Method and system for constructing vascular organoid based on human induced pluripotent stem cells

Abstract

The invention relates to the technical field of human induced pluripotent stem cells, and discloses a method and a system for constructing vascular organs based on human induced pluripotent stem cells, and the method comprises the following steps: inducing human induced pluripotent stem cells to aggregate into cell aggregates, and identifying aggregate characteristics of the cell aggregates; determining mesoderm induction molecules of the cell aggregate to construct a mesoderm induction environment of the cell aggregate, and performing induced differentiation on the human induced pluripotent stem cells in the cell aggregate; detecting the expression level of the mesoderm marker gene in the mesoderm cells, constructing a vascular induction culture medium of the mesoderm cells, and inducing the mesoderm cells to differentiate into vascular lineage cells; constructing a three-dimensional extracellular matrix of the vascular lineage cells to differentiate the vascular lineage cells into a vascular network; and calculating the permeability and branch density of the vascular network to determine the fluid shear force and pulse frequency of the vascular network, and culturing the vascular network into a vascular organ. The construction stability and functionality of the vascular organoid can be improved.

Description

Technical Field

[0001] This invention relates to a method and system for constructing vascular organoids based on human induced pluripotent stem cells, belonging to the field of human induced pluripotent stem cell technology. Background Technology

[0002] The construction of vascular organoids from human induced pluripotent stem cells (i.e., vascular organoids) refers to a technical method that uses human i.e., induced pluripotent stem cells as starting cells and, through a series of precisely controlled in vitro culture and induced differentiation steps, gradually differentiates them into three-dimensional micro-organs (i.e., "vascular organoids") with vascular structures, cellular composition, and physiological functions. Human i.e., vascular organoids can serve as seed cells and functional vascular structures for tissue engineering and angiogenesis, providing new ideas for the treatment of ischemic diseases (such as myocardial infarction and peripheral artery disease).

[0003] Traditional methods for constructing vascular organoids involve two-dimensional endothelial cell culture. Endothelial cells are seeded onto the surface of a culture plate and cultured in a two-dimensional environment to form a monolayer. Angiogenic factors are added to promote cell proliferation and migration, resulting in simple tubular structures. However, the resulting vascular network structure is relatively simple, lacking the complexity and hierarchy of blood vessels in vivo. Furthermore, the vessel wall structure is incomplete, lacking sufficient encapsulation by smooth muscle cells and pericytes, making it difficult to simulate the stability of blood vessels in vivo. Summary of the Invention

[0004] This invention provides a method and system for constructing vascular organoids based on human induced pluripotent stem cells, the main purpose of which is to improve the stability and functionality of vascular organoids constructed from human induced pluripotent stem cells.

[0005] To achieve the above objectives, the present invention provides a method for constructing vascular organoids based on human induced pluripotent stem cells, comprising: A cell culture environment for human induced pluripotent stem cells was constructed to induce the human induced pluripotent stem cells to aggregate into cell aggregates, and the aggregate characteristics of the cell aggregates were identified. The mesodermal inducing molecules of the cell aggregates are identified to construct a mesodermal induction environment for the cell aggregates. Based on the mesodermal induction environment and the characteristics of the aggregates, human induced pluripotent stem cells in the cell aggregates are induced to differentiate to obtain mesodermal cells. The expression levels of mesodermal marker genes in the mesodermal cells were detected, angiogenesis induction culture medium for the mesodermal cells was constructed, and the mesodermal cells were induced to differentiate into vascular lineage cells based on the expression levels and the angiogenesis induction culture medium. A three-dimensional extracellular matrix of the vascular lineage cells was constructed, and the vascular lineage cells were embedded in the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network; The permeability and branch density of the vascular network are calculated to determine the fluid shear force and pulse frequency of the vascular network. Based on the fluid shear force and the pulse frequency, the vascular network is cultured into vascular organoids.

[0006] Optionally, calculating the permeability and branch density of the vascular network includes: Identify fluorescent markers for the vascular network; Time-series images of the diffusion of the fluorescent marker from the lumen of the corresponding blood vessel in the vascular network to the extracellular matrix were acquired. The time-series images were segmented into vascular regions and extracellular matrix regions; Calculate the vascular surface area of ​​the vascular region; The fluorescence intensity of the vascular region and the matrix fluorescence intensity of the extracellular matrix region were measured separately. Based on the fluorescence intensity of the vascular region and the fluorescence intensity of the matrix, the concentration difference between the inside and outside of the vascular region and the extracellular matrix region is calculated. The permeability of the vascular network is calculated based on the concentration difference between the inside and outside of the blood vessel. Extract vascular network samples from the vascular network; Calculate the sample volume of the vascular network sample; Extract the vascular skeleton of the vascular network sample to calculate the number of branch points of the vascular network sample; The complexity of the vascular network sample is quantified, and the branch density of the vascular network is calculated based on the complexity, the number of branch points, and the sample volume.

[0007] Optionally, the cell culture environment for constructing human induced pluripotent stem cells includes: Configure the extremely low adhesion culture plate for the aforementioned human induced pluripotent stem cells; The coating matrix of the extremely low adhesion culture plate is determined so as to coat the extremely low adhesion culture plate to obtain a coated extremely low adhesion culture plate. The culture conditions for the human induced pluripotent stem cells are constructed, wherein the culture conditions include: a sterile environment, temperature and humidity, and gas conditions; Prepare the culture medium for aggregates of the aforementioned human induced pluripotent stem cells; Based on the aforementioned coated low-attachment culture plate, the aforementioned culture conditions, and the aforementioned aggregate culture medium, an integrated cell culture environment for human induced pluripotent stem cells is created.

[0008] Optionally, inducing the human induced pluripotent stem cells to aggregate into cell aggregates includes: Construct a single-cell suspension of the aforementioned human induced pluripotent stem cells; The single-cell suspension was subjected to a technique to obtain the cell count; Determine the target cell density of the single-cell suspension, and adjust the density of the single-cell suspension based on the cell number and the target cell density to obtain an adjusted single-cell suspension; Based on the cell culture environment corresponding to the human induced pluripotent stem cells, the adjusted single-cell suspension is cultured to obtain cell clusters; The cell clusters are induced to form cell aggregates using an aggregate culture medium corresponding to the cell culture environment.

[0009] Optionally, the mesodermal induction environment for constructing the cell aggregates includes: Determine the basic culture medium formulation for the cell aggregates; The formulation of the inducing factor for the cell aggregate is determined based on the mesodermal inducing molecule corresponding to the cell aggregate. Based on the basic culture medium formula and the inducing factor formula, a mesodermal induction culture medium for the cell aggregates was prepared. The culture parameters of the cell aggregates are determined, wherein the culture parameters include: culture time, culture temperature and humidity, and culture medium replacement cycle; The mesodermal induction environment of the cell aggregates is determined based on the mesodermal induction medium and the culture parameters.

[0010] Optionally, detecting the expression level of mesodermal marker genes in the mesodermal cells includes: The mesodermal cells were sampled to obtain cell samples; The cell sample was lysed to obtain a cell lysate; RNA molecules were extracted from the cell lysate; The RNA molecule was reverse transcribed to obtain a cDNA molecule. Identify the target gene primers and internal reference gene primers for the mesoderm marker genes in the cDNA molecule; Based on the target gene primer and the internal reference gene primer, the cDNA molecule was detected by qRT-PCR to obtain the detection results; The CT values ​​of the target gene and the internal reference gene in the detection results are extracted to calculate the expression level of the mesodermal marker gene.

[0011] Optionally, inducing the mesodermal cells to differentiate into vascular lineage cells based on the expression level and the angiogenesis induction culture medium includes: When the expression level reaches a preset expression level threshold, the mesodermal cells are plated to obtain plated cells. The plated cells were cultured without induction to obtain adherent cells; Establish the angiogenesis induction culture conditions for the adherent cells; The adherent cells were induced and cultured according to the described vascular induction culture conditions and the described vascular induction culture medium to obtain cells in culture. Real-time acquisition of morphological change data of cells in culture is used to adjust the concentration of angiogenic factors in the angiogenic induction medium. Based on the concentration of the angiogenesis-inducing factor, the cultured cells are differentiated into angiogenesis lineage cells.

[0012] Optionally, the three-dimensional extracellular matrix for constructing the vascular lineage cells includes: The matrix construction material for the vascular lineage cells is configured, wherein the matrix construction material comprises: biomaterials and cell culture reagents; Determine the vessel diameter, vessel branches, and vessel porosity of the vascular lineage cells; The three-dimensional vascular structure of the vascular lineage cells is determined based on the vessel diameter, vessel branches, and vessel porosity. Based on the three-dimensional vascular structure, a three-dimensional scaffold matrix for the vascular lineage cells is constructed using the matrix construction material. The vascular lineage cells' vascular growth factor was added to the three-dimensional scaffold matrix to obtain the three-dimensional extracellular matrix of the vascular lineage cells.

[0013] Optionally, embedding the vascular lineage cells into the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network includes: Analyze the mixing ratio of the vascular lineage cells and the three-dimensional extracellular matrix; Based on the mixing ratio, the vascular lineage cells are embedded in the three-dimensional extracellular matrix to obtain a cell-matrix mixture; Matrix curing conditions for the cell-matrix mixture were constructed to cure the cell-matrix mixture, resulting in a cell-cured matrix mixture; Monitor the angiogenesis status of the cell-stabilized matrix mixture during differentiation; Based on the angiogenesis status, the dynamic culture parameters of the cell-stabilized matrix mixture are adjusted, wherein the dynamic culture parameters include: growth factor concentration, bioreactor rotation speed, and nutrient solution concentration; Based on the dynamic culture parameters, the cell-stabilized matrix mixture is dynamically cultured to obtain a vascular network.

[0014] To address the aforementioned problems, the present invention also provides a system for constructing vascular organoids based on human induced pluripotent stem cells, the system comprising: A cell aggregate construction module is used to construct a cell culture environment for human induced pluripotent stem cells to induce the human induced pluripotent stem cells to aggregate into cell aggregates and to identify the aggregate characteristics of the cell aggregates; The mesodermal cell culture module is used to determine the mesodermal inducing molecules of the cell aggregates to construct the mesodermal induction environment of the cell aggregates. Based on the mesodermal induction environment and the characteristics of the aggregates, human induced pluripotent stem cells in the cell aggregates are induced to differentiate to obtain mesodermal cells. A vascular lineage cell differentiation module is used to detect the expression level of mesodermal marker genes in the mesodermal cells, construct angiogenesis induction culture medium for the mesodermal cells, and induce the mesodermal cells to differentiate into vascular lineage cells according to the expression level and the angiogenesis induction culture medium. A vascular network differentiation module is used to construct a three-dimensional extracellular matrix of the vascular lineage cells, embedding the vascular lineage cells into the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network; The vascular organoid culture module is used to calculate the permeability and branch density of the vascular network to determine the fluid shear force and pulse frequency of the vascular network, and to culture the vascular network into vascular organoids based on the fluid shear force and the pulse frequency.

[0015] Compared to the problems described in the background art, this invention constructs a cell culture environment for human induced pluripotent stem cells (iPSCs), inducing them to aggregate into cell aggregates with specific characteristics. Combined with a multi-stage induction and three-dimensional culture strategy, it can efficiently and controllably differentiate into functional vascular organoids, offering significant technical and application advantages. First, by optimizing the formation conditions of cell aggregates, this invention enhances intercellular interactions, improving the efficiency and uniformity of subsequent mesodermal induction. Second, based on the precise regulation of mesodermal induction molecules according to aggregate characteristics, it can directionally induce human iPSCs to differentiate into mesodermal cells, significantly increasing the expression levels of mesodermal marker genes and laying a solid foundation for vascular lineage differentiation. Furthermore, by constructing a vascular induction culture medium and combining it with dynamic regulation of gene expression levels, this invention can efficiently induce mesodermal cells to differentiate into vascular lineage cells such as vascular endothelial cells and smooth muscle cells, improving differentiation efficiency and cell functionality. Subsequently, embedding vascular lineage cells into a three-dimensional extracellular matrix simulates the in vivo microenvironment, promoting their self-organization into a vascular network with complex branching structures, significantly improving the maturity and physiological relevance of the vascular network. This invention, by calculating the permeability and branch density of vascular networks, can accurately determine fluid shear force and pulse frequency, optimize dynamic culture conditions, and further enhance the functional characteristics of vascular organoids, such as barrier function, contractility, and blood flow responsiveness. This invention not only achieves efficient construction of vascular organoids but also provides a highly controllable and reproducible in vitro model for bioengineering, tissue model construction, and basic biological research, promoting technological progress and application expansion in related fields. Therefore, the method for constructing vascular organoids based on human induced pluripotent stem cells provided in this invention can improve the stability and functionality of vascular organoids constructed from human induced pluripotent stem cells. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating a method for constructing vascular organoids based on human induced pluripotent stem cells according to an embodiment of the present invention. Figure 2 This is a schematic diagram of the modules of a system for constructing vascular organoids based on human induced pluripotent stem cells, provided in an embodiment of the present invention.

[0017] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] This application provides a method for constructing vascular organoids based on human induced pluripotent stem cells. The execution entity of this method includes, but is not limited to, at least one of the following electronic devices that can be configured to execute the method provided in this application: a server, a terminal, etc. In other words, the method for constructing vascular organoids based on human induced pluripotent stem cells can be executed by software or hardware installed on a terminal device or a server device. The server includes, but is not limited to, a single server, a server cluster, a cloud server, or a cloud server cluster.

[0020] Reference Figure 1 The diagram shown is a flowchart illustrating a method for constructing vascular organoids based on human induced pluripotent stem cells according to an embodiment of the present invention. In this embodiment, the method for constructing vascular organoids based on human induced pluripotent stem cells includes: S1. Construct a cell culture environment for human induced pluripotent stem cells to induce the human induced pluripotent stem cells to aggregate into cell aggregates, and identify the aggregate characteristics of the cell aggregates.

[0021] This invention, through the construction of a cell culture environment for human induced pluripotent stem cells (iPSCs), can maintain the pluripotency of cells, prevent spontaneous differentiation, and significantly improve the survival rate and proliferation capacity of iPSCs in a three-dimensional culture environment. The cell culture environment refers to an in vitro culture system used to support the growth of iPSCs, maintain their pluripotency, and induce them to spontaneously aggregate into three-dimensional cell aggregates.

[0022] As an embodiment of the present invention, the cell culture environment for constructing human induced pluripotent stem cells includes: Configure the extremely low adhesion culture plate for the aforementioned human induced pluripotent stem cells; The coating matrix of the extremely low adhesion culture plate is determined so as to coat the extremely low adhesion culture plate to obtain a coated extremely low adhesion culture plate. The culture conditions for the human induced pluripotent stem cells are constructed, wherein the culture conditions include: a sterile environment, temperature and humidity, and gas conditions; Prepare the culture medium for aggregates of the aforementioned human induced pluripotent stem cells; Based on the aforementioned coated low-attachment culture plate, the aforementioned culture conditions, and the aforementioned aggregate culture medium, an integrated cell culture environment for human induced pluripotent stem cells is created.

[0023] The ultra-low attachment culture plate refers to a cell culture container with a hydrophilic polymer coating on its surface, which prevents cell adhesion and promotes spontaneous aggregation of cells into three-dimensional cell aggregates in a suspended state, such as the Corning® Costar® Ultra-Low Attachment 6-well or 96-well plate. The coating matrix refers to the biomaterial used to modify the surface of the ultra-low attachment culture plate; in this invention, Matrigel is used. The coated ultra-low attachment culture plate refers to an ultra-low attachment culture plate treated with a coating matrix, its surface covered with a specific proportion of Matrigel to form a bioactive culture interface. The culture conditions refer to the external environmental parameters required to maintain the growth, proliferation, and differentiation of human induced pluripotent stem cells. The sterile environment refers to the operating space and conditions for eliminating and controlling microbial contamination during cell culture through physical and chemical means. The temperature and humidity refer to the temperature and relative humidity maintained in the incubator, such as a temperature of 37°C and a humidity of 95%. The gas conditions refer to the gas composition and concentration maintained in the incubator to support cellular respiration and pH stability, such as a CO2 concentration of 5%. The aggregate culture medium refers to a specialized culture medium used to support the growth, proliferation, and formation of three-dimensional aggregates of human induced pluripotent stem cells in a very low adhesion culture plate.

[0024] Optionally, the coating matrix of the ultra-low adhesion culture plate can be determined by a high-throughput screening platform, such as microarray matrix screening or multi-parameter evaluation models.

[0025] Optionally, the aggregate culture medium can be prepared using basal medium (DMEM / F12), serum substitutes (such as KSR or B27), growth factors (such as bFGF, TGF-β), and additives (such as non-essential amino acids, β-mercaptoethanol, GlutaMAX, etc.). This invention, through inducing human induced pluripotent stem cells to aggregate into cell aggregates, can simulate the microenvironment of close intercellular contact during embryonic development, activate cell self-organization mechanisms, and lay the foundation for subsequent angiogenesis. The cell aggregates refer to cell clusters with a three-dimensional spatial structure induced from a large number of human induced pluripotent stem cells under three-dimensional culture conditions.

[0026] As an embodiment of the present invention, the induction of the human induced pluripotent stem cells to aggregate into cell aggregates includes: Construct a single-cell suspension of the aforementioned human induced pluripotent stem cells; The single-cell suspension was subjected to a technique to obtain the cell count; Determine the target cell density of the single-cell suspension, and adjust the density of the single-cell suspension based on the cell number and the target cell density to obtain an adjusted single-cell suspension; Based on the cell culture environment corresponding to the human induced pluripotent stem cells, the adjusted single-cell suspension is cultured to obtain cell clusters; The cell clusters are induced to form cell aggregates using an aggregate culture medium corresponding to the cell culture environment.

[0027] The single-cell suspension refers to a liquid system formed by digesting human induced pluripotent stem cells from a culture plate, dispersing them into single cells through enzymatic hydrolysis, and suspending them in a culture medium. The cell number refers to the total number of cells in the single-cell suspension. The target cell density refers to the number of cells that should be contained in a unit volume of culture medium, as determined according to the experimental design, such as 1 × 10⁻⁶ cells / mL. 6 The adjusted single-cell suspension refers to a suspension whose cell density is adjusted to meet the experimental design requirements by diluting or concentrating the original single-cell suspension according to the target cell density. The cell clusters refer to the early three-dimensional structures formed by the spontaneous aggregation of single cells after the adjusted single-cell suspension has undergone preliminary incubation (usually 24 hours) in a culture container (such as a low-absorption plate).

[0028] Optionally, the number of cells can be obtained by cell counting methods, such as hemocytometers, automated cell counters, etc.

[0029] Optionally, the adjusted single-cell suspension can be obtained by cell density adjustment methods, such as dilution or concentration.

[0030] This invention, by identifying the aggregate characteristics of the cell aggregates, can help screen out aggregates with optimal morphology and function for subsequent culture. The aggregate characteristics refer to a series of indicators that systematically describe and evaluate cell aggregates from multiple dimensions, including morphology, structure, function, and molecular biology, such as the aggregate's diameter, volume, and shape.

[0031] Alternatively, the aggregate features can be identified using imaging and morphological analysis techniques.

[0032] S2. Determine the mesoderm-inducing molecules of the cell aggregates to construct the mesoderm-inducing environment of the cell aggregates. Based on the mesoderm-inducing environment and the characteristics of the aggregates, induce human induced pluripotent stem cells in the cell aggregates to differentiate into mesodermal cells.

[0033] This invention demonstrates that by identifying mesodermal inducing molecules in the cell aggregates, suitable signaling molecules and regulatory factors can be selected to directionally induce hiPSCs to differentiate into mesoderm, thereby providing a high-quality cell source for the subsequent construction of vascular organoids. Specifically, the mesodermal inducing molecules refer to a class of key signaling molecules, such as CHIR99021, that can directionally regulate the differentiation of human induced pluripotent stem cells into the mesodermal lineage.

[0034] Optionally, the mesodermal inducing molecules of the human induced pluripotent stem cells can be determined by high-throughput screening techniques, such as small molecule compound library screening and CRISPR gene editing screening.

[0035] This invention, through the construction of a mesodermal induction environment for the aforementioned cell aggregates, can simulate the microenvironment of early embryonic development, thereby achieving efficient and precise mesodermal differentiation. This induction environment not only affects the efficiency of differentiation but also determines the structure and function of subsequent vascular organoids. Specifically, the mesodermal induction environment refers to a three-dimensional microenvironment system capable of efficiently and precisely inducing human induced pluripotent stem cells to differentiate into mesodermal cells and their subsequent lineages (such as blood vessels, myocardium, and bone).

[0036] As an embodiment of the present invention, the mesodermal induction environment for constructing the cell aggregates includes: Determine the basic culture medium formulation for the cell aggregates; The formulation of the inducing factor for the cell aggregate is determined based on the mesodermal inducing molecule corresponding to the cell aggregate. Based on the basic culture medium formula and the inducing factor formula, a mesodermal induction culture medium for the cell aggregates was prepared. The culture parameters of the cell aggregates are determined, wherein the culture parameters include: culture time, culture temperature and humidity, and culture medium replacement cycle; The mesodermal induction environment of the cell aggregates is determined based on the mesodermal induction medium and the culture parameters.

[0037] The basic culture medium formulation refers to the basic nutrient solution components used to support the growth and maintenance of cell aggregates (such as organoids or embryoids), such as DMEM / F12, amino acids, vitamins, and inorganic salts. The inducing factor formulation refers to a specific combination of signaling molecules used to guide the differentiation of human induced pluripotent stem cells into mesoderm, such as CHIR99021, BMP4, and bFGF. The mesoderm induction medium refers to a complete culture system combining the basic culture medium formulation and the inducing factor formulation. The culture parameters refer to the external conditions required for cell aggregates to undergo mesoderm induction. The culture time refers to the total duration from cell aggregate formation to the completion of mesoderm induction, such as 1-5 days. The culture temperature and humidity refer to the constant temperature and humidity conditions required to maintain cell aggregate growth, such as a temperature of 37°C and a humidity of 95%. The culture medium replacement cycle refers to the time interval for regularly replacing the culture medium with fresh medium during the induction process, such as replacing the medium every 24–48 hours.

[0038] Optionally, the formulation of the inducing factor for the cell aggregates can be determined by orthogonal experimental design, such as by testing the effect of different small molecule combinations on the mesoderm induction efficiency through orthogonal experimental design, in order to determine the formulation of the inducing factor for the cell aggregates.

[0039] Optionally, the culture parameters of the cell aggregates can be determined using microfluidic chip technology.

[0040] This invention, through inducing differentiation of human induced pluripotent stem cells (i.e., stem cells) within the cell aggregates based on the mesodermal induction environment and aggregate characteristics, yields mesodermal cells that significantly enhance the vascularization of organoids, forming functional microvascular networks. The mesodermal cells refer to cell types belonging to the mesodermal lineage, obtained from human i.e., stem cells through directed differentiation.

[0041] S3. Detect the expression level of mesodermal marker genes in the mesodermal cells, construct angiogenesis induction culture medium for the mesodermal cells, and induce the mesodermal cells to differentiate into vascular lineage cells according to the expression level and the angiogenesis induction culture medium.

[0042] This invention, through detecting the expression levels of mesodermal marker genes in mesodermal cells, can confirm whether cultured cells have successfully entered the mesoderm stage, excluding undifferentiated and heterogeneous cell contamination. The mesodermal marker genes refer to a class of genes specifically expressed in mesodermal cells or their precursor cells during human induced pluripotent stem cell differentiation, such as Brachyury and Mixl1. The expression level refers to the degree of transcriptional or translational activity of the mesodermal marker genes at specific cells, tissues, and time points.

[0043] As an embodiment of the present invention, detecting the expression level of mesodermal marker genes in the mesodermal cells includes: The mesodermal cells were sampled to obtain cell samples; The cell sample was lysed to obtain a cell lysate; RNA molecules were extracted from the cell lysate; The RNA molecule was reverse transcribed to obtain a cDNA molecule. Identify the target gene primers and internal reference gene primers for the mesoderm marker genes in the cDNA molecule; Based on the target gene primer and the internal reference gene primer, the cDNA molecule was detected by qRT-PCR to obtain the detection results; The CT values ​​of the target gene and the internal reference gene in the detection results are extracted to calculate the expression level of the mesodermal marker gene.

[0044] The cell sample refers to a cell population extracted from cultured mesodermal cells for subsequent experiments. The cell lysis buffer is the liquid obtained after treating the cell sample with a lysis reagent (such as TRIzol or RIPA buffer). The RNA molecule refers to ribonucleic acid isolated and purified from the cell lysis buffer. The cDNA molecule refers to complementary DNA synthesized using RNA as a template via reverse transcriptase. The target gene primer refers to an oligonucleotide sequence that specifically amplifies a mesodermal marker gene. The internal reference gene primer refers to an oligonucleotide sequence that amplifies an internal reference gene. The detection result refers to the amplification curve and CT value data output by the qRT-PCR instrument. The target gene CT value refers to the number of cycles in which the target gene amplification signal reaches a threshold. The internal reference gene CT value refers to the number of cycles in which the internal reference gene amplification signal reaches a threshold.

[0045] Optionally, the RNA molecules in the cell lysate can be extracted by solid-phase extraction, such as using a silica membrane or a glass fiber filter membrane.

[0046] Optionally, the target gene primer and the internal reference gene primer can be determined using a high-throughput primer screening platform, such as the Fluidigm BioMark HD system or the WaferGen SmartChip system.

[0047] This invention, through the construction of a mesodermal cell angiogenesis induction medium, can efficiently induce mesodermal stem cells to differentiate into vascular endothelial cells and promote the formation of luminal structures and the expression of maturation markers in endothelial cells. The angiogenesis induction medium refers to an in vitro culture system used to induce the directed differentiation of mesodermal stem cells into vascular lineage cells (including vascular endothelial cells, perivascular cells, smooth muscle cells, etc.).

[0048] Optionally, the angiogenesis induction culture medium can be constructed using angiogenesis induction factors such as VEGF and FGF2.

[0049] This invention, through inducing mesodermal cells to differentiate into vascular lineage cells based on the stated expression level and the stated angiogenesis induction medium, promotes the spontaneous formation of a three-dimensional vascular network by mesodermal cells in the angiogenesis induction medium, mimicking the in vivo angiogenesis process, thereby achieving efficient and targeted differentiation of mesodermal cells into vascular lineage cells. The vascular lineage cells refer to a series of cells with vascular structural and functional characteristics differentiated from mesodermal stem cells, such as vascular endothelial cells, vascular smooth muscle cells, and perivascular cells.

[0050] As an embodiment of the present invention, the step of inducing the mesodermal cells to differentiate into vascular lineage cells according to the expression level and the angiogenesis induction culture medium includes: When the expression level reaches a preset expression level threshold, the mesodermal cells are plated to obtain plated cells. The plated cells were cultured without induction to obtain adherent cells; Establish the angiogenesis induction culture conditions for the adherent cells; The adherent cells were induced and cultured according to the described vascular induction culture conditions and the described vascular induction culture medium to obtain cells in culture. Real-time acquisition of morphological change data of cells in culture is used to adjust the concentration of angiogenic factors in the angiogenic induction medium. Based on the concentration of the angiogenesis-inducing factor, the cultured cells are differentiated into angiogenesis lineage cells.

[0051] The preset expression level threshold refers to the minimum expression level standard of mesodermal marker genes (such as Brachyury, MIXL1, T, etc.) set before inducing differentiation of mesodermal cells into vascular lineage cells. The post-plating cells refer to mesodermal cells (that have reached the preset expression level threshold) that have been removed from their original culture environment and re-seeded (plated) at a specific density into new culture plates or dishes for subsequent induction of differentiation. The adherent cells refer to cells that have been plated and cultured in non-induction medium for a period of time (usually 24-48 hours) to fully adhere to the surface of the culture plate and spread out, forming a stable adherent state. The vascularization induction culture conditions refer to the comprehensive culture environment set to promote the differentiation of adherent cells into vascular lineage cells, including the culture medium formulation, gas environment (e.g., 5% CO2), temperature (37℃), humidity (95%), and physical stimuli (e.g., fluid shear force, hypoxia). The cells in culture refer to cells undergoing differentiation under vascularization induction culture conditions. The morphological change data refers to the cell morphological characteristics information acquired in real time during the induction culture process using a microscope or high-content imaging system, including cell shape, size, arrangement, and luminal structure formation. The angiogenesis induction factor concentration refers to the specific concentration ratio of key growth factors (such as VEGF, FGF2, BMP4, etc.) in the angiogenesis induction culture medium.

[0052] Optionally, the angiogenesis induction culture conditions for the adherent cells can be constructed using microfluidic chip technology.

[0053] Optionally, the concentration of angiogenic factors can be determined by using the morphological change data to determine the differentiation efficiency and direction of cells in the culture, in order to calculate the concentration of angiogenic factors in the angiogenic induction medium.

[0054] S4. Construct a three-dimensional extracellular matrix for the vascular lineage cells, and embed the vascular lineage cells into the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network.

[0055] This invention, through the construction of a three-dimensional extracellular matrix for vascular lineage cells, can simulate the biomechanical microenvironment of different tissues (such as the brain, liver, and myocardium), thereby influencing the migration, differentiation, and lumen formation of vascular lineage cells. It also supports the formation of tight junctions, gap junctions, and adhesion junctions between cells, promoting the synergistic effect of endothelial cells and pericytes / smooth muscle cells to form a stable vascular structure. The three-dimensional extracellular matrix refers to a three-dimensional structural scaffold capable of simulating the in vivo tissue microenvironment.

[0056] As an embodiment of the present invention, the construction of the three-dimensional extracellular matrix of the vascular lineage cells includes: The matrix construction material for the vascular lineage cells is configured, wherein the matrix construction material comprises: biomaterials and cell culture reagents; Determine the vessel diameter, vessel branches, and vessel porosity of the vascular lineage cells; The three-dimensional vascular structure of the vascular lineage cells is determined based on the vessel diameter, vessel branches, and vessel porosity. Based on the three-dimensional vascular structure, a three-dimensional scaffold matrix for the vascular lineage cells is constructed using the matrix construction material. The vascular lineage cells' vascular growth factor was added to the three-dimensional scaffold matrix to obtain the three-dimensional extracellular matrix of the vascular lineage cells.

[0057] The term "matrix construction material" refers to the complete combination of materials used to construct the three-dimensional extracellular matrix. "Biomaterials" refers to natural and synthetic materials used to construct the three-dimensional scaffold, possessing biocompatibility and capable of supporting cell attachment, proliferation, and differentiation, such as collagen, fibrin, hyaluronic acid, gelatin, and sodium alginate. "Cell culture reagents" refers to chemical and biological reagents used to support cell growth, differentiation, and functional maintenance, such as DMEM, F12, and RPMI-1640. "Vascular diameter" refers to the width of the vascular lumen, such as capillaries (approximately 5-10 micrometers in diameter). "Vascular branching" refers to the bifurcation structure in the vascular network, including the number, angle, and hierarchy of branches. "Vascular porosity" refers to the volume percentage of pores in the three-dimensional scaffold. "Three-dimensional vascular structure" refers to a three-dimensional model of a vascular network designed based on parameters such as vascular diameter, branching, and porosity. "Three-dimensional scaffold matrix" refers to a physical scaffold constructed using matrix construction materials (biomaterials and cell culture reagents) based on the three-dimensional vascular structure, providing a three-dimensional environment for cell attachment, growth, and differentiation. The term "vascular growth factor" refers to proteins that can promote the proliferation, migration, and angiogenesis of vascular endothelial cells, such as vascular endothelial growth factor and basic fibroblast growth factor.

[0058] Optionally, the vessel diameter, vessel branches, and vessel porosity of the vascular lineage cells can be determined using vascular skeletonization algorithms, such as ImageJ, MATLAB, and Amira.

[0059] Optionally, the three-dimensional scaffold matrix of the vascular lineage cells can be constructed using 3D bioprinting technology.

[0060] This invention, through embedding the vascular lineage cells into the three-dimensional extracellular matrix, differentiates the vascular lineage cells into a vascular network. This activates the differentiation signaling pathways of vascular endothelial cells, promoting their differentiation into a functional vascular network, thereby further enhancing the functionality of the vascular network and achieving blood flow similar to that in vivo. The vascular network refers to a three-dimensional structural system composed of cell types such as vascular endothelial cells, smooth muscle cells, and pericytes.

[0061] As an embodiment of the present invention, the step of embedding the vascular lineage cells into the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network includes: Analyze the mixing ratio of the vascular lineage cells and the three-dimensional extracellular matrix; Based on the mixing ratio, the vascular lineage cells are embedded in the three-dimensional extracellular matrix to obtain a cell-matrix mixture; Matrix curing conditions for the cell-matrix mixture were constructed to cure the cell-matrix mixture, resulting in a cell-cured matrix mixture; Monitor the angiogenesis status of the cell-stabilized matrix mixture during differentiation; Based on the angiogenesis status, the dynamic culture parameters of the cell-stabilized matrix mixture are adjusted, wherein the dynamic culture parameters include: growth factor concentration, bioreactor rotation speed, and nutrient solution concentration; Based on the dynamic culture parameters, the cell-stabilized matrix mixture is dynamically cultured to obtain a vascular network.

[0062] The mixing ratio refers to the volume ratio of vascular lineage cells to three-dimensional extracellular matrix material during mixing. The cell-matrix mixture refers to a mixed system formed by uniformly suspending vascular lineage cells in a liquid three-dimensional extracellular matrix. The cell-stabilized matrix mixture refers to a solid three-dimensional structure formed after solidifying the matrix in the cell-matrix mixture. The angiogenesis state refers to the biological state of vascular lineage cells gradually forming tubular structures, branching networks, and functional blood vessels during differentiation in the cell-stabilized matrix mixture, such as lumen diameter, number of branch points, and network connectivity. The dynamic culture parameters refer to adjustable environmental variables during dynamic culture. The growth factor concentration refers to the molar concentration of pro-angiogenic factors added to the dynamic culture system. The bioreactor rotation speed refers to the set value of stirring in a rotary bioreactor. The nutrient solution concentration refers to the concentration of key nutrients in the culture medium, including glucose, amino acids, vitamins, and serum.

[0063] Optionally, the mixing ratio of the vascular lineage cells and the three-dimensional extracellular matrix can be analyzed by using a deep learning model to perform microscopic image analysis on the mixed cell-matrix complex.

[0064] Optionally, the dynamic cultivation parameters can be adjusted using optimization algorithms, such as Bayesian optimization algorithms, genetic algorithms, etc.

[0065] S5. Calculate the permeability and branch density of the vascular network to determine the fluid shear force and pulse frequency of the vascular network, and culture the vascular network into vascular organoids based on the fluid shear force and the pulse frequency.

[0066] This invention, through calculating the permeability and branch density of the vascular network, reflects the tightness of connections between vascular endothelial cells and measures the complexity and maturity of the vascular network, thus providing a scientific basis for optimizing vascular culture parameters, constructing vascular organoids, and clinical applications. Permeability refers to the ability of the vascular endothelial barrier to allow substances (such as water, ions, proteins, and drug molecules) to pass through. Branch density is an important parameter for measuring the complexity and spatial distribution of the vascular network.

[0067] As an embodiment of the present invention, the calculation of the permeability and branch density of the vascular network includes: Identify fluorescent markers for the vascular network; Time-series images of the diffusion of the fluorescent marker from the lumen of the corresponding blood vessel in the vascular network to the extracellular matrix were acquired. The time-series images were segmented into vascular regions and extracellular matrix regions; Calculate the vascular surface area of ​​the vascular region; The fluorescence intensity of the vascular region and the matrix fluorescence intensity of the extracellular matrix region were measured separately. Based on the fluorescence intensity of the vascular region and the fluorescence intensity of the matrix, the concentration difference between the inside and outside of the vascular region and the extracellular matrix region is calculated. The permeability of the vascular network is calculated based on the concentration difference between the inside and outside of the blood vessel. Extract vascular network samples from the vascular network; Calculate the sample volume of the vascular network sample; Extract the vascular skeleton of the vascular network sample to calculate the number of branch points of the vascular network sample; The complexity of the vascular network sample is quantified, and the branch density of the vascular network is calculated based on the complexity, the number of branch points, and the sample volume.

[0068] The fluorescent markers refer to molecules with fluorescent signals used to track the diffusion behavior of substances in vascular networks, such as FITC-dextran and sodium fluorescein. The time-series images refer to a sequence of fluorescent images acquired continuously at different time points within the same field of view, used to dynamically observe the diffusion process of fluorescent markers from the vascular lumen to the extracellular matrix. The vascular region refers to the area in the image surrounded by vascular endothelial cells and possessing a vascular lumen structure. The extracellular matrix region refers to the three-dimensional matrix structure outside the vascular lumen. The vascular surface area refers to the total surface area of ​​vascular endothelial cells in contact with the external environment within the vascular network. The fluorescence intensity of the vascular region refers to the total signal intensity of the fluorescent markers within the vascular lumen. The matrix fluorescence intensity refers to the signal intensity of the fluorescent markers in the extracellular matrix region. The concentration difference between the inside and outside of the blood vessel refers to the concentration difference of the fluorescent markers within the vascular lumen and in the extracellular matrix. The vascular network sample refers to a representative vascular structural unit used for branch density analysis. The sample volume refers to the three-dimensional spatial volume occupied by the vascular network sample. The vascular skeleton refers to the central line structure of the vascular network after skeletonization. The number of branch points refers to the number of nodes where three or more blood vessels intersect in the vascular skeleton. The complexity refers to the irregularity and branching richness of the vascular network structure.

[0069] Optionally, the time-series images of vascular regions and extracellular matrix regions can be obtained using threshold segmentation methods, such as global threshold segmentation or local adaptive threshold segmentation.

[0070] Optionally, the surface area of ​​the blood vessels in the vascular region can be calculated using surface area calculation methods based on contour fitting, such as OpenCV, ImageJ, etc.

[0071] Optionally, the complexity of the vascular network sample can be quantified by topological and geometric quantification indicators, such as fractal dimension method, average path length method, etc.

[0072] Optionally, the sample volume of the vascular network sample can be calculated by pixel statistics, such as by counting the pixels occupied by the vascular region layer by layer, multiplying by the layer thickness, and summing them to obtain the total volume.

[0073] As another implementation, the permeability is calculated using the following formula: ; Where T(t) represents the permeability of the vascular network at time t (unit: m / s), T0 represents the initial permeability (unit: m / s), e represents an exponential function with base e, μ represents the time decay coefficient (unit: 1 / s), t represents time (unit: s), and K represents the diffusion constant (unit: m). 2 / s), This represents the concentration difference between the inside and outside of the vascular network at time t (unit: mol / m³). 3 M represents the vascular surface area of ​​the vascular network (unit: m²). 2 ), d represents the vascular thickness of the vascular network (unit: m), and c0 represents the initial concentration difference (unit: mol / m). 3 ).

[0074] It should be explained that in this application, μ represents the time decay coefficient, which refers to the rate at which vascular barrier function deteriorates over time, and has a value of [0.01, 0.5] min. -1 If you need to simulate rapid decline (such as acute injury to the vascular network), the upper limit can be set to 0.5 minutes. -1 If simulating slow decline (e.g., a normal vascular network), a lower limit of 0.01 min can be selected. -1 K represents the diffusion constant, which refers to the diffusion ability of the fluorescent label in the blood vessel wall and matrix, and has a value of [300, 700] μm. 2 / s, if using medium-sized molecules (such as 70 kDa dextran), an intermediate value of 500 μm can be selected. 2 / s, if the molecular weight of the labeled substance is large (e.g., 200 kDa) and the diffusion is slow, a lower limit of 300 μm can be selected. 2 / s, if the molecular weight is small (e.g., 4 kDa) and diffusion is fast, an upper limit of 700 μm can be selected. 2 / s.

[0075] Optionally, the time decay coefficient can be calculated by monitoring the accumulation of fluorescent markers (such as FITC-dextran) outside blood vessels in an in vitro model in real time, recording the fluorescence intensity over time, and then fitting a time decay curve.

[0076] In another implementation, the branch density is calculated using the following formula: ; Where D represents the branch density (unit: 1 / m) 3 F represents complexity, N represents the number of branch points, and V represents the sample volume (unit: m). 3 ).

[0077] This invention, through determining the fluid shear force and pulse frequency of the vascular network, can activate mechanoreceptors (such as PECAM-1, VE-cadherin, and integrins) in endothelial cells, promoting cell polarity and tight junction protein expression, thereby enhancing vascular barrier function, reducing permeability, and mimicking the physiological state of blood vessels in vivo. Here, the fluid shear force refers to the frictional force exerted by the flowing liquid on the surface of the vascular network. The pulse frequency refers to the frequency at which the flow direction and velocity of the fluid in the vascular network exhibit periodic changes.

[0078] Optionally, the fluid shear force and pulse frequency of the vascular network can be determined by computational fluid dynamics simulation and machine learning algorithms. For example, a three-dimensional model of the vascular network can be constructed using software such as COMSOL and ANSYS Fluent to simulate the fluid dynamic behavior of the vascular network. Based on the fluid dynamic behavior, the fluid shear force and pulse frequency of the vascular network can be determined by machine learning algorithms.

[0079] In this embodiment of the invention, by culturing the vascular network into vascular organoids based on the fluid shear force and the pulse frequency, the vascular endothelial cell barrier function can be enhanced, thereby improving vascular contraction and dilation functions, and enhancing substance exchange and permeability regulation.

[0080] Compared to the problems described in the background art, this invention constructs a cell culture environment for human induced pluripotent stem cells (iPSCs), inducing them to aggregate into cell aggregates with specific characteristics. Combined with a multi-stage induction and three-dimensional culture strategy, it can efficiently and controllably differentiate into functional vascular organoids, offering significant technical and application advantages. First, by optimizing the formation conditions of cell aggregates, this invention enhances intercellular interactions, improving the efficiency and uniformity of subsequent mesodermal induction. Second, based on the precise regulation of mesodermal induction molecules according to aggregate characteristics, it can directionally induce human iPSCs to differentiate into mesodermal cells, significantly increasing the expression levels of mesodermal marker genes and laying a solid foundation for vascular lineage differentiation. Furthermore, by constructing a vascular induction culture medium and combining it with dynamic regulation of gene expression levels, this invention can efficiently induce mesodermal cells to differentiate into vascular lineage cells such as vascular endothelial cells and smooth muscle cells, improving differentiation efficiency and cell functionality. Subsequently, embedding vascular lineage cells into a three-dimensional extracellular matrix simulates the in vivo microenvironment, promoting their self-organization into a vascular network with complex branching structures, significantly improving the maturity and physiological relevance of the vascular network. This invention, by calculating the permeability and branch density of vascular networks, can accurately determine fluid shear force and pulse frequency, optimize dynamic culture conditions, and further enhance the functional characteristics of vascular organoids, such as barrier function, contractility, and blood flow responsiveness. This invention not only achieves efficient construction of vascular organoids but also provides a highly controllable and reproducible in vitro model for bioengineering, tissue model construction, and basic biological research, promoting technological progress and application expansion in related fields. Therefore, the method for constructing vascular organoids based on human induced pluripotent stem cells provided in this invention can improve the stability and functionality of vascular organoids constructed from human induced pluripotent stem cells.

[0081] like Figure 2 The diagram shown is a system functional block diagram of a vascular organoid constructed based on human induced pluripotent stem cells according to the present invention.

[0082] The system 200 for constructing vascular organoids based on human induced pluripotent stem cells, as described in this invention, can be installed in an electronic device. Depending on the functions implemented, the system may include a cell aggregate construction module 201, a mesodermal cell culture module 202, a vascular lineage cell differentiation module 203, a vascular network differentiation module 204, and a vascular organoid culture module 205. The modules described in this invention can also be referred to as units, which are a series of computer program segments that can be executed by the processor of an electronic device and perform a fixed function, stored in the memory of the electronic device.

[0083] In this embodiment of the invention, the functions of each module / unit are as follows: The cell aggregate construction module 201 is used to construct a cell culture environment for human induced pluripotent stem cells to induce the human induced pluripotent stem cells to aggregate into cell aggregates and to identify the aggregate characteristics of the cell aggregates. The mesodermal cell culture module 202 is used to determine the mesodermal inducing molecules of the cell aggregates to construct the mesodermal induction environment of the cell aggregates. Based on the mesodermal induction environment and the characteristics of the aggregates, human induced pluripotent stem cells in the cell aggregates are induced to differentiate to obtain mesodermal cells. The vascular lineage cell differentiation module 203 is used to detect the expression level of mesodermal marker genes in the mesodermal cells, construct the vascularization induction culture medium for the mesodermal cells, and induce the mesodermal cells to differentiate into vascular lineage cells according to the expression level and the vascularization induction culture medium. The vascular network differentiation module 204 is used to construct a three-dimensional extracellular matrix of the vascular lineage cells and embed the vascular lineage cells into the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network. The vascular organoid culture module 205 is used to calculate the permeability and branch density of the vascular network to determine the fluid shear force and pulse frequency of the vascular network, and to culture the vascular network into vascular organoids based on the fluid shear force and the pulse frequency.

[0084] In detail, the modules in the system 200 for constructing vascular organoids based on human induced pluripotent stem cells described in this embodiment of the invention employ the same methods as described above. Figure 1 The method described herein is the same as the one used to construct vascular organoids based on human induced pluripotent stem cells, and can produce the same technical effects, so it will not be repeated here.

[0085] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0086] Finally, it should be noted that in the above embodiments, each embodiment can be combined with each other or independent. Deleting any one of them will not affect the technical implementation of other embodiments. The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing vascular organoids based on human induced pluripotent stem cells, characterized in that, The method includes: A cell culture environment for human induced pluripotent stem cells was constructed to induce the human induced pluripotent stem cells to aggregate into cell aggregates, and the aggregate characteristics of the cell aggregates were identified. The mesodermal inducing molecules of the cell aggregates are identified to construct a mesodermal induction environment for the cell aggregates. Based on the mesodermal induction environment and the characteristics of the aggregates, human induced pluripotent stem cells in the cell aggregates are induced to differentiate to obtain mesodermal cells. The expression levels of mesodermal marker genes in the mesodermal cells were detected, angiogenesis induction culture medium for the mesodermal cells was constructed, and the mesodermal cells were induced to differentiate into vascular lineage cells based on the expression levels and the angiogenesis induction culture medium. A three-dimensional extracellular matrix of the vascular lineage cells was constructed, and the vascular lineage cells were embedded in the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network; The permeability and branch density of the vascular network are calculated to determine the fluid shear force and pulse frequency of the vascular network. Based on the fluid shear force and the pulse frequency, the vascular network is cultured into vascular organoids.

2. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The calculation of the permeability and branch density of the vascular network includes: Identify fluorescent markers for the vascular network; Time-series images of the diffusion of the fluorescent marker from the lumen of the corresponding blood vessel in the vascular network to the extracellular matrix were acquired. The time-series images were segmented into vascular regions and extracellular matrix regions; Calculate the vascular surface area of ​​the vascular region; The fluorescence intensity of the vascular region and the matrix fluorescence intensity of the extracellular matrix region were measured separately. Based on the fluorescence intensity of the vascular region and the fluorescence intensity of the matrix, the concentration difference between the inside and outside of the vascular region and the extracellular matrix region is calculated. The permeability of the vascular network is calculated based on the concentration difference between the inside and outside of the blood vessel. Extract vascular network samples from the vascular network; Calculate the sample volume of the vascular network sample; Extract the vascular skeleton of the vascular network sample to calculate the number of branch points of the vascular network sample; The complexity of the vascular network sample is quantified, and the branch density of the vascular network is calculated based on the complexity, the number of branch points, and the sample volume.

3. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The cell culture environment for constructing human induced pluripotent stem cells includes: Configure the extremely low adhesion culture plate for the aforementioned human induced pluripotent stem cells; The coating matrix of the extremely low adhesion culture plate is determined so as to coat the extremely low adhesion culture plate to obtain a coated extremely low adhesion culture plate. The culture conditions for the human induced pluripotent stem cells are constructed, wherein the culture conditions include: a sterile environment, temperature and humidity, and gas conditions; Prepare the culture medium for aggregates of the aforementioned human induced pluripotent stem cells; Based on the aforementioned coated low-attachment culture plate, the aforementioned culture conditions, and the aforementioned aggregate culture medium, an integrated cell culture environment for human induced pluripotent stem cells is created.

4. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The induction of the human induced pluripotent stem cells to aggregate into cell aggregates includes: Construct a single-cell suspension of the aforementioned human induced pluripotent stem cells; The single-cell suspension was subjected to a technique to obtain the cell count; Determine the target cell density of the single-cell suspension, and adjust the density of the single-cell suspension based on the cell number and the target cell density to obtain an adjusted single-cell suspension; Based on the cell culture environment corresponding to the human induced pluripotent stem cells, the adjusted single-cell suspension is cultured to obtain cell clusters; The cell clusters are induced to form cell aggregates using an aggregate culture medium corresponding to the cell culture environment.

5. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The mesodermal induction environment for constructing the cell aggregates includes: Determine the basic culture medium formulation for the cell aggregates; The formulation of the inducing factor for the cell aggregate is determined based on the mesodermal inducing molecule corresponding to the cell aggregate. Based on the basic culture medium formula and the inducing factor formula, a mesodermal induction culture medium for the cell aggregates was prepared. The culture parameters of the cell aggregates are determined, wherein the culture parameters include: culture time, culture temperature and humidity, and culture medium replacement cycle; The mesodermal induction environment of the cell aggregates is determined based on the mesodermal induction medium and the culture parameters.

6. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The detection of the expression level of mesoderm marker genes in the mesodermal cells includes: The mesodermal cells were sampled to obtain cell samples; The cell sample was lysed to obtain a cell lysate; RNA molecules were extracted from the cell lysate; The RNA molecule was reverse transcribed to obtain a cDNA molecule. Identify the target gene primers and internal reference gene primers for the mesoderm marker genes in the cDNA molecule; Based on the target gene primer and the internal reference gene primer, the cDNA molecule was detected by qRT-PCR to obtain the detection results; The CT values ​​of the target gene and the internal reference gene in the detection results are extracted to calculate the expression level of the mesodermal marker gene.

7. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The step of inducing the mesodermal cells to differentiate into vascular lineage cells based on the expression level and the angiogenesis induction medium includes: When the expression level reaches a preset expression level threshold, the mesodermal cells are plated to obtain plated cells. The plated cells were cultured without induction to obtain adherent cells; Establish the angiogenesis induction culture conditions for the adherent cells; The adherent cells were induced and cultured according to the described vascular induction culture conditions and the described vascular induction culture medium to obtain cells in culture. Real-time acquisition of morphological change data of cells in culture is used to adjust the concentration of angiogenic factors in the angiogenic induction medium. Based on the concentration of the angiogenesis-inducing factor, the cultured cells are differentiated into angiogenesis lineage cells.

8. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The three-dimensional extracellular matrix for constructing the vascular lineage cells includes: The matrix construction material for the vascular lineage cells is configured, wherein the matrix construction material comprises: biomaterials and cell culture reagents; Determine the vessel diameter, vessel branches, and vessel porosity of the vascular lineage cells; The three-dimensional vascular structure of the vascular lineage cells is determined based on the vessel diameter, vessel branches, and vessel porosity. Based on the three-dimensional vascular structure, a three-dimensional scaffold matrix for the vascular lineage cells is constructed using the matrix construction material. The vascular lineage cells' vascular growth factor was added to the three-dimensional scaffold matrix to obtain the three-dimensional extracellular matrix of the vascular lineage cells.

9. The method for constructing vascular organoids based on human induced pluripotent stem cells as described in claim 1, characterized in that, The step of embedding the vascular lineage cells into the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network includes: Analyze the mixing ratio of the vascular lineage cells and the three-dimensional extracellular matrix; Based on the mixing ratio, the vascular lineage cells are embedded in the three-dimensional extracellular matrix to obtain a cell-matrix mixture; Matrix curing conditions for the cell-matrix mixture were constructed to cure the cell-matrix mixture, resulting in a cell-cured matrix mixture; Monitor the angiogenesis status of the cell-stabilized matrix mixture during differentiation; Based on the angiogenesis status, the dynamic culture parameters of the cell-stabilized matrix mixture are adjusted, wherein the dynamic culture parameters include: growth factor concentration, bioreactor rotation speed, and nutrient solution concentration; Based on the dynamic culture parameters, the cell-stabilized matrix mixture is dynamically cultured to obtain a vascular network.

10. A system for constructing vascular organoids based on human induced pluripotent stem cells, characterized in that, The system includes: A cell aggregate construction module is used to construct a cell culture environment for human induced pluripotent stem cells to induce the human induced pluripotent stem cells to aggregate into cell aggregates and to identify the aggregate characteristics of the cell aggregates; The mesodermal cell culture module is used to determine the mesodermal inducing molecules of the cell aggregates to construct the mesodermal induction environment of the cell aggregates. Based on the mesodermal induction environment and the characteristics of the aggregates, human induced pluripotent stem cells in the cell aggregates are induced to differentiate to obtain mesodermal cells. A vascular lineage cell differentiation module is used to detect the expression level of mesodermal marker genes in the mesodermal cells, construct angiogenesis induction culture medium for the mesodermal cells, and induce the mesodermal cells to differentiate into vascular lineage cells according to the expression level and the angiogenesis induction culture medium. A vascular network differentiation module is used to construct a three-dimensional extracellular matrix of the vascular lineage cells, embedding the vascular lineage cells into the three-dimensional extracellular matrix to differentiate the vascular lineage cells into a vascular network; The vascular organoid culture module is used to calculate the permeability and branch density of the vascular network to determine the fluid shear force and pulse frequency of the vascular network, and to culture the vascular network into vascular organoids based on the fluid shear force and the pulse frequency.

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