% Triton X-100) (primary antibody dilution ratio 1:100) and incubated at 4°C for 24 h. Then, the cells were washed three times with PBST (PBS + 0.1% Tween-20) to remove unbound primary antibodies, 10 min each time. The fluorescently labeled secondary antibody (DyLight 488 and 549 bound) was diluted with PBST (PBS + 0.1% Triton X-100). Cell nuclei were stained with DAPI (Sigma) (secondary antibody dilution ratio 1:200) and incubated at room temperature for 2 h 30 min, followed by washing with PBST three times for 10 min each time to remove unbound secondary antibody.

[0173] 4. Finally, dilute the DAPI stock solution to 1 μg / mL with PBS. After staining the cell nuclei with DAPI for 10-15 minutes, wash with PBS for 5 minutes before use for detection. The corresponding detection results are as follows: Figure 7 As shown, the results indicate that by day 7, the 3D bioprinted human vascularized organoids contained a thicker and more continuous interconnected tubular network, and were strongly positive for CD31. After 14 days of culture, the vascular network structure of the human vascularized organoids became denser and more complex.

[0174] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A kit A for bioprinting human vascular organoids, comprising composition 1, composition 2, composition 3 and printing composition 4; The composition 1 comprises the following components: serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The composition 2 comprises the following components: serum albumin, transferrin, vitamin C, sodium selenite, platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β); The composition 3 comprises the following components: serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β). The printing composition 4 includes methacrylic hydrogel (GelMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Matrigel, serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β).

2. The reagent kit A according to claim 1, characterized in that: In the composition 1, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) is (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (10-250) ng : (2-50) ng; And / or, in the composition 2, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) is (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (5-10) ng : (1-2) ng; And / or, in the composition 3, the mass ratio of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) is (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (10-50) ng : (2-20) ng : (5-10) ng : (1-2) ng; And / or, in the printing composition 4, the proportions of methacrylic hydrogel (GelMA), phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) solution, Matrigel, serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor β (TGF-β) are respectively (0.05-0.3)g : (50-100)μL : (50-100)μL : (0.1-2.5)mg : (1-25)μg : (0.04-1)mg : (2-50)ng : (10-50)ng : (2-20)ng : (5-10)ng : (1-2)ng.

3. The reagent kit A according to claim 1 or 2, characterized in that: The kit A further includes composition 5; composition 5 comprises the following components: serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor; In the composition 5, the dosage ratios of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and glycogen synthase kinase 3 (GSK3) inhibitor are (0.1-2.5) mg : (1-25) μg : (0.04-1) mg : (2-50) ng : (2-50) ng : (1-10) mmol.

4. The reagent kit A according to any one of claims 1-3, characterized in that: The kit A also contains culture medium VI-1 and / or culture medium VI-2; The culture medium VI-1 is TeSR-E8 complete culture medium or other culture medium used for culturing human pluripotent stem cells; The culture medium VI-2 is a complete TeSR-E8 culture medium containing 2-10 μM ROCK inhibitor.

5. A kit B for bioprinting human vascular organoids, comprising culture medium I, culture medium II, culture medium III, and printing solution IV; The culture medium I comprises composition 1 as described in claim 1 or 2 and a serum-free basic culture medium; The culture medium II comprises composition 2 as described in claim 1 or 2 and a serum-free basic culture medium; The culture medium III comprises the composition 3 according to claim 1 or 2 and a serum-free basic culture medium; The printing solution IV comprises the printing composition 4 as described in claim 1 or 2 and a serum-free basic culture medium.

6. The reagent kit B according to claim 5, characterized in that: Each 1 mL of the culture medium I comprises 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 10-250 ng VEGF, 2-50 ng bFGF, and the remainder is the serum-free basal culture medium; the pH of the culture medium I is 7.0-7.6; And / or, each 1 mL of the culture medium II comprises 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 5-10 ng PDGF-BB, 1-2 ng TGF-β, with the remainder being the serum-free basal culture medium; the pH of the culture medium II is 7.0-7.6; And / or, each 1 mL of the culture medium III comprises 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 10-50 ng VEGF, 2-20 ng bFGF, 5-10 ng PDGF-BB, and 1-2 ng TGF-β, with the remainder being the serum-free basal culture medium; the pH of the culture medium III is 7.0-7.6; And / or, each 1 mL of the printing solution IV comprises (0.05-0.3) g GelMA, (50-100) μL LAP solution, (50-100) μL Matrigel, 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 10-50 ng VEGF, 2-20 ng bFGF, 5-10 ng PDGF-BB and 1-2 ng TGF-β, with the remainder being the serum-free basal culture medium; the pH of the printing solution IV is 7.0-7.

6.

7. The reagent kit B according to claim 5 or 6, characterized in that: The kit B also includes culture medium V, which comprises composition 5 of claim 3 and serum-free basal culture medium; Each 1 mL of the culture medium V comprises 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 2-50 ng BMP4, 1-10 mmol GSK3 inhibitor, and the remainder is the serum-free basal culture medium; the pH of the culture medium V is 7.0-7.

6.

8. The reagent kit B according to any one of claims 5-7, characterized in that: The kit B also contains culture medium VI-1 and / or culture medium VI-2; The culture medium VI-1 is TeSR-E8 complete culture medium or other culture medium used for culturing human pluripotent stem cells; The culture medium VI-2 is a complete TeSR-E8 culture medium containing 2-10 μM ROCK inhibitor.

9. A method for bioprinting human vascular organoids, comprising the following steps: (a) Preparing vascular endothelial cells using human pluripotent stem cells, culture medium V as described in claim 7, and culture medium I as described in claim 5 or 6; Vascular smooth muscle cells were prepared using human pluripotent stem cells, culture medium V as described in claim 7, and culture medium II as described in claim 5 or 6. (b) Collect the vascular endothelial cells and vascular smooth muscle cells obtained in step (a), mix the two types of cells and add them to the printing solution IV described in claim 5 or 6, mix well and add to the printing syringe, and store at 4°C for 30s to 5min. (c) Using a bio-3D printer, the mixture in the printing syringe is used as the bio-ink for 3D printing; (d) The printed vascularized organoids were cultured in culture medium III to obtain human vascular organoids.

10. The preparation method according to claim 9, characterized in that: The human pluripotent stem cells are any one of b1)-b6): b1) Human embryonic stem cell line; b2) Human induced pluripotent stem cell lines; b3) Human embryonic stem cell line H1; b4) Human embryonic stem cell line H7; b5) Human embryonic stem cell line H9; b6) Human induced pluripotent stem cell line CD34-iPSC.

11. The preparation method according to claim 9 or 10, characterized in that: In step (b), the concentration of endothelial cell precursor cells in the resulting mixture is 1 × 10⁻⁶. 6 -2×10 6 The concentration of smooth muscle cell precursor cells was 5 × 10⁶ cells / mL. 5 -1×10 6 cells / mL; And / or, in step (b), the temperature of the printing liquid IV is 25-37°C; And / or, in step (c), the bio-3D printer is a SunP BioMaker 4 bio-3D printer; the printer parameters of the SunP BioMaker 4 bio-3D printer are set as follows: printhead temperature 18-20℃, platform temperature 4-10℃, printing speed 2.5-3mm / s, extrusion speed 0.7-1mm. 3 / s, retraction distance 0.2-0.3mm, 405nm ultraviolet light intensity 15%, each layer of printed structure photocuring time 5-10s, last layer time 10-15s, printer photocuring light distance 30mm, light mode 1; And / or, in step (d), the culture temperature is 36℃-38℃, and the culture time is 7-14 days.

12. The preparation method according to claim 9 or 10, characterized in that: In step (a), the method for preparing vascular endothelial cells includes the following steps: (3) Human pluripotent stem cells are seeded into the culture medium V described in claim 7 and cultured to obtain mesodermal cells; (4-1) After completing step (3), the mesodermal cells are seeded into the culture medium I described in claim 5 or 6 for culture; vascular endothelial cells are obtained; And / or, in step (a), the method for preparing vascular smooth muscle cells includes the following steps: (3) Human pluripotent stem cells are seeded into the culture medium V described in claim 7 and cultured to obtain mesodermal cells; (4-2) After completing step (3), mesodermal cells are seeded into culture medium II as described in claim 5 or 6 and cultured to obtain vascular smooth muscle cells.

Images