Vascular organ with lymphatic vessel and construction method thereof

Abstract

The invention belongs to the technical field of preparation of organoid, and particularly relates to a vessel organoid with lymphatic vessels and a construction method of the vessel organoid. According to the construction method provided by the invention, the lymphatic vessel structure formed by the lymphatic endothelial cells is successfully prepared for the first time. According to the organ with lymphatic vessels constructed by the invention, an obvious network-shaped vascular structure is formed in the functional lymphatic vessel organ; and different acellular matrixes or artificially synthesized hydrogel can be used for culturing functional vessel organs. The constructed perfect vessel organ with the lymphatic vessel and vascular structure has very important significance in exploration of organ development, drug screening, pathogenesis and tissue regeneration, and has a huge application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid preparation, and specifically relates to a vascular organoid with lymphatic vessels and a construction method thereof. Background Art

[0002] The lymphatic system plays a crucial role in host defense mechanisms, including promoting immune cell maturation, lipid reabsorption, and interstitial fluid homeostasis. Compared to capillaries, lymphatic microcapillaries are composed of a discontinuous basement membrane and lack an extensive pericyte coverage. Furthermore, lymphatic vessels exhibit irregular shapes or collapsed lumens and lack red blood cells. Lymphatic structures are generally classified as capillaries (10-60 µm in diameter) or collecting lymphatic vessels (50-200 µm in diameter). Similarly, blood vessels can be categorized as capillaries (4-10 µm) or larger structures such as arterioles and venules (10-100 µm). Although the blood and lymphatic vasculature are functionally closely interconnected, the two systems are anatomically separated to ensure cellular nutrient supply and tissue homeostasis. However, due to the scarcity of in vivo lymphatic endothelial cells (LECs), an optimized LEC differentiation system is needed to study the mechanisms underlying LEC development in vivo. The lack of a functional vascular network in vitro is a major drawback of many bioengineered tissues and a common cause of necrosis in vivo. While most previous studies have focused on the vascular system, recent discoveries have led to increasing interest in the role of lymphatic vessels and their potential role in tissue regeneration. However, to date, only a few studies have attempted to co-engineer blood and lymphatic microcapillaries. Therefore, there is a need for bioengineering methods to increase capillary size through in vitro self-assembly.

[0003] The vasculature (Vascular and Lymphatic System) is a network of tubes responsible for the transport of body fluids (blood and lymph) within the human body. It primarily consists of the vascular system and the lymphatic system. Its primary function is to transport substances, namely, oxygen taken in by the lungs and nutrients absorbed by the digestive system to organs, tissues, and cells throughout the body. It also transports metabolic products (such as CO2, uric acid, urea, and creatinine) to organs such as the lungs, kidneys, and skin for excretion, thereby maintaining normal metabolism. Hormones secreted by the body's endocrine organs, endocrine tissues, and scattered endocrine cells are also transported through the vasculature to target organs and cells, achieving fluid regulation. Furthermore, the vasculature plays a vital role in maintaining the relative stability of the human internal environment and the body's defenses. Vascularization is a technical challenge that remains to be addressed in various organoids. Currently constructed organoids lack a vascular network, resulting in necrosis of internal cells as they grow in size due to a lack of vascular nutrient delivery. Reports of using vascularized organoids to construct various vascularized organs have been published, and the technology is maturing. However, no reports of vascularized organoids have been reported to date. Methods such as constructing microvasculature using lymphatic endothelial cells (LECs), vascular endothelial cells (VECs), and mesenchymal stem cells (MSCs); and simulating developmental biology to activate vascular cell induction pathways face challenges such as tissue heterogeneity, complex technical methods, the formation of irregular endothelial cell colonies in organoids, and the inability to effectively simulate realistic vascular structures. Vascularization is crucial for understanding organ development, drug screening, and disease modeling.

[0004] Numerous studies have demonstrated that lymphatic vessels, as a crucial component of the immune system, play a crucial role in physiological processes. Lymphatic vessels, which co-exist with blood vessels to form the vasculature, are crucial for organ development and the maturation of cellular function, structure, and metabolism. Therefore, constructing more complete vascular organoids with both lymphatic and vascular structures is crucial for exploring organ development, drug screening, pathogenic mechanisms, and tissue regeneration, and holds enormous potential for application. Summary of the Invention

[0005] In response to the research and development gaps in the prior art, the present invention provides a method for constructing vascular organoids with lymphatic vessels.

[0006] Another object of the present invention is to provide vascular organoids having lymphatic vessels obtained by the above-mentioned construction method.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows: The present invention provides a method for constructing a lymphatic vessel organoid, comprising the following steps: S1 uses a suspension low-adsorption culture method to culture iPSCs, which have the potential to differentiate into vascular lineage cells, to form aggregates, namely embryoid bodies; S2: Transfer the aggregates into stem cell culture medium to induce the aggregates to differentiate into spherical vascular organoids of mesoderm; S3 then adds the obtained spherical vascular organoids into vascular cell sphere induction medium to induce vascular lineage cell differentiation and obtain organoid precursors; S4 encapsulates the organoid sphere precursors and then performs three-dimensional culture differentiation to obtain organoids with tubular structures; S5 encapsulates the organoid sphere precursors for three-dimensional culture, then peels the organoids and cultures them in ultra-low adhesion well plates to form mature organoid spheres.

[0008] Preferably, in step S1, the pluripotent stem cells (iPSCs) are selected from induced pluripotent stem cells, embryonic stem cells, or adult stem cells of humans or animals.

[0009] Preferably, in step S1, the specific process of the suspension low-adsorption culture method is: resuspending in an ultra-low adsorption well plate with 10 μM stem cell mTesR or Essential 8 complete culture medium, and culturing at 37°C and 5% CO2 for 1-2 days.

[0010] Preferably, in step S2, the medium for inducing mesoderm differentiation is N2B27 medium, and 3-12µM CHIR99021 and 5-30 ng / mL BMP4 are added to form a mesoderm induction medium, 2 mL of mesoderm induction medium is slowly added along the side wall of each well, and culture is carried out for 3 days.

[0011] Preferably, in step S3, the medium for inducing differentiation into vascular lineage cells is N2B27 medium, and the specific process is: transferring the aggregates formed in step S2 into N2B27 medium, adding 10-100 ng / ml VEGF165, 0.01-2.0 mM pyruvate, 10-100 ng / ml VEGFC / VEGFD, 10 3 -10 6 Induce vascular lineage cell differentiation with LIF and 0.5-5.0 μM Forskolin and culture at 37°C and 5% CO2 for 2-3 days.

[0012] Preferably, in step S4, the mixed culture medium for the encapsulated culture is StemPro-34 SFM complete culture medium, supplemented with 10-150 ng / ml VEGF165, 10-150 ng / ml FGF2, 10-150 ng / ml VEGF165, 5%-30% FBS and 20-100 ng / ml VEGFC / VEGFD, and the culture time is 4-6 days.

[0013] Preferably, in step S4, the specific process is: (1) Remove the culture medium of the organoid precursors, wash with PBS, centrifuge to remove the supernatant, and collect the organoids; (2) Resuspend the organoids in 4°C pre-cooled Matrigel to obtain an organoid Matrigel suspension; (3) Plate the organoid matrix gel suspension on a culture plate preheated at 37°C, incubate in an incubator for 10 minutes, then invert the culture plate and incubate for another 20 minutes; add organoid culture medium and change the medium every 2 days to obtain the target organoid for subculture; (4) After peeling the organoid cultured three-dimensionally in matrix gel, transfer it to a low-adhesion well plate for culture for 5-7 days to obtain multi-lumen spherical vascular organoids.

[0014] Preferably, in step S4, the matrix glue is a composite glue formed by an immune organ decellularized matrix or a polymer with good biocompatibility and its derivatives with different functional modifications and biological proteins in a mass ratio of 1-3:1; the biological protein is one or more of collagen (I, II, III), elastin, fibronectin and laminin; the immune organ decellularized matrix is a decellularized lymph node matrix, a decellularized spleen matrix, a decellularized bone marrow matrix, a decellularized thymus matrix or a sarcoma decellularized matrix (Matrigel); the polymer with good biocompatibility and its derivatives with different functional modifications are one or more of gelatin, GelMA, hyaluronic acid, silk fibroin, chitosan, sodium alginate and polyethylene glycol; the organoid culture medium is StemPro-34 SFM complete medium supplemented with 10-150 ng / ml VEGF165, 10-150 ng / ml FGF2, 10-150 ng / ml VEGF165, 5%-30% FBS and 20-100 ng / ml VEGFC / VEGFD.

[0015] Preferably, in step S5, the ultra-low adhesion plate is treated with Anti-Adherence Rinsing Solution (Stemcell, 07010) for half an hour in advance.

[0016] The present invention also provides a vascular organoid having a lymphatic vessel structure prepared using the above-mentioned construction method. The vascular organoid has a lymphatic vessel structure inside and also has a vascular network structure.

[0017] Furthermore, human induced pluripotent stem cells (iPSCs) were cultured using a specialized stem cell culture medium (mTeSR Basal Medium, Product No. 85851; mTeSRTM1 Supplement 5X, Product No. 85852) on stem cell Matrigel (Xiamen Model Biotechnology Co., Ltd., Gold Matrigel Stem Cells, Product No. 082777). For subculture, Accutase Cell Dispersion Solution (Gibco Product No. ES0017) and penicillin-streptomycin solution (purchased from Hyclone, USA) were used.

[0018] Preferably, the pluripotent stem cells are maintained at a culture density of 80-90% before passage using a stem cell culture medium.

[0019] Preferably, hiPSCs are digested into a single-cell suspension using Accutase digestion solution or 0.5-1.5 mM EDTA solution. The digestion conditions are preferably room temperature or 37°C, and the digestion time is 3-5 minutes, until the cells float from the bottom plate. mTesR complete culture medium is added to terminate the digestion.

[0020] Furthermore, the preparation process of the vascular cell spheres in step 2 is to induce the stem cells to differentiate into vascular cells in a low-adhesion well plate and then aggregate them into spheres; or to culture the stem cells to form pseudo-spheroids and then differentiate them into vascular cell spheres.

[0021] Preferably, in step S2, 5-15 μM CHIR99021 is added to the N2B27 culture medium; immediately in step S3, biological factors such as VEGF are added to the N2B27 culture medium, and the culture medium is changed every two days.

[0022] Preferably, an induction system with clear components is constructed to induce EBs into vascular cell spheres.

[0023] The vascular organoids with lymphatic vessels constructed by the present invention are used in the fields of exploring organ development, drug screening, pathogenic mechanisms and tissue regeneration.

[0024] The beneficial effects of the present invention are: 1. The construction method provided by the present invention successfully prepared a lymphatic vessel structure formed by lymphatic endothelial cells for the first time.

[0025] 2. The present invention constructs vascular organoids with lymphatic vessels, forming a distinct network of vascular structures within these functional lymphatic and vascular organoids; these functional vascular organoids can be cultured using various acellular matrices. The comprehensive vascular organoids constructed by the present invention, with their lymphatic and vascular structures, are of great significance for exploring organ development, drug screening, pathogenic mechanisms, and tissue regeneration, and possess enormous potential for application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Timeline for culturing vascular organoids with lymphatic vessels; Figure 2 These are light microscopic images of the organoid morphology at different culture time periods in Example 1; Figure 3 Encapsulated immunofluorescence staining images (lymphatic marker Prox1); Figure 4 Immunofluorescence staining images of Prox1 and CD31 (lymphatic marker Prox1 and vascular endothelial CD31); Figure 5 Immunofluorescence staining images of Prox1 and α-SMA (lymphoid marker Prox1 and smooth muscle α-SMA). DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Culture timeline of vascular organoids with lymphatic vessel structure Figure 1 shown.

[0030] Example 1 A method for preparing a vascular organoid having a lymphatic vessel structure comprises the following steps: (1) Digest hiPSCs into single-cell suspensions, passage them into ultra-low attachment plates at a ratio of 1:3, resuspend them in complete stem cell culture medium (mTesR), and culture them in the presence of Y-27632 (10 μM) in ultra-low attachment plates at 37°C and 5% CO2 for 1 day to allow embryonic stem cells or induced pluripotent stem cells to form embryoid bodies (EBs). (2) After EBs are formed, they are induced to differentiate into mesoderm. N2B27 medium is added with 6 μM CHIR99021 and 20 ng / mL BMP4 to form a mixed medium. 1.0 mL of this medium is added to gently resuspend the aggregates. 1.0 mL of this medium is slowly added to each well along the side wall. The culture is kept at 37°C and 5% CO2 for 2 days. (3) On the 4th day, discard the old culture medium and prepare the vascular lineage induction medium in a 15 mL centrifuge tube: add 20 ng / ml VEGF165, 0.5 mM pyruvate, 20 ng / ml VEGFC, 10 4 U LIF, 2.0 μM Forskolin culture induced vascular lineage cell differentiation, and cultured at 37°C, 5% CO2 for 2 days; (4) Subsequently, commercial matrix gel Matrige and type I collagen were mixed in a 1:1 ratio to form a gel solution, and then the organoid spheres were wrapped with the gel solution for 3D culture and incubated at 37°C for 2 hours to allow the gel to solidify. The culture time was about 6 days. The obtained lymphatic vascular organoids were expressed by immunofluorescence staining of the lymphatic endothelial cell-specific marker Prox1. Figure 3 shown.

[0031] Example 2 A method for preparing a vascular organoid having a lymphatic vessel structure comprises the following steps: (1) Digest hiPSCs into single-cell suspensions, passage them into ultra-low attachment plates at a ratio of 1:3, resuspend them in complete stem cell culture medium (mTesR), and culture them in the presence of Y-27632 (10 μM) in ultra-low attachment plates at 37°C and 5% CO2 for 1 day to allow embryonic stem cells or induced pluripotent stem cells to form embryoid bodies (EBs). (2) After EBs are formed, they are induced to differentiate into mesoderm. N2B27 medium is added with 6 μM CHIR99021 and 20 ng / mL BMP4 to form a mixed medium. 1.0 mL of this medium is added to gently resuspend the aggregates. 1.0 mL of this medium is slowly added to each well along the side wall and cultured for 2 days. (3) On the 4th day, discard the old culture medium and prepare the vascular lineage induction medium in a 15 mL centrifuge tube: add 20 ng / ml VEGF165, 0.5 mM pyruvate, 20 ng / ml VEGFD, 10 4 U LIF, 2.0 μM Forskolin culture induced vascular lineage cell differentiation, and cultured at 37°C, 5% CO2 for 2 days; (4) Subsequently, commercial Matrigel and type I collagen were mixed in a 1:1 ratio to form a gel solution. The organoids were then wrapped with the gel solution for 3D culture and incubated at 37°C for 2 h to allow the gel to solidify. The culture time was approximately 6 days.

[0032] (5) The obtained lymphatic vascular organoids were shown to express the lymphatic endothelial cell-specific marker Prox1 by immunofluorescence staining.

[0033] Example 3 A method for three-dimensional (3D) culturing of vascular organoids having lymphatic vessel structures in Matrigel comprises the following steps: (1) Digest hiPSCs into single-cell suspensions, passage them into ultra-low attachment plates at a ratio of 1:3, and culture them in stem cell culture medium for 1 day to allow embryonic stem cells or induced pluripotent stem cells to form embryoid bodies (EBs); (2) After EBs are formed, they are induced to differentiate into mesoderm. N2B27 medium is added with 6 μM CHIR99021 and 20 ng / mL BMP4 to form a mixed medium. 1.0 mL of this medium is added to gently resuspend the aggregates. 1.0 mL of this medium is slowly added to each well along the side wall and cultured for 2 days. (3) On the 4th day, discard the old culture medium and prepare the vascular lineage induction medium in a 15 mL centrifuge tube: add 20 ng / ml VEGF165, 0.5 pyruvate, 20 ng / ml VEGFD, 10 4 U LIF, 2.0 μM Forskolin culture induced vascular lineage cell differentiation, and cultured at 37°C, 5% CO2 for 2 days; (4) Subsequently, commercial matrix gel and type I collagen were mixed in a 1:1 ratio to form a gel solution. The organoid spheres were then wrapped with the gel solution for 3D culture and incubated at 37°C for 2 h to allow the gel to solidify. The culture time was approximately 7 days. (5) The obtained lymphatic vascular organoids were stained with immunofluorescence for the specific markers of lymphatic endothelial cells Prox1 and vascular endothelial CD31 in the organoids. Figure 4 shown.

[0034] Example 4 A method for three-dimensional (3D) culturing of vascular organoids having lymphatic vessel structures in Matrigel comprises the following steps: (1) Digest hiPSCs into single-cell suspensions, passage them into ultra-low attachment plates at a ratio of 1:3, and culture them in stem cell culture medium for 1 day to allow embryonic stem cells or induced pluripotent stem cells to form embryoid bodies (EBs); (2) After EBs are formed, they are induced to differentiate into mesoderm. N2B27 medium is added with 6 μM CHIR99021 and 20 ng / mL BMP4 to form a mixed medium. 1.0 mL of this medium is added to gently resuspend the aggregates. 1.0 mL of this medium is slowly added to each well along the side wall and cultured for 2 days. (3) On the 4th day, discard the old culture medium and prepare the vascular lineage induction medium in a 15 mL centrifuge tube: add 20 ng / ml VEGF165, 0.5 mM pyruvate, 20 ng / ml VEGFD, 10 4 U LIF, 2.0 μM Forskolin culture induced vascular lineage cell differentiation, cultured at 37°C, 5% CO2 for 2 days; (4) Subsequently, commercial matrix gel and type I collagen were mixed in a 1:1 ratio to form a gel solution. The organoid spheres were then wrapped with the gel solution for 3D culture and incubated at 37°C for 2 h to allow the gel to solidify. The culture time was approximately 6 days. (5) The organoids were then peeled and placed in a low-adhesion well plate for culture, and then cultured for 5-7 days to obtain multi-lumen network organoid spheroids.

[0035] (6) The obtained lymphatic vascular spheroid organoids were stained with fluorescent staining and found to express the lymphatic endothelial cell-specific marker Prox1 and the smooth muscle marker α-SMA. Figure 5 shown.

[0036] Example 5 A mature method for culturing vascular organoid spheres with lymphatic vessel structures comprises the following steps: (1) Digest hiPSCs into single-cell suspensions, passage them into ultra-low attachment plates at a ratio of 1:3, and culture them in stem cell culture medium for 1 day to allow embryonic stem cells or induced pluripotent stem cells to form embryoid bodies (EBs); (2) After EBs are formed, they are induced to differentiate into mesoderm. N2B27 medium is added with 6 μM CHIR99021 and 20 ng / mL BMP4 to form a mixed medium. 1.0 mL of this medium is added to gently resuspend the aggregates. 1.0 mL of this medium is slowly added to each well along the side wall and cultured for 2 days. (3) On the 4th day, discard the old culture medium and prepare the vascular lineage induction medium in a 15 mL centrifuge tube: add 20 ng / ml VEGF165, 0.5 mM pyruvate, 20 ng / ml VEGFC, 10 4 LIF, 2.0 μM Forskolin were used to induce vascular lineage cell differentiation and cultured at 37°C, 5% CO2 for 2 days; (4) Subsequently, a 1:1 mixture of decellularized spleen matrix gel and type I collagen was used to form a gel solution. The organoid spheres were then wrapped with the gel solution for 3D culture and incubated at 37°C for 2 h to allow the gel to solidify. The culture time was approximately 5-7 days. (5) The organoids are then peeled and placed in a low-adhesion well plate for culture. After 7 days of culture, multi-lumen network organoid spheres can be obtained.

[0037] (6) The obtained lymphatic vascular sphere organoids were stained with fluorescent staining and found to express the lymphatic endothelial cell-specific marker Prox1 and the smooth muscle marker α-SMA.

[0038] Example 6 A mature method for culturing vascular organoid spheres with lymphatic vessel structures comprises the following steps: (1) Digest hiPSCs into single-cell suspensions, passage them into ultra-low attachment plates at a ratio of 1:3, and culture them in stem cell culture medium for 1 day to allow embryonic stem cells or induced pluripotent stem cells to form embryoid bodies (EBs); (2) After EBs are formed, they are induced to differentiate into mesoderm. N2B27 medium is added with 6 μM CHIR99021 and 20 ng / mL BMP4 to form a mixed medium. 1.0 mL of this medium is added to gently resuspend the aggregates. 1.0 mL of this medium is slowly added to each well along the side wall and cultured for 2 days. (3) On the 4th day, discard the old culture medium and prepare the vascular lineage induction medium in a 15 mL centrifuge tube: add 20 ng / ml VEGF165, 0.5 mM pyruvate, 20 ng / ml VEGFD, 10 4 U LIF, 2.0 μM Forskolin culture induced vascular lineage cell differentiation, and cultured at 37°C, 5% CO2 for 2 days; (4) The organoid spheres were encapsulated with a gel formed by mixing GelMA and type I collagen at a weight-volume concentration of 3:1 for 3D culture, irradiated with 405 nm or 365 nm blue light for 30 seconds to solidify the gel, and then cultured with culture medium for 6 days; (5) The organoids are then peeled and placed in a low-adhesion well plate for culture. After 5-7 days of culture, multi-lumen network organoid spheres can be obtained.

[0039] (6) The obtained lymphatic vascular sphere organoids were stained with fluorescent staining and found to express the lymphatic endothelial cell-specific marker Prox1 and the smooth muscle marker α-SMA.

[0040] Comparative Example 1 (1) Digest hiPSCs into single-cell suspensions, passage them into ultra-low attachment plates at a ratio of 1:3, resuspend them in complete stem cell culture medium (mTesR), and culture them in the presence of Y-27632 (10 μM) in ultra-low attachment plates at 37°C and 5% CO2 for 1 day to allow embryonic stem cells or induced pluripotent stem cells to form embryoid bodies (EBs). (2) After EBs are formed, they are induced to differentiate into mesoderm. N2B27 medium is added with 6 μM CHIR99021 and 20 ng / mL BMP4 to form a mixed medium. 1.0 mL of this medium is added to gently resuspend the aggregates. 1.0 mL of this medium is slowly added to each well along the side wall. The culture is kept at 37°C and 5% CO2 for 2 days. (3) On the 4th day, discard the old culture medium and prepare the vascular lineage induction medium in a 15 mL centrifuge tube: add 20 ng / ml VEGF165 and 2.0 μM Forskolin to the N2B27 culture medium to induce vascular lineage cell differentiation, and culture at 37°C and 5% CO2 for 2 days; (4) Subsequently, commercial matrix gel and type I collagen were mixed in a 1:1 ratio to form a gel solution. The organoid spheres were then wrapped with the gel solution for 3D culture and incubated at 37°C for 2 h to allow the gel to solidify. The culture time was approximately 6 days. (5) The organoids were then peeled and placed in low-adhesion well plates for culture. At this stage, 0.5 mM pyruvate, 20 ng / ml VEGFD, and 10 4 U LIF and subsequent culture for 6 days can obtain multi-lumen network organoid spheroids.

[0041] The organoids obtained in this control group lacked a lymphatic vessel network.

[0042] Finally, it should be noted that the above specific embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications may be made to the technical solutions described in the above embodiments, or some or all of the technical features therein may be replaced with equivalents. However, such modifications or replacements do not deviate from the essence of the corresponding technical solutions within the scope of the technical solutions of the embodiments of the present invention. Therefore, such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for constructing vascular organoids with lymphatic vessels, characterized in that: The following steps are involved: S1 uses a suspension low-adsorption culture method to culture iPSCs, which have the potential to differentiate into vascular lineage cells, to form aggregates, namely embryoid bodies; S2: Transfer the aggregates into stem cell culture medium to induce the aggregates to differentiate into spherical vascular organoids of mesoderm; S3 then adds the obtained spherical vascular organoids into vascular cell sphere induction medium to induce vascular lineage cell differentiation and obtain organoid precursors; S4 encapsulates the organoid sphere precursors and then performs three-dimensional culture differentiation to obtain organoids with tubular structures; S5 encapsulates the organoid sphere precursors for three-dimensional culture, then peels the organoids and cultures them in ultra-low adhesion well plates to form mature organoid spheres.

2. The construction method according to claim 1, characterized in that In step S1, the pluripotent stem cells (iPSCs) are selected from induced pluripotent stem cells, embryonic stem cells, or adult stem cells of humans or animals.

3. The construction method according to claim 1, characterized in that In step S1, the specific process of the suspension low-adsorption culture method is: resuspending the cells in an ultra-low-adsorption well plate with 10 μM stem cell mTesR or Essential 8 complete culture medium, and culturing at 37° C. and 5% CO 2 for 1-2 days.

4. The construction method according to claim 1, characterized in that In step S2, the medium for inducing mesoderm differentiation is N2B27 medium, and 3-12 µM CHIR99021 and 5-30 ng / mL BMP4 are added to form a mesoderm induction medium. 2 mL of mesoderm induction medium is slowly added along the side wall of each well and cultured for 3 days.

5. The construction method according to claim 1, characterized in that In step S3, the medium for inducing differentiation into vascular lineage cells is N2B27 medium, and the specific process is: the aggregates formed in step S2 are transferred into N2B27 medium, 10-100 ng / ml VEGF165, 0.01-2.0 mM pyruvate, 10-100 ng / ml VEGFC / VEGFD, 10 3 -10 6 Induce vascular lineage cell differentiation with LIF and 0.5-5.0 μM Forskolin and culture at 37°C, 5% CO2 for 2-3 days.

6. The construction method according to claim 1, characterized in that In step S4, the mixed culture medium for the encapsulated culture is StemPro-34 SFM complete culture medium, supplemented with 10-150 ng / ml VEGF165, 10-150 ng / ml FGF2, 10-150 ng / ml VEGF165, 5%-30% FBS and 20-100 ng / ml VEGFC / VEGFD, and the culture time is 4-6 days.

7. The construction method according to any one of claims 1 to 6, characterized in that: In step S4, the specific process is: (1) Remove the culture medium of the organoid precursors, wash with PBS, centrifuge to remove the supernatant, and collect the organoids; (2) Resuspend the organoids in 4°C pre-cooled Matrigel to obtain an organoid Matrigel suspension; (3) Plate the organoid matrix gel suspension on a culture plate preheated at 37°C, incubate in an incubator for 10 minutes, then invert the culture plate and incubate for another 20 minutes; add organoid culture medium and change the medium every 2 days to obtain the target organoid for subculture; (4) After peeling the organoids cultured in three-dimensional matrix gel, transfer them to low-adhesion well plates for culture for 5-7 days to obtain multi-lumen spherical vascular organoids.

8. The construction method according to claim 7, wherein in step S4, the matrix glue is a composite glue formed by an immune organ decellularized matrix or a polymer with good biocompatibility and its derivatives with different functional modifications and a biological protein in a mass ratio of 1-3:1; the biological protein is one or more of collagen (I, II, III), elastin, fibronectin and laminin; the immune organ decellularized matrix is a decellularized lymph node matrix, a decellularized spleen matrix, a decellularized bone marrow matrix, a decellularized thymus matrix or a sarcoma decellularized matrix (Matrigel); the polymer with good biocompatibility and its derivatives with different functional modifications are one or more of gelatin, hyaluronic acid, silk fibroin, chitosan, sodium alginate and polyethylene glycol; the organoid culture medium is StemPro-34 SFM complete medium supplemented with 10-150 ng / ml VEGF165, 10-150 ng / ml FGF2, 10-150 ng / ml VEGF165, 5%-30% FBS and 20-100 ng / ml VEGFC / VEGFD.

9. The construction method according to claim 1, characterized in that: In step S5, the ultra-low adhesion plate is treated with Anti-Adherence Rinsing Solution (Stemcell, 07010) for half an hour in advance.

10. A vascular organoid having lymphatic vessels prepared by the construction method according to any one of claims 1 to 9, characterized in that: The vascular organoid has a lymphatic vessel structure and a vascular network structure inside.

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