Venous vascular organoids induced by human pluripotent stem cells and their construction methods

Abstract

The present invention relates to a method for constructing venous vascular organoids induced by human pluripotent stem cells, comprising the following steps: Step S1, culturing human pluripotent stem cells in a well plate using mTESR1 medium; Step S2, preparing human pluripotent stem cells into a single-cell suspension and forming uniform cell aggregates in an ultra-low adsorption culture plate; Step S3, inducing mesodermal cell spheroids, collecting the cell aggregates, discarding the supernatant, and adding a first culture medium containing Glutamax additive, L-ascorbic acid, CHIR99021, and BMP4, culturing for 2-3 days to induce the formation of mesodermal cell spheroids; Step S4, differentiating venous vascular organoids, adding a second culture medium to promote further differentiation of mesodermal cells into vascular lineages, inducing the formation of a venous vascular network. This invention achieves the directed differentiation of human pluripotent stem cells into venous vascular organoids, successfully constructing microvascular organoids with venous characteristics.

Description

Technical Field

[0001] This invention belongs to the field of organoid construction technology, specifically relating to venous vascular organoids induced by human pluripotent stem cells and their construction methods. Background Technology

[0002] In recent years, the incidence and disease burden of vascular diseases in my country have shown a continuous upward trend, encompassing small vessel disease, microcirculatory disorders, tumor-related vascular abnormalities, diabetic vascular disease, and immune-mediated vascular diseases. These diseases have become the leading cause of death among Chinese residents, highlighting the urgent need to strengthen research on the pathogenesis of vascular diseases and accelerate the development of innovative diagnostic and treatment technologies. As a crucial component of the vascular system, abnormal vein function can lead to deep vein thrombosis, chronic venous diseases, and venous malformations, causing pain, ulcers, tissue hypoxia, and multi-organ complications such as pulmonary embolism and skin abnormalities, severely impacting patients' health and exacerbating the medical burden. However, due to a lack of precise research models, progress in pathological mechanism research and treatment development has been slow.

[0003] While vascular organoid technology, a significant breakthrough in biomedical engineering, has made remarkable progress in vascular biology research, key technological gaps remain in the study of venous system-specific conditions. Currently, mainstream vascular organoid models suffer from the following limitations: First, the organoid technology pioneered by Wimmer's team, as described in *Nature* (Vol. 565, No. 7740, 2019, pp. 505-510), while capable of mimicking capillary networks, lacks the ability to autonomously differentiate into arteries and veins, requiring in vivo transplantation to form a functional vascular network. Second, while the CTGF regulatory mechanism discovered by Romeo's team based on this model provides new insights into diabetic microvascular complications, it cannot specifically mimic the unique pathological features of the venous system. Third, although existing biomaterial construction strategies have made progress in vascular graft development, the lack of a natural vascular differentiation process makes them difficult to apply to studies of venous development and disease mechanisms. These technological limitations severely restrict the study of mechanisms and drug development for venous-specific diseases such as deep vein thrombosis and venous malformations.

[0004] Therefore, establishing vascular organoid models with clearly defined venous characteristics and achieving specific differentiation and functional reconstruction of venous endothelial cells and pericytes is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] The first objective of this invention is to provide a method for constructing venous vascular organoids based on human pluripotent stem cells, addressing the problems in the prior art.

[0006] Therefore, the above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0007] The method for constructing venous vascular organoids based on human pluripotent stem cells includes the following steps:

[0008] Step S1, human pluripotent stem cell culture: human pluripotent stem cells are cultured in well plates using mTESR1 medium.

[0009] Step S2, cell aggregate formation: The human pluripotent stem cells cultured in step S1 are prepared into a single-cell suspension and formed into uniform cell aggregates in an ultra-low adsorption culture plate.

[0010] Step S3: Mesodermal cell spheroid induction. Collect cell aggregates, discard the supernatant, and add the first culture medium containing Glutamax additive, L-ascorbic acid, CHIR99021, and BMP4. Culture for 2-3 days to induce the formation of mesodermal cell spheroids.

[0011] Step S4, differentiation of venous vascular organoids: Collagen I and matrix gel are mixed in a 3:1 ratio to prepare a mixed gel. A portion of the mixed gel is solidified to form a lower gel. Another portion of the mixed gel is resuspended in mesodermal cell spheres and seeded onto the surface of the lower gel to form an upper gel. After the upper gel solidifies, a second culture medium is added and the cells are cultured in an incubator for 6-8 days to promote the continued differentiation of mesodermal cells into vascular lineages and induce the formation of a venous vascular network. The second culture medium contains: Glutamax additive, L-ascorbic acid, VEGFα, bFGF and 10% fetal bovine serum.

[0012] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions:

[0013] As a preferred technical solution of the present invention: the cell confluence of human pluripotent stem cells cultured in step S1 is 80%-90%.

[0014] As a preferred embodiment of the present invention: In step S2, the human pluripotent stem cells cultured in step S1 are washed with PBS, digested with Accutase enzyme, and then prepared into a single-cell suspension, with the cell density adjusted to 2.0 × 10⁻⁶. 5 -5.0×10 5 Cells / mL were seeded into ultra-low adsorption culture plates. In ultra-low adsorption 6-well plates, cells were cultured in mTESR1 medium containing 10 μM Y27632 for 20-24 h to form uniform cell aggregates.

[0015] As a preferred technical solution of the present invention: In step S3, when collecting cell aggregates, after centrifuging at 500 r / min for 1 min and discarding the supernatant, the first culture medium is added.

[0016] As a preferred technical solution of the present invention: In step S4, collagen I solution is prepared first, and then a mixed gel solution is prepared according to the ratio of collagen I and matrix gel 3:1. The lower layer of mixed gel is first laid in the well plate, and then the well plate is placed in a 37°C incubator for 20-30 minutes to solidify. After solidification, the cell spheres are resuspended in the mixed gel and laid on top of the lower gel to form an upper gel that encapsulates the cell spheres. Then the well plate is placed in a 37°C incubator to solidify. After the upper gel has also solidified, a second culture medium is added and cultured for 6-8 days to form a venous network.

[0017] As a preferred technical solution of the present invention: Collagen I solution is composed of collagen I stock solution, 10X Ham's F-12 solution and buffer solution, with a composition ratio of 8:1:1. The buffer solution is an ultrapure aqueous solution containing 2.2g NaHCO3, 0.05N NaOH and 200mM HEPES.

[0018] As a preferred embodiment of the present invention, the first culture medium comprises: Glutamax additive, 60 μg / mL L-ascorbic acid, 12 μM CHIR99021 and 30 ng / mL BMP4.

[0019] As a preferred embodiment of the present invention, the second culture medium comprises: Glutamax additive, 60 μg / mL L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF and 10% fetal bovine serum.

[0020] The second objective of this invention is to provide a venous vascular organoid derived from the directed differentiation of human pluripotent stem cells, addressing the problems in the prior art.

[0021] Therefore, the above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0022] The venous vascular organoids differentiated from human pluripotent stem cells have a distinct vascular network, vascular endothelial cells and pericytes, with pericytes covering the endothelial cells, the endothelial cells forming the lumen, and specifically expressing venous markers. The vascular diameter is between 15μm and 25μm, exhibiting the characteristics of microveins in the human body.

[0023] Compared with existing technologies, the venous vascular organoids induced by human pluripotent stem cells and their construction method of the present invention have the following beneficial effects: The present invention achieves efficient and targeted differentiation of venous vascular organoids through a two-stage culture medium induction of mesodermal differentiation and venous formation, combined with a three-dimensional culture microenvironment formed by a collagen I / matrix mixed matrix; it synergistically activates the mesodermal pathway by combining the GSK3 inhibitor CHIR99021 with BMP4 and L-ascorbic acid; and it promotes endothelial cell proliferation and angiogenesis by combining VEGFα with bFGF, co-inducing the venous phenotype and forming a network structure, establishing a vascular organoid model with clear venous characteristics, realizing the specific differentiation and functional reconstruction of venous endothelial cells and pericytes, which is urgently needed in current vascular biology research and will also provide a revolutionary research platform for precision medicine of venous diseases.

[0024] This invention constructs venous vascular organoids based on human pluripotent stem cells. Through the time-series induction of complex growth factors and the regulation of a three-dimensional microenvironment, it achieves the directed differentiation of human pluripotent stem cells into venous vascular organoids and successfully constructs microvascular organoids with venous characteristics. This provides a highly biomimetic three-dimensional biological model of venous vascular organoids for vascular disease research and provides an innovative platform for revealing the pathophysiological mechanisms of the venous system, advancing high-throughput screening of targeted drugs, and developing personalized precision intervention programs. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the construction method of venous vascular organoids based on human pluripotent stem cells according to the present invention;

[0026] Figure 2 Morphological observations of venous vascular organoids at different differentiation times in Example 1;

[0027] Figure 3 This is an image showing the immunofluorescence staining results of the venous vascular organoids in Example 1, where CD31 represents vascular endothelial cells and PDGFRβ represents vascular pericytes.

[0028] Figure 4 The images show the RT-qPCR results, diameter statistics, and immunofluorescence staining of the venous vascular organoids in Example 1. Image A shows the RT-qPCR results of the vascular organoids, Image B shows the diameter statistics of the vascular organoids, and Image C shows the immunofluorescence staining of the vascular organoids.

[0029] Figure 5 The images show the immunofluorescence staining results and branch point counts of vascular organoids after high sugar and high fat stimulation in Examples 2 and 3. Image A shows the immunofluorescence staining results of vascular organoids after high sugar and high fat stimulation, and Image B shows the branch point counts of vascular organoids after high sugar and high fat stimulation.

[0030] Figure 6 Figure A shows the RT-qPCR results of oxidative stress-related genes in vascular organoids after high glucose and high lipid stimulation in Examples 2 and 3; Figure B shows the RT-qPCR results of oxidative stress-related genes in vascular organoids after high glucose stimulation.

[0031] Figure 7 This is a bright-field morphology diagram of the venous organoid in Comparative Example 1. Detailed Implementation

[0032] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

[0033] like Figure 1 As shown, the method for constructing venous vascular organoids based on human pluripotent stem cells according to the present invention includes the following steps:

[0034] Step S1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium;

[0035] mTESR1 medium is a feeder-free, chemically defined medium specifically designed for the in vitro culture of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

[0036] Step S2: Once the confluence of human pluripotent stem cells cultured in Step 1 reaches 80%-90%, wash the cells twice with PBS, digest them with Accutase enzyme for 5 min, and stop digestion by adding mTESR1 medium. Collect the cells in a centrifuge tube and centrifuge at 1000 r / min for 3 min, discarding the supernatant. Resuspend the cells in cell aggregation medium to obtain a single-cell suspension. Then adjust the cell density of the suspension and seed the above 2.5 × 10⁵ single cells into ultra-low adsorption six-well plates. Culture in cell aggregation medium for 20-24 h to obtain cell aggregates. The cell aggregation medium is mTESR1 medium containing 10 μM Y27632 inhibitor.

[0037] PBS is phosphate buffer.

[0038] In this invention, apoptosis is reduced by combining an ultra-low adsorption plate with the ROCK inhibitor Y27632, thereby promoting the formation of uniform aggregates.

[0039] Step S3: Collect cell aggregates, centrifuge at 500 r / min for 1 min, discard the supernatant, add the first culture medium, and culture for 2-3 days to carry out mesodermal differentiation. The first culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021 and 30 ng / mL BMP4.

[0040] Among them, Advanced DMEM / F12 is a high-performance cell culture medium optimized for the in vitro culture of primary cells and stem cells.

[0041] Glutamax is a stable L-glutamine substitute, chemically named L-alanyl-L-glutamine.

[0042] CHIR99021 is a highly efficient selective inhibitor of glycogen synthase kinase-3 (GSK-3).

[0043] BMP4 is bone morphogenetic protein 4.

[0044] Step S4: First, prepare the collagen I solution, then prepare a mixed gel solution at a ratio of collagen I to matrix gel of 3:1. First, lay the lower layer of the mixed gel in the well plate, then place the plate in a 37°C incubator for 20-30 minutes to allow it to solidify. After solidification, resuspend the cell spheres in the mixed gel and lay it on top of the lower gel to form an upper gel encapsulating the cell spheres. Then, place the plate in a 37°C incubator to allow it to solidify. After the upper gel has also solidified, add the second culture medium and incubate for 6-8 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0045] Among them, bFGF refers to basic fibroblast growth factor;

[0046] VEGFα refers to vascular endothelial growth factor α.

[0047] In this invention, collagen I and matrix gel in a 3:1 ratio are used to simulate the in vivo basement membrane, thereby enhancing the self-assembly ability of the vascular network.

[0048] Specifically, the collagen I solution consists of collagen I stock solution, 10X Ham's F-12 solution and buffer solution in a ratio of 8:1:1. The buffer solution is an ultrapure aqueous solution containing 2.2g NaHCO3, 0.05N NaOH and 200mM HEPES, wherein HEPES is 4-hydroxyethylpiperazine ethanesulfonic acid.

[0049] The reagents and abbreviations involved in this invention are as follows:

[0050] 10×Ham's F-12 solution refers to a 10-fold concentrated solution of Ham's F-12 culture medium, containing nutrients such as amino acids, vitamins, and inorganic salts.

[0051] BMP4: Bone morphogenetic protein 4;

[0052] bFGF: Basic fibroblast growth factor;

[0053] VEGFα: Vascular endothelial growth factor α.

[0054] According to the present invention, L-ascorbic acid plays a beneficial role in endothelial growth, survival and maintenance of vascular responsiveness and integrity, and can also promote the formation of new blood vessels in tumors and induce tumor vascular normalization. In the present invention, the addition of L-ascorbic acid achieves the induction of differentiation of vascular organoids.

[0055] Example 1

[0056] 1.1 Culture of human pluripotent stem cells

[0057] Step 1: Embed the base adhesive in an ice box and place it in a 4°C freezer overnight to melt. Pre-cool the nozzle and EP tube at -20°C in advance.

[0058] Step 2: Dispense the melted matrix gel from Step 1 into EP tubes on ice, 500 μL per EP tube;

[0059] Step 3: Dilute the matrix gel dispensed in Step 2 at a ratio of 1:100 in 6 mL of pre-cooled PBS at 4°C and place it in an ice box for later use.

[0060] Step 4: Culture cells using a six-well plate. Add 1 mL of diluted matrix gel to each well of the six-well plate, rotate the plate to spread the gel evenly, and then fix the six-well plate at 37°C for 40 min. Discard the supernatant from the six-well plate.

[0061] Step 5: Collect the culture supernatant from Step 4, rinse the cells with PBS, add 0.5 mL of Accutase digestive enzyme to each well, and digest for 3-5 min; use a P1000 pipette tip to pipette the digested cells twice to separate the cell colonies into single cells, add culture supernatant to neutralize the digestive enzyme, and centrifuge at 300×g at room temperature for 5 min.

[0062] Step 6: Resuspend the cells obtained in step 5 in mTESR1 medium containing 10 μM Y27632 inhibitor, and then passage them at 37°C. Change the medium 24 hours after passage.

[0063] 1.2 Induction of human pluripotent stem cells into venous vascular organoids:

[0064] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium;

[0065] Step 2: Once the confluence of human pluripotent stem cells cultured in Step 1 reaches 80%-90%, wash the cells twice with PBS, digest them with digestive enzymes for 5 minutes, and stop digestion by adding mTESR1 medium. Collect the cells in a centrifuge tube and centrifuge at 1000 rpm for 3 minutes, discarding the supernatant. Resuspend the cells in cell aggregation medium to obtain a single-cell suspension. Then, adjust the cell density of the suspension by adding 2.5 × 10⁻⁶ cells. 5 Single cells were seeded into ultra-low adsorption six-well plates and cultured in cell aggregation medium for 24 h to obtain cell aggregates. The cell aggregation medium was mTESR1 medium containing 10 μM Y27632 inhibitor.

[0066] Step 3: Collect cell aggregates, centrifuge at 500 r / min for 1 min, discard the supernatant, add the first culture medium, and culture for 3 days to carry out mesodermal differentiation. The first culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021 and 30 ng / mL BMP4.

[0067] Step 4: First, prepare the collagen I solution, then prepare a mixed gel solution according to the ratio of collagen I to matrix gel of 3:1. First, lay the lower layer of mixed gel in the well plate, then place the well plate in a 37°C incubator for 20-30 minutes to allow it to solidify. After solidification, resuspend the cell spheres in the mixed gel and lay it on top of the lower gel to form an upper gel that encapsulates the cell spheres. After the upper gel also solidifies, add the second culture medium and culture for 7 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF and 10% fetal bovine serum.

[0068] This embodiment uses an inverted microscope to observe the morphology of various stages of vascular organoid development, and the results are as follows: Figure 2 As shown, hiPSCs gradually form embryoid bodies, relatively dense mesodermal cell spheres, and eventually form a distinct venous network.

[0069] This embodiment uses immunofluorescence staining technology to identify vascular organoids, such as... Figure 3As shown, the vascular organoids contain endothelial cells and pericytes, and can spontaneously assemble to form a vascular network. At the same time, pericytes are seen covering the vascular endothelial cells, and there are vascular lumens between the endothelial cells, indicating that the vascular organoids of the present invention are quite similar to blood vessels in the body.

[0070] This embodiment uses quantitative reverse transcription polymerase chain reaction (RT-qPCR) to identify vascular organoids and statistically analyze vascular diameters, such as... Figure 4 As shown, the vascular organoids highly express venous markers NRP2, EphB4, and COUP-TFII, while expressing low levels of arterial marker SOX17 and arteriole marker DLL4. The average diameter of the vessels in the vascular organoids is approximately 21 μm, close to that of human venules. Further immunofluorescence staining showed that the vascular organoids highly express the venule marker EphB4, indicating that the vascular organoids constructed in this embodiment are human venule vascular organoids.

[0071] Example 2

[0072] High sugar levels stimulate vascular organoids:

[0073] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium;

[0074] Step 2: Once the confluence of human pluripotent stem cells cultured in Step 1 reaches 80%-90%, wash the cells twice with PBS, digest them with digestive enzymes for 5 minutes, and stop digestion by adding mTESR1 medium. Collect the cells in a centrifuge tube and centrifuge at 1000 rpm for 3 minutes, discarding the supernatant. Resuspend the cells in cell aggregation medium to obtain a single-cell suspension. Then, adjust the cell density of the suspension by adding 2.5 × 10⁻⁶ cells. 5 Single cells were seeded into ultra-low adsorption plates and cultured in cell aggregation medium for 24 h to obtain cell aggregates. The cell aggregation medium was mTESR1 medium containing 10 μM Y27632 inhibitor.

[0075] Step 3: Collect cell aggregates, centrifuge at 500 r / min for 1 min, discard the supernatant, add the first culture medium, and culture for 3 days to carry out mesodermal differentiation. The first culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021 and 30 ng / mL BMP4.

[0076] Step 4: First, prepare the collagen I solution, then prepare a mixed gel solution according to the ratio of collagen I to matrix gel of 3:1. First, lay the lower layer of mixed gel in the well plate, then place the well plate in a 37°C incubator for 20-30 minutes to solidify it. After solidification, resuspend the cell spheres in the mixed gel and lay it on top of the lower gel to form an upper gel that encapsulates the cell spheres. After the upper gel also solidifies, add the second culture medium and culture for 6 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF and 10% fetal bovine serum.

[0077] Step 5: Replace the second culture medium with high-glucose medium 3 and culture for 5 days, changing the medium every 24 hours. Simultaneously, the control group continues to culture in control medium 5 for 5 days, changing the medium every 24 hours. High-glucose medium 3 consists of Advanced DMEM / F12, 1× Glutamax additive, 100 ng / mL VEGFα, 100 ng / mL bFGF, 10% fetal bovine serum, and 25 mM D(+) glucose. Control medium 5 consists of Advanced DMEM / F12, 1× Glutamax additive, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0078] After the high-sugar stimulation treatment in step 5, the following tests were performed:

[0079] Immunofluorescence staining: Immunofluorescence staining detection of vascular organoids after high glucose stimulation;

[0080] Statistics on the number of branch points in the CD31+ vascular network: Statistical analysis of the number of branch points in the CD31+ vascular network;

[0081] RT-qPCR: RT-qPCR experiments were performed on vascular organoids stimulated with high glucose.

[0082] This embodiment uses immunofluorescence staining technology to identify vascular organoids, such as... Figure 5 As shown, the number of vascular branching points decreased significantly after high glucose stimulation, indicating that the high glucose environment inhibited angiogenesis in vascular organoids.

[0083] This embodiment uses RT-qPCR to identify vascular organoids, such as Figure 6 As shown in Figure A, the expression levels of oxidative stress-related genes in vascular organoids increased significantly after high glucose stimulation, indicating that vascular organoids can respond to oxidative stress in a high glucose environment.

[0084] Example 3

[0085] High-fat diets stimulate vascular organoids:

[0086] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium;

[0087] Step 2: Once the confluence of human pluripotent stem cells cultured in Step 1 reaches 80%-90%, wash the cells twice with PBS, digest them with digestive enzymes for 5 minutes, and stop digestion by adding mTESR1 medium. Collect the cells in a centrifuge tube and centrifuge at 1000 rpm for 3 minutes, discarding the supernatant. Resuspend the cells in cell aggregation medium to obtain a single-cell suspension. Then, adjust the cell density of the suspension by adding 2.5 × 10⁻⁶ cells. 5 Single cells were seeded into ultra-low adsorption plates and cultured in cell aggregation medium for 24 h to obtain cell aggregates. The cell aggregation medium was mTESR1 medium containing 10 μM Y27632 inhibitor.

[0088] Step 3: Collect cell aggregates, centrifuge at 500 r / min for 1 min, discard the supernatant, add the first culture medium, and culture for 3 days to carry out mesodermal differentiation. The first culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021 and 30 ng / mL BMP4.

[0089] Step 4: First, prepare the collagen I solution, then prepare a mixed gel solution at a ratio of collagen I to matrix gel of 3:1. First, lay the lower layer of mixed gel in the well plate, then place the well plate in a 37°C incubator for 20-30 minutes to allow it to solidify. After solidification, resuspend the cell spheres in the mixed gel and lay them on top of the lower gel to form an upper gel encapsulating the cell spheres. After the upper gel also solidifies, add the second culture medium and culture for 6 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 60 μg / ml L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF and 10% fetal bovine serum.

[0090] Step 5: Replace the second culture medium with high-fat medium 4 and incubate for 5 days, changing the medium every 24 hours. Simultaneously, replace the control medium with control medium 5 and continue incubation for 5 days, changing the medium every 24 hours. High-fat medium 4 consists of Advanced DMEM / F12, 1× Glutamax additive, 100 ng / mL VEGFα, 100 ng / mL bFGF, 10% fetal bovine serum, and 0.5 mM palmitic acid. Control medium 5 consists of Advanced DMEM / F12, 1× Glutamax additive, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0091] After the high-fat stimulation treatment in step 5, the following tests were performed:

[0092] Immunofluorescence staining: Immunofluorescence staining detection of vascular organoids after high-fat stimulation;

[0093] Statistics on the number of vascular branch points: for CD31 + Statistical analysis of the number of branch points in the vascular network;

[0094] RT-qPCR: RT-qPCR experiments were performed on vascular organoids stimulated with high lipid levels.

[0095] This embodiment uses immunofluorescence staining technology to identify vascular organoids, such as... Figure 5 As shown, the number of vascular branching points decreased significantly after high-fat stimulation, indicating that the high-fat environment inhibited angiogenesis in vascular organoids.

[0096] This embodiment uses RT-qPCR to identify vascular organoids, such as Figure 6 As shown in Figure B, the expression levels of oxidative stress-related genes in vascular organoids increased significantly after high-fat stimulation, indicating that vascular organoids can respond to oxidative stress in a high-fat environment.

[0097] Comparative Example 1

[0098] This comparative example provides a method for preparing vascular organoids, which differs from the method in Example 2 in that L-ascorbic acid is not added to the first and second culture media. The specific operation is as follows:

[0099] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium;

[0100] Step 2: Once the confluence of human pluripotent stem cells cultured in Step 1 reaches 80%-90%, wash the cells twice with PBS, digest them with digestive enzymes for 5 min, and stop digestion by adding mTESR1 medium. Collect the cells in a centrifuge tube and centrifuge at 1000 r / min for 3 min, discarding the supernatant. Resuspend the cells in cell aggregation medium to obtain a single-cell suspension. Then, adjust the cell density of the suspension and seed the above 2.5 × 10⁵ single cells into ultra-low adsorption six-well plates. Culture in cell aggregation medium for 24 h to obtain cell aggregates. The cell aggregation medium is mTESR1 medium containing 10 μM Y27632 inhibitor.

[0101] Step 3: Collect cell aggregates, centrifuge at 500 r / min for 1 min, discard the supernatant, add the first culture medium, and culture for 3 days to carry out mesodermal differentiation. The first culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 12 μM CHIR99021 and 30 ng / mL BMP4.

[0102] Step 4: First, prepare the collagen I solution, then prepare a mixed gel solution at a ratio of collagen I to matrix gel of 3:1. First, lay the lower layer of mixed gel in the well plate, then place the well plate in a 37°C incubator for 20-30 minutes to allow it to solidify. After solidification, resuspend the cell spheres in the mixed gel and lay it on top of the lower gel to form an upper gel that encapsulates the cell spheres. After the upper gel also solidifies, add the second culture medium and culture for 7 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax additive, 100 ng / mL VEGFα, 100 ng / mL bFGF and 10% fetal bovine serum.

[0103] This comparative example uses an inverted microscope to observe the morphology of vascular organoids, such as... Figure 7 As shown in the comparative example, without the addition of L-ascorbic acid, the vascular organoids could not form a vascular branching network, demonstrating that L-ascorbic acid plays an important role in angiogenesis.

[0104] Experimental Example 1

[0105] Immunofluorescence staining for identification of vascular organoids:

[0106] Step 1: Use a P1000 pipette to aspirate the culture medium from the well plate and incubate the sample with 4% tissue cell fixative for 20 min at room temperature. Discard the 4% tissue cell fixative and then add PBS.

[0107] Step 2: Under a microscope, use ultrafine scissors and fine tweezers to cut the gel obtained in Step 1 into small pieces, avoiding cutting the vascular network. Use tweezers to transfer the gel fragments into fresh 2 mL EP tubes, add blocking buffer, and allow to permeate the vascular network at room temperature for 2 hours. For the vascular network, add 2 mL of blocking buffer to each tube and the rock tube rack (placed vertically on a vibrating track screen) to allow for proper permeation.

[0108] Step 3: Remove the blocking buffer and add 50-100 μL of primary antibody diluted in the blocking buffer. Incubate the vascular network upright on a shaker overnight at 4°C.

[0109] Step 4: On the second day, wash the sample three times in PBS-T solution for 10-15 minutes each time. For vascular networks, place the tube rack vertically on the track vibrating screen for cleaning.

[0110] Step 5: Add secondary antibody to the sample from step 4 in blocking buffer and incubate at room temperature for 2 hours; wash the sample twice in PBS-T solution for 10-15 minutes each time; then add DAPI solution (1:1000 in PBS) and incubate for 10-15 minutes; finally wash the sample in PBS for 10-15 minutes.

[0111] Step 6: Transfer the sample to a glass slide, cover it with a coverslip, and then take a photograph.

[0112] The immunofluorescence staining markers used in the above method are as follows: CD31 represents vascular endothelial cells, PDGFRβ represents vascular pericytes, and EphB4 represents venous endothelial cells.

[0113] Experiment Example 2

[0114] RT-qPCR:

[0115] Step 1: Transfer the cultured vascular organoids to a 15ml centrifuge tube, add fresh PBS up to the top of the tube, centrifuge for 5 minutes at 1000 rpm, and discard the supernatant.

[0116] Step 2: Add 1 ml of Trizol and repeatedly pipette to ensure complete lysis of the sample;

[0117] Step 3: Add 200 μl of chloroform, shake for 15 seconds, let stand at room temperature for 5 minutes, then centrifuge at 4°C for 15 minutes at a speed of 12000 g;

[0118] Step 4: Transfer the supernatant to a new 1.5ml enzyme-free centrifuge tube, add an equal volume of isopropanol, invert to mix, let stand at room temperature for 10 minutes, centrifuge at 4℃ for 10 minutes; the speed is 12000g, and discard the supernatant.

[0119] Step 5: Slowly add 1 ml of 75% ethanol solution along the wall of the centrifuge tube, invert the tube to wash, centrifuge at 4°C for 5 min at 12000 g, and discard the supernatant;

[0120] Step 6: After drying at room temperature for 5 minutes, add an appropriate amount of enzyme-free water to fully dissolve the RNA;

[0121] Step 7: Add RNA solution and reverse transcriptase in proportion to prepare a reverse transcription reaction mixture. Incubate at 37°C for 15 min, then at 85°C for 5 s to obtain the sample cDNA solution.

[0122] Step 8: Using the obtained cDNA as a template, add primers for the target gene, and use a real-time quantitative fluorescence kit and a real-time quantitative PCR instrument to detect the expression level of the target gene.

[0123] The above specific embodiments are used to explain and illustrate the present invention, and are only preferred embodiments of the present invention, not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for constructing venous vascular organoids based on human pluripotent stem cells, characterized in that, Includes the following steps: Step S1: Culture human pluripotent stem cells in well plates using mTESR1 medium; Step S2, cell aggregate formation: The human pluripotent stem cells cultured in step S1 are prepared into a single-cell suspension and formed into uniform cell aggregates in an ultra-low adsorption culture plate. Step S3: Mesodermal cell spheroidization, cell aggregates are collected, the supernatant is discarded and the first culture medium is added. The first culture medium contains: Glutamax additive, 60 μg / mL L-ascorbic acid, 12 μM CHIR99021 and 30 ng / mL BMP4. Step S4: Differentiation of venous vascular organoids. Collagen I and matrix gel are mixed in a 3:1 ratio to prepare a mixed gel. A portion of the mixed gel is solidified to form a lower gel layer. Another portion of the mixed gel is resuspended in mesodermal cell spheres and seeded onto the surface of the lower gel layer to form an upper gel layer. After the upper gel solidifies, a second culture medium is added, and the cells are cultured in an incubator for 6-8 days to promote the continued differentiation of mesodermal cells into vascular lineages and induce the formation of a venous vascular network. The second culture medium contains: Glutamax additive, 60 μg / mL L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum. The venous vascular organoids have a distinct vascular network, with vascular endothelial cells and pericytes. Pericytes cover endothelial cells, and endothelial cells form an inner lumen. They specifically express venous markers, and the vascular diameter is between 15 μm and 25 μm, exhibiting the characteristics of microveins in the human body.

2. The method for constructing venous vascular organoids based on human pluripotent stem cells as described in claim 1, characterized in that: The cell confluence of human pluripotent stem cells cultured in step S1 is 80%-90%.

3. The method for constructing venous vascular organoids based on human pluripotent stem cells as described in claim 1, characterized in that: In step S2, the human pluripotent stem cells cultured in step S1 are washed with PBS, digested with Accutase enzyme to prepare a single-cell suspension, and the cell density is adjusted to 2.0 × 10⁻⁶. 5 -5.0×10 5 Cells / mL were seeded into ultra-low adsorption culture plates. In ultra-low adsorption 6-well plates, cells were cultured in mTESR1 medium containing 10 μM Y27632 for 20-24 hours to form uniform cell aggregates.

4. The method for constructing venous vascular organoids based on human pluripotent stem cells as described in claim 1, characterized in that: In step S3, when collecting cell aggregates, the supernatant is discarded after centrifugation at 500 r / min for 1 min, and the first culture medium is added.

5. The method for constructing venous vascular organoids based on human pluripotent stem cells as described in claim 1, characterized in that: In step S4, collagen I solution is prepared first, and then a mixed gel solution is prepared according to the ratio of collagen I to matrix gel of 3:

1. The lower layer of mixed gel is first laid in the well plate, and then the well plate is placed in a 37°C incubator for 20-30 minutes to solidify. After solidification, the cell spheres are resuspended in the mixed gel and laid on top of the lower gel to form an upper gel that encapsulates the cell spheres. The well plate is then placed in a 37°C incubator to solidify. After the upper gel has also solidified, the second culture medium is added and cultured for 6-8 days to form a venous network.

6. The method for constructing venous vascular organoids based on human pluripotent stem cells as described in claim 1, characterized in that: Collagen I solution consists of collagen I stock solution, 10× Ham's F-12 solution and buffer solution in a ratio of 8:1:

1. The buffer solution is an ultrapure aqueous solution containing 2.2g NaHCO3, 0.05N NaOH and 200mM HEPES.

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