Multi-cell barrier tissue model of bionic sequential development mechanism

Abstract

A multi-cell barrier tissue model of a bionic sequential development mechanism has a multi-layer multi-cell structure simulating a physiological barrier, related cells of different barriers are distributed in different layers according to the physiological structure, and the development time and sequence of the cells of different layers are regulated and controlled according to natural barrier multi-cell development physiological characteristics; the multi-cell three-dimensional structure of in-vivo barrier tissue can be simulated, regulation and control of cell development behaviors are achieved by regulating and controlling the matrix environment, and meanwhile, the model has enough overall mechanical support through the soft / hard chimeric structural design so as to adapt to perfusion culture and operation requirements; the model can realize functional bionics of natural barrier tissues at the same time in the spatial structure dimension and the time development dimension so as to realize application in the aspects of disease modeling, drug screening and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue engineering, biomaterials and organ-on-chip technology, and particularly relates to a multi-cell barrier tissue model simulating time-sequential development mechanism. BACKGROUND

[0002] Physiological barrier tissues (such as blood-brain barrier, alveolar-capillary barrier, intestinal epithelial barrier, etc.) are composed of multiple cells, forming a membrane structure with tight junctions and selective permeability, which is of great significance in maintaining human homeostasis and drug screening research. For example, the "neurovascular unit" of the blood-brain barrier is composed of brain microvascular endothelial cells, pericytes and astrocytes, etc., and each cell develops in order and is coupled to form a coaxial multi-layer structure with a highly selective barrier. The existing in vitro models and manufacturing technologies still have limitations in simulating the spatial multi-cell hierarchical complex structure and time-sequential growth pattern of the barrier tissue.

[0003] Currently, there are patent applications that propose to separate and culture different types of cells based on the "Transwell" porous membrane method to construct physiological barrier models, such as the patent application entitled "Blood-brain barrier model" (Publication No. CN109790520B), the patent application entitled "Neural-blood brain barrier co-culture model and its construction method and application" (Publication No. CN119351217A), the patent application entitled "Plug-in type bionic physiological barrier dynamic culture device" (Publication No. CN114410470B), the patent application entitled "In vitro blood-brain barrier model with tight junction structure and its application" (Publication No. CN116790472A), and the patent application entitled "In vitro co-culture alveolar-capillary model system" (Publication No. CN119899790A), which are used to construct blood-brain barrier models, alveolar-capillary models, etc. However, most of these models are two-dimensional planar configurations, and the cells lack the three-dimensional extracellular matrix support similar to that in vivo, and the endothelial cell layer lacks the blood flow stimulation similar to that in vivo, so they cannot well reproduce the structure and physiological function of the in vivo barrier. Some patent applications also use microfluidic chip technology, micro-nano manufacturing technology, etc. to introduce a microfluidic system based on the porous membrane barrier model to provide controllable fluid stimulation and more precise multi-cell spatial location relationship, such as the patent application entitled "MICROFLUDIC CHIPS AND MICROPHYSIOLOGICAL SYSTEMS USING THE SAME" (Publication No. US20230147702A1), the patent application entitled "Chip for simulating cerebral ischemia-reperfusion pathological model" (Publication No. CN111621419B), the patent application entitled "Vascularized organoid chip based on stamp microstructure patterning and preparation method thereof" (Publication No. CN119432597A), and the patent application entitled "Continuous fiber support barrier for engineering vascular networks" (Publication No. CN119013055A). However, these models still lack the natural barrier coaxial tubular three-dimensional configuration, and the cells also lack the support of the three-dimensional matrix microenvironment.There are also patent applications that propose to construct three-dimensional barrier models by embedding cells in hydrogels, such as the patent application entitled "A Method for Constructing an In Vitro Placental Barrier Model Based on a Hydrogel System" (Publication No. CN118147048A) which uses methacrylated gelatin and ordinary gelatin as a three-dimensional hydrogel scaffold and introduces cells to construct an in vitro placental barrier model; the patent application entitled "BLOOD BRAIN BARRIER MODEL AND METHODS OF MAKING AND USING THE SAME" (Publication No. US11230702B2) proposes a blood-brain barrier model formed by a coaxial multi-layer ring surrounded by multiple cells, which has approached the natural barrier characteristics in physiological structure, but the above models are "static" models, i.e., the matrix properties, cell types and arrangement methods are fixed when the model is constructed, and multiple cells are synchronized to grow and develop during culture, so the "dynamic" characteristics of the natural barrier in which multiple cells develop and grow in a certain order cannot be reproduced, and the spatiotemporal activation and differentiation process which is crucial in the morphogenesis of tissue morphology cannot be simulated.

[0004] Taking the blood-brain barrier as an example, the maturation of the blood-brain barrier in vivo is a process strictly regulated by time. Early on, pericytes are recruited to support endothelial cell stability, followed by the gradual involvement of astrocytes, which complete the establishment of barrier function and neural vascular coupling. If this sequence is disrupted, the maturation and stability of the barrier will be destroyed. In addition, the microenvironment of the blood-brain barrier also undergoes extracellular matrix remodeling and mechanical diversification - the overall brain tissue maintains low stiffness (~1 kPa), while the vascular area bears dynamic stress from blood flow and blood pressure. This mechanical heterogeneity can regulate cell fate through mechanical transduction, but existing in vitro systems generally lack the ability to integrate such temporal dynamic cues.

[0005] In recent years, researchers have also used the self-organizing ability of stem cells to construct barrier organoids, such as the patent application entitled "A Method for Constructing a Vascularized Placental Organoid Model Based on an Organ Chip" (Publication No. CN117305223A), the patent application entitled "Choroid Plexus Organoid and Methods of Producing the Same" (Publication No. CN113631701B), and the patent application entitled "Human Nasal Organoid and Methods of Making and Using the Same" (Publication No. CN119790137A). Since the models constructed by this method are based on the differentiation and development of stem cells, they can preserve the temporal cues of multicellular growth, but the size and structure of the models generated by this method are poorly controllable, often only forming limited-diameter spherical structures, which do not conform to the hollow coaxial multi-layer tubular structure of natural barrier tissues.

[0006] In summary, the prior art lacks a barrier tissue model that can simultaneously achieve: (1) multi-cell three-dimensional coaxial layered co-culture structure; (2) multi-cell ordered development growth pattern; (3) substrate mechanical property heterogenization construction and dynamic regulation. The prior art cannot truly simulate the spatial structure and temporal development of in vivo barrier characteristics, and meet the demand for high-simulation in vitro barrier model in the fields of drug screening, disease modeling, etc. SUMMARY

[0007] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a multi-cell barrier tissue model with a bionic temporal development mechanism, which can simulate the multi-cell three-dimensional structure of in vivo barrier tissue, and realize the regulation of cell development behavior by regulating the substrate environment, and at the same time, through the structure design of soft / hard embedding, the model has sufficient overall mechanical support to adapt to perfusion culture and operation requirements; the model can realize the functionalization of natural barrier tissue in spatial structure dimension and time development dimension at the same time, so as to realize the application in disease modeling, drug screening, etc.

[0008] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0009] A multi-cell barrier tissue model 1 with a bionic temporal development mechanism has a multi-layer multi-cell structure of a bionic physical barrier, and related cells of different barriers are distributed in different layers according to physiological structure, and the development time and order of cells in different layers are regulated according to the physiological characteristics of natural barrier multi-cell development.

[0010] The multi-cell barrier tissue model 1 is a hollow coaxial multi-layer tubular structure or a multi-layer superposition structure, and has four cell layers from inside to outside, which are a cavity endothelium layer 2, a tube wall regulation layer 3, a wall external interaction layer 4 and a tissue parenchyma layer 5. The cavity endothelium layer 2 is composed of a single layer of endothelial cells 2-1 directly contacting with blood flow or lumen content, and the cell selection types include continuous endothelial cells, windowed endothelial cells and discontinuous endothelial cells. The tube wall regulation layer 3 is composed of a three-dimensional hydrogel layer containing wall cells 3-1, and the cell selection types include pericytes and smooth muscle cells. The wall external interaction layer 4 is composed of a three-dimensional hydrogel layer containing tissue-specific supporting cells 4-1, and the cell selection types include astrocytes, Muller glial cells, type I / II alveolar epithelial cells and podocytes. The tissue parenchyma layer 5 is composed of a three-dimensional hydrogel layer containing tissue-specific functional cells 5-1, and the cell selection types include neurons, oligodendrocytes, photoreceptor cells, retinal ganglion cells, alveolar macrophages and renal tubular epithelial cells. The above-mentioned cells are added with corresponding cell types in different cell layers according to different barrier model types to be constructed, and primary cells, cell lines or stem cell induced cells are selected. In addition, for disease models, corresponding diseased cells or tumor cells are selected and added in the corresponding layer according to the disease characteristics.

[0011] The constituting material of the pipe wall regulation layer 3, the wall outer interaction layer 4 and the tissue substance layer 5 is a soft phase hydrogel 6, which is a hydrogel material with time-sequential regulation of mechanical properties; the soft phase hydrogel 6 is a double network structure composed of a cleavable support network material 6-1 and a cell-friendly protein network material 6-2; the soft phase hydrogel 6 can be converted from a dense, high modulus and strong binding state before cleavage to a loose, low modulus and weak binding state after cleavage by cleavage stimulation 6-3, so that the cells in it are converted from a resting spherical state to an active spreading state.

[0012] The cleavable support network material 6-1 is one or more combinations of sodium alginate, methacrylated gelatin, methacrylated hyaluronic acid, disulfide cross-linked polyethylene glycol, and ortho-nitrobenzyl modified polyethylene glycol; the cell-friendly protein network material 6-2 is one or more combinations of collagen, fibrinogen, Matrigel, methacrylated gelatin, RGD modified sodium alginate and other cell adhesion domain containing materials; the cleavage mechanism of the cleavable support network material 6-1 selects the corresponding cleavage conditions, including enzymes, ph, reducing agents, light, etc.

[0013] The soft phase hydrogel 6 of the pipe wall regulation layer 3, the wall outer interaction layer 4 and the tissue substance layer 5 selects the same type of material or different types of material; if the same type of material is selected, the concentration and ratio of the cleavable support network material 6-1 and the cell-friendly protein network material 6-2 in the soft phase hydrogel 6 of different layers are different; if different types of materials are selected, the type of the cleavable support network material 6-1 in the soft phase hydrogel 6 of different layers is different, and the corresponding cleavage mechanism is also different.

[0014] When the strength of the soft phase hydrogel 6 of a certain layer of the pipe wall regulation layer 3, the wall outer interaction layer 4 and the tissue substance layer 5 is too low after cleavage, a hard phase scaffold 7 is selectively embedded in the layer to form a soft / hard hybrid structure; the hard phase scaffold 7 is composed of high modulus, non-degradable polymers or hydrogel materials, and the material of the hard phase scaffold 7 is one or more combinations of PLA, PCL, ABS, PLGA, sodium alginate, methacrylated gelatin, methacrylated hyaluronic acid, chitosan, nanocellulose, nanoclay, polyethylene glycol diacrylate and other materials, and the concentration selection and material ratio meet the following conditions: the Young's modulus is not less than 10 kPa, the degradation rate is less than 20% within 14 days, and the Young's modulus drop rate is not more than 10%; the hard phase scaffold 7 adopts a spiral scaffold structure 7-1 or a grid scaffold structure 7-2.

[0015] The matrix mechanical properties and cell activation development of different cell layers of the multi-cell barrier tissue model 1 can be time-sequentially regulated; wherein the cell activation development mode of the lumen endothelial layer 2 is to apply dynamic perfusion 2-2 inside the lumen to make the endothelial cells of the lumen form a tightly connected cell layer; the cell activation development mode of the pipe wall regulation layer 3, the wall outer interaction layer 4 and the tissue parenchyma layer 5 is to adjust the cleavable support network material 6-1 in the soft phase hydrogel 6 of the layer by external cleavage stimulation, reduce the mechanical properties and spatial constraints of the soft phase hydrogel, and trigger the state activation and functional remodeling of the cells in the soft phase hydrogel; the activation time and activation sequence of the cells in different layers are time-sequentially controlled in imitation of the development process of natural barrier tissues.

[0016] The cell time-sequentially activation method in the soft phase hydrogel 6 of the pipe wall regulation layer 3, the wall outer interaction layer 4 and the tissue parenchyma layer 5 is as follows:

[0017] (i) The response speed of a certain selected cell to the cleavage intensity of a certain soft phase hydrogel 6 is determined, which is evaluated by parameters such as cell spreading, migration and protein expression; the cleavage intensity includes the size of the cleavage condition parameter and the cleavage time;

[0018] (ii) The soft phase hydrogel 6 of each layer is selected according to the response speed and the required multi-cell time-sequential development target; if the soft phase hydrogel 6 of the same material type is selected, the material concentration and ratio need to be selected according to the response speed difference of different cells embedded in each layer to different material concentrations, ratios and cleavage intensities under the same cleavage mechanism; if the soft phase hydrogel 6 of different material types is selected, the material type needs to be selected according to the response speed of different cells embedded in each layer under different cleavage mechanisms and their cleavage intensities;

[0019] (iii) The cleavage stimulation is applied to the model at a specific time point according to the required multi-cell development timeline with a clear cleavage mechanism and cleavage intensity; if the soft phase hydrogel 6 of the same material type is selected, the same cleavage mechanism is applied in several times with different cleavage intensities to realize time-sequential development based on the response speed difference of different cells to different material concentrations, ratios and cleavage intensities; if the soft phase hydrogel 6 of different material types is selected, different cleavage mechanisms and cleavage intensities are applied in several times to realize time-sequential development based on the response difference of different cells to different cleavage mechanisms corresponding to different material types.

[0020] The barrier tissue model 1 is used for imitating the construction of blood-brain barrier, blood-cerebrospinal fluid barrier, blood-retinal barrier, blood-cerebrospinal fluid barrier, placental barrier, small intestinal epithelial barrier or other barrier tissues with selective permeability.

[0021] The multi-cell barrier tissue model 1 has two independent and unconnected spaces inside and outside the barrier, i.e., inside the cavity endothelial layer and outside the tissue parenchyma layer, different culture environments such as culture solution and gas are applied inside and outside the barrier, dynamic perfusion is carried out inside the barrier, and metabolic products inside and outside the barrier are extracted for analysis and detection.

[0022] The multi-cell barrier tissue model 1 performs real-time transendothelial resistance monitoring, optical observation, fluorescence particle permeability monitoring and other functional evaluations, and realizes drug permeability, barrier integrity, physiological response, multi-cell interaction and other applications.

[0023] The multi-layer multi-cell hydrogel structure of the multi-cell barrier tissue model 1 is manufactured by multi-material 3D printing or multi-layer pouring; if a layer needs to contain a hard phase support 7, a step-by-step pouring forming method is used to manufacture the hard phase support 7 first, or an integrated 3D printing method is used to manufacture.

[0024] Compared with the prior art, the beneficial effects of the present application are: the present application integrates dynamic timing mechanical regulation, multi-cell layered co-culture and soft / hard composite scaffold, provides a more realistic and more functional in vitro barrier tissue model than the prior art, and has the following advantages:

[0025] (1) The accuracy of the development function simulation of the tissue multi-cell is improved by dynamic timing regulation of the matrix. By adjusting the mechanical properties of the water-based matrix at different times of culture, the change of the mechanical microenvironment of the tissue in the development, damage repair or disease process can be simulated. For example, in the construction of the blood-brain barrier, a higher stiffness can be provided in the initial stage to promote the formation of tight junctions of endothelial cells to form a barrier, and then the stiffness is reduced in the mature stage to simulate the stable soft environment, which in turn promotes the role of pericytes and glial cells in consolidating the blood-brain barrier; or the matrix microenvironment is regulated again after the barrier matures to simulate the change of the microenvironment caused by fibrosis or inflammation, and then the change of the barrier function is evaluated, realizing the fine control of dynamic mechanical stimulation, providing a more biologically in vitro tissue model, and providing a powerful tool for in-depth study of the influence of mechanical factors on the formation and regulation of barrier tissues.

[0026] (2) Multicellular co-culture recreates the complex cell interactions of tissues in vivo. Arranging various cell types in three dimensions according to their anatomical positions in vivo preserves intercellular interactions (such as endothelial-pericillin signaling and epithelial-mesenchymal cell interactions) in vitro. This layered structure significantly improves barrier function indicators; for example, higher TEER values ​​and lower permeability were measured in the BBB model containing pericytes and glial cells. In the alveolar model, bilateral co-culture of epithelium and endothelium more realistically reflects changes in permeability caused by inflammatory factors and the cell damage repair process. Multicellular co-culture endows model tissues with specific physiological and pathological responses, making them more predictive in drug screening and mechanism studies.

[0027] (3) High versatility and customizability, applicable to research in multiple fields. The material composition and structural design of this invention are flexible and can be adjusted according to the characteristics of different barrier tissues. Therefore, the same platform can support the construction of various barrier tissue models such as the blood-brain barrier, blood-cerebrospinal fluid barrier, blood-retinal barrier, blood-cerebrospinal fluid barrier, placental barrier, and small intestinal epithelial barrier, with the advantage of "one platform for multiple uses", reducing R&D costs and improving the reproducibility and standardization of experiments. Models constructed using human cells (including primary cells and stem cell-differentiated cells) are expected to more accurately predict human responses and have higher relevance for drug screening. Similarly, the introduction of patient-specific cells to construct models can be used for personalized medicine research; the addition of tumor or immune cells can explore complex processes such as cancer cell metastasis and immune cell crossing of barriers. This invention has broad application prospects in many fields such as basic research, drug development, and toxicology testing. In summary, this invention maintains good operability and multifunctionality while enhancing the physiological relevance of the model, providing a systematic solution to the technical challenge of in vitro reconstruction of tissue barriers, and has important innovative significance and practical value. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the hierarchical structure and temporal development of the multicellular barrier tissue model and its temporal construction according to the present invention.

[0029] Figure 2 This is a schematic diagram illustrating the changes in network and cell states mediated by the material composition and lysis mechanism of the dual-network soft phase hydrogel of this invention.

[0030] Figure 3 This is a schematic diagram of the rigid phase support of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with embodiments. The described embodiments are one embodiment of the present invention, but not all embodiments.

[0032] Embodiment: a multi-cell barrier tissue model of a biomimetic time-sequential development mechanism, a blood-brain barrier tissue model for glioma drug screening;

[0033] (1) Model design:

[0034] This embodiment realizes the screening of brain glioma candidate drugs by constructing a multi-level, time-sequential development in-vitro blood-brain barrier (BBB) model, referring to Figure 1 , the multi-cell barrier tissue model 1 is a hollow coaxial multi-layer tubular structure or a multi-layer superimposed structure, and has four cell layers from inside to outside, which are a cavity endothelium layer 2, a tube wall regulation layer 3, an outer wall interaction layer 4 and a tissue parenchyma layer 5, wherein the cavity endothelium layer 2 is composed of a single layer of endothelial cells 2-1 directly contacting with blood flow or lumen content, the cells are selected from brain microvascular endothelial cells, and are covered on the cavity surface of the cavity endothelium layer 2; the tube wall regulation layer 3 is composed of a three-dimensional hydrogel layer containing wall cells 3-1, and the cells are selected from pericytes; the outer wall interaction layer 4 is composed of a three-dimensional hydrogel layer containing tissue-specific supporting cells 4-1, and the cells are selected from astrocytes; the tissue parenchyma layer 5 is composed of a three-dimensional hydrogel layer containing tissue-specific functional cells 5-1, and the cells are selected from glioma cells; the above structure is the basic composition of a physiological blood-brain barrier, and a glioma cell layer is additionally embedded on the outside, which is used for simulating a glioma invasion environment.

[0035] Referring to Figure 1 , Figure 2 , the constituting materials of the tube wall regulation layer 3, the outer wall interaction layer 4 and the tissue parenchyma layer 5 are soft phase hydrogels 6, and the soft phase hydrogel 6 is a hydrogel material with time-sequential controllable mechanical properties; the soft phase hydrogel 6 is a double network structure composed of a cleavable support network material 6-1 and a cell-friendly protein network material 6-2; the soft phase hydrogel 6 can make the double network structure thereof change from a dense, high modulus and strong binding state before cleavage to a loose, low modulus and weak binding state after cleavage through cleavage stimulation 6-3, so that the cells in the soft phase hydrogel 6 change from a resting spherical state to an active spreading state.

[0036] The cleavable support network material 6-1 is sodium alginate, and the cell-friendly protein network material 6-2 is collagen; the sodium alginate network can be cleaved on demand by a sodium alginate cleavage enzyme to reduce the rigidity of the matrix; the tube wall regulation layer 3, the outer wall interaction layer 4 and the tissue parenchyma layer 5 adopt sodium alginate / collagen hydrogels with different concentration ratios.

[0037] Referring to Figure 1 , Figure 3 , the outer wall interaction layer 4 is embedded with a hard phase scaffold 7, and the hard phase scaffold 7 adopts a spiral scaffold structure 7-1 or a grid scaffold structure 7-2, and the hard phase scaffold 7 of the utility model adopts the spiral scaffold structure 7-1.

[0038] Through experiments, the speed rule of cell activation and growth of pericytes, astrocytes and glioma cells under a certain lysis intensity of different soft phase hydrogel 6 concentration ratio materials is determined, and the material ratio of each layer and the lysis intensity are determined.

[0039] (2) Preparation process:

[0040] Preparation of hard phase scaffold 7: take photosensitive cross-linked gelatin (GelMA) material, and prepare a spiral scaffold structure 7-1 by three-dimensional light curing printing; during printing, adjust the light curing parameters to make the scaffold have good structural stability and biocompatibility, and provide mechanical support for the subsequent soft phase hydrogel 6.

[0041] Construction of hollow coaxial multilayer hydrogel structure: a thin-walled tubular structure containing pericytes, i.e. the tube wall regulation layer 3, is constructed by using bioprinting technology; then, the wall outer interaction layer 4 containing astrocytes with embedded hard phase scaffold 7 is prepared outside the tube wall regulation layer 3 by using the casting method, and the tissue parenchyma layer 5 containing glioma cells is prepared outside the wall outer interaction layer 4 by using the casting method;

[0042] Endothelial layer inoculation and functionalization: after the construction of the above multilayer structure is completed, the brain microvascular endothelial cell suspension is injected into the central lumen of the model, and the endothelial cells are attached to the inner wall of the lumen and form a tightly connected functional endothelial cell layer 2 by continuous dynamic perfusion 2-2 culture;

[0043] Multilayer hydrogel multi-cell time sequence activation: to simulate the time sequence development process of cells, the concentration and action time of sodium alginate lyase are adjusted in this embodiment to perform layered lysis on each soft phase hydrogel 6; specifically, a lower concentration of enzyme can be used to lyse the low-concentration sodium alginate network in the tube wall regulation layer 3 to preferentially activate pericytes; then, the enzyme concentration and reaction time are increased to lyse the medium-concentration sodium alginate network in the wall outer interaction layer 4 to activate astrocytes; finally, the remaining high-concentration sodium alginate network in the tissue parenchyma layer 5 is completely lysed to activate glioma cells; through the stage-by-stage enzyme lysis treatment and the differential material ratio of each layer of soft phase hydrogel, the time sequence functionalization of pericytes, astrocytes and glioma cells in the model is realized.

[0044] (3) Functionalized model and drug screening evaluation:

[0045] After the above construction and functionalization, the multi-cell barrier tissue model can be used for glioma drug screening, and the barrier integrity can be evaluated in real time through indicators such as transendothelial electrical resistance (TEER) and fluorescent glucose permeation rate; during screening, the drug to be tested is added to the liquid flow in the lumen of the model, and the drug passes through the barrier tissue through continuous perfusion; by monitoring indicators such as glioma cell activity (such as cell survival rate or proliferation), morphological changes, TEER value changes, and the rate and degree of drug penetration from the lumen to the outside, the penetration efficiency and therapeutic effect of the drug can be comprehensively evaluated, and the above indicators help to determine the killing effect of the drug on glioma cells and the influence on the blood-brain barrier, providing a quantitative basis for drug efficacy screening.

Claims

1. A multicellular barrier tissue model with a biomimetic temporal development mechanism (1), characterized in that: It has a multi-layered, multi-cellular structure that mimics physiological barriers. The cells related to different barriers are distributed in different layers according to the physiological structure. The development time and order of cells in different layers are regulated according to the physiological characteristics of multi-cellular development of natural barriers.

2. The multicellular barrier tissue model according to claim 1, characterized in that: The multicellular barrier tissue model (1) is a hollow coaxial multilayer tubular structure or a multilayer superimposed structure with four cell layers, from the inside to the outside: the luminal endothelial layer (2), the tube wall regulation layer (3), the extramural interaction layer (4), and the tissue parenchyma layer (5). The luminal endothelial layer (2) is composed of a single layer of endothelial cells (2-1) that are in direct contact with blood flow or lumen contents. The cell types selected include continuous endothelial cells, fenestrated endothelial cells, or discontinuous endothelial cells. The tube wall regulation layer (3) is composed of a three-dimensional hydrogel layer containing wall cells (3-1). The cell types selected include pericytes or smooth muscle cells. The extramural interaction layer (4) is composed of tissue-specific... The three-dimensional hydrogel layer consists of sex-supporting cells (4-1), and the cell types selected include astrocytes, Müller glial cells, type I / II alveolar epithelial cells, or podocytes; the tissue parenchyma layer (5) consists of a three-dimensional hydrogel layer containing tissue-specific functional cells (5-1), and the cell types selected include neurons, oligodendrocytes, photoreceptor cells, retinal ganglion cells, alveolar macrophages, or renal tubular epithelial cells; the above cells are added to different cell layers according to the different barrier models to be constructed, and primary cells, cell lines, or stem cell-induced cells are selected; or corresponding diseased cells or tumor cells are added to the corresponding layers according to the disease characteristics.

3. The multicellular barrier tissue model according to claim 2, characterized in that: The wall regulation layer (3), the external interaction layer (4), and the tissue matrix layer (5) are composed of soft phase hydrogel (6). Soft phase hydrogel (6) is a hydrogel material whose mechanical properties can be controlled in time. Soft phase hydrogel (6) is a double network structure composed of a cleavable support network material (6-1) and a cell-friendly protein network material (6-2). Soft phase hydrogel (6) can change its double network structure from a dense, high-modulus, and strongly bound state before cleavage to a loose, low-modulus, and weakly bound state after cleavage through cleavage stimulation (6-3), thereby changing the cells in it from a resting spherical state to an active spreading state.

4. The multicellular barrier tissue model according to claim 3, characterized in that: The cleavable support network material (6-1) is one or more combinations of sodium alginate, methacrylated gelatin, methacrylated hyaluronic acid, disulfide-crosslinked polyethylene glycol, and o-nitrobenzyl-modified polyethylene glycol; the cell-friendly protein network material (6-2) is one or more combinations of collagen, fibrinogen, matrix gelatin, methacrylated gelatin, and RGD-modified sodium alginate containing cell adhesion domains; the cleavage mechanism of the cleavable support network material (6-1) is selected from appropriate cleavage conditions, including enzymes, pH, reducing agents, or light.

5. The multicellular barrier tissue model according to claim 3, characterized in that: The soft phase hydrogels (6) of the tube wall regulation layer (3), the external wall interaction layer (4), and the tissue substance layer (5) are selected from the same type of material or different types of material; If the same type of material is selected, the concentration and ratio of the cleavable support network material (6-1) and the cell-friendly protein network material (6-2) in the soft phase hydrogel (6) of different layers will be different; If different types of materials are selected, the types of pyrolytic support network materials (6-1) in the soft phase hydrogels (6) of different layers will be different, and their corresponding pyrolysis mechanisms will also be different.

6. The multicellular barrier tissue model according to claim 3, characterized in that: When the strength of a certain soft phase hydrogel (6) in the tube wall regulation layer (3), the external wall interaction layer (4), and the tissue substance layer (5) is too low after lysis, a hard phase scaffold (7) is embedded in the layer to form a soft / hard intercalation structure. The hard phase scaffold (7) is composed of high modulus, non-degradable polymer or hydrogel material. The material of the hard phase scaffold (7) is one or more of PLA, PCL, ABS, PLGA, sodium alginate, methacrylated gelatin, methacrylated hyaluronic acid, chitosan, nanocellulose, nanoclay, and polyethylene glycol diacrylate. The concentration selection and material ratio meet the following conditions: Young's modulus is not less than 10 kPa, the degradation rate is less than 20% within 14 days, and the Young's modulus decrease rate is not more than 10%. The hard phase scaffold (7) adopts a spiral scaffold structure (7-1) or a grid scaffold structure (7-2).

7. The multicellular barrier tissue model according to claim 3, characterized in that: The matrix mechanical properties and cell activation and development of different cell layers in the multicellular barrier tissue model (1) can be regulated in a time-sequential manner. Among them, the cell activation and development of the luminal endothelial layer (2) is achieved by applying dynamic perfusion (2-2) inside the lumen to form a tightly connected cell layer of endothelial cells on the luminal surface. The cell activation and development of the tube wall regulation layer (3), the wall-external interaction layer (4), and the tissue parenchyma layer (5) are achieved by regulating the pyrolytic support network material (6-1) in the soft phase hydrogel (6) of the layer through external lysis stimulation, thereby reducing the mechanical properties and spatial constraints of the soft phase hydrogel and triggering the activation and functional remodeling of cells in the soft phase hydrogel. The activation time and activation sequence of cells in different layers are controlled in a time-sequential manner, mimicking the development process of natural barrier tissues.

8. The multicellular barrier tissue model according to claim 7, characterized in that: The cell-time activation method in the soft phase hydrogel (6) of the tube wall regulation layer (3), the extracellular interaction layer (4), and the tissue parenchyma layer (5) is as follows: (i) Determine the response rate of a selected cell to the lysis intensity of a soft phase hydrogel (6), and evaluate it through parameters of cell spreading, migration, and protein expression; the lysis intensity includes the magnitude of lysis condition parameters and lysis time; (ii) Select the soft phase hydrogel (6) for each layer according to the response rate and the required multicellular time-series development goal; if the same type of soft phase hydrogel (6) is selected, the material concentration and ratio need to be selected according to the difference in response rate of different cells embedded in each layer to different material concentrations, ratios and lysis intensities under the same lysis mechanism. If different types of soft phase hydrogels are selected (6), the material type needs to be selected according to the response rate of different cells embedded in each layer under different lysis mechanisms and lysis intensities. (iii) Apply lysis stimulation to the model at specific time points with a clear lysis mechanism and lysis intensity according to the required multicellular development timeline; if the same type of soft phase hydrogel (6) is selected, apply the same lysis mechanism stimulation in stages with different lysis intensities, and realize time-series development based on the difference in response speed of different cells to different material concentrations, ratios and lysis intensities; if different types of soft phase hydrogel (6) are selected, apply different lysis mechanisms and lysis intensities stimulation in stages, and realize time-series development based on the difference in response of different cells to different lysis mechanisms corresponding to different material types.

9. The multicellular barrier tissue model according to claim 2, characterized in that: The multicellular barrier tissue model (1) has two independent, non-connected spaces: the inner side of the luminal endothelial layer and the outer side of the tissue parenchyma layer. Different culture media and gas environments are applied inside and outside the barrier. Dynamic perfusion is performed inside the barrier, and metabolites inside and outside the barrier are extracted for analysis and detection. The multicellular barrier tissue model (1) performs functional evaluations such as real-time transendothelial resistance monitoring, optical observation, and fluorescent particle permeability monitoring, realizing the application of drug permeability, barrier integrity, physiological response, and multicellular interaction.

10. The multicellular barrier tissue model according to any one of claims 1-9, characterized in that: The multi-layer multi-cell hydrogel structure of the multi-cell barrier tissue model (1) is manufactured by multi-material 3D printing or multi-layer casting. If a certain layer needs to contain a rigid phase scaffold (7), the rigid phase scaffold (7) is manufactured first and then manufactured by step casting, or by an integrated 3D printing method.

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