A gradient blood vessel tissue model construction method based on multi-level modular assembly and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a gradient blood vessel tissue model construction method based on multi-stage modular assembly and application thereof. The present application utilizes a multi-stage modular assembly strategy, uses a hydrogel sheet containing endothelial cell microspheres as a basic unit for modular assembly, constructs a novel in-vitro tissue model with a multi-scale blood vessel structure, wherein the cell microsphere type, density and spatial structure of each layer are controllable and adjustable, and the in-vitro tissue model can be applied to tissue development mechanism research and drug screening.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing a gradient vascular tissue model based on multi-level modular assembly and its application. Background Technology

[0002] Current in vitro tissue models are still mainly two-dimensional cell models. These models are easy to prepare and relatively inexpensive, and have been widely used in early-stage research in tissue engineering. However, due to the inherent limitations of two-dimensional cell models, they still have shortcomings in areas such as three-dimensional biomimetic structures and microenvironment modeling. While animal models have solved the problems of three-dimensional structure and some microenvironment modeling, issues such as interspecies immune differences and batch-to-batch stability hinder their further development. Animal models cannot truly replicate human diseases, and drug responses and toxicity differ between animals and humans.

[0003] On the other hand, the development of bio-3D printing technology in recent years has provided a powerful tool for constructing in vitro biomimetic tissue models. Introducing bioactive materials into 3D printing allows for the successful construction of complex structures, including blood vessels, and enables precise control of cell placement, providing a more biomimetic physiological environment for in vitro models.

[0004] 3D-printed vascularized scaffold models and monolayer 3D vascular network microfluidic chips have overcome the limitations of traditional two-dimensional cell and animal models, but they still cannot meet all needs. For example, while 3D-printed vascularized scaffold models can exhibit certain vascular structures and functions, they cannot effectively promote endogenous angiogenesis and tissue growth due to the limitations of the bio-inks used. Furthermore, the limited printing materials typically require long culture times. In contrast, while monolayer 3D vascular network microfluidic chips can cultivate well-structured and functional monolayer microvascular networks within 7 days, their chip size limits their ability to culture only small-scale tissues, preventing the cultivation of larger tissues.

[0005] In addition, due to limitations such as the mass transfer efficiency of hydrogels, in large-volume hydrogel 3D printed structures, cells often die on a large scale due to oxygen and nutrient supply before they have fully matured, making it difficult to achieve the unification of millimeter-scale perfusionable vascular structures and endothelial microvascular networks.

[0006] Vascular networks in real tissues typically exhibit a hierarchical structure, including blood vessels of different scales and sometimes heterogeneous tissue. This hierarchical structure and heterogeneous tissue significantly influence physiological and pathological processes, such as thrombosis and vascular tumor embolism. Therefore, there is an urgent need to construct vascular tissue models with hierarchical vascular network structures composed of blood vessels of different scales and with heterogeneous tissue to simulate the complex vascular system in the human body. Summary of the Invention

[0007] This invention provides a method for constructing a gradient vascular tissue model based on multi-level modular assembly and its application. Addressing the problems existing in various in vitro tissue models, this invention combines a multi-level modular assembly strategy with bio-3D printing technology. It uses hydrogel sheets containing endothelial cell microspheres (including endothelial cell aggregates, endothelial cell clusters, or vascular organoids, etc.) as basic units for modular assembly, aiming to construct a novel in vitro tissue model with multi-scale vascular structures. The types, densities, and spatial structures of cell microspheres in each layer are controllable and adjustable, and can be applied to the study of tissue development mechanisms and drug screening.

[0008] This invention provides a hydrogel layer containing endothelial cell microspheres, the raw materials of which include: endothelial cell microspheres (including cell spheroids including cell aggregates, clusters or organoid structures), dispersed endothelial cells, crosslinking agent and hydrogel prepolymer solution. According to the cell-containing microsphere hydrogel layer, the crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoyl phosphite (LAP); preferably, the amount of the crosslinking agent is 0.15-0.4% w / v; And / or, the hydrogel is one or more of sodium alginate, methacrylated gelatin, and gelatin; preferably, the amount of the hydrogel used is 3-8% w / v; Preferably, its raw materials include 0.5×10 6 ~2×10 6 / ml dispersed endothelial cells, 0.15–0.4% w / v LAP and 3–8% w / v methacrylated gelatin.

[0009] The present invention also provides a method for preparing a cell-containing microsphere hydrogel layer, wherein the raw materials are mixed as a bio-ink and directly extruded or formed using bio-3D printing.

[0010] This invention uses a mixture of dispersed cells and cell microspheres with a hydrogel prepolymer solution as a bio-ink. A monolayer hydrogel sheet containing cell microspheres is prepared by direct extrusion curing in a well plate or by bio-3D printing. A microvascular network connects after 48-72 hours of incubation. Preferably, extruding directly into the well plate using a 1ml pipette tip and curing to form a circular sheet shortens the preparation time.

[0011] The method for preparing the cell-containing microsphere hydrogel layer includes: (1) Obtain endothelial cell microspheres, dispersed endothelial cells, crosslinking agent, and hydrogel prepolymer solution, mix them to obtain bio-ink containing endothelial cell microspheres and dispersed endothelial cells, and directly extrude or use bio-3D printing to form a primary hydrogel layer containing endothelial cell microspheres. (2) The primary hydrogel layer containing endothelial cell microspheres obtained in step (1) is subjected to a cross-linking reaction; (3) The hydrogel layer containing endothelial cell microspheres after the cross-linking reaction in step (2) is cultured for cell culture.

[0012] The present invention also provides a vascularized tissue model, comprising an alternating layer of cell-containing microsphere hydrogel and a channel-containing hydrogel layer; the channel-containing hydrogel layer has perfusionable channels; The channel opening of the channel-containing hydrogel layer is in contact with the adjacent cell-containing microsphere hydrogel layer.

[0013] The raw materials for the cell-containing microsphere hydrogel layer include endothelial cell microspheres and dispersed endothelial cells.

[0014] The channel-containing hydrogel layer may contain cellular microspheres and dispersed endothelial cells, or may only have a channel structure without cellular components.

[0015] Preferably, the cell microspheres are spherical structures containing endothelial cells, including endothelial cell aggregates, endothelial cell clusters, or vascular organoids.

[0016] Preferably, after the channel-containing hydrogel is constructed, a high-density endothelial cell suspension can be added into the channel to perform endothelialization treatment on the channel surface to improve biomimicry.

[0017] Preferably, the high-density endothelial cell suspension refers to 1.0 × 10⁻⁶ cells / mL. 7 5.0 x 10⁻⁵ cells / ml 7 per ml.

[0018] This invention uses a hydrogel layer containing cell microspheres as the basic building unit, and constructs millimeter-scale perfusionable vascular channels, microvascular network connections of endothelial cells, and tissue parenchymal cell microsphere microenvironments in different layers, and realizes the construction of vascularized tissue models through modular assembly.

[0019] The gradient vascularized tissue model based on multi-level modular assembly contains N hydrogel layers, where N is any natural number ≥ 2.

[0020] Preferably, N is 3, 4, 5, 6, 10, etc.

[0021] Preferably, there is at least one channel-containing hydrogel layer between the cell-containing microsphere hydrogel layers.

[0022] Preferably, taking a three-layer structure assembly as an example, the middle layer is a channeled hydrogel layer, and the top and bottom layers contain cell microspheres. The three layers are stacked sequentially in a 24-well plate, and a small amount of methacrylated gelatin prepolymer solution is added along the sidewall to fill the gaps between the layers. After cross-linking, the three layers are fixed as a whole. Then, a punch is used to penetrate the three layers at the channel positions to facilitate perfusion culture. Structures with more than three layers can be prepared using the same approach.

[0023] Based on the vascularized tissue model, the cell-containing microsphere hydrogel layer is utilized.

[0024] The present invention also provides a method for preparing the vascularized tissue model, wherein through-holes are constructed between the cell-containing microsphere hydrogel layers, so that they are connected with the channels of the channel-containing hydrogel layer, forming a perfusion channel that can perfuse the entire layer of the vascularized tissue model.

[0025] According to the method for preparing the vascularized tissue model, a channel-containing hydrogel layer is prepared by using a sacrificial template method or by suspension printing in a hydrogel prepolymer solution.

[0026] Preferably, the method of sacrificing the template includes: placing a model that can form a channel into the hydrogel prepolymer solution before crosslinking, and removing the model after crosslinking; more preferably, the model is a metal rod, specifically a short stainless steel rod.

[0027] Preferably, Prönnik F127 is used as the sacrificial material to print sacrificial channels internally, and the material is removed after construction to form the channels.

[0028] The method for preparing the vascularized tissue model includes the following steps: (1) Preparation of microporous array culture plate: Prepare microporous array culture plate and perform surface hydrophobic treatment on microporous array culture plate; preferably, add the required cell suspension (endothelial cells, fibroblasts, parenchymal cells, etc.) into microporous array culture plate, and utilize its microporous structure and surface hydrophobic effect to form dense cell microspheres after 12h to 24h of culture.

[0029] (2) Preparation of endothelial cell microspheres: Endothelial cell microspheres are obtained by inoculating endothelial cell suspension in microwell array culture plates or by inoculating stem cells to induce differentiation to obtain vascular organoids.

[0030] Preferably, the endothelial cell suspension is a culture medium solution containing human endothelial cells, and a certain proportion of culture medium solutions of other cells may be added to improve the functionality of the microspheres. The endothelial cells include human umbilical vein endothelial cells (HUVECs), human microvascular endothelial cells (HMECs), human coronary artery endothelial cells (HCAECs), etc., and the other cells include human embryonic lung fibroblasts (MRC-5), mouse embryonic fibroblasts (NIH-3T3), mesenchymal stem cells (MSCs), etc.

[0031] Preferably, the endothelial cell microspheres can be vascular organoids. Stem cells that can differentiate into endothelial cells, such as mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and endothelial progenitor cells (EPCs), are added to a microporous array culture plate, and vascular organoids are obtained under the action of related factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).

[0032] Preferably, the culture time is 12h to 48h.

[0033] (3) Preparation of monolayer hydrogel sheets.

[0034] Cell microspheres were collected and prepared, and mixed with dispersed endothelial cells in a suitable density and ratio with a hydrogel prepolymer solution to form a bio-ink. The bio-ink was then prepared by extrusion 3D printing to create a single-layer hydrogel sheet with a specific shape and spatial structure. After culturing (preferably 48h to 72h), a vascular network was formed between the cell microspheres and the dispersed cells. (4) Modular assembly of multilayer hydrogel sheets: Assemble a cellular microsphere hydrogel sheet with pre-formed vascular connections, ensuring that at least a perfusionable vascular channel layer (preferably millimeter-scale), a microvascular connection layer with endothelial cell microspheres, and a microenvironment layer mainly composed of parenchymal cell microspheres are present. Preferably, the type of cell microspheres, the shape of the hydrogel layer, and the spatial structure of each hydrogel layer can be individually adjusted; (5) Gradient vascular structure construction: After culturing, the resulting structure forms a vascular connection between the hydrogel layer containing cell microspheres and the hydrogel layer containing perfusion vascular channels (preferably at the millimeter level), thus realizing the construction of multi-scale vascular structures.

[0035] The method for preparing the vascularized tissue model includes the following steps: (1) Rapid and stable preparation of endothelial cell microspheres based on microporous array culture plates. The microporous array culture plates used include, but are not limited to, oxygen-permeable microporous array culture plates based on polydimethylsiloxane (PDMS), silicone rubber, or hydrogel materials, as well as commercially available microporous array culture plates. Two or more cell types, including endothelial cells (such as endothelial cells, fibroblasts, etc.), are added to the microporous array culture plate in a suitable ratio and density. Due to the hydrophobic effect of the micropore surface, dense cell microspheres are formed after culturing in a 37°C incubator for 12-48 hours. Traditional methods for constructing microspheres using cell self-assembly are time-consuming and inefficient. The microporous array culture plate technology used in this invention can achieve rapid and stable preparation of cell microspheres. The cell composition (usually containing endothelial cells, fibroblasts, etc.), ratio, and microsphere size (76μm, 126μm, 200μm, and 326μm) are controllable and adjustable, and dense cell microspheres can be formed in 12-48 hours.

[0036] (2) Preparation of monolayer cell-containing microsphere hydrogels. Endothelial cell microspheres are collected and mixed with dispersed cells in a suitable ratio and density with bio-ink (e.g., sodium alginate, methacrylated gelatin, etc.). The mixture is then prepared into monolayer cell-containing hydrogel sheets of a specific shape (containing perfusionable vascular channel structures) by direct extrusion or bio-3D printing. After culturing for 48-72 hours, the dispersed endothelial cells in the hydrogel connect with the endothelial cell microspheres to form a vascular network structure. Traditional methods typically involve directly embedding cell microspheres into the hydrogel for culture, resulting in difficulty in forming connections between microspheres or a long time required for such connections to form. This invention simultaneously adds cell microspheres and dispersed cells to the hydrogel at a suitable density and ratio, promoting the formation of connections between the microspheres and the surrounding dispersed endothelial cells, thus achieving rapid generation of vascular structures within the hydrogel.

[0037] (3) Modular assembly of multilayer hydrogels. Multilayer hydrogel sheets containing cell microspheres are assembled, with different layers containing different types of cell microspheres, and specific geometric shapes and spatial structures can be designed individually. After assembly and culturing for 48-72 hours, vascular connections are formed between cells and cell microspheres in adjacent layers. Traditional methods directly construct vascular structures using 3D printing, but it is difficult to achieve anastomosis between microvessels and millimeter-scale perfusionable vessels. This invention modularly assembles multilayer hydrogel sheets composed of specific shapes, spatial structures, and cell microspheres, where hydrogel layers with millimeter-scale vascular channel structures form microvascular connections with adjacent layers, realizing the construction of a gradient vascular structure, and providing sufficient oxygen and nutrient supply through perfusion culture.

[0038] The tissue model of the present invention, which features a biomimetic vascular microenvironment, can provide a more realistic tissue growth environment and can be used as a research model for tissue development.

[0039] Furthermore, since this invention has a gradient vascular structure as a tissue model, it can also be used to develop vascularized tumor models and serve as an in vitro drug evaluation model for drug delivery processes and tumor-killing / anti-angiogenic drugs.

[0040] The present invention also provides the cell-containing microsphere hydrogel layer, the preparation method of the cell-containing microsphere hydrogel layer, the vascularized tissue model, and the application of the preparation method of the vascularized tissue model in the preparation of vascularized tissue or biomedical materials.

[0041] The beneficial effects of this invention are: (1) This invention enables the rapid generation of vascular structures in hydrogels. This invention simultaneously incorporates cellular microspheres and dispersed cells into the hydrogel, promoting the formation of connections between the microspheres and the surrounding dispersed endothelial cells, thereby achieving the rapid generation of vascular structures.

[0042] (2) The preparation method of the cell-containing microsphere hydrogel layer of the present invention is simple and low in cost. In addition to using bio-3D printing technology for molding, the cell-containing microsphere hydrogel layer of the present invention can be directly extruded and fixed, which is simple to operate and greatly reduces the preparation cost.

[0043] (3) This invention enables the construction of in vitro tissue models with multi-scale vascular structures and can also meet the needs for heterogeneous tissue construction. Traditional in vitro tissue models have limitations in the construction of multi-scale vascular structures, making it difficult to simultaneously generate micron-scale microvascular networks and millimeter-scale perfusionable vascular structures. This invention assembles cultured cell-containing microsphere hydrogel sheets layer by layer, where each layer can be customized with cell microsphere types, hydrogel sheet shapes, spatial structures, etc. After the assembled structure is cultured, vascular connections are formed between adjacent layers. By customizing different layers into blood vessels of different scales, a hierarchical vascular network structure composed of blood vessels of different scales can be constructed. By designing some layers to contain heterogeneous cells, a vascular tissue model containing heterogeneous tissue can be constructed. In this invention, some hydrogel layers in the multi-scale vascular structure have designed millimeter-scale perfusionable vascular structures, which can form vascular connections with endothelial cells / cell microspheres in adjacent layers after modular assembly, realizing the generation of gradient vascular structures.

[0044] (4) The vascularized tissue model of the present invention solves the problem of large-scale cell death in large-volume hydrogel printed structures. The present invention improves oxygen transport efficiency and solves the defects of hydrogel bio-3D printed structures by arranging cell-containing microsphere hydrogel layers and channel-containing hydrogel layers alternately.

[0045] (5) This invention provides a large-volume vascularized tissue model. Traditional vascularized models are generally at the millimeter level, but because the model of this invention has an assembly structure, it can provide a large vascularized tissue model at the centimeter level.

[0046] (6) This invention can provide a large-volume vascularized tissue model in a short time. Existing technologies lack large-volume vascularized models. The model culture time of this invention is short, allowing for the construction of a large-volume vascularized tissue model in a short time, even shorter than the culture time of traditional small-volume 3D vascularized tissue models.

[0047] (7) This invention improves the biomimeticity and heterogeneity of the basic units of in vitro tissue models. Traditional in vitro tissue models use two-dimensional planar cells or cell microspheres as basic building blocks, which have limited biomimeticity and microenvironment shaping capabilities. This invention constructs a hydrogel layer containing cell microspheres and specific spatial structures, providing a biomimetic microenvironment for cells. At the same time, endothelial cells, parenchymal cells, etc. are introduced into different layers, thereby improving the biomimeticity of the model. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0049] Figure 1 Endothelial cell microspheres were prepared for the microporous culture plate provided in Example 1 of this invention; A mixed cell suspension of endothelial cells and fibroblasts was added to the microporous array shown in the figure at an appropriate density and ratio. After 24–48 hours of culture, dense co-cultured cell microspheres were generated.

[0050] Figure 2 The hydrogel layer containing cell microspheres provided in Example 1 of the present invention; The solution of GelMA prepolymer containing cell microspheres was extruded, printed, and solidified in a 24-well plate, or perfusion channels were constructed by suspension printing in a GelMA hydrogel layer, followed by the addition of culture medium and incubation in an incubator for 3–4 days. Cells are removed from a monolayer hydrogel after they have matured. Two types of hydrogel layers were obtained: one containing cell microspheres that developed into a vascular network, and the other containing no cell microspheres, but perfusion channels ranging from hundreds of micrometers to millimeters were constructed using a sacrificial printing method.

[0051] Figure 3 This is a stacked assembly with a sacrificial channel layer provided in Embodiment 1 of the present invention; Taking the three-layer structure assembly as an example, the middle layer is a channel hydrogel layer, and the upper and lower layers contain cell microspheres. The three layers are stacked in sequence in a 24-well plate, and a small amount of GelMA prepolymer solution is added along the side wall to fill the gaps between the layers. After cross-linking, the three layers are fixed as a whole. Then, a punch is used to penetrate the three layers at the channel position to facilitate perfusion culture.

[0052] Figure 4 This is a schematic diagram of the stacking of multilayer hydrogel layers provided in Embodiment 1 of the present invention.

[0053] Figure 5 This is an image showing the results of CD31 immunostaining of endothelial cell microspheres provided in Example 1 of the present invention.

[0054] Figure 6 This is a diagram showing the microvascular connections in the microsphere-containing hydrogel layer and its immunostaining results provided in Example 1 of the present invention. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. In the examples of this invention, polydimethylsiloxane was purchased from Dow Corning (USA); polyquaternium salt was purchased from Nippon Oil Co., Ltd. (NOF, Japan); F127 material was purchased from Shanghai Puhua Biotechnology Co., Ltd. (Beijing); and human umbilical vein endothelial cells (HUVEC), human hepatocellular carcinoma cells (HepG2), and human embryonic lung fibroblasts (MRC-5) were purchased from the National Experimental Cell Resource Sharing Platform (Beijing).

[0057] This invention provides a method for constructing a vascularized tissue model based on multi-level modular assembly. This method can effectively introduce gradient vascular structures while constructing an in vitro tissue model and improve the biomimetic microenvironment of the in vitro tissue model.

[0058] Specifically, the method includes: (1) Fabrication of oxygen-permeable microporous array: Oxygen-permeable microporous array films were prepared using PDMS (polydimethylsiloxane), and PMB (polyquaternary ammonium salt) was added after surface oxygen plasma treatment for surface hydrophobic treatment.

[0059] (2) Preparation of endothelial cell microspheres: Add endothelial cells at an appropriate density according to the micropore size (a certain proportion of fibroblasts can be added for co-culture at the same time), and dense cell microspheres will form after 12h to 48h of culture.

[0060] (3) Preparation of monolayer hydrogel sheets: The prepared multi-cell co-culture microspheres (containing endothelial cells) were collected and mixed with dispersed endothelial cells in a hydrogel prepolymer solution (such as sodium alginate, methacrylated gelatin, etc.) at appropriate density and ratio to form a bio-ink. The bio-ink was then prepared into a monolayer hydrogel sheet with a specific shape and spatial structure by extrusion 3D printing in a 48-well plate. After 48h to 72h of culture, a vascular network connection was formed between the cell microspheres and the dispersed cells.

[0061] (4) Modular assembly of multilayer hydrogel sheets: Assemble a cellular microsphere hydrogel sheet with pre-formed vascular connections. The type of cellular microspheres, the shape of the hydrogel layer, and the spatial structure of each hydrogel layer can be individually adjusted to ensure that there is at least a perfusionable vascular channel layer at the millimeter level, a microvascular connection layer with endothelial cell microspheres, and a microenvironment layer mainly composed of parenchymal cell microspheres.

[0062] (5) Gradient vascular structure construction: After a period of culture, the resulting structure forms a vascular connection between the hydrogel layer containing cell microspheres (including endothelial cell microspheres and parenchymal cell microspheres) and the hydrogel layer with millimeter-scale perfusion vascular channels, thus realizing the construction of multi-scale vascular structures.

[0063] Example 1: An in vitro vascularized tissue model constructed based on a multi-level modular assembly method.

[0064] (1) Cell Acquisition: Human umbilical vein endothelial cells (HUVECs) and human embryonic lung fibroblasts (MRC-5) were pre-cultured and, after digestion and counting, prepared into a mixed cell suspension at a cell density ratio of 4:1, with HUVECs and MRC-5 cells having a cell density of 1.2 × 10⁻⁶. 6 / ml and 0.3×10 6 / ml.

[0065] (2) Preparation of microporous array films: PDMS and curing agent were mixed at a mass ratio of 10:1 and stirred evenly to ensure full contact. The mixture was then placed in a vacuum pump to remove air bubbles generated during stirring. The treated PDMS liquid was poured into a pre-made silicon plate mold with a honeycomb-like structure and placed in an 80℃ oven for curing for 2 hours. After curing, the PDMS sheet was peeled off, and a PDMS membrane with a diameter of 16 mm was obtained using a punch, exhibiting a 126 μm honeycomb-like groove structure. To reduce cell adhesion to the membrane, a Plasma oxygen plasma processor was used at 75 W for 15 s to improve the hydrophilicity of the membrane surface, allowing the surface treatment agent to better bind with PDMS. Lipidure-PMB (often called "artificial cell membrane," possessing a phospholipid molecular structure similar to cell membranes) and anhydrous ethanol were then mixed at a volume ratio of 1:1.5 and the mixture was used to completely immerse the membrane. After removing air bubbles from the membrane surface, it was placed in a 70℃ oven until completely dried for subsequent cell inoculation.

[0066] (3) Assembly of oxygen-permeable microwell array culture plate: After assembling the autoclaved microwell array culture plate components, place them in a laminar flow hood for 30 minutes of UV irradiation for sterilization. Then, wash the membranes 2-3 times in the laminar flow hood using 75% alcohol and sterile PBS (by volume). After washing, place the membranes into the microwell array culture plate, add 1 ml of DMEM cell culture medium (purchased from Gibco) to keep the surface moist, and wait for subsequent use.

[0067] (4) Preparation of cell microspheres: 1 ml of the multi-cell suspension obtained in step (1) was added to each well of the oxygen-permeable microporous array culture plate assembled in step (3), with a total cell count of approximately 1.5 × 10⁻⁶ cells per well. 6 The culture mold was then placed in an incubator at 37°C and 5% CO2 for 24–48 hours to obtain tightly packed cell microspheres of HUVEC and MRC-5 co-culture. Before using the microspheres, the remaining culture medium in the wells was slowly aspirated with a 1 ml pipette, followed by the addition of 1 ml of fresh culture medium and gentle pipetting several times to transfer all the cell microspheres in the microwell array to the culture medium. The culture medium containing the cell microspheres was placed in a 15 ml centrifuge tube and allowed to stand for 3–5 minutes until stratification occurred. The supernatant was then removed to obtain the desired cell microspheres.

[0068] (5) Preparation of hydrogel material: Weigh 0.5g of sponge-like GelMA solid and dissolve it in 8ml of sterile PBS. Place it in a 70℃ oven and heat for 30-45min. Remove it and shake it evenly to obtain a 6.25% (w / v) GelMA prepolymer solution. Store the hydrogel in a 4℃ refrigerator. Before use, preheat it in an oven to convert it into a liquid state.

[0069] (6) Preparation of crosslinking agent: Weigh 50mg LAP powder and dissolve it in 2ml sterile PBS. Place it in a 70℃ oven in the dark and heat for 30-45min. Then take it out to obtain 2.5% (w / v) LAP crosslinking agent.

[0070] (7) Preparation of the cell-containing microsphere hydrogel layer: Obtain the HUVEC cell suspension again and adjust its density to 1.0 × 10⁻⁶. 7 / ml, then mixed evenly with the LAP crosslinking agent obtained in step (6) and the GelMA prepolymer solution obtained in step (5) at a ratio of 1:1:8 (volume ratio), the final solution containing 1.0×10 6 / ml of endothelial cells, 0.25% (w / v) LAP, and 5% (w / v) GelMA were dispersed. The cell microspheres obtained in step (4) were then resuspended by pipetting using the mixture, and 120ul was pipetted into each well of a standard 48-well plate to form a thin sheet (note that the wells need to be rinsed with sterile PBS first, otherwise the surface tension may not be able to completely cover the sheet). The sheet was then cross-linked and cured for 15s using a 405nm light source to form a stable structure. Finally, culture medium was added and the plate was placed in an incubator for incubation.

[0071] (8) Preparation of channel-containing hydrogel layer: For the preparation process of cell-microsphere-containing hydrogel layer in step (7), cell microspheres can be omitted. Instead, perfusionable channels can be prepared in GelMA using a sacrificial template method. This structure is called channel-containing hydrogel layer. Specifically, taking a simple straight channel as an example, a 1 mm diameter stainless steel short rod can be placed in the GelMA prepolymer solution before crosslinking. After crosslinking at a 405 nm light source for 15 s, it can be removed to obtain a perfusionable channel structure with an inner diameter of 1 mm. For more complex branched channel structures, sacrificial channels can be printed internally using F127 material using a suspension printing method, and removed after curing to construct the structure. Furthermore, after the structure is completed, holes can be punched at both ends of the channel using a puncher to facilitate perfusion culture of adjacent layers in the subsequent multilayer hydrogel structure.

[0072] (9) Assembly of multilayer hydrogel structures: Several hydrogel thin layers containing cell microspheres were prepared according to the above method and cultured in an incubator for a period of time to mature. Before assembly, the remaining culture medium in the wells was slowly aspirated with a pipette and 1 ml of sterile mineral oil was added. After gentle tapping, the bottom of the hydrogel layer was slowly blown to detach it from the bottom surface of the well plate. Then, the hydrogel thin layer was carefully picked up with plastic flat-tipped forceps and transferred to another thin layer. The two layers were stacked together, and a small amount of GelMA prepolymer solution was added from the side wall of the round well to fill the gap and crosslink and fix it. Finally, culture medium was added and the plate was placed in an incubator for incubation.

[0073] Example 2: An in vitro vascularized tumor model constructed based on tumor cell lines and a multi-level modular assembly method.

[0074] (1) Cell Acquisition: Human hepatocellular carcinoma cells (HepG2) and human umbilical vein endothelial cells (HUVECs) were pre-cultured and, after digestion and counting, prepared into a mixed cell suspension at a cell density ratio of 4:1, with HepG2 and HUVEC cells having a cell density of 1.2 × 10⁻⁶ cells per cell. 6 / ml and 0.3×10 6 / ml.

[0075] (2) Preparation of microporous array films: PDMS and curing agent were mixed at a mass ratio of 10:1 and stirred evenly to ensure full contact. The mixture was then placed in a vacuum pump to remove air bubbles generated during stirring. The treated PDMS liquid was poured into a pre-made silicon plate mold with a honeycomb-like structure and placed in an 80℃ oven for curing for 2 hours. After curing, the PDMS sheet was peeled off, and a PDMS membrane with a diameter of 16 mm was obtained using a punch, exhibiting a 126 μm honeycomb-like groove structure. To reduce cell adhesion to the membrane, a Plasma oxygen plasma processor was used at 75 W for 15 s to improve the hydrophilicity of the membrane surface, allowing the surface treatment agent to better bind with PDMS. Lipidure-PMB (often called "artificial cell membrane," possessing a phospholipid molecular structure similar to cell membranes) and anhydrous ethanol were then mixed at a volume ratio of 1:1.5 and the mixture was used to completely immerse the membrane. After removing air bubbles from the membrane surface, it was placed in a 70℃ oven until completely dried for subsequent cell inoculation.

[0076] (3) Assembly of oxygen-permeable microwell array culture plate: After assembling the autoclaved microwell array culture plate components, place them in a laminar flow hood for 30 minutes of UV irradiation for sterilization. Then, wash the membranes 2-3 times in the laminar flow hood using 75% alcohol and sterile PBS. After washing, place the membranes into the microwell array culture plate, add 1 ml of cell culture medium to keep the surface moist, and wait for subsequent use.

[0077] (4) Preparation of cell microspheres: 1 ml of the multi-cell suspension obtained in step (1) was added to each well of the oxygen-permeable microporous array culture plate assembled in step (3), with a total cell count of approximately 1.5 × 10⁻⁶ cells per well. 6The culture mold was then placed in an incubator at 37°C and 5% CO2 for 24–48 hours to obtain pre-vascularized tumor cell microspheres co-cultured with HepG2 and HUVEC. Before using the cell microspheres, the remaining culture medium in the wells was slowly aspirated using a 1 ml pipette, followed by the addition of 1 ml of fresh culture medium and gentle pipetting several times to transfer all the microspheres in the microarray to the culture medium. The culture medium containing the microspheres was transferred to a 15 ml centrifuge tube and allowed to stand for 3–5 minutes until stratification occurred. The supernatant was then removed to obtain the desired pre-vascularized tumor microspheres.

[0078] (4) Preparation of hydrogel material: Weigh 0.5g of sponge-like GelMA solid and dissolve it in 8ml of sterile PBS. Place it in a 70℃ oven and heat for 30-45min. Remove it and shake it evenly to obtain a 6.25% (w / v) GelMA prepolymer solution. Store the hydrogel in a 4℃ refrigerator. Before use, preheat it in an oven to convert it into a liquid state.

[0079] (5) Preparation of crosslinking agent: Weigh 50mg LAP powder and dissolve it in 2ml sterile PBS. Place it in a 70℃ oven in the dark and heat for 30-45min. Then take it out to obtain 2.5% (w / v) LAP crosslinking agent.

[0080] (6) Preparation of the tumor microsphere-containing hydrogel layer: The HUVEC cell suspension was obtained again and its density was adjusted to 1.0 × 10⁻⁶. 7 / ml, then mixed evenly with the LAP crosslinking agent obtained in step (6) and the GelMA prepolymer solution obtained in step (5) at a ratio of 1:1:8 (volume ratio), the final solution containing 1.0×10 6 / ml of endothelial cells, 0.25% (w / v) LAP, and 5% (w / v) GelMA were dispersed. The cell microspheres obtained in step (4) were then resuspended by pipetting using the mixture, and 120ul was pipetted into each well of a standard 48-well plate to form a thin sheet (note that the wells need to be rinsed with sterile PBS first, otherwise the surface tension may not be able to completely cover the sheet). The sheet was then cross-linked and cured for 15s using a 405nm light source to form a stable structure. Finally, culture medium was added and the plate was placed in an incubator for incubation.

[0081] (7) Preparation of channel-containing hydrogel layer: For the preparation process of the cell-microsphere-containing hydrogel layer in step (7), cell microspheres can be omitted. Instead, a permeable channel can be prepared in GelMA using a sacrificial template method. This structure is called a channel-containing hydrogel layer. Specifically, taking a simple straight channel as an example, a 1 mm diameter stainless steel short rod can be placed in the GelMA prepolymer solution before crosslinking. After crosslinking at a 405 nm light source for 15 s, it can be removed to obtain a permeable channel structure with an inner diameter of 1 mm. For more complex branched channel structures, sacrificial channels can be printed internally using F127 material using a suspension printing method, and removed after curing to construct the structure. Furthermore, after the structure is completed, holes can be punched at both ends of the channel using a puncher to facilitate perfusion culture of adjacent layers in the subsequent multilayer hydrogel structure.

[0082] (8) Assembly of multilayer hydrogel structures: Several hydrogel thin layers containing prevascularized tumor microspheres were prepared according to the above method and cultured in an incubator for a period of time to mature. Before assembly, the remaining culture medium in the wells was slowly aspirated with a pipette, followed by the addition of 1 ml of sterile mineral oil. After gentle tapping, the bottom of the hydrogel layer was slowly blown to detach it from the bottom surface of the well plate. Then, the hydrogel thin layer was carefully picked up with plastic flat-tipped forceps and transferred to another thin layer. The two layers were stacked together, and a small amount of GelMA prepolymer solution was added from the side wall of the round well to fill the gap and crosslink and fix it. Finally, culture medium was added and the plate was placed in an incubator for incubation.

[0083] Example 3: An in vitro vascularized tissue model constructed based on degradable microgels and a multi-level modular assembly method.

[0084] (1) Cell acquisition: Human umbilical vein endothelial cells (HUVECs) were pre-cultured and counted after digestion at a rate of 1.5 × 10⁻⁶. 6 Prepare a cell suspension at a density of / ml for later use.

[0085] (2) Preparation of hydrogel materials: Weigh 0.5g of sponge-like GelMA solid and dissolve it in 8ml of sterile PBS. Place it in a 70℃ oven and heat for 30-45min. Remove and shake evenly to obtain a 6.25% (w / v) GelMA prepolymer solution. Prepare a 10% gelatin prepolymer solution using the same procedure. Both hydrogel materials should be stored in a 4℃ refrigerator and preheated in an oven to convert them into a liquid state before use.

[0086] (3) Preparation of crosslinking agent: Weigh 50mg LAP powder and dissolve it in 2ml sterile PBS. Place it in a 70℃ oven in the dark and heat for 30-45min. Then take it out to obtain 2.5% (w / v) LAP crosslinking agent.

[0087] (4) Preparation of microporous array films: PDMS and curing agent were mixed at a mass ratio of 10:1 and stirred evenly to ensure full contact. The mixture was then placed in a vacuum pump to remove air bubbles generated during stirring. The treated PDMS liquid was poured into a pre-made silicon plate mold with a honeycomb-like structure and placed in an 80℃ oven for curing for 2 hours. After curing, the PDMS sheet was peeled off, and a PDMS membrane with a diameter of 16 mm was obtained using a punch, exhibiting a 126 μm honeycomb-like groove structure. To reduce cell adhesion to the membrane, a Plasma oxygen plasma processor was used at 75 W for 15 s to improve the hydrophilicity of the membrane surface, allowing the surface treatment agent to better bind with PDMS. Lipidure-PMB (often called "artificial cell membrane," possessing a phospholipid molecular structure similar to cell membranes) and anhydrous ethanol were then mixed at a volume ratio of 1:1.5 and the mixture was used to completely immerse the membrane. After scraping off air bubbles from the membrane surface, it was placed in a 70℃ oven until completely dried for subsequent cell inoculation.

[0088] (5) Assembly of oxygen-permeable microwell array culture plate: After assembling the autoclaved microwell array culture plate components, place them in a laminar flow hood for 30 minutes of UV irradiation for sterilization. Then, wash the membranes 2-3 times in the laminar flow hood using 75% alcohol and sterile PBS. After washing, place the membranes into the microwell array culture plate, add 1 ml of cell culture medium to keep the surface moist, and wait for subsequent use.

[0089] (6) Preparation of cell microspheres: 1 ml of the cell suspension obtained in step (1) was added to each well of the oxygen-permeable microporous array culture plate assembled in step (3), with a total cell count of approximately 1.5 × 10⁻⁶ cells per well. 6 The culture mold was then placed in an incubator with 5% CO2 at 37°C and cultured for 24–48 hours to obtain HUVEC cell microspheres. Before using the cell microspheres, the remaining culture medium in the wells was slowly aspirated using a 1 ml pipette, followed by the addition of 1 ml of fresh culture medium and gentle pipetting several times to transfer all the microspheres in the microarray to the culture medium. The culture medium containing the microspheres was transferred to a 15 ml centrifuge tube and allowed to stand for 3–5 minutes until stratification occurred. The supernatant was then removed to obtain the desired endothelial cell microspheres.

[0090] (7) Preparation of biodegradable microgels: 10% gelatin was preheated in a 37°C incubator to melt and transform into a liquid. The endothelial cell microspheres obtained in step (6) were then resuspended in the liquid to obtain a gelatin prepolymer solution containing cell microspheres. Subsequently, a common simple microfluidic device was used to prepare gelatin microgels containing cell microspheres. The gelatin prepolymer solution was an aqueous phase, and mineral oil containing 5% Span 80 was used as the oil phase for shearing to obtain gelatin microgels containing cell microspheres. The collected microspheres were stored at 4°C for a short time and used as soon as possible. (8) Preparation of hydrogel layer containing sacrificial microgel: Obtain HUVEC cell suspension again and adjust its density to 1.0 × 10⁻⁶. 7 / ml, then mixed evenly with the LAP crosslinking agent obtained in step (3) and the GelMA prepolymer solution obtained in step (2) at a ratio of 1:1:8 (volume ratio), and the final solution contains 1.0×10 6 / ml of endothelial cells, 0.25% (w / v) LAP, and 5% (w / v) GelMA were dispersed. The gelatin microgel obtained in step (7) was then resuspended using this mixture, and 120µl was pipetted into each well of a standard 48-well plate to form a thin sheet (note that the wells need to be rinsed with sterile PBS first, otherwise surface tension may prevent complete coverage). The gelatin microgel was then crosslinked for 15s using a 405nm light source to solidify the GelMA and form a stable structure. Finally, culture medium was added and the plate was incubated. During this process, the gelatin microgel in the structure degraded upon heating, creating interconnected void regions within the hydrogel structure, and the cell microspheres encapsulated in the gelatin microspheres were released and remained within the hydrogel voids for growth.

[0091] (9) Preparation of channel-containing hydrogel layer: For the preparation process of the cell-microsphere-containing hydrogel layer in step (7), cell microspheres can be omitted. Instead, perfusionable channels can be prepared in GelMA using a sacrificial template method. This structure is called a channel-containing hydrogel layer. Specifically, taking a simple straight channel as an example, a 1 mm diameter stainless steel short rod can be placed in the GelMA prepolymer solution before crosslinking. After crosslinking at a 405 nm light source for 15 s, it can be removed to obtain a perfusionable channel structure with an inner diameter of 1 mm. For more complex branched channel structures, sacrificial channels can be printed internally using F127 material using a suspension printing method, and removed after curing to construct the structure. Furthermore, after the structure is completed, holes can be punched at both ends of the channel using a puncher to facilitate perfusion culture of adjacent layers in the subsequent multilayer hydrogel structure.

[0092] (10) Assembly of multilayer hydrogel structures: Several hydrogel thin layers containing endothelial cell microspheres were prepared according to the above method and cultured in an incubator for a period of time to mature. Before assembly, the remaining culture medium in the wells was slowly aspirated with a pipette, followed by the addition of 1 ml of sterile mineral oil. After gentle tapping, the bottom of the hydrogel layer was slowly blown to detach it from the bottom surface of the well plate. Then, the hydrogel thin layer was carefully picked up with plastic flat-tipped forceps and transferred to another thin layer. The two layers were stacked together, and a small amount of GelMA prepolymer solution was added from the side wall of the round well to fill the gap and crosslink and fix it. Finally, culture medium was added and the plate was placed in an incubator for incubation.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a gradient vascularized tissue model based on multi-level modular assembly, characterized in that, The method includes the following steps: (1) Preparation of microporous array culture plate: Microporous array culture plate was prepared and its surface was hydrophobically treated; (2) Preparation of endothelial cell microspheres: Inoculate endothelial cells or induced pluripotent stem cells in a microwell array culture plate to culture endothelial cell aggregates, endothelial cell clusters, or induce differentiation into vascular organoids; (3) Preparation of hydrogel layer containing endothelial cell microspheres: The prepared endothelial cell microspheres were collected and mixed with dispersed endothelial cells in a hydrogel prepolymer solution to form a bio-ink. A monolayer hydrogel sheet with a specific shape and spatial structure was prepared by extrusion 3D printing. After culture, a vascular network connection was formed between the endothelial cell microspheres and the dispersed endothelial cells. (4) Preparation of channel-containing hydrogel layer: The channel-containing hydrogel layer is prepared by sacrificial template method or by suspension printing method; the sacrificial template method includes: placing a model that can form channels before crosslinking the hydrogel prepolymer solution, and removing the model after crosslinking; the suspension printing method includes: printing sacrificial channels inside the hydrogel, and removing them after curing to construct injectable channels; (5) Modular assembly of multilayer hydrogel sheets: The endothelial cell microsphere hydrogel layer and the channel-containing hydrogel layer are arranged alternately and assembled so that the channel openings of the channel-containing hydrogel layer are in contact with the adjacent endothelial cell microsphere hydrogel layer. Through-holes are constructed between the endothelial cell microsphere hydrogel layers to connect with the channels of the channel-containing hydrogel layer, forming a perfusion channel that can perfuse the entire layer of the vascularized tissue model. The assembled structure ensures that there is at least a perffusionable vascular channel layer and a microvascular connection layer with endothelial cell microspheres. (6) Gradient vascular structure construction: After being cultured, the resulting structure forms a vascular connection between the hydrogel layer containing endothelial cell microspheres and the channel-containing hydrogel layer with perfusion vascular channels, thus realizing the construction of multi-scale vascular structures.

2. A gradient vascularized tissue model based on multi-level modular assembly, characterized in that, It is prepared by the preparation method described in claim 1; the gradient vascularized tissue model based on multi-level modular assembly contains N hydrogel layers, where N is any natural number ≥ 2; Between the endothelial cell microsphere hydrogel layers, there is at least one channel-containing hydrogel layer.

3. The gradient vascularized tissue model based on multi-level modular assembly according to claim 2, characterized in that, N is 3, 4, 5, 6 or 10.

4. The gradient vascularized tissue model based on multi-level modular assembly according to claim 2 or 3, characterized in that, The hydrogel layer containing endothelial cell microspheres is made from the following raw materials: endothelial cell microspheres, dispersed endothelial cells, crosslinking agent, and hydrogel prepolymer solution.

5. The gradient vascularized tissue model based on multi-level modular assembly according to claim 4, characterized in that, The crosslinking agent is LAP; And / or, the hydrogel is one or more of sodium alginate, methacrylated gelatin, and gelatin.

6. The application of the method for preparing a gradient vascularized tissue model based on multi-level modular assembly as described in claim 1 in the preparation of vascularized tissue.

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