A human pluripotent stem cell-induced hepatic sinusoid-like blood vessel organoid and a method for constructing the same

Abstract

The present application relates to the field of stem cell differentiation and tissue engineering, in particular, it relates to a kind of liver sinusoid-like vascular organoid based on human pluripotent stem cell induction and its construction method, including the following steps, culture human pluripotent stem cell;Form cell embryoid body;Mesodermal cell sphere induction;Vascular progenitor cell ball differentiation;Induction differentiation of liver sinusoidal vascular network;Liver sinusoidal vascular network matures, i.e. obtain liver sinusoid-like vascular organoid.Combined with the three-dimensional culture microenvironment formed by liver acellular matrix hydrogel, synergistic effect with specific VEGF subtype (VEGF-121) is realized, and high-efficiency directional differentiation and maintenance of liver sinusoid-like vascular organoid are realized.

Description

Technical Field

[0001] This invention relates to the field of stem cell differentiation and tissue engineering, and more specifically, to a liver sinusoidal vascular organoid induced by human pluripotent stem cells and a method for constructing the same. Background Technology

[0002] The vascular system is one of the most widely distributed systems in the human body, responsible for the transport of oxygen and nutrients and the removal of metabolic waste, playing a vital role in maintaining tissue homeostasis. With the rapid development of stem cell technology and tissue engineering, the in vitro construction of three-dimensional vascular organoids using human pluripotent stem cells has become a hot topic in biomedical research. These vascular organoids can reproduce the three-dimensional luminal structure, basement membrane formation, and some physiological functions of human blood vessels in vitro, providing advanced models superior to traditional two-dimensional cell culture for research in vascular developmental biology, the analysis of pathological mechanisms of vascular-related diseases (such as diabetic vascular complications), and drug screening.

[0003] While general techniques for constructing vascular organoids have made some progress, most existing induction protocols generate non-specific vascular networks, often lacking the vascular heterogeneity specific to the organ's microenvironment. Vascular endothelial cells in different organs exhibit high structural and functional heterogeneity to adapt to the specific functions of their respective organs. For example, cerebral blood vessels have a tight blood-brain barrier, while glomerular blood vessels have a unique filtration function. Currently, research on how to precisely induce and maintain organ-specific vascular phenotypes (such as those of the liver, kidneys, and brain) remains relatively scarce, which significantly limits the depth of application of vascular organoids in organ-specific disease models.

[0004] As the largest metabolic and detoxification organ in the human body, the liver possesses a highly specialized vascular network—the hepatic sinusoids. Hepatic sinusoidal endothelial cells are unique capillary endothelial cells in the liver, with phenotypic characteristics significantly different from ordinary vascular endothelial cells: they lack a continuous basement membrane, possess characteristic fenestrated structures, and highly express specific scavenger receptors (such as LYVE-1, STAB2, FCGR2B, etc.), playing a central role in hepatic substance exchange, immune tolerance, and regeneration.

[0005] However, hepatic sinusoidal endothelial cells are extremely unstable in vitro, readily dedifferentiating and rapidly losing their unique fenestration structure and molecular markers, transforming into a common vascular endothelial phenotype. Existing vascular organoid induction systems mainly rely on universal matrix gels (such as Matrigel) and conventional growth factors (such as VEGF-165), which are insufficient to provide the liver-specific extracellular matrix microenvironment and specific signal induction. Consequently, the generated organoids cannot effectively acquire or maintain the characteristic phenotypes of hepatic sinusoidal endothelial cells (such as high expression of LYVE-1 and FCGR2B).

[0006] To address the aforementioned technical bottlenecks, there is an urgent need to develop a method for constructing vascular organoids that can simulate the liver microenvironment and efficiently induce and maintain hepatic sinusoidal characteristics. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing hepatic sinusoidal vascular organoids induced by human pluripotent stem cells. By combining the three-dimensional culture microenvironment formed by decellularized liver matrix hydrogel with the synergistic effect of a specific VEGF subtype (VEGF-121), the efficient directed differentiation and maintenance of hepatic sinusoidal vascular organoids are achieved.

[0008] Another objective of this invention is to provide a hepatic sinusoidal vascular organoid induced by human pluripotent stem cells, which has a distinct vascular network, CD31+ vascular endothelial cells, and hepatic sinusoidal endothelial cells expressing LYVE-1+FCGR2B+.

[0009] The technical problem solved by this invention is achieved by the following technical solution.

[0010] Step S1: Culture human pluripotent stem cells in well plates using mTeSR1 medium; Step S2: Prepare the pluripotent stem cells from step S1 into a single-cell suspension and form uniformly sized cell embryoids in the well plates of an Aggrewell-800. Step S3: Mesodermal cell spheroid induction. Collect cell embryoids, discard the supernatant, and add mesodermal induction medium, which includes BMP-4 and CHIR99021. Culture for 3 days to induce the formation of mesodermal cell spheroids. Step S4, differentiation of vascular progenitor cell spheroids: add vascular progenitor cell spheroid differentiation medium, which includes VEGF-165 and FGF-2, and culture for 2 days to induce the formation of vascular progenitor cell spheroids; Step S5, Induction and differentiation of hepatic sinusoidal vascular network: vascular progenitor cell spheres are embedded in liver decellularized matrix hydrogel, and the first induction medium for hepatic sinusoidal vascular organoids is added for induction for 6 days. The first induction medium for hepatic sinusoidal vascular organoids includes VEGF-121 and FGF-2. Step S6: Maturation of the hepatic sinusoidal vascular network. The culture medium is replaced with the second induction medium for hepatic sinusoidal vascular organoids. Induction is performed for 4-8 days to promote and maintain the maturation of the hepatic sinusoidal vascular network. The second induction medium for hepatic sinusoidal vascular organoids includes SB431542 and 8-Br-cAMP.

[0011] As a preferred technical solution of the present invention: in step S1, the cell confluence of the cultured human pluripotent stem cells is 70%-80%.

[0012] As a preferred embodiment of the present invention: In step S2, the human pluripotent stem cells cultured in step S1 are washed twice with PBS, digested with TrypLE for 3-4 min to prepare a single-cell suspension, and the cell density is adjusted to 1.5 × 10⁻⁶ cells / mL. 5 Cells / mL were seeded into 24-well plates of Aggrewell-800 treated with ultra-low adhesion, centrifuged at 200×g for 3 min, and the cells settled into the wells. The cells were cultured in mTeSR1 medium containing 10 μM Y27632 for 24 hours to form uniformly sized cell embryoids.

[0013] As a preferred technical solution of the present invention: In step S3, cell embryoids are collected in a 15 mL centrifuge tube, the supernatant is discarded by natural sedimentation, the cell embryoids are washed with DPBS, and then the culture medium is replaced with mesotherapy medium.

[0014] As a preferred technical solution of the present invention: in step S5, the protein concentration of the liver decellularized matrix hydrogel is 5 mg / mL.

[0015] As a preferred embodiment of the present invention, the mesotherapy medium comprises: DMEM / F12 (50%, v / v), Neurobasal Medium (50%, v / v), 1×N2, 1×B27, 10 μM CHIR99021 and 25 ng / mL BMP-4.

[0016] As a preferred embodiment of the present invention, the vascular progenitor cell differentiation medium comprises: DMEM / F12 (50%, v / v), Neurobasal Medium (50%, v / v), 100 ng / mL VEGF-165 and 100 ng / mL FGF-2.

[0017] As a preferred embodiment of the present invention, the first induction culture medium for hepatic sinusoidal vascular organoids comprises: StemPro-34 SFM (50%, v / v), advanced-DMEM / F12 (50%, v / v), 2 mM L-Glutamine, 10 ng / mL VEGF-121 and 30 ng / mL FGF-2.

[0018] As a preferred embodiment of the present invention, the second induction culture medium for liver sinusoidal vascular organoids comprises: StemPro-34 SFM (50%, v / v), advanced-DMEM / F12 (50%, v / v), 2 mM L-Glutamine, 30 ng / mLFGF-2, 6 μM SB431542 and 1 mM 8-Br-cAMP.

[0019] The second objective of this invention is to address the problems in the prior art by providing a hepatic sinusoidal vascular organoid derived from the directed differentiation of human pluripotent stem cells. This hepatic sinusoidal vascular organoid, prepared by the above method, has a distinct vascular network, CD31+ vascular endothelial cells, and also LYVE-1+ FCGR2B+ hepatic sinusoidal endothelial cells.

[0020] Compared with the prior art, the liver sinusoidal vascular organoids induced by human pluripotent stem cells and their construction method of the present invention have the following beneficial effects: This invention achieves efficient and targeted differentiation and maintenance of hepatic sinusoidal vascular organoids through culture medium induction of four stages: mesodermal differentiation, vascular progenitor cell spheroid formation, hepatic sinusoidal vascular organoid formation and maturation. This is achieved in conjunction with a three-dimensional culture microenvironment formed by decellularized liver matrix hydrogel, synergistically with a specific VEGF isoform (VEGF-121). The mesodermal pathway is synergistically activated by the GSK3 inhibitor CHIR99021 combined with BMP4. VEGF-121 combined with FGF-2 promotes the differentiation of generalized endothelial cells into LVYE-1+ hepatic sinusoidal endothelial cells and angiogenesis. Furthermore, the introduction of the cAMP signaling pathway activator 8-Br-cAMP and the TGF-β inhibitor SB431542 combined with FGF-2 induces the maturation of LVYE-1+ hepatic sinusoidal endothelial cells and the expression of the FCGR2B marker, forming a network structure and establishing a vascular organoid model with distinct hepatic sinusoidal characteristics. Hepatic sinusoidal vascular organoids with high expression of characteristic markers such as LYVE-1 and FCGR2B and similar morphology were successfully constructed.

[0021] This invention constructs hepatic sinusoidal vascular organoids based on human pluripotent stem cells. Through complex growth factor temporal regulation and the preparation of a three-dimensional microenvironment, the construction of hepatic sinusoidal vascular organoids based on human pluripotent stem cells has been achieved. The microvascular network organoids with hepatic sinusoidal characteristics have been successfully constructed, filling the gap in the field of organ-specific vascular organoids. It also provides a research platform that is closer to the in vivo physiological state for in vitro modeling of liver diseases, liver toxicology testing, and liver regenerative medicine. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the construction method of hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to the present invention; Figure 2 Morphological comparison of hepatic sinusoidal vascular organoids induced by different VEGF subtypes (VEGF165 or VEGF121); Figure 3 Immunofluorescence of hepatic sinusoidal vascular organoids induced by different VEGF subtypes (VEGF165 or VEGF121); Figure 4 Comparison of qPCR results for hepatic sinusoidal vascular organoids induced by different VEGF subtypes (VEGF165 or VEGF121). Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0026] like Figure 1 As shown, the method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to the present invention includes the following steps: Step S1: Culture human pluripotent stem cells in well plates using mTeSR1 medium; 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).

[0027] Step S2: When the iPSC confluence in step S1 reaches 70%-80%, wash the cells twice with PBS, digest with TrypLE for 3-4 minutes, and terminate digestion with mTeSR1 medium to obtain a single-cell suspension. Then adjust the cell density to 1.5 × 10⁶ cells / mL. 5 Cells were seeded into Aggrewell-800 well plates, and the plates were then centrifuged at 200×g in three groups to promote single-cell settling and aggregation, forming uniformly sized cell embryoids. The cell aggregation medium was mTeSR1 medium containing 10 μMY27632.

[0028] PBS is phosphate-buffered saline. Y27632 is a ROCK inhibitor that can reduce apoptosis and promote embryoid formation.

[0029] Step S3: After discarding the supernatant from the Aggrewell-800 plate in S2, add mesodermal induction medium and culture for 3 days to induce the formation of mesodermal cell spheroids. The mesodermal medium consists of DMEM / F12 (50%, v / v), Neurobasal Medium (50%, v / v), 1×N2, 1×B27, 10 μM CHIR99021 and 25 ng / mL BMP-4.

[0030] DMEM / F12 is a common cell culture medium that provides the nutrients needed by cells; Neurobasal Medium is a neuronal culture medium that can provide rich nutrients for primary cells and stem cells; N2 and B27 are serum-free supplements with well-defined nutrient compositions that provide rich nutrients for primary cells and stem cells; CHIR99021 is a highly efficient selective inhibitor of glycogen synthase kinase-3 (GSK-3); and BMP-4 is bone morphogenetic protein 4.

[0031] Step S4: Discard the mesotherapy medium from S3, add vascular progenitor cell spheroid differentiation medium, and culture for 2 days to induce the formation of vascular progenitor cell spheroids. The vascular progenitor cell spheroid differentiation medium consists of DMEM / F12 (50%, v / v), Neurobasal Medium (50%, v / v), 100 ng / mL VEGF-165, and 100 ng / mL FGF-2.

[0032] VEGF-165 is endothelial growth factor 165; FGF-2 is basic fibroblast growth factor.

[0033] In step S5, first, layer the lower layer of decellularized liver matrix hydrogel in the well plate, then place the well plate in a 37°C incubator for 20 minutes until it is almost solidified. Simultaneously, aspirate the vascular progenitor cell spheroids from step S4 into a 15 mL centrifuge tube, allow them to settle naturally, discard the supernatant, and wash twice with PBS. Resuspend the vascular progenitor cell spheroids in the decellularized liver matrix hydrogel, and layer them on top of the lower gel to form an upper gel encapsulating the cell spheroids. Then, place the well plate in a 37°C incubator until it is completely solidified. Add the first induction medium for hepatic sinusoidal vascular organoids and induce induction for 6 days. The first induction medium for hepatic sinusoidal vascular organoids consists of StemPro-34 SFM (50%, v / v), advanced-DMEM / F12 (50%, v / v), 2 mM L-Glutamine, 10 ng / mL VEGF-121, and 30 ng / mL FGF-2.

[0034] StemPro-34 SFM is a serum-free culture medium suitable for the development of artificial blood cells in the culture system; advanced-DMEM / F12 is a high-performance cell culture medium optimized for the in vitro culture of primary cells and stem cells; L-Glutamine is L-glutamine, which participates in the synthesis of cellular proteins and the production of glucose; VEGF-121 is endothelial growth factor 121.

[0035] The synergistic effect of decellularized liver matrix hydrogel and VEGF-121 positively promotes the differentiation and morphological formation of hepatic sinusoidal vascular organoids from vascular progenitor cell spheroids. In this invention, the budding and tube formation of hepatic sinusoidal vascular organoids were achieved using decellularized liver matrix hydrogel, while VEGF-121 facilitated the expression of characteristic morphology and biomarkers of hepatic sinusoids.

[0036] Step S6: The culture medium is replaced with the second induction medium for hepatic sinusoidal vascular organoids, and induction is performed for 4-8 days to promote and maintain the maturation of the hepatic sinusoidal vascular network. The second induction medium for hepatic sinusoidal vascular organoids consists of StemPro-34 SFM (50%, v / v), advanced-DMEM / F12 (50%, v / v), 2 mM L-Glutamine, 30 ng / mL FGF-2, 6 μM SB431542 and 1 mM 8-Br-cAMP.

[0037] SB431542 is an inhibitor of the TGF-beta signaling pathway, and inhibition of this pathway helps induce the expression of STAB2 and FCGR2B. 8-Br-cAMP is an activator of the cAMP signaling pathway, and activation of this pathway can upregulate the expression of LVYE-1, STAB2, and FCGR2B. SB431542 and 8-Br-cAMP play a positive role in the growth, maturation, and maintenance of hepatic sinusoidal vascular organoids.

[0038] In step S5, the method for preparing the decellularized liver matrix hydrogel includes the following steps: Obtaining biological matrix materials: Animal-derived liver tissue was taken, and the hepatic artery, vein and bile duct of the liver tissue were cut off. The liver tissue was perfused with heparinized PBS for 15 min, followed by washing with sterile PBS three times for 1 h each time to obtain the processed liver tissue. Decellularization: The treated liver tissue is cut into 1-3 mm pieces. 3 Small tissue pieces were incubated with PBS containing sodium dodecyl sulfate and Triton X-100, rinsed with deionized water by shaking, and the water was changed every 40-60 min for a total of 15-25 times. The tissues were then freeze-dried under vacuum to obtain lyophilized tissues. Digestion into gel: The freeze-dried tissue was ground into fragments, pepsin was added, and the mixture was stirred at a constant temperature to digest the tissue and obtain a liver extracellular matrix solution. Under ice bath conditions, the pH of the liver extracellular matrix solution was adjusted to neutral with 1 M sodium hydroxide solution, and then incubated at 37°C to form a gel, thus obtaining a decellularized liver matrix hydrogel.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments. Example 1 1. Culture of human pluripotent stem cells Step 1: Melt the matrix gel overnight at 4°C, then dilute it in DMEM / F12 medium at a ratio of 1:100 and incubate overnight at 4°C. Step 2: Spread the diluted matrix adhesive from Step 1 into a six-well plate, fix at 37°C for 1 hour, and then remove the matrix adhesive. Step 3: Passage human pluripotent stem cells into well plates from Step 2 and culture until confluence reaches 70%-80%. Wash the cells with PBS, add 0.5 mL of RelesR to each well for digestion for 3-4 minutes. Use a P1000 pipette tip to pipette the digested cells twice to form cell debris. Add mTeSR1 to neutralize the digestive enzymes and centrifuge at 300×g for 3 minutes.

[0040] Step 4: Resuspend the cells from Step 3 in mTeSR1 medium, and then passage them at 37 °C. Change the medium 24 h after passage.

[0041] 2. Human pluripotent stem cells induced to differentiate into hepatic sinusoidal vascular organoids Step 1: Culture human pluripotent stem cells in a six-well plate using mTeSR1 medium; Step 2: When the cells from Step 1 have reached a confluence of 70%-80%, wash the cells twice with PBS, add 1 mL of TrypLE to each well for 3-4 minutes of digestion; use a P1000 pipette tip to pipette the digested cells to form single cells, add mTeSR1 to neutralize the digestive enzymes, and centrifuge at 300×g for 3 minutes. Adjust the cell density of the suspension to 1.5×10⁻⁶ cells / well. 5 Cells / mL were seeded into Aggrewell-800 plates treated with low adhesion medium, centrifuged at 200×g for 3 minutes to force cell sedimentation and aggregation, and then cultured in mTeSR1 medium containing 10 μM Y27632 for 24 hours. Step 3: After discarding the supernatant from the Aggrewell-800 plate in Step 2, add mesotherapy induction medium and culture for 3 days to induce the formation of mesotherapy cell spheroids; the mesotherapy medium consists of DMEM / F12 (50%, v / v), Neurobasal Medium (50%, v / v) 1×N2, 1×B27, 10 μM CHIR99021 and 25 ng / mL BMP-4; Step 4: Discard the mesotherapy medium from Step 3, add vascular progenitor cell spheroid differentiation medium, and culture for 2 days to induce the formation of vascular progenitor cell spheroids. The vascular progenitor cell spheroid differentiation medium consists of DMEM / F12 (50%, v / v), Neurobasal Medium (50%, v / v), 100 ng / mL VEGF-165, and 100 ng / mL FGF-2.

[0042] Step 5: First, lay the lower layer of decellularized liver matrix hydrogel in the well plate, then place the well plate in a 37°C incubator for 20 minutes until it is almost solidified. Simultaneously, aspirate the vascular progenitor cell spheroids from Step 4 into a 15 mL centrifuge tube, allow them to settle naturally, discard the supernatant, and wash twice with PBS. Resuspend the vascular progenitor cell spheroids in the decellularized liver matrix hydrogel and lay them on top of the lower gel layer to form an upper gel layer encapsulating the cell spheroids. Then place the well plate in a 37°C incubator until it is completely solidified. Add the first induction medium for hepatic sinusoidal vascular organoids and induce induction for 6 days. The first induction medium for hepatic sinusoidal vascular organoids consists of StemPro-34 SFM (50%, v / v), advanced-DMEM / F12 (50%, v / v), 2 mM L-Glutamine, 30 ng / mL LFGF-2, and 10 ng / mL VEGF-121. Step 6: Replace the culture medium from Step 5 with the second induction medium for hepatic sinusoidal vascular organoids. Induce for 4-8 days to promote and maintain the maturation of the hepatic sinusoidal vascular network, thus obtaining hepatic sinusoidal vascular organoids. The second induction medium for hepatic sinusoidal vascular organoids consists of StemPro-34 SFM (50%, v / v), advanced-DMEM / F12 (50%, v / v), 2 mM L-Glutamine, 30 ng / mL FGF-2, 6 μM SB431542, and 1 mM 8-Br-cAMP.

[0043] All of the above-mentioned cultivation steps were carried out under normoxic conditions.

[0044] In step 5, the method for preparing the decellularized liver matrix hydrogel is as follows: Obtaining biological matrix materials: Animal-derived liver tissue was taken, and the hepatic artery, vein and bile duct of the liver tissue were cut off. The liver tissue was perfused with heparinized PBS for 15 min, followed by washing with sterile PBS three times for 1 h each time to obtain the processed liver tissue. Decellularization: The treated liver tissue was cut into small pieces of 1-3 mm3, incubated with PBS containing sodium dodecyl sulfate and Triton X-100, rinsed with deionized water by shaking, changing the water every 40-60 min for a total of 15-25 times, and then freeze-dried under vacuum to obtain lyophilized tissue. Digestion into gel: The freeze-dried tissue was ground into fragments, pepsin was added, and the mixture was stirred at a constant temperature to digest the tissue and obtain a liver extracellular matrix solution. Under ice bath conditions, the pH of the liver extracellular matrix solution was adjusted to neutral with 1 M sodium hydroxide solution, and then incubated at 37°C to form a gel, thus obtaining a decellularized liver matrix hydrogel.

[0045] Comparative Example The difference from Example 1 is that in step 5, 10 ng / mL VEGF-165 was used instead of 10 ng / mL VEGF-121 in the first induction culture medium for hepatic sinusoidal vascular organoids for comparison.

[0046] Morphological observations of various stages of hepatic sinusoidal vascular organoid development in this embodiment and the comparative example were performed using a microscope, and the results are as follows: Figure 2 As shown. From Figure 2 As can be seen, vascular progenitor cell spheroids, when cultured with VEGF-121 (example), were able to bud and form tubes normally, and the induced blood vessel morphology was more similar in thickness to the hepatic sinusoidal structure in vivo, eventually gradually forming a morphology-specific hepatic sinusoidal vascular network. However, under culture conditions with 10 ng / mL VEGF-165 (comparative example), no morphology-specific hepatic sinusoidal vascular network was formed.

[0047] The hepatic sinusoidal vascular organoids of this embodiment and the comparative example were identified using immunofluorescence staining technology, such as... Figure 3 As shown; from the appendix Figure 3 It can be concluded that the vascular organoids in the embodiments contain CD31+ endothelial cells and LYVE-1+FCGR2B+ hepatic sinusoidal endothelial cells, and can spontaneously grow to form a vascular network, indicating that the hepatic sinusoidal vascular organoids of the present invention are quite similar to hepatic sinusoidal vessels in vivo. In contrast, the hepatic sinusoidal vascular organoids in the comparative examples did not form sufficient LYVE-1+FCGR2B+ hepatic sinusoidal endothelial cells.

[0048] The expression of classic genes in hepatic sinusoidal vascular organoids of this embodiment and the comparative embodiment was identified using RT-qPCR technology. Figure 4As shown, VEGF-121 subtype-induced hepatic sinusoidal endothelial cells express higher levels of hepatic sinusoidal specific markers, such as CD14, STAB2, FCGR2B, LYVE-1, and CD36, further indicating that the hepatic sinusoidal vascular organoids of the present invention are more similar to hepatic sinusoidal vessels in vivo.

[0049] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for constructing hepatic sinusoidal vascular organoids induced by 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: Pluripotent stem cells are prepared into a single-cell suspension and formed into cell embryoids in the well plates of Aggrewell-800. Step S3, Mesodermal cell spheroid induction: Collect cell embryoids, discard the supernatant, add mesodermal induction medium, and culture for 3 days to induce the formation of mesodermal cell spheroids; Step S4, differentiation of vascular progenitor cell spheroids: Add vascular progenitor cell spheroid differentiation medium, culture for 2 days, and induce the formation of vascular progenitor cell spheroids; Step S5, Induction and differentiation of hepatic sinusoidal vascular network: Embedded vascular progenitor cell spheres into decellularized liver matrix hydrogel, added to the first induction medium for hepatic sinusoidal vascular organoids, and induced for 6 days; Step S6, maturation of the hepatic sinusoidal vascular network: The culture medium is replaced with the second induction medium for hepatic sinusoidal vascular organoids, and induction is performed for 4-8 days to promote the maturation of the hepatic sinusoidal vascular network, thus obtaining hepatic sinusoidal vascular organoids.

2. The method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to claim 1, characterized in that, Step S2 includes the following steps: The human pluripotent stem cells cultured in step S1 were washed twice with PBS, digested with TrypLE for 3-4 min to prepare a single-cell suspension, and the cell density was adjusted to 1.5 × 10⁻⁶. 5 Cells / mL were seeded into 24-well plates of Aggrewell-800 treated with ultra-low adhesion, centrifuged at 200×g for 3 min, and the cells settled into the wells. The cells were cultured in mTeSR1 medium containing 10 μM Y27632 for 24 hours to form cell embryoids.

3. The method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to claim 1, characterized in that, The mesodermal induction medium in step S3 includes: 50% (v / v) basal medium DMEM / F12, 50% (v / v) Neurobasal Medium, 1×N2, 1×B27, 10 μM CHIR99021 and 25 ng / mL BMP-4.

4. The method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to claim 1, characterized in that, The vascular progenitor cell differentiation medium in step S4 includes: 50% (v / v) basal medium DMEM / F12, 50% (v / v) Neurobasal Medium, 100 ng / mL VEGF-165 and 100 ng / mL FGF-2.

5. The method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to claim 1, characterized in that, The method for preparing the decellularized liver matrix hydrogel in step S5 includes the following steps: Obtaining biological matrix materials: Animal-derived liver tissue was taken, and the hepatic artery, vein and bile duct of the liver tissue were cut off. The liver tissue was perfused with heparinized PBS for 15 min, followed by washing with sterile PBS three times for 1 h each time to obtain the processed liver tissue. Decellularization: The treated liver tissue is cut into 1-3 mm pieces. 3 Small tissue pieces were incubated with PBS containing sodium dodecyl sulfate and Triton X-100, rinsed with deionized water by shaking, and the water was changed every 40-60 min for a total of 15-25 times. The tissues were then freeze-dried under vacuum to obtain lyophilized tissues. Digestion into gel: The freeze-dried tissue was ground into fragments, pepsin was added, and the mixture was stirred at a constant temperature to digest the tissue and obtain a liver extracellular matrix solution. Under ice bath conditions, the pH of the liver extracellular matrix solution was adjusted to neutral with 1 M sodium hydroxide solution, and then incubated at 37°C to form a gel, thus obtaining a decellularized liver matrix hydrogel.

6. The method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to claim 1, characterized in that, Step S5, embedding vascular progenitor cell spheres into a decellularized liver matrix hydrogel, includes the following steps: First, a layer of decellularized liver matrix hydrogel is laid in the well plate. Then, the well plate is placed in a 37°C incubator for 20 minutes to allow it to partially solidify. Next, vascular progenitor cell spheres are resuspended in the decellularized liver matrix hydrogel and laid on top of the partially solidified decellularized liver matrix hydrogel to form an upper gel layer that encapsulates the cell spheres. The well plate is then placed in a 37°C incubator to allow it to solidify.

7. The method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to claim 1, characterized in that, In step S5, the first induction culture medium for the liver sinusoidal vascular organoids includes: 50% (v / v) basal culture medium StemPro-34, 50% (v / v) SFMadvanced-DMEM / F12, 2 mM L-Glutamine, 10 ng / mL VEGF-121 and 30 ng / mL FGF-2.

8. The method for constructing hepatic sinusoidal vascular organoids based on human pluripotent stem cells according to claim 1, characterized in that, The second induction culture medium for hepatic sinusoidal vascular organoids mentioned in step S6 includes: 50% (v / v) StemPro-34 SFM basal medium, 50% (v / v) advanced-DMEM / F122, mM L-Glutamine, 30 ng / mL FGF-2, 6 μM SB431542 and 1 mM 8-Br-cAMP.

9. A liver sinusoidal vessel organoid induced by human pluripotent stem cells, characterized in that: The organoids were prepared using the method for constructing hepatic sinusoidal vessels induced by human pluripotent stem cells as described in any one of claims 1-8.

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