Multi-layer organization for in-situ tumor modeling, and preparation method therefor and use thereof

Abstract

Provided are a multi-layer organization for in-situ tumor modeling, and a preparation method therefor and the use thereof. The multi-layer organization comprises an adhesive layer, a sealing and protecting layer, and tumor cells and / or tumor mass encapsulated between the adhesive layer and the sealing and protecting layer; the edges of the adhesive layer and the sealing and protecting layer are attached to prevent the tumor cells from permeating out from a side edge or the sealing and protecting layer; and the adhesive layer is used for attaching to target organ tissue by means of the adhesive property of the film itself or by means of a medical adhesive. By means of the multi-layer organization, the construction process of an in-situ / metastatic tumor model on multiple organs / tissues of an animal can become a standardized process, the obstacle in modeling thin-walled organ tissues of small animals is broken through, and the present invention involves a simple and efficient process, has a low cost, and has high repeatability and a high modeling rate.

Description

A multilayer structure for in situ tumor modeling and its preparation method and application

[0001] Cross-references

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office, with application number 202311155887.7, application date September 8, 2023, and application name “A multilayer structure for in situ tumor modeling, its preparation method and application”. The entire contents of the application are incorporated by reference into this application. Technical Field

[0003] The present application relates to the field of biomaterial technology, and in particular to a multilayer structure for in situ tumor modeling, a preparation method thereof, and an application thereof. Background Art

[0004] Transplanting tumor cells (or tissues) into animals and using them as models to study tumorigenesis and treatment is a common approach in current cancer research. Most current approaches to cancer treatment require testing in animal tumor models. The simplest tumor model is the subcutaneous tumor model, in which cells (cell-derived xenograft, CDX) or tissue (tissue-derived xenograft, TDX) derived from human tumors (xenograft models) or mouse tumors (syngeneic transplant models) are inoculated subcutaneously into mice, allowing for visual monitoring of tumor growth. However, subcutaneous tumor models fail to reflect the complexity of the tumor and its native tissue environment, presenting significant deficiencies. Orthotopic tumor models are a further development of subcutaneous tumor models. These involve orthotopic transplantation of tumor cells or tissue fragments into the corresponding tissues and organs of the modeling animal, allowing tumors to develop and spontaneously metastasize. Orthotopic transplantation models better simulate the in vivo microenvironment in which tumor cells grow, simulating the processes of tumor growth and even metastasis. In recent years, patient-derived xenograft models have garnered significant interest. These models are established by directly transplanting fresh patient tumor tissue into mice. Compared to CDX and TDX models, PDX models utilize specimens directly derived from human tumor tissue, stably preserving the genetic, histological, and phenotypic characteristics of the tumor—i.e., tumor heterogeneity. The tumor microenvironment more closely resembles the human body, and drug screening results offer better clinical predictability. However, these models suffer from limitations such as high tumor tissue viability requirements, short sample preparation times, and limited tumorigenicity. Currently, PDX models primarily utilize subcutaneous implantation, with a limited use of in situ seeding. It is clear that in situ seeding offers greater clinical significance than subcutaneous seeding.

[0005] Currently, the construction of orthotopic tumor models faces numerous obstacles. The key is modeling efficiency, which involves ensuring tumor cell and tissue viability, as well as various experimental conditions. In particular, most current in situ modeling methods involve direct implantation of cell suspensions, tumor tissue homogenates, or tumor masses into the corresponding organs or tissues via puncture. This places very high demands on the initial sample preparation and implantation process, and several technologies address this, such as invention patents CN202111119755 and CN202111123981. However, direct inoculation inevitably leads to leakage of cell fluid or tissue fragments, resulting in unnecessary passive transfer, a problem that is difficult to avoid with direct inoculation. To improve success rates and reduce leakage, some researchers have employed methods to improve sample stability and survival by immobilizing cells or tissues within biomaterials. Currently, these methods primarily utilize 3D printing prefabrication and biomaterial scaffold culturing, such as invention patents CN202010094635 and CN202111661435. These methods are complex and require high-quality equipment, making them particularly challenging to effectively apply in the construction of PDX in situ models. In addition, it also brings another problem, that is, the difficulty of fixing materials containing cells or tissues on the target tissue. Most biological materials do not have sufficient stickiness to the wet tissue surface or need to be fixed by other adhesives, otherwise they will fall off, which will still lead to modeling failure or unnecessary transfer. Another problem is that the various methods currently involved, especially the puncture method, will cause serious secondary damage to animal organs or tissues. Although there are many inventions that try to reduce damage, for example, CN202120096586 and CN202022527066, secondary damage is still unavoidable in in situ modeling operations; in addition, puncture modeling is almost impossible to complete effectively on thin-walled organs such as the gastrointestinal tract and bladder, as well as small organs, and it is very easy to cause perforation and other problems that endanger animal life.

[0006] Therefore, appropriate materials and reliable configurations are needed to overcome various challenges in establishing orthotopic tumor models, minimizing the impact of environmental and environmental factors. Numerous patents exist for two-dimensional materials used in biomedicine, such as CN201310241402 and CN202210705366. However, there are no patents publicly available for using multi-layered two-dimensional materials to maintain tumor cells and tissues for the construction of animal tumor models.

[0007] Summary of the Invention

[0008] Purpose of the Invention

[0009] To overcome the above-mentioned deficiencies, the purpose of the present application is to provide a multilayer structure for in situ tumor modeling, and a preparation method and application thereof.

[0010] The multi-layer structure of the present application can make the construction process of animal in situ / metastatic tumor models a standardized process, breaking through the obstacles of modeling thin-walled organ tissues of small animals. The process is simple and efficient, low-cost, highly repeatable, and has a high modeling rate, and has good application prospects.

[0011] Solution

[0012] To achieve the purpose of this application, the technical solutions adopted in this application are as follows:

[0013] In the first aspect, the present application provides a multi-layer structure for in situ tumor modeling, in which the tumor cells and tumor masses are encapsulated between an adhesion layer and a protective layer, the edges are completely bonded, and the surface of the protective layer is cross-linked to prevent tumor cells from leaking out from the sides and the protective layer. The adhesion layer relies on the membrane's own adhesive properties or is bonded to the target organ tissue through a medical adhesive. During the application process, a protective layer can be optionally covered on one side of the treated surface of the protective layer.

[0014] Furthermore, the number of membrane layers ranges from 2 to 6.

[0015] Furthermore, the thickness of the protective layer film is 5nm~2mm, optionally 50nm~2mm, optionally 50nm~1mm, optionally 40nm~1.5μm, optionally 90nm~2μm, optionally 90nm~200nm, optionally 500nm~20μm, optionally 500nm~1mm, optionally 10μm~500μm, optionally 100μm~1mm.

[0016] Generally speaking, the thickness of the protective layer depends on the material of the membrane. For example, the thickness of the protein membrane can be thinner, which can be at the nm level. When targeting large animals, the thickness of the protein membrane can also be increased by controlling the protein concentration. Due to the limitations of the electrospinning process, the thickness of the electrospun membrane is thicker, which can be 500nm~1mm.

[0017] Furthermore, the thickness of the adhesion layer is 40 nm to 10 μm, optionally 90 nm to 1 mm, optionally 40 nm to 200 nm, optionally 1 μm to 10 μm.

[0018] Furthermore, the protective layer film or adhesive layer film is cut to a suitable size according to different animal organ tissues and tumor cells or tissue samples, and the film area can be optionally 0.5mm 2 ~9cm 2 .

[0019] Furthermore, in the protective layer film or the adhesive layer film, the pore size of the adhesive layer film is not smaller than that of the protective layer film, and can be optionally 1 nm to 2 μm.

[0020] Furthermore, in the protective layer film or the adhesive layer film, the Young's modulus of the film is 1 GPa to 12 GPa, optionally 1 GPa to 2 GPa, optionally 3 GPa to 5 GPa, or optionally 6 GPa to 12 GPa.

[0021] Furthermore, the toughness of the protective layer film or the adhesive layer film is 0.1 kJ / m 2 ~8kJ / m 2 , optionally 0.1 kJ / m 2 ~3.4kJ / m 2 , optionally 4.1 kJ / m 2 ~6kJ / m 2 , optionally 6.1 kJ / m 2 ~8kJ / m 2 .

[0022] Furthermore, in the protective layer film or the adhesive layer film, the adhesion strength of the film is 0.1 kPa to 300 kPa, optionally 0.1 kPa to 10 kPa, optionally 11 kPa to 100 kPa, optionally 100 kPa to 300 kPa.

[0023] Furthermore, the edges of the protective layer film and the adhesive layer film are adhered by the film's own viscosity or adhesive, and then sealed with an oxidant (the oxidant can make the protective layer film denser to prevent tumor cells from penetrating).

[0024] Furthermore, the protective layer film and / or the adhesive layer film is a protein film, and optionally, the protein is lactoferrin, lysozyme, bovine serum albumin, insulin, α-lactalbumin, human serum albumin, fibrinogen, β-amyloid protein, soy protein, zein, wheat alcohol-soluble protein, horseradish peroxidase, hemoglobin, hemocyanin histone, gluten, collagenase, ovalbumin, soy protein, transferrin, thyroglobulin, trypsin, pepsin, chymotrypsin, One or more of mucins; optionally, the cross-linking agent is selected from any one or more of 4-hydroxyethylpiperazineethanesulfonic acid, dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride, cysteine, reduced glutathione, dimercaptosuccinic acid, sodium sulfite, glutaraldehyde, genipin, transglutaminase, and carbodiimide; optionally, the protein film also contains metal nanoparticles, optionally, the protein film contains nanosilver; optionally, the protein film itself has viscosity. Optionally, the protein film is a protein film formed by self-assembly of a protein aqueous solution and a cross-linking agent aqueous solution at the gas-liquid interface. The protein film can also be prepared by spin coating or electrospinning. Optionally, the thickness of the protective layer of the protein film is 5nm to 2mm, optionally the adhesion layer is 90nm to 1mm, and the thickness of the protective layer is 10nm to 100nm.

[0025] And / or, the sealing layer membrane and / or the adhesion layer membrane is an electrospun two-dimensional film. Optionally, the material of the electrospun two-dimensional film is selected from one or more of polyvinyl pyrrolidone, polyethylene oxide, polyester, polyamide, polyimide, polypropylene, polyethylene, collagen, silk fibroin, chitosan, and gelatin; optionally, the electrospun film is thicker. Optionally, the thickness of the sealing layer is 100μm to 1mm, and the thickness of the protective layer is 20nm to 100μm.

[0026] And / or, the protective layer film and / or the adhesive layer film is a vapor deposition film. Optionally, the material of the vapor deposition film is selected from one or more of carbon nanotube film, graphene, molybdenum disulfide, etc. Optionally, the protective layer of the vapor deposition film has a thickness of 90 nm to 200 nm. The thickness of the protective layer is 10 nm to 100 nm.

[0027] And / or, the protective layer and / or the adhesive layer are spin-coated films. Optionally, the spin-coated film is made of a polysaccharide, and may be made of one or more soluble polymers selected from chitosan, hydroxymethyl cellulose, sodium alginate, hyaluronic acid, and carrageenan. The protective layer of the spin-coated film has a thickness of 40 nm to 1.5 μm. The protective layer has a thickness of 200 nm to 1.5 μm.

[0028] And / or, a protective layer is further attached to the sealing layer film. Optionally, the protective layer is made of the same material as the sealing layer film.

[0029] Optionally, the thickness of the protective layer is 10 nm to 100 μm, optionally 20 nm to 100 μm, optionally 10 nm to 20 μm, optionally 200 nm to 1.5 μm, optionally 10 nm to 100 nm, optionally 500 nm to 20 μm.

[0030] Furthermore, it also includes a support for placing it in a culture solution or maintenance solution for further culture or maintenance;

[0031] Optionally, the support is one or more of hydrogel, plastic, and metal;

[0032] Optionally, the hydrogel is any one or more of small molecules, natural polymers and synthetic polymers;

[0033] Optionally, the small molecule includes one or any combination of fluorenyl dipeptide, octapeptide, and oligopeptide derivatives;

[0034] Optionally, the natural polymer includes polysaccharides, peptides, and nucleic acids; the polysaccharides include one or more of starch, cellulose, alginic acid, hyaluronic acid, chitosan, K2 carrageenan, locust bean gum, gelatin, and agarose; and the polypeptides include one or more of collagen, fibrin, poly-L-lysine, and poly-L-glutamic acid;

[0035] Optionally, the synthetic polymer includes one or more of polyacrylic acid and its derivatives, polyethers, and polyesters; optionally, the polyethers include one or any several of polypropylene oxide, polyethylene glycol, and polyethylene oxide; optionally, the polyacrylic acid and its derivatives include one or more of polyacrylamide, polyisopropylacrylamide, polyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone, and polyvinyl alcohol; optionally, the polyesters include one or more of polyhydroxyethyl methacrylate, polypropylene fumarate, polycaprolactone, and polydimethylaminoethyl methacrylate;

[0036] Optionally, the plastics include one or more of thermoplastic plastics, thermosetting plastics, general plastics, engineering plastics, functional plastics, crystalline plastics, and non-crystalline plastics;

[0037] Optionally, the plastic component includes one or more of polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, polyamide, polycarbonate, acrylonitrile-butadiene-styrene copolymer, polymethyl methacrylate, acrylonitrile-styrene copolymer, polyester, phenolic resin, epoxy resin, amino resin, alkyd resin, allyl resin, urea-formaldehyde resin, melamine resin, unsaturated polyester, silicone resin, and polyurethane; optionally, the polyester is selected from one or more of PETP polybutylene terephthalate and PBTP polyethylene terephthalate;

[0038] Optionally, the metal material includes one or more of stainless steel, gold, silver, and copper;

[0039] Optionally, the culture medium / maintenance medium includes BME medium, MEM medium, DMEM medium, IMDM medium, PRMI1640 medium, HAM F10 / F12 medium, 199 medium, CHO medium, Vero medium, NS0 medium, PFM medium, UW tissue preservation medium, HBSS tissue preservation medium, GALA tissue preservation medium, One or any combination of FRS tissue preservation solution, MACS tissue preservation solution, phosphate buffered saline, normal saline, and isotonic glucose solution;

[0040] Optionally, the culture medium contains serum, including one or any combination of calf serum, new bovine serum, fetal bovine serum, horse serum, and human serum;

[0041] Optionally, the culture / maintenance temperature range includes 2°C-37°C;

[0042] Alternatively, the culture environment may contain 0-5% CO2.

[0043] Furthermore, the multilayer structure is used for implantation into a target organ or tissue in a corresponding animal body, so that the target organ or tissue adheres firmly.

[0044] Optionally, the target organ or tissue is one or more of various tumors in organs or tissues such as the brain, heart, pericardium, blood vessels, pleura, peritoneum, liver, gallbladder, bile duct, pancreas, spleen, lung, kidney, bladder, prostate, urethra, oral cavity, gums, tongue, nasal cavity, throat, ear canal, thyroid, esophagus, stomach, intestine, anus, ovary, uterus, penis, testicles, cartilage, bone tissue, bone marrow, nerve tissue, eyeball, eyelid, breast, lymph node, muscle, tendon, skin, fat body, etc.;

[0045] Optionally, the animal includes one or any combination of mice, rats, guinea pigs, golden hamsters, rabbits, dogs, pigs, monkeys, and gorillas.

[0046] Furthermore, the multilayer structure adheres to the tissue through its own adhesion or an adhesive; optionally, the adhesive is selected from one or any combination of cyanoacrylates, hydrogels, melanins, melanin-like substances, celluloses, proteins, medical resins, sugars, and nucleic acids.

[0047] In a second aspect, a method for constructing an animal model of multi-organ tissue tumors in situ / metastasis is provided, comprising the following steps:

[0048] 1) preparing a protective layer membrane, optionally, the protective layer membrane is selected from one or more of a protein membrane, a fiber membrane, a synthetic macromolecular membrane, a metal membrane, and an inorganic substrate membrane;

[0049] 2) Implanting tumor cells or tumor tissue on the protective layer membrane;

[0050] 3) preparing an adhesive layer, covering the surface of the protective layer membrane containing tumor cells or tumor tissue, and gluing and sealing the edges to form a multilayer structure;

[0051] 4) The multilayer structure is surgically attached to the corresponding animal organ or tissue using its own adhesiveness or adhesive to prepare the corresponding animal model.

[0052] Furthermore, the multi-layer structure in step 4) adopts the multi-layer structure described in the first aspect.

[0053] Optionally, in step 3), the multilayered fabric is placed on a support for supporting or facilitating attachment to an organ or tissue, with the non-adhesive layer in contact with the support. Optionally, the fabric is placed in a culture medium or maintenance medium for culture or maintenance. Optionally, in step 4), the multilayered fabric is attached to the corresponding animal organ or tissue and then the support is removed. The purpose of the support is to facilitate the preparation of the multilayered fabric and subsequent attachment to the corresponding animal organ or tissue.

[0054] In a third aspect, a multi-organ tissue tumor in situ / metastatic tumor animal model prepared by the preparation method is provided.

[0055] In a fourth aspect, a method is provided for using the multi-layered structure described in the first aspect in preparing an animal model of multi-organ tissue tumors in situ / metastasis.

[0056] This application discloses a method for constructing an animal model of multi-organ tissue tumor in situ / metastasis. First, a membrane-shaped material with permeability, degradability, toughness, and viscosity that can be used in vivo is used to establish a multi-layer structure that can be used for encapsulation, culture, or maintenance of tumor cells and tissues. The tumor cells or tissues are encapsulated in the multi-layer structure, and the edges of the structure are sealed to prevent leakage. The internal cells or tissues can be further cultured with culture fluid, and maintenance fluid can also be used to maintain the internal cells or tissues to keep them active. Finally, the multi-layer structure is firmly attached to the target organ or tissue of the modeling animal by a wet adhesion method, allowing the cells and tissues to infiltrate into normal tissues, thereby realizing low-cost, large-scale, and high-efficiency construction of animal tumor in situ / metastasis models. This overcomes the problems existing in current modeling methods and realizes the comprehensive construction of a broad-spectrum tumor model. Beneficial effects

[0057] (1) In the present application, a multilayer structure is established that can culture or maintain tumor cells / tissues and achieve firm adhesion of animal organ tissues, thereby achieving large-scale construction of animal in situ / metastatic tumor models.

[0058] (2) The method established in this application first breaks through the technical barrier of modeling thin-walled organ tissues, making it possible to construct in situ models of areas such as the esophagus, stomach, intestines, and bladder on a large scale. Furthermore, the modeling is highly reproducible and the film formation rate is high.

[0059] (3) The method established in this application reduces the difficulty and cost of modeling micro-organ tissues, making tumor modeling in the prostate, ovary, lymph, ear, nose and throat area, eye and other areas more effective and with a higher tumor formation rate.

[0060] (4) The method established in this application reduces secondary damage during the modeling process of in situ / metastatic tumor models of other organs and tissues, increases the survival rate of animals, and further improves the modeling rate.

[0061] (5) The method established in this application also greatly reduces unnecessary passive metastasis and prevents the implanted cells or tissues from colonizing and growing outside the target tissue.

[0062] (6) The method established in this application can improve the modeling efficiency of clinical PDX models and effectively ensure the survival rate of human samples.

[0063] (7) The method established in this application can make the construction process of animal in situ / metastatic tumor models a standardized process. The process is simple and efficient, low cost, highly reproducible, and has a high modeling rate, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0065] FIG1 is a schematic diagram of the multi-layer structure assembly process of the present application.

[0066] FIG2 is a photo of the multilayer structure of Example 1 of the present application.

[0067] FIG3 is a SEM photograph of the multilayer structure of Example 1 of the present application.

[0068] FIG4 is a photograph of the multilayered structure in vitro tissue application according to Example 1 of the present application.

[0069] FIG5 is a photograph of the multilayer structure of the loaded cell in Example 1 of the present application.

[0070] FIG6 is a fluorescent photograph of live and dead cell staining after culture of the cell-loaded multilayered tissue in Example 1 of the present application.

[0071] FIG7 is a photograph of the results of applying the multi-layer structure to the main organs and tissues in an animal body according to an embodiment of the present application.

[0072] FIG8 is a photograph showing the results of constructing a mouse liver orthotopic tumor model using the cell-loaded multilayer structure of Example 1 of the present application.

[0073] FIG9 shows the pathological results of the cell-loaded multilayer structure of Example 2 of the present application used in an in situ tumor model of the mouse gastric wall.

[0074] FIG10 is a photograph showing the results of constructing a mouse uterine orthotopic tumor model using the multilayered structure of the loaded tissue according to Example 3 of the present application. DETAILED DESCRIPTION

[0075] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0076] In addition, in order to better illustrate the present application, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present application can be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0077] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0078] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0079] In this specification, references to "embodiment" and the like mean that the particular elements (e.g., features, properties, and / or characteristics) described in connection with that embodiment are included in at least one embodiment described herein and may or may not be present in other embodiments. Additionally, it should be understood that the elements may be combined in any suitable manner in the various embodiments.

[0080] This specification involves a large number of experiments and operating techniques that are commonly used in biological and medical research. It should be understood that the main purpose of protection is to protect the content and spirit of the method, rather than the general experimental method.

[0081] In the following examples, the detection method is:

[0082] The testing method of Young's modulus and toughness is as follows: the membrane material is cut into 1cm×4cm strips, and then a universal tensile testing machine is used to record the stress (σ)-strain (ε) curve.

[0083] Young's modulus (E) can be calculated using the following formula:

[0084] E=σ / ε

[0085] The value of ε does not exceed 1%.

[0086] The calculation formula of toughness (T) is as follows:

[0087] T=F / A×L

[0088] Where F is the fracture strength, A is the cross section at the fracture, and L is the physical length of the fracture.

[0089] Adhesion strength was tested using the pull-off method: a 20 mm diameter spindle was attached to the membrane using epoxy adhesive (DP420, 3M). Excess adhesive was removed and then cured at 60°C for 5 hours. The spindle was then pulled from the substrate, and the force required to separate the coating from the substrate was recorded.

[0090] Example 1

[0091] 1) Protein Film Preparation: 4-HEPES was used as a crosslinker, and lysozyme was used as the crosslinker. The lysozyme concentrations were 2, 4, and 8 mg / ml, respectively, and the amount of 4-HEPES was 15 mg / ml. The two were mixed in a 1:1 ratio by volume. After standing, adhesive two-dimensional protein films were prepared using a vapor phase interface method with thicknesses of 10 nm, 80 nm, and 150 nm, respectively.

[0092] Protein membranes with thicknesses of 10 nm, 80 nm, and 150 nm (the thickness of the protein membrane can be controlled by protein concentration or increased by regrowth) were cut separately. The membrane area was approximately 0.5 cm 2 The liquid phase interface is a non-adhesive surface, and the gas phase interface is an adhesive interface; the Young's modulus is between 1GPa-3GPa; the toughness range is 0.1-1.4kJ / m 2 Adhesion strength of the adhesive surface is 120-300kPa;

[0093] 2) Preparation of the Protective Layer: The 150 nm thick protein film prepared in step 1) was immobilized using 1% formaldehyde (oxidizing agent) to form a protective layer. The 10 nm thick protein film prepared in step 1) was then adhered to the non-adhesive layer of the solidified 150 nm thick protective layer using its own adhesive properties to form a double-layer membrane. This double-layer membrane was then placed on an agarose gel block, with the 10 nm thick protective layer directly contacting the agar, i.e., the 10 nm thick protective layer was on the bottom and the 150 nm thick protective layer on the top. A suspension of human hepatoma cell line BEL-7402 cells (from ATCC) was then added to the center of the 150 nm thick protective layer membrane surface. After deposition, the culture medium was aspirated.

[0094] 3) Preparation of Adhesion Layer: The 80 nm thick protein film from step 1) was overlaid on the surface of the 150 nm thick protective layer film from step 2) on which the BEL-7402 cells were deposited, utilizing the membrane's own adhesion. The edges were bonded using its own adhesion and treated with 0.5% peracetic acid (which could be applied dropwise and then smeared) to complete the edge sealing, resulting in a three-layer multilayer structure (see Figure 1).

[0095] 4) The agar gel block containing the BEL-7402 cell multilayer construct from step 3) above was placed in a 3.5 cm culture dish. DMEM high-glucose medium supplemented with 10% fetal bovine serum was added, with the medium soaking but not covering the multilayer construct. The culture was incubated at 37°C, 5% CO₂ for 18-24 h. After the cells were stably viable and adhered to the membrane, they were used for in vivo modeling.

[0096] 5) Six-week-old BALB / c-nu / nu nude mice (purchased from Beijing Huafukang Co., Ltd.) were anesthetized by isoflurane inhalation and fixed in a supine position on a sterilized small animal operating table while maintaining anesthetic gas. The abdomen of the mouse was extensively disinfected with iodine tincture and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum below the xiphoid process of the mouse sternum along the skin grain. The liver was carefully turned out and the entire liver lobe was separated. The surface moisture of the liver was absorbed with a sterile cotton swab.

[0097] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried liver surface from step 5), allowing the membrane to adhere to the liver surface through its own adhesive properties. The liver is carefully folded up and disinfected, followed by suture of the peritoneum with 6-0 absorbable sutures and the integument with 4-0 sutures. The wound is then disinfected, lidocaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0098] The multilayered construct constructed in this example is shown in Figures 2 and 3 . Its macroscopic and ultrastructural structures are visible, and it firmly adheres to tissues (results shown in Figure 4 ). Loaded cells can effectively grow within the construct and maintain high viability (test results shown in Figures 5 and 6 ). The results of a multi-tissue universal application test are shown in the liver photograph in Figure 7 . The cell-containing multilayered construct prepared in this example, applied to the surface of a mouse liver, formed orthotopic liver cancer tumors approximately one week after application, as shown in Figure 8 . Repeated attempts at the method described in this example consistently achieved an orthotopic liver modeling rate exceeding 95% in mice, with the rate of unnecessary passive metastasis kept below 10%.

[0099] Example 2

[0100] 1) Protein Film Preparation: β-mercaptoethanol was used as a crosslinker and BSA was used as a crosslinker. The BSA concentrations were 2, 4, and 10 mg / ml. The β-mercaptoethanol aqueous solution was mixed at a 1:1 volume ratio. Adhesive two-dimensional protein films with thicknesses of 20 nm, 60 nm, and 180 nm were prepared via a vapor-phase interface method.

[0101] Protein membranes with thicknesses of 20 nm, 60 nm, and 180 nm (the thickness of the protein membrane can be controlled by protein concentration or increased by regrowth) were cut separately. The membrane area was approximately 0.2 cm 2 The liquid phase interface is a non-adhesive surface, and the gas phase interface is an adhesive interface; the Young's modulus is between 1GPa-3GPa; the toughness range is 0.1-1.9kJ / m 2 ; The adhesion strength of the adhesive surface is 100-300kPa;

[0102] 2) Preparation of the Protective Layer: The 180 nm thick protein film from step 1) was immobilized with 2% glutaraldehyde (oxidizing agent) to form a protective layer. A 20 nm thick protein film from step 1) was then adhered to the non-adhesive layer of the solidified 180 nm protective layer film via its own adhesive properties to form a double-layer membrane. This double-layer membrane was then placed on an agarose gel block, with the 20 nm thick protective layer contacting the agar, i.e., the 20 nm thick protective layer was on the bottom and the 180 nm thick protective layer on the top. A suspension of human gastric cancer cell line BGC-823 cells (from ATCC) was then added to the center of the surface of the 180 nm protective layer membrane, and after deposition, the culture medium was aspirated.

[0103] 3) Preparation of Adhesion Layer: Using the membrane's own adhesion, the 60 nm thick protein film from step 1) was overlaid on the surface of the 180 nm protective layer membrane from step 2) deposited with BGC-823 cells. The edges were bonded using its own adhesion and treated with 3% hydrogen peroxide (which could be applied dropwise and smeared) to complete the edge sealing, resulting in a three-layer multilayer structure (see Figure 1).

[0104] 4) The agar gel block containing the cell multilayered construct from step 3) above was placed in a 3.5 cm culture dish. DMEM high-glucose medium containing 10% fetal bovine serum was added, and the medium was infiltrated into but not covered the multilayered construct. The culture was incubated at 37°C, 5% CO2 for 18-24 hours. After the cells were stably viable and adhered to the membrane, they were used for in vivo modeling.

[0105] 5) Six-week-old BALB / c-nu / nu nude mice (purchased from Beijing Huafukang Company) were anesthetized with isoflurane inhalation and fixed in a supine position on a sterilized small animal operating table while maintaining anesthetic gas. The abdomen of the mouse was extensively disinfected with iodine tincture and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum below the xiphoid process of the mouse sternum along the skin grain. The gastric pouch was carefully exposed, and the surface moisture of the gastric wall was absorbed with a sterile cotton swab.

[0106] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried stomach wall from step 5), allowing adhesion to the stomach wall surface by the membrane's own adhesive properties. The stomach pouch is carefully folded up, disinfected, and the peritoneum is sutured with 6-0 absorbable sutures and the epithelium is sutured with 4-0 sutures. The wound is disinfected, lidocaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0107] After the cell-containing multilayered construct prepared in this example was applied to the mouse stomach wall, the results of a multi-tissue universal application test are shown in the stomach photograph in Figure 7. In approximately ten days, an orthotopic gastric cancer tumor was formed, as shown in Figure 9. The method of this example can increase the success rate of orthotopic gastric tumor models in mice to over 90%, while keeping the rate of unnecessary passive metastasis below 10%.

[0108] Example 3

[0109] 1) Protein film preparation: Using dimercaptosuccinic acid as a crosslinker and lactoferrin as the material, the lactoferrin concentrations were 1, 2, and 7 mg / ml, respectively, and the dimercaptosuccinic acid dosage was 13 mg / ml. The two were mixed in a 1:1 volume ratio to prepare adhesive two-dimensional protein films via a vapor phase interface method. The thicknesses were 10 nm, 40 nm, and 120 nm, respectively.

[0110] Protein membranes with thicknesses of 10 nm, 40 nm, and 120 nm (the thickness of the protein membrane can be controlled by protein concentration or increased by regrowth) with an area of ​​approximately 40 mm 2 The liquid phase interface is a non-adhesive surface, and the gas phase interface is an adhesive interface; the Young's modulus is between 1GPa-2GPa; the toughness range is 0.1-1.2kJ / m 2 ; The adhesion strength of the adhesive surface is 20-100kPa;

[0111] 2) Preparation of the Protective Layer: The 120 nm thick protein film from step 1) was immobilized using 6 mg / ml tris(2-carboxyethyl)phosphine hydrochloride to form a protective layer membrane. The 10 nm thick protein film from step 1) was then adhered to the non-adhesive layer of the solidified 120 nm protective layer membrane via its own adhesive properties to form a double-layer membrane. The double-layer membrane was placed on a chitosan gel block, with the 10 nm thick protective layer membrane contacting the agar, i.e., the 10 nm thick protective layer was on the bottom and the 120 nm thick protective layer membrane was on the top. A human cervical cancer cell line HeLa cell suspension (from ATCC) was then added to the center of the surface of the 120 nm protective layer membrane, and after deposition, the culture medium was aspirated;

[0112] 3) Preparation of Adhesion Layer: Using the membrane's own adhesion, the 40 nm thick protein membrane from step 1) was covered on the surface of the 120 nm protective layer membrane from step 2) on which the HeLa cells were deposited. The edges were bonded using its own viscosity and the edges were quickly treated with a 1% dilute oxalic acid aqueous solution (which can be treated by dripping and smearing) to complete the edge sealing, thereby preparing a multilayer structure with three membrane layers (see Figure 1).

[0113] 4) The agar gel block containing the cell multilayered construct from step 3) above was placed in a 3.5 cm culture dish. DMEM high-glucose medium containing 10% fetal bovine serum was added, and the medium was infiltrated into but not covered the multilayered construct. The culture was incubated at 37°C, 5% CO2 for 18-24 hours. After the cells were stably viable and adhered to the membrane, they were used for in vivo modeling.

[0114] 5) Six-week-old BALB / c-nu / nu nude mice were anesthetized by isoflurane inhalation and secured in a supine position on a sterilized small animal operating table under anesthesia. The abdomen of the mouse was extensively disinfected with iodine tincture and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum along the dermal grain 0.5 cm above the opening of the mouse's reproductive tract. The bladder was carefully exposed. The Y-shaped uterus was located and exposed along the subureteral path. The outer surface of the uterus was then dried with a sterile cotton swab.

[0115] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried uterus from step 5), allowing the membrane to adhere to the uterine surface through its own adhesive properties. The uterus and bladder are carefully retracted, and after disinfection, the peritoneum is sutured with 6-0 absorbable sutures, and the integument is sutured with 4-0 sutures. The wound is then disinfected, lidocaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0116] After the cell-containing multilayered construct prepared in this example was applied to the mouse uterus, the results of a multi-tissue universal application test are shown in the uterine photograph in Figure 7. In approximately one week, orthotopic uterine cancer tumors were formed in mice, as shown in Figure 10. The method of this example achieved a success rate of over 90% for orthotopic uterine cancer models in mice, while keeping the rate of unnecessary passive metastasis below 10%.

[0117] Example 4

[0118] 1) Using polyvinyl pyrrolidone K30 as the material and anhydrous ethanol as the solvent at a concentration of 100 mg / mL, a two-dimensional film was prepared by electrospinning technology with a set speed of 50 μL / h and a voltage of 10 kV.

[0119] Two-dimensional films with thicknesses of 40 μm, 80 μm, and 200 μm were prepared (the membrane thickness was adjusted by adjusting the electrospinning parameters), and the membrane area was approximately 4 cm 2 ; Young's modulus between 2GPa-8GPa; toughness range 2.4-4.5kJ / m 2 ; Adhesion strength is 0.1-10kPa;

[0120] 2) Preparation of a protective layer: The 40 μm thick two-dimensional film from step 1) was applied to the 200 μm thick two-dimensional film from step 1) using a cyanoacrylate adhesive. The two-layer film was fixed with dilute hydrochloric acid to form a protective layer film, which was then placed on a PE plastic base, with the 40 μm thick two-dimensional film directly contacting the PE plastic base, i.e., the 40 μm thick two-dimensional film was on the bottom and the 200 μm thick two-dimensional film was on the top. A rat pancreatic cancer cell line DSL-6A / C1 cell suspension was then added to the center of the surface of the 200 μm thick protective layer film, and the culture medium was aspirated after deposition;

[0121] 3) Preparation of Adhesion Layer: Using mucin, a 40 μm thick two-dimensional film from step 1) was applied to the 80 μm film from step 1) to form an adhesion layer. This layer was then covered on the surface of the protective layer film from step 2) on which the DSL-6A / C1 cells were deposited. The edges of the two films were completely bonded together using a cyanoacrylate adhesive. The edges of the films were further treated with 1% dilute hydrochloric acid (which can be applied by dripping and smearing) to complete the edge sealing, thereby producing a multilayered structure.

[0122] 4) Place the PE plastic block containing the cell-containing multilayered construct from step 3) above in a 10 cm culture dish and add DMEM high-glucose medium containing 10% fetal bovine serum. The medium should soak into but not cover the multilayered construct. Incubate at 37°C, 5% CO2 for 18-24 h. Once the cells are stably viable and adhere to the membrane, they can be used for in vivo modeling.

[0123] 5) Male SD rats weighing approximately 200 g (purchased from Beijing Huafukang Company) were anesthetized by isoflurane inhalation and fixed in a supine position on a sterilized small animal operating table under anesthetic gas. The rats' abdomens were first shaved and skinned. The abdomens were extensively disinfected with iodine and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum along the dermal lines on the left side of the rat's upper abdomen. The spleen was located and the pancreas was carefully exposed and exposed. The outer surface of the pancreas was dried with a sterile cotton swab.

[0124] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried pancreas from step 5), and the tissue surfaces are bonded using cyanoacrylate adhesive. The pancreas and spleen are carefully repositioned, and after disinfection, the peritoneum is sutured with 4-0 absorbable sutures, and the integument is sutured with 2-0 sutures. The wound is disinfected, lidocaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0125] After the cell-containing multilayered structure prepared in this example was applied to the rat pancreas, the results of the multi-tissue universal application test are shown in the pancreas photograph of FIG7 . In about one week, an in situ pancreatic cancer tumor was formed in the rat.

[0126] Example 5

[0127] 1) Using polyethylene oxide as the material, a two-dimensional film was prepared by electrospinning technology. The solvent was methanol with a concentration of 100 mg / mL. The two-dimensional film was prepared by electrospinning technology with a set speed of 50 μL / h and a voltage of 10 kV.

[0128] Two-dimensional films with membrane thicknesses of 20 μm, 100 μm, and 1 mm were prepared (the membrane thickness was adjusted by adjusting the electrospinning parameters), and the membrane area was about 0.5 cm 2 ; Young's modulus between 1GPa-8GPa; toughness range 0.4-6.9kJ / m 2 ; Adhesion strength 2-6.1kPa;

[0129] 2) Preparation of the protective layer: One surface of the 1 mm thick two-dimensional film from step 1) was oxidized with dilute chromic acid. Chromic acid was then dripped onto the film surface for 20 seconds and then quickly rinsed. This formed the protective layer film, which was then placed on a PP plastic base, with the oxidized surface of the protective layer film in direct contact with the PP plastic base, i.e., the oxidized surface facing downward and the unoxidized surface facing upward. A rat cancer cell line CBRH-7919 cell suspension was added to the unoxidized surface of the film, and the culture medium was aspirated after deposition.

[0130] 3) Preparation of the adhesion layer: Using polydopamine gel, the 20 μm thick two-dimensional film of step 1) is applied to the 100 μm thick two-dimensional film to form an adhesion layer, which is then covered on the surface of the cell-deposited protective layer membrane of step 2). The edges of the two films are completely bonded using a glucose-citric acid adhesive, and the edges are treated with 1% dilute chromic acid (which can be treated by dripping and smearing) to complete the edge sealing and prepare a multilayer structure.

[0131] 4) Place the PP plastic block containing the cell-containing multilayered construct from step 3) above in a 3.5 cm culture dish and add DMEM high-glucose medium containing 10% fetal bovine serum. The medium should soak but not cover the construct. Incubate at 37°C, 5% CO2 for 18-24 h. Once the cells are stably viable and adhere to the membrane, they can be used for in vivo modeling.

[0132] 5) Male SD rats weighing approximately 200 g (purchased from Beijing Huafukang Company) were anesthetized by isoflurane inhalation and fixed in a supine position on a sterilized small animal operating table under anesthetic gas. The rats' abdomens were first shaved and skin prepared. The abdomens of the rats were extensively disinfected with iodine and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum along the dermal lines at the lateral position of the mid-abdomen of the rats. The kidneys were carefully exposed, and the outer surface of the kidneys was dried with a sterile cotton swab.

[0133] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried kidney from step 5), and the kidney surface is adhered using surgical glue. The kidney is carefully repositioned, and after disinfection, the peritoneum is sutured with 4-0 absorbable sutures, and the epidermis is sutured with 2-0 sutures. The wound is disinfected, procaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0134] After the cell-containing multilayered structure prepared in this example was applied to the rat kidney, the results of the multi-tissue universal application test are shown in the kidney photograph of Figure 7. In about eight days, an in situ tumor of rat renal cancer was formed.

[0135] Example 6

[0136] 1) Two-dimensional thin films were prepared using chitosan by spin coating. Chitosan was ultrasonically dispersed at a concentration of 5 mg / ml in a 3% aqueous acetic acid solution. One to two drops of chitosan were then added to a smooth stainless steel surface. The spin coating was performed at a starting speed of 600 rpm and a constant speed of 2200 rpm for 8 seconds and 20 seconds, respectively, repeated 3 to 5 times. The thin films were then dried at 37°C for 12 hours to prepare the thin films.

[0137] Two-dimensional films with thicknesses of 100 nm and 200 nm were prepared (the film thickness was controlled by the number of coatings), and the area of ​​the cut film was about 0.4 cm 2; Young's modulus between 5GPa-8GPa; toughness range 1.8-7.4kJ / m 2 ; Adhesion strength 10-54kPa;

[0138] 2) Preparation of a protective layer: One surface of the 200 nm thick two-dimensional film prepared in step 1) was oxidized with sodium percarbonate, subjected to a post-treatment for 1 minute, and quickly rinsed to form a protective layer. The film was then placed on a stainless steel base, with the oxidized surface of the film directly in contact with the base (i.e., with the oxidized surface facing downward). A rat cancer cell line MADB106 cell suspension was added to the unoxidized surface of the film, and the culture medium was aspirated after deposition.

[0139] 3) Using mussel protein, two 100 nm thick two-dimensional films from step 1) are attached to form an adhesion layer, which is then covered on the surface of the cell-deposited protective layer membrane from step 2). The edges of the membranes are completely bonded using a high-sugar adhesive and treated with 0.5% dilute nitric acid (which can be treated by dripping and smearing) to complete the edge sealing and prepare a multilayer structure.

[0140] 4) Place the stainless steel base block containing the cell-multilayered construct from step 3) above in a 3.5 cm culture dish and add MEM high-glucose medium containing 10% fetal bovine serum. The medium should soak but not cover the construct. Incubate at 37°C, 5% CO2 for 18-24 hours. Once the cells are stably viable and adhere to the membrane, they can be used for in vivo modeling.

[0141] 5) Male SD rats weighing approximately 200 g (purchased from Beijing Huafukang Company) were anesthetized by intraperitoneal injection of 5% chloral hydrate solution and fixed in a supine position on a sterilized small animal operating table under anesthesia. The rats' abdomens were first shaved and the skin prepared. The upper abdomen of the rats was extensively disinfected with iodine and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum along the skin grain at the inferior edge of the xiphoid process of the rats. The stomach pouch was carefully identified, and the esophagus was explored upward. The esophagus was fixed with a stent, and the outer surface of the esophagus was dried with a sterile cotton swab.

[0142] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried esophagus from step 5), and the esophageal surface is adhered using surgical glue. The esophagus is carefully repositioned and confirmed to be open. After disinfection, the peritoneum is sutured with 4-0 absorbable sutures, and the epithelium is sutured with 2-0 sutures. The wound is disinfected, procaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0143] After the cell-containing multilayered structure prepared in this example was applied to the rat esophagus, the results of the multi-tissue universal application test are shown in the esophagus photograph in FIG7 . In about one week, an in situ tumor of the rat esophageal cancer was formed.

[0144] Example 7

[0145] 1) Using methane as the raw material, ferrocene and sulfur powder as auxiliary agents at 65-85°C, and a two-dimensional carbon nanotube film was prepared by vapor deposition at 600°C.

[0146] Two-dimensional films with thicknesses of 20 nm, 120 nm, and 160 nm were prepared (thickness was controlled by reaction time), and the area of ​​the film was approximately 0.6 cm 2 ; Young's modulus between 10GPa-12GPa; toughness range 0.1-0.7kJ / m 2 ; Adhesion strength is 0.1-0.2kPa;

[0147] 2) Preparation of a protective layer: The 160 nm thick and 20 nm thick two-dimensional films from step 1) were bonded together using a carboxymethyl cellulose adhesive. The 160 nm thick two-dimensional film was then oxidized with sodium dichromate to form a protective layer. The 20 nm thick two-dimensional film was used as a protective layer and placed on a stainless steel base, with the 20 nm thick two-dimensional film directly contacting the base, i.e., the 20 nm thick two-dimensional film facing downward and the 160 nm thick two-dimensional film facing upward. A rat cancer cell line CBRH-7919 cell suspension was added to the surface of the 160 nm film, and after deposition, the culture medium was aspirated;

[0148] 3) Using pullulan adhesive, the 20 nm thick two-dimensional film of step 1) and the 100 nm thick two-dimensional film of step 1) are attached to form an adhesion layer, and then one side of the 100 nm thick two-dimensional film is covered on the surface of the protective layer membrane on which the cells are deposited. The edges of the membranes are completely bonded and sealed using a 1.5% pullulan adhesive, and the edges are treated with a 2% sodium dichromate solution (which can be treated by dripping and smearing) to complete the edge sealing and prepare a multilayer structure.

[0149] 4) Place the stainless steel base block containing the cell-based multilayered construct from step 3) above in a 3.5 cm culture dish and add 1640 culture medium containing 10% fetal bovine serum. The culture medium should soak the construct but not cover it. Incubate at 37°C, 5% CO2 for 18-24 hours. Once the cells are stably viable and adhere to the membrane, they can be used for in vivo modeling.

[0150] 5) Male SD rats weighing approximately 200 g (purchased from Beijing Huafukang Company) were anesthetized by intraperitoneal injection of sodium pentobarbital and fixed in a supine position on a sterilized small animal operating table under anesthesia. The rats' abdomens were first shaved and the skin prepared. The lower abdomen of the rats was extensively disinfected with iodine and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum 1 cm above the opening of the rat's reproductive tract according to the skin grain. The colon was carefully identified and secured with a support. The outer surface of the colon was dried with a sterile cotton swab.

[0151] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried colon from step 5), and the colon surface is adhered using surgical glue. The colon is carefully repositioned and confirmed to be permeable. After disinfection, the peritoneum is sutured with 4-0 absorbable sutures, and the integument is sutured with 2-0 sutures. The wound is disinfected, procaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0152] After the cell-containing multilayered structure prepared in this example was applied to the rat colon, the results of the multi-tissue universal application test are shown in the colon photograph of FIG7 . In about twelve days, an in situ tumor of the rat colon cancer was formed.

[0153] Example 8

[0154] 1) A two-dimensional thin film was prepared by spin coating using hydroxymethyl cellulose at a concentration of 5 mg / ml. After ultrasonic dispersion in a 30% ethanol aqueous solution, 1 to 2 drops were added to a smooth stainless steel surface. The film was rotated at a starting speed of 800 rpm and a constant speed of 2800 rpm for 5 seconds and 30 seconds, respectively, for 3 to 5 times, and then dried at 37°C for 24 hours.

[0155] The thickness was controlled by the number of droplets, and films with thicknesses of 40 nm, 200 nm, and 1.5 μm were prepared, respectively. The area of ​​the films was approximately 4 cm 2 ; Young's modulus between 2GPa-4GPa; toughness range 1.4-4.8kJ / m 2 ; Adhesion strength 10-19kPa;

[0156] 2) Preparation of a protective layer: Using a protein adhesive, the 40 nm thick two-dimensional film from step 1) and the 1.5 μm thick two-dimensional film from step 1) were bonded together. The 1.5 μm thick two-dimensional film was then oxidized with dilute sulfuric acid to form a protective layer. The 40 nm thick protective layer was placed on a stainless steel base, with the 40 nm thick two-dimensional film directly contacting the base, i.e., the 40 nm thick two-dimensional film facing downward and the 1.5 μm thick two-dimensional film facing upward. A rabbit cancer cell line VX2 cell suspension was added to the surface of the 1.5 μm two-dimensional film, and after deposition, the culture medium was aspirated;

[0157] 3) Using a protein adhesive, the 40 nm thick film of step 1) and the 200 nm thick two-dimensional film of step 1) are attached to form an adhesion layer, which is then covered on the surface of the protective layer film on which the cells are deposited, wherein the 200 nm thick two-dimensional film is in direct contact with the 1.5 μm thick film of step 2) on which the cells are deposited. The edges of the membranes are completely bonded using a protein adhesive and treated with 2% dilute sulfuric acid (which can be treated by dripping and smearing) to complete the edge sealing, thereby preparing a multilayer structure.

[0158] 4) Place the stainless steel base block containing the cell-based multilayered construct from step 3) above in a 10 cm culture dish and add HAM F10 / F12 culture medium containing 10% fetal bovine serum. Add the medium to soak but not cover the construct. Incubate at 37°C, 5% CO2 for 18-24 hours. Once the cells adhere to the membrane, use for in vivo modeling.

[0159] 5) Male large-eared white rabbits weighing approximately 3000 g (purchased from Chongqing Tengxin Biotechnology Co., Ltd.) were anesthetized by isoflurane inhalation and secured in a supine position on a sterilized small animal operating table under gas-maintained anesthesia. The rabbit's lower abdomen was shaved and the skin prepared. The lower abdomen was extensively disinfected with iodine and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum along the skin grain 2 cm above the opening of the rabbit's reproductive tract, exposing the bladder. The bladder was secured with a support device, and the outer surface of the bladder was blotted dry with a sterile cotton swab.

[0160] 6) The cultured cell-containing multilayered adhesive layer from step 4) is applied to the dried bladder from step 5) and adhered to the bladder surface using surgical glue. After disinfection, the peritoneum is sutured with 2-0 absorbable sutures, and the integument is sutured with 1-0 sutures. The wound is disinfected, procaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0161] After the cell-containing multilayered structure prepared in this example was applied to the rabbit bladder, the results of the multi-tissue universal application test are shown in the bladder photograph of Figure 7. In about two weeks, an in situ tumor of rabbit bladder cancer was formed.

[0162] Example 9

[0163] 1) 4-HEPES was used as a crosslinker and lactoferrin was used as a crosslinker. Lactoferrin was mixed at a volume ratio of 1:1 with 13 mg / ml of 4-HEPES at a concentration of 1:1. Adhesive two-dimensional protein films with thicknesses of 10 nm, 50 nm, and 200 nm were prepared via a vapor phase interface method.

[0164] Protein films with thicknesses of 10 nm, 50 nm, and 200 nm were cut, and the area of ​​the films was approximately 0.5 cm 2 The liquid phase interface is a non-adhesive surface, and the gas phase interface is an adhesive interface; the Young's modulus is between 1GPa-2GPa; the toughness range is 0.1-1.2kJ / m 2 ; The adhesion strength of the adhesive surface is 20-100kPa;

[0165] 2) Preparation of a protective layer: The 200 nm thick protein film from step 1) was immobilized with β-mercaptoethanol to form a protective layer. A 10 nm thick protein film was then applied to the non-adhesive layer of the 200 nm thick protein film via its own adhesive properties to serve as a protective layer. The bilayer was then placed on an agarose gel block, with the 10 nm thick protein film contacting the agar at the bottom and the 200 nm thick protein film at the top. A cultured mouse breast cancer cell line 4T1 tumor was then added to the center of the surface of the 200 nm thick protein film. The tumor tissue was stored in UW tissue preservation medium at 8°C.

[0166] 3) Using the membrane's own adhesion, the 10 nm thick protein film from step 1) was applied to the hydrophilic surface of the 50 nm thick protein film from step 1) to form an adhesion layer. This adhesion layer was then covered on the surface of the protective layer of the membrane carrying the tumor tissue. The edges were bonded using the membrane's own adhesion and treated with 1% dilute oxalic acid (which could be treated by dripping and smearing) to complete the edge sealing. A multilayered structure loaded with tumor tissue was prepared (structure shown in Figure 1). The multilayered structure was stored in phosphate buffer, with the liquid level not exceeding the multilayered tissue adhesive layer.

[0167] 4) Six-week-old female BALB / c mice (purchased from Beijing Huafukang Company) were anesthetized with isoflurane inhalation and fixed in a supine position on a sterilized small animal operating table under anesthesia. The abdomen of the mouse was extensively disinfected with iodine tincture and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum below the xiphoid process of the mouse sternum along the skin grain. The liver was carefully turned out and the entire liver lobe was separated. The surface moisture of the liver was absorbed with a sterile cotton swab.

[0168] 5) The multilayered adhesive layer containing the tumor tissue from step 3) is applied to the dry liver surface, allowing the membrane to adhere to the liver surface through its own adhesive properties. The liver is carefully folded up and disinfected, followed by suturing the peritoneum with 6-0 absorbable sutures and the integument with 4-0 sutures. The wound is then disinfected, lidocaine analgesic is injected, and the wound is sealed with medical surgical glue.

[0169] After the cell-containing multilayered structure prepared in this example was applied to the liver of a mouse, an orthotopic liver tumor was formed in about five days.

[0170] Example 10

[0171] 1) Adhesive two-dimensional metal hybrid films with thicknesses of 25 nm, 40 nm, and 90 nm were prepared by mixing silver nanoparticles and collagen with 4-hydroxyethylpiperazineethanesulfonic acid as a crosslinker. The silver nanoparticles were mixed at a volume ratio of 1:1:1 at a concentration of 15 mg / mL, collagen at concentrations of 2, 4, and 7 mg / mL, and 4-hydroxyethylpiperazineethanesulfonic acid at a concentration of 13 mg / mL. The films were then prepared via a vapor phase interface method.

[0172] Thin films with thicknesses of 25 nm, 40 nm, and 90 nm (the thickness of the protein film can be controlled by protein concentration or increased by regrowth) were cut separately. The area of ​​the film was about 0.5 cm 2 ; Young's modulus between 4GPa-8GPa; toughness range 0.1-1.0kJ / m 2 ; The adhesion strength of the adhesive surface is 1-23kPa;

[0173] 2) Preparation of a protective layer: The 20 nm thick film from step 1) was applied as a protective layer to the 90 nm thick film from step 1) via its own adhesive properties. The hydrophilic surface of the 90 nm thick film from step 1) was fixed with dilute nitric acid to form the protective layer. The film was then placed on an agarose gel block, with the 20 nm film contacting the agar at the bottom and the 90 nm film at the top. A human gastric cancer tumor mass was added to the center of the 90 nm film surface. The tumor mass was maintained in GALA tissue preservation solution.

[0174] 3) Preparation of Adhesion Layer: Using the membrane's own adhesion, the 90 nm thick membrane from step 1) is applied to the outside of the 40 nm thin film to form an adhesion layer. This layer is then covered on the surface of the cell-deposited protective layer membrane. The edges are bonded using its own adhesion and treated with dilute hydrochloric acid to complete edge sealing. A multilayer construct is prepared and immersed in MACS tissue preservation solution at 4°C for subsequent in vivo modeling. During the immersion process, the liquid must not cover the construct adhesion layer.

[0175] 4) Six-week-old BALB / c-nu / nu nude mice were anesthetized by isoflurane inhalation and secured in a supine position on a sterilized small animal operating table under anesthesia. The abdomen of the mouse was extensively disinfected with iodine tincture and covered with a sterile surgical drape. A No. 10 scalpel blade was used to incise the skin and peritoneum below the xiphoid process of the sternum, following the cutaneous grain. The gastric pouch was carefully exposed, and the surface moisture of the pouch was absorbed with a sterile cotton swab.

[0176] 5) Apply the multilayered adhesive layer containing the tumor tissue to the dry surface of the stomach wall, allowing it to adhere to the surface through its own adhesive properties. Carefully retract the membrane, disinfect it, and suture the peritoneum with 6-0 absorbable sutures and the epidermis with 4-0 sutures. Disinfect the wound, inject lidocaine as an analgesic, and seal it with medical surgical glue.

[0177] After the cell-containing multilayered structure prepared in this example was applied to the stomach wall of mice, an in situ gastric cancer tumor was formed in about eight days.

[0178] Example 11

[0179] 1) Using gold nanoparticles and lysozyme as materials, 4-hydroxyethylpiperazineethanesulfonic acid as a crosslinker, the gold nanoparticles were mixed at a volume ratio of 1:1:1 at a concentration of 15 mg / mL, lysozyme at concentrations of 2, 3, and 7 mg / ml, and 4-hydroxyethylpiperazineethanesulfonic acid at a crosslinking agent. The thicknesses of the mixed films were 15 nm, 60 nm, and 130 nm, respectively, using a vapor phase interface method.

[0180] Protein membranes with thicknesses of 15 nm, 60 nm, and 130 nm (the thickness of the protein membrane can be controlled by protein concentration or increased by regrowth) were cut separately. The membrane area was approximately 0.3 cm 2 ; Young's modulus between 1GPa-7GPa; toughness range 0.1-1.6kJ / m 2 ; Adhesion strength of the adhesive surface 0.1-43kPa;

[0181] 2) Preparation of a Protective Layer: The 15 nm thick membrane from step 1) was applied as a protective layer to the 130 nm thick membrane from step 1) via self-adhesion. The hydrophilic surface of the 130 nm thick membrane from step 1) was fixed with dilute chromic acid to form a protective layer. The membrane was then placed on a gelatin block, with the 15 nm thick membrane contacting the agar at the bottom and the 130 nm thick membrane at the top. A human prostate tumor mass was added to the outer surface of the membrane. The tumor mass was maintained in HBSS tissue preservation solution at 2°C.

[0182] 3) Using the membrane's own adhesion, the 60 nm thick membrane from step 1) was overlaid on the surface of the 130 nm thick protective layer containing the tumor tissue. The edges were bonded using the membrane's own adhesion and treated with dilute chromic acid to complete the edge sealing. The resulting multilayered construct was then immersed in GALA tissue preservation fluid for subsequent in vivo modeling. During the immersion process, the liquid did not cover the construct's adhesion layer.

[0183] 4) Six-week-old male BALB / c-nu / nu nude mice were anesthetized by isoflurane inhalation and secured in a supine position on a sterilized small animal operating table while maintaining anesthesia. The mouse's lower abdomen was extensively disinfected with iodine tincture and covered with a sterile surgical drape. Using a No. 10 scalpel blade, the skin and peritoneum were incised along the dermal lines 0.5 cm above the opening of the mouse's reproductive tract. The bladder and coagulation glands were carefully exposed, and the prostate was then located and removed with a sterile cotton swab, after which the surface moisture was blotted dry.

[0184] 5) Apply the multilayered adhesive layer containing the tumor tissue to the dry surface of the prostate, allowing the membrane to adhere to the surface through its own adhesive properties. Carefully retract the membrane, disinfect it, and suture the peritoneum with 6-0 absorbable sutures and the epidermis with 4-0 sutures. Disinfect the wound, inject lidocaine as an analgesic, and seal it with medical surgical glue.

[0185] After the cell-containing multilayered structure prepared in this example was applied to the prostate of a mouse, a prostate orthotopic tumor was formed in about a week.

[0186] Example 12

[0187] 1) Prepare a two-dimensional gold nano-protein composite film by ion sputtering. The 10 nm protein film obtained in Example 1 was placed in an ion sputtering instrument with a vacuum degree of 2×10 -3 Pa, voltage is 500 V, and spraying time is 3 minutes.

[0188] By controlling the spraying time, films with thicknesses of 500 nm, 10 μm, and 100 μm were prepared, and the area of ​​the films was approximately 4 cm 2 ; Young's modulus between 10GPa-12GPa; toughness range 0.4-3.8kJ / m 2 ; Adhesion strength is 0.1-0.6kPa;

[0189] 2) Preparation of the protective layer: The 100 μm thick membrane from step 1) and the 10 μm thick membrane from step 1) were fixed with chromic acid and then bonded with a medical resin adhesive to form a protective layer. The layer was then placed on a PF plastic matrix block, with the 10 μm membrane contacting the matrix block at the bottom and the 100 μm membrane at the top. A canine colon cancer tumor was placed in the center of the 100 μm membrane surface. The tumor tissue was maintained in FRS tissue preservation solution.

[0190] 3) Using a medical resin adhesive, the 500 nm thick membrane from step 1) was applied to the tumor tissue-bearing side of the 100 μm thick membrane from step 1) to form an adhesion layer. The edges were treated with chromic acid to complete the edge sealing, thereby preparing a multilayer structure. The structure was then immersed in FRS tissue preservation solution at 4°C for subsequent in vivo modeling.

[0191] 4) A female beagle dog (purchased from Chongqing Tengxin Biotechnology Co., Ltd.) was anesthetized with isoflurane inhalation and secured in a supine position on a sterilized small animal operating table while maintaining anesthetic gas. A large area of ​​the dog's lower abdomen was prepared and disinfected using iodine tincture. A sterile surgical drape was used to cover the dog. A No. 10 scalpel blade was used to incise the skin and peritoneum along the dermal lines below the reproductive tract on one side. The colon was carefully exposed, and the surface moisture of the colon was absorbed with a sterile cotton swab.

[0192] 5) Apply the adhesive layer of the multilayered construct containing colon cancer tissue to the dry colon surface and adhere the construct to the colon surface using surgical glue. Carefully retract the construct and, after disinfection, suture the peritoneum with 2-0 absorbable sutures and the integument with 2-0 sutures. Disinfect the wound, inject lidocaine as an analgesic, and seal the wound with surgical glue.

[0193] After the multilayered tumor tissue-loaded structure prepared in this example was applied to the dog's colon, colon cancer in situ tumors were formed in about three weeks.

[0194] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial Applicability

[0195] The present application provides a multilayer structure for in situ tumor modeling, its preparation method, and application, which includes an adhesive layer and a protective layer, and tumor cells and / or tumor masses encapsulated between the adhesive layer and the protective layer; the edge bonding of the adhesive layer and the protective layer is used to prevent tumor cells from leaking out from the side or the protective layer; the adhesive layer is used to adhere to the target organ tissue by relying on the membrane's own adhesive properties or through a medical adhesive. The multilayer structure of the present application can make the construction process of in situ / metastatic tumor models on multiple organs / tissues of animals a standardized process, breaking through the obstacles of modeling thin-walled organs and tissues of small animals. The process is simple and efficient, low-cost, highly repeatable, and has a high modeling rate, and has good application prospects.

Claims

1. A multilayered structure for in situ tumor modeling, characterized in that: It comprises an adhesion layer and a sealing layer, and tumor cells and / or tumor masses encapsulated between the adhesion layer and the sealing layer; The edge bonding of the adhesive layer and the sealing layer is used to prevent tumor cells from penetrating from the side or the sealing layer; The adhesive layer is used to adhere to the target organ tissue by relying on the membrane's own adhesive properties or through a medical adhesive.

2. The multilayer structure according to claim 1, characterized in that: The thickness of the protective layer film is 5nm-2mm, optionally 50nm-2mm, optionally 50nm-1mm, optionally 40nm-1.5μm, optionally 90nm-2μm, optionally 90nm-200nm, optionally 500nm-20μm, optionally 500nm-1mm, optionally 10μm-500μm, optionally 100μm-1mm; and / or, the thickness of the adhesion layer is 40 nm to 10 μm, optionally 90 nm to 1 mm, optionally 40 nm to 200 nm, optionally 1 μm to 10 μm; And / or, in the sealing layer film or the adhesive layer film, the film is cut to a suitable size according to different animal organ tissues and tumor cells or tissue samples, and the film area can be optionally 0.5mm 2 ~9cm 2 ; And / or, in the sealing layer membrane or the adhesive layer membrane, the pore size of the adhesive layer membrane is not smaller than that of the sealing layer membrane, and the optional pore size is 1 nm to 2 μm; And / or, in the sealing layer film or the adhesive layer film, the Young's modulus of the film is 1 Gpa to 12 Gpa, optionally 1 Gpa to 2 Gpa, optionally 3 Gpa to 5 Gpa, optionally 6 Gpa to 12 Gpa; And / or, in the sealing layer film or the adhesive layer film, the toughness of the film is 0.1 kJ / m 2 ~8kJ / m 2 , optionally 0.1 kJ / m 2 ~3.4kJ / m 2 , optionally 4.1 kJ / m 2 ~6kJ / m 2 , optionally 6.1 kJ / m 2 ~8kJ / m 2 ; And / or, in the protective layer film or the adhesive layer film, the adhesion strength of the film is 0.1 kPa to 300 kPa, optionally 0.1 kPa to 10 kPa, optionally 11 kPa to 100 kPa, optionally 100 kPa to 300 kPa.

3. The multilayer structure according to claim 1 or 2, characterized in that: The edges of the sealing layer film and the adhesive layer film are adhered by the film's own viscosity or adhesive, and then sealed by an oxidant; Optionally, the oxidant for edge sealing treatment includes one or more of hydrogen peroxide, formaldehyde, peracetic acid, sulfuric acid, hydrochloric acid, nitric acid, oxalic acid, sodium dichromate, chromic acid, potassium permanganate, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, bromine, and iodine; And / or, the sealing layer film and / or the adhesive layer film is a protein film, and optionally, the protein is lactoferrin, lysozyme, bovine serum albumin, insulin, α-lactalbumin, human serum albumin, fibrinogen, β-amyloid protein, soybean protein, zein, wheat prolamin, horseradish peroxidase, hemoglobin, hemocyanin histone, glutathione, One or more of protein, collagenase, ovalbumin, soy protein, transferrin, thyroglobulin, trypsin, pepsin, chymosin, and mucin; optionally, the cross-linking agent is selected from any one or more of 4-hydroxyethylpiperazineethanesulfonic acid, dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride, cysteine, reduced glutathione, dimercaptosuccinic acid, sodium sulfite, glutaraldehyde, genipin, glutamine aminotransferase, carbodiimide, and epichlorohydrin; optionally, the protein film itself has viscosity; optionally, the protein film is a protein film formed by self-assembly of a protein aqueous solution and a cross-linking agent aqueous solution at a gas-liquid interface; And / or, the sealing layer film and / or the adhesion layer film is an electrospun two-dimensional film, and optionally, the material of the electrospun two-dimensional film is selected from one or more of polyvinyl pyrrolidone, polyethylene oxide, polyester, polyamide, polyimide, polypropylene, polyethylene, collagen, silk fibroin, chitosan, and gelatin; And / or, the sealing layer film and / or the adhesion layer film is a vapor deposition film, and optionally, the material of the vapor deposition film is selected from one or more of carbon nanotube film, graphene, molybdenum disulfide, etc.; And / or, the sealing layer film and / or the adhesion layer film is a spin-coated film, and optionally, the material of the spin-coated film is selected from polysaccharides, and optionally selected from one or more of chitosan, hydroxymethyl cellulose, sodium alginate, hyaluronic acid, and carrageenan soluble polymers; And / or, a protective layer is also attached to the sealing layer film. Optionally, the protective layer is made of the same material as the sealing layer film. Optionally, the thickness of the protective layer is 10nm~100μm, optionally 20nm~100μm, optionally 10nm~20μm, optionally 200nm~1.5μm, optionally 10nm~100nm, optionally 500nm~20μm.

4. The multilayer structure according to any one of claims 1 to 3, characterized in that: It also includes a support for further culturing or maintaining the product in a culture solution or a maintenance solution; Optionally, the support is one or more of hydrogel, plastic, and metal; Optionally, the hydrogel is any one or more of small molecules, natural polymers and synthetic polymers; Optionally, the small molecule includes one or any several of fluorenyl dipeptide, octapeptide, and oligopeptide derivatives; Optionally, the natural polymer includes polysaccharides, peptides, and nucleic acids; the polysaccharides include one or any combination of starch, cellulose, alginate, hyaluronic acid, chitosan, k2 carrageenan, locust bean gum, gelatin, and agarose; and the polypeptides include one or more of collagen, fibrin, poly-L-lysine, and poly-L-glutamic acid; Optionally, the synthetic polymer includes one or more of polyacrylic acid and its derivatives, polyethers, and polyesters; optionally, the polyethers include one or any of polypropylene oxide, polyethylene glycol, and polyethylene oxide; optionally, the polyacrylic acid and its derivatives include one or more of polyacrylamide, polyisopropylacrylamide, polyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone, and polyvinyl alcohol; optionally, the polyester includes one or more of polyhydroxyethyl methacrylate, polypropylene fumarate, polycaprolactone, and polydimethylaminoethyl methacrylate. Several kinds; Optionally, the plastics include one or more of thermoplastic plastics, thermosetting plastics, general plastics, engineering plastics, functional plastics, crystalline plastics, and non-crystalline plastics; Optionally, the plastic component includes one or more of polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, polyamide, polycarbonate, acrylonitrile-butadiene-styrene copolymer, polymethyl methacrylate, acrylonitrile-styrene copolymer, polyester, phenolic resin, epoxy resin, amino resin, alkyd resin, allyl resin, urea-formaldehyde resin, melamine resin, unsaturated polyester, silicone resin, polyurethane; Optionally, the polyester is selected from one or more of PETP polybutylene terephthalate and PBTP polyethylene terephthalate; Optionally, the metal material includes one or more of stainless steel, gold, silver, and copper; Optionally, the culture medium / maintenance medium includes BME medium, MEM medium, DMEM medium, IMDM medium, PRMI1640 medium, HAM F10 / F12 medium, 199 medium, CHO medium, Vero medium, NS0 medium, PFM medium, UW tissue preservation medium, HBSS tissue preservation medium, GALA tissue preservation medium, One or any combination of FRS tissue preservation solution, MACS tissue preservation solution, phosphate buffer, physiological saline, and isotonic glucose solution; Optionally, the culture medium contains serum, including one or any combination of calf serum, new bovine serum, fetal bovine serum, horse serum, and human serum; Optionally, the culture / maintenance temperature range includes 2°C-37°C; Optionally, the culture environment may contain 0-5% CO2.

5. The multilayer structure according to any one of claims 1 to 4, characterized in that: The multilayer structure is used for implantation into a target organ or tissue in a corresponding animal body to make it firmly adhered; Optionally, the target organ or tissue is selected from one or any combination of various tumors of organs or tissues such as the brain, heart, pericardium, blood vessels, pleura, peritoneum, liver, gallbladder, bile duct, pancreas, spleen, lung, kidney, bladder, prostate, urethra, oral cavity, gums, tongue, nasal cavity, throat, ear canal, thyroid, esophagus, stomach, intestine, anus, ovary, uterus, penis, testicle, cartilage, bone tissue, bone marrow, nerve tissue, eyeball, eyelid, breast, lymph node, muscle, tendon, skin, fat body, etc.; Optionally, the animal includes one or any several of mice, rats, guinea pigs, golden hamsters, rabbits, dogs, pigs, monkeys, and gorillas.

6. The multilayer structure according to claim 5, characterized in that: The multilayer structure adheres to the tissue through its own adhesion or an adhesive; optionally, the adhesive is selected from one or any combination of cyanoacrylates, hydrogels, melanins, melanin-like substances, celluloses, proteins, medical resins, sugars, and nucleic acids.

7. A method for constructing an animal model of multi-organ tissue tumor in situ / metastasis, characterized in that: The steps include: 1) preparing a protective layer film, optionally, the protective layer film is selected from one or more of a protein film, a fiber film, a synthetic macromolecular film, a metal film, and an inorganic substrate film; 2) Placing tumor cells or tumor tissue on the sealing layer film; 3) preparing an adhesive layer, covering the surface of the protective layer film containing tumor cells or tumor tissues, and bonding and sealing the edges to form a multi-layer structure; 4) The multilayer structure is adhered to the corresponding animal organ or tissue by using its own adhesion or adhesive through surgery to prepare the corresponding animal model.

8. The construction method according to claim 7, characterized in that: The multilayer structure in step 4) adopts the multilayer structure described in any one of claims 1 to 6; Optionally, in step 3), the multilayer structure is placed on a support for supporting or conveniently attaching to organ tissue, and the contact surface is a non-adhesive layer in contact with the support; optionally, it is placed in a culture medium or maintenance medium for culture or maintenance; optionally, in step 4), the multilayer structure is adhered to the corresponding animal organ or tissue and then the support is removed.

9. An animal model of multi-organ tissue tumor in situ / metastasis prepared by the preparation method according to claim 7 or 8.

10. Use of the multilayer structure according to any one of claims 1 to 6 in preparing an animal model of multi-organ tissue tumor in situ / metastasis.

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