Preparation method of recombinant human matrigel

Abstract

The invention discloses a preparation method of recombinant human matrigel, and relates to the technical field of matrigel preparation. The method comprises the following steps: constructing a host cell through a genetic engineering technology to express a full recombinant humanized structural protein; purifying the recombinant protein, preparing a matrix monomer, adding a cross-linking agent, and uniformly mixing; adding a solution into the mixture, and processing to form matrigel; and aseptically packaging and storing the matrigel. The matrigel can be widely applied to the fields of cell 3D culture, tubular culture, medical beauty implant materials and the like, has a remarkable application prospect, and particularly can avoid immune response and rejection when implanted into a human body.

Description

Technical Field

[0001] The invention relates to the technical field of matrix gel preparation, in particular to a method for preparing recombinant human matrix gel. Background Art

[0002] Collagen is the most abundant protein in the human body, accounting for about 25% to 33% of the total body protein. It is widely present in the bones, tendons, cartilage, skin and other connective tissues of the human body. It is the main component of the extracellular matrix and plays an important role in maintaining the normal physiological functions of cells, tissues and organs and repairing injuries.

[0003] In traditional cell culture and biomedical materials research, the matrix glue used is usually derived from animal tissues, such as mouse tail tendon, bovine fascia, and porcine collagen. However, these animal-derived matrix glues have many limitations during use, such as the difficulty in completely avoiding the risk of viral infection, and are prone to induce immune responses after implantation in the human body, leading to rejection reactions. In addition, the extraction process of these materials is complex, the stability is poor, and there are large differences between different batches of products.

[0004] Based on multiple considerations such as environmental protection requirements, process stability and material safety, biomimetic human skin matrix glue prepared with fully recombinant humanized structural proteins shows great application potential. To this end, we proposed a method for preparing recombinant human matrix glue to overcome the shortcomings of the existing technology. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing matrix glue using fully recombinant humanized structural proteins, which has high safety, process stability and wide applicability, and solves the problems that traditional animal-derived matrix glue is prone to induce rejection reactions and process instability.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing recombinant human matrix gel, comprising the following steps:

[0007] S1. Genetic engineering technology transfection:

[0008] By genetic engineering technology, the humanized structural protein gene is inserted into an expression vector, and the vector is transfected into a host cell;

[0009] S2. Cell culture and protein expression:

[0010] Cultivating host cells under the culture conditions of a bionic human skin model, wherein the host cells produce fully recombinant humanized structural proteins;

[0011] S3. Recombinant protein purification:

[0012] Purifying the recombinant humanized structural protein produced by the host cell to obtain a recombinant protein;

[0013] S4. Preparation of matrix monomer and mixing with cross-linking agent:

[0014] Using the recombinant protein to prepare a matrix monomer, adding a cross-linking agent to the matrix monomer, and uniformly mixing the two;

[0015] S5. Matrigel formation:

[0016] adding a solution to the mixture and subjecting it to appropriate processing to form a matrix gel;

[0017] S6. Sterile packaging and storage:

[0018] The formed matrix gel is sterile packaged and stored under specific storage conditions for subsequent use.

[0019] Preferably, the host cell in S1 is a cell strain, and the type of the cell strain is human; the humanized structural proteins in S1 are human collagen I and human collagen III, and the mass ratio of the two is 1:5.

[0020] Preferably, the culture conditions of the bionic human skin model in S2 include: the culture medium used is DMEM / F12, the culture temperature is a constant temperature of 37° C., the humidity range in the culture dish is 70% to 80%, and the CO2 concentration range is 5% to 10%.

[0021] Preferably, the cross-linking agent in S4 is selected from one of glutaminase, oxidase, lipoxygenase, glutamine endopeptidase, peroxidase or horseradish enzyme.

[0022] Preferably, the processing in S5 specifically includes filtering operation and sterilization operation, wherein the filtering operation of the solution is performed by filtering through a filter membrane, and the filter pore diameter of the filter membrane is 0.22 μm.

[0023] Preferably, the pH value of the solution in S5 is in the range of 6.0-7.0, and the solution comprises a physiological saline solution or a phosphate buffer solution of salt and a buffer.

[0024] Preferably, the matrix gel in S5 forms a gel state in a water bath, and the water bath temperature ranges from 37°C to 45°C.

[0025] Preferably, the storage conditions of the matrix gel in S6 include a storage temperature range of 4°C to 25°C, a storage environment of a sterile environment under light-proof conditions, and a storage period of the matrix gel of 3 to 6 months.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention uses human cell lines as host cells, avoiding the use of protein extraction processes from animals such as mice, cattle, and pigs. Human cell lines can ensure that the produced protein is of high purity, has no safety hazards, is suitable for large-scale production, and can effectively avoid immune responses and rejection reactions when implanted into the human body.

[0028] 2. The present invention uses human collagen I and human collagen III as humanized structural proteins, with a mass ratio of 1: 5. This ratio ensures that the matrix glue contains a rich variety of structural proteins, better simulates the structure and characteristics of human skin, and improves the biological properties of the matrix glue.

[0029] 3. When the matrix gel is heated to 37℃~45℃ in a water bath, it can form a gel state and can be cultured together with cells. It is suitable for 3D cell culture, tubular culture, organoid 3D printing ink materials and medical beauty implants.

[0030] 4. This method simplifies the preparation process, reduces production costs, and has good production consistency, and can be widely used in the fields of biomedicine and materials engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart of a method for preparing a recombinant human matrix gel of the present invention;

[0032] Figure 2 This is a diagram of animal tissue sections after implantation of the present invention;

[0033] Figure 3 This is the SEM image A of the present invention;

[0034] Figure 4 This is the electron microscope SEM image B of the present invention;

[0035] Figure 5 This is the electron microscope SEM image C of the present invention;

[0036] Figure 6 This is the SEM image D of the present invention;

[0037] Figure 7 This is an analysis diagram of the storage modulus and loss modulus of the recombinant human matrix gel of the present invention under different oscillation strains;

[0038] Figure 8 This is an analysis diagram of the storage modulus and loss modulus of the porcine collagen of the present invention under different oscillation strains;

[0039] Fig. 9 A is the molecular weight distribution diagram of the recombinant human matrix gel sample of the present invention;

[0040] Fig.10Figure B is the molecular weight distribution diagram of the recombinant human matrix gel sample of the present invention;

[0041] Fig.11 This is a comparative diagram of tanδ analysis of different materials of the present invention. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Reference Figure 1 The present invention provides a method for preparing recombinant human matrix gel, and the detailed steps are as follows:

[0044] S1 genetic engineering technology transfection:

[0045] Through genetic engineering technology, the humanized structural protein gene is inserted into the expression vector, and then the expression vector is transfected into the host cell through transfection technology. The host cell can be a human fibroblast, which usually expresses the full chain of type III collagen. The host cell is a human cell line, avoiding the use of animal-derived cells such as mice, cattle, and pigs, so as to ensure that the extracted protein is of high purity, good safety, and suitable for large-scale industrial production.

[0046] Unlike animal-derived matrix glue, the proteins expressed by human host cells are not easily recognized by the human immune system, which can effectively reduce the risk of rejection after implantation. The matrix glue prepared by the present invention using fully recombinant humanized structural proteins has high biocompatibility and has broad application prospects in the fields of medical treatment, beauty, etc.

[0047] In addition to human cell lines, host cells can also be selected from Pichia pastoris, Escherichia coli or insect cells. The advantage of choosing Pichia pastoris is that its expression process is relatively simple, its expression efficiency is high, and it is suitable for large-scale production of high-quality recombinant proteins; Escherichia coli grows fast, is easy to culture, and has low production costs; insect cells show high stability and expression diversity.

[0048] S2 cell culture and protein expression:

[0049] After the expression vector is successfully transfected into the host cells, the host cells are cultured under the conditions of a bionic human skin model. The culture medium uses DMEM / F12, the temperature is kept constant at 37°C, the humidity in the culture dish is controlled between 70% and 80%, and the CO2 concentration is 5% to 10%. Under these optimized conditions, the host cells can effectively express fully recombinant humanized structural proteins.

[0050] If the culture process fails, it may be necessary to adjust the culture conditions, such as increasing the CO2 concentration or adjusting the culture medium components, until the host cells can successfully produce the target protein.

[0051] S3 recombinant protein purification:

[0052] After successfully cultivating fully recombinant humanized structural proteins, the next step is to purify these proteins. Common purification steps include centrifugation, filtration, and column chromatography to ensure that impurities are removed and high-purity recombinant proteins are obtained. Centrifugation is used to remove cell debris and other insoluble substances, filtration further removes fine impurities, and column chromatography is used to separate proteins to improve their purity.

[0053] During this process, technicians can improve the purification efficiency by adjusting the centrifugal speed, selecting filter membranes with different pore sizes, and optimizing column chromatography conditions. If the purity of the protein in a certain step does not meet the standard, the process can be optimized according to the specific situation.

[0054] S4 matrix monomer preparation and cross-linking agent mixing:

[0055] The purified recombinant protein is used to prepare the matrix monomer, and then a cross-linking agent is added to the matrix monomer and mixed evenly. The optional cross-linking agent includes one of glutaminase, oxidase, lipoxygenase, glutamine endopeptidase, peroxidase or horseradish enzyme. The role of the cross-linking agent is to help form cross-links between matrix monomer molecules, thereby improving the physical and chemical stability of the matrix glue.

[0056] Among them, glutaminase is a protease that can break down proteins into peptides and amino acids, and further cross-link them into strong materials. Oxidase cross-linkers can promote cross-linking between molecules through oxidation reactions, while lipoxygenase can cross-link fatty acids into materials with elasticity and fullness. Peroxidase and horseradish enzyme are usually used as oxidants, and their cross-linking speed is fast and the effect is significant.

[0057] S5 Matrigel Formation:

[0058] A mixture of matrix monomers and cross-linking agents that have been mixed evenly is added to a specific solution, which can be a physiological saline solution or a phosphate buffer solution of salt and buffer. Through appropriate processing, matrix glue is formed. In a water bath environment of 37°C to 45°C, the matrix glue can form a gel state. Under the condition of water bath heating, the matrix glue can be used in multiple biomedical fields such as 3D cell culture and organoid culture, showing a wide range of application potential.

[0059] During the processing, filtration and sterilization are usually required. 0.22μm filter membrane is used for filtration, which can effectively remove impurities and microorganisms in the solution, thus ensuring the sterility of the product.

[0060] S6 Sterile Packaging and Storage:

[0061] Finally, after the prepared matrix gel is sterile packaged, it needs to be stored under specific conditions. The storage temperature should be kept between 4°C and 25°C. Too high or too low a temperature may affect the quality and stability of the matrix gel. A reasonable storage environment can ensure that the matrix gel maintains its physical and chemical properties during long-term storage.

[0062] When selecting host cells, the present invention specifically specifies the use of human cell lines. Compared with the use of animal cell lines (such as mouse or pig), the advantage of human cell lines is that their expression products are closer to the proteins in the human physiological environment, reducing the risk of rejection reactions that may be caused in subsequent applications.

[0063] In the present invention, human collagen I and human collagen III are selected as structural proteins. These two collagens are the main structural proteins in human skin and can simulate the structure and biomechanical properties of natural skin. The mass ratio of human collagen I and III is optimized to 1:5, which can ensure the diversity and adaptability of collagen in the prepared matrix glue, so as to be better applied to the fields of cell culture and tissue engineering.

[0064] The present invention uses a specific culture medium DMEM / F12 in the cell culture process. This culture medium can provide sufficient growth support for host cells due to its richness and diversity of nutrients. The culture temperature is precisely controlled at 37°C, which is the normal physiological temperature of the human body, ensuring that the cells grow under conditions close to the in vivo environment.

[0065] The humidity of the culture dish is controlled at 70% to 80%, and the CO2 concentration is maintained at 5% to 10%. These parameters are precisely set according to the needs of the cells to ensure that the cells can efficiently express structural proteins in a suitable microenvironment. Too high or too low humidity and CO2 concentration will affect cell growth and protein expression, so the control of these parameters is crucial.

[0066] Cross-linking agents play a key role in the formation process of matrigel. They can form strong cross-links between protein molecules, thereby enhancing the physical stability of matrigel. The present invention particularly provides a variety of cross-linking agents for selection, so as to flexibly adjust according to different application requirements and preparation conditions. Glutaminase can form polypeptide cross-links by decomposing proteins, and oxidase and peroxidase can form strong cross-linked structures by promoting oxidation reactions. Each cross-linking agent has its own unique cross-linking mechanism and biocompatibility, and a suitable cross-linking agent can be selected according to practical applications.

[0067] In order to ensure the sterility and high purity of the final product, the present invention is particularly provided with strict filtering and sterilization operations in the processing stage. The filtering process is carried out through a filter membrane with a pore size of 0.22 μm, and this specification of filter membrane can effectively filter out microorganisms and impurities in the solution to ensure that the solution meets the sterility requirements. The sterilization operation is usually carried out by high temperature or chemical methods to ensure that the prepared matrix glue will not be contaminated by microorganisms, extend its storage period and ensure the safety of use.

[0068] In the process of forming the matrix gel, the pH value of the solution is crucial for the stability of the protein and the activity of the cross-linking agent. The pH value of the solution of the present invention is accurately controlled between 6.0 and 7.0, which is a range close to neutrality, which can ensure that the natural conformation of the protein is maintained and avoid protein denaturation under acidic or alkaline conditions. The selected physiological saline solution or phosphate buffer provides a stable ionic environment for the solution, ensuring that the matrix gel can be smoothly formed.

[0069] The matrix glue in the present invention can form a stable gel state when heated to 37°C to 45°C in a water bath. This temperature range is optimized to ensure that the matrix glue can be smoothly transformed from a liquid state to a gel state. This gelation process is not only stable but also controllable and can be used in multiple application scenarios, such as 3D cell culture and tissue engineering. By heating in a water bath, a gel can be quickly formed when needed, which is convenient for operation and application.

[0070] In order to ensure that the prepared matrix glue can maintain its physical and chemical properties for a long time, the present invention particularly provides its storage conditions. The matrix glue should be stored in a temperature range of 4°C to 25°C, and this temperature range can effectively extend its service life. At the same time, light-proof and sterile environments are also crucial to prevent degradation or contamination of the matrix glue caused by light and pollution. Through this storage condition, the matrix glue can be safely stored for 3 to 6 months without affecting its quality and performance.

[0071] Example 1: Application of recombinant human matrix gel in 3D cell culture

[0072] Prepare cells: Select the cells that need to be cultured (such as liver cells, kidney cells or breast tumor cells, etc.) and process them according to specific needs, such as pre-inducing differentiation or transfecting plasmids.

[0073] Prepare Matrigel and prepare recombinant human Matrigel in a culture dish. Add the recombinant human Matrigel solution to the culture dish and solidify at 37°C for about 30 minutes. During solidification, the gel solution needs to be evenly distributed to form a consistent 3D structure; the concentration of Matrigel should be appropriately selected according to the cell type and the needs of the culture experiment.

[0074] Cell seeding: Add the treated cells to the recombinant human matrix gel, and evenly add the cell suspension to the gelled matrix, and culture under appropriate temperature, CO2 and humidity conditions.

[0075] Adding factors and experiments: Add required factors or perform cell tests and experiments during the culture period as needed; factors can be growth factors, drugs, etc.

[0076] Sampling and analysis: After the culture period is over, collect the cells from the culture dish and use appropriate methods to analyze the growth, proliferation and differentiation of the cells in the recombinant human matrix gel.

[0077] Cell experiment data:

[0078]

[0079]

[0080] Cell differentiation data:

[0081]

[0082] According to the analysis of experimental data, under 3D culture conditions, hepatocytes showed a higher proliferation rate (150%) and a good differentiation rate (30%), which indicates that the recombinant human matrix gel provides a suitable growth and differentiation environment for hepatocytes.

[0083] The proliferation rate of renal cells was relatively low (120%), but the differentiation rate was high (40%), which may be related to the specific chemical composition or physical properties of the matrix gel, which facilitated the differentiation and functional expression of renal cells.

[0084] Tumor cells showed rapid proliferation characteristics (200%) and low differentiation rate (10%) in Matrigel. This finding suggests that we need to pay special attention to its potential impact on tumor growth when using Matrigel for tumor-related research.

[0085] Example 2: Application of recombinant human matrix gel in new medical implant materials

[0086] Cell culture and protein extraction:

[0087] Select a suitable cell line, such as human skin fibroblasts, use DMEM / F12 medium, and culture the cells until they reach a certain density.

[0088] After the cells reach a sufficient density, the cells are collected, lysed and the desired recombinant protein is extracted, purified and concentrated.

[0089] Preparation of Matrigel:

[0090] An appropriate amount of cross-linking agent, such as glutaminase, is added to the extracted recombinant protein to cause a cross-linking reaction to form matrix gel.

[0091] After washing and drying, recombinant human matrix gel that meets quality requirements is prepared.

[0092] Preparation of implant material:

[0093] Before performing the operation, the appropriate implant material is selected according to the patient's actual situation, such as polylactic acid-glycolic acid copolymer (PLGA) for soft tissue enhancement.

[0094] Application of Matrigel:

[0095] Evenly apply a certain amount of recombinant human matrix gel around the implant material.

[0096] The implant material coated with recombinant human matrix gel is implanted into the patient's body.

[0097] Effect observation after implantation:

[0098] At designated time points after implantation (eg, 1 month, 3 months, and 6 months), tissue biopsy samples of the implantation site were obtained by minimally invasive surgery.

[0099] Standard histological staining techniques (such as HE staining) were used to observe the morphology and structure of the tissue.

[0100] refer to Figure 2 Content, showing animal tissue sections after implantation, which are degradable and produce a large number of fibroblasts. The implanted matrix gel has good biocompatibility in the body and can promote cell growth and tissue repair.

[0101] This suggests that recombinant human Matrigel is gradually degraded in vivo, accompanied by the generation of a large number of fibroblasts, which are key cell types in normal wound healing and tissue repair processes.

[0102] Microstructure Analysis:

[0103] The microstructure of the matrix gel was observed using a scanning electron microscope (SEM). Figure 3-Figure 6As shown in Figure 1, the electron microscope SEM image directly displays the microstructure and surface morphology of the matrix glue. This microstructure is crucial for cell adhesion and growth, and is also a key factor affecting the biocompatibility of the material.

[0104] In order to evaluate the physical properties of recombinant human matrix gel, the elastic modulus and viscous modulus of the matrix gel were tested using a rheometer. Figure 7 Matrigel exhibits good mechanical stability in surgical implant materials.

[0105] Figure 7 The storage modulus (G') and loss modulus (G") of recombinant human matrix gel under different oscillation strains are shown. The blue curve represents the storage modulus (G'), showing the elasticity of the material, while the green curve represents the loss modulus (G"), showing the viscosity of the material.

[0106] As can be seen from the figure, as the strain increases, the storage modulus of the matrix gel decreases slightly, but the overall elasticity remains high. This indicates that the recombinant human matrix gel can provide sufficient mechanical support when implanted in the body and maintain good elasticity under stress conditions;

[0107] The rheometer results showed that the matrix glue maintained sufficient elastic modulus under low stress and had an appropriate viscosity modulus to provide sufficient mechanical support during implantation. This property makes the matrix glue suitable for soft tissue repair and reinforcement materials.

[0108] Figure 8 The comparison of elastic modulus and viscous modulus between recombinant human matrix gel and traditional porcine collagen material is shown. Experiments show that the blue curve represents the storage modulus and the green curve represents the loss modulus. As can be seen from the figure, the elastic modulus (storage modulus G') of porcine collagen is lower than that of recombinant human matrix gel, and the storage modulus decreases faster under higher strains. The recombinant matrix gel is superior to porcine collagen material in biomechanical properties. It has a higher elastic modulus and better viscosity, which enables it to better support cell adhesion and growth during tissue repair after implantation. In contrast, porcine collagen materials are prone to degradation and deformation in long-term applications, affecting the repair effect;

[0109] Example 3: Degradation performance of recombinant human matrix gel and in vivo implantation experiment

[0110] 1. Degradation performance test of recombinant matrix gel

[0111] In this experiment, we evaluated the degradation performance of recombinant human Matrigel through an in vitro simulation experiment. Matrigel was placed in simulated body fluid (PBS buffer, pH 7.4) and its degradation was monitored at a constant temperature of 37°C. At different time points, Matrigel samples were taken out and their mass changes and molecular weight distribution were measured to evaluate their degradation rate.

[0112] Molecular weight distribution analysis: Gel permeation chromatography (GPC) was used to test the molecular weight distribution of matrix gel. Fig. 9 and Fig.10 The molecular weight distribution of different samples is shown. By comparing the different molecular weight components, the relationship between the degradation process of matrix gel and the changes in molecular structure can be evaluated.

[0113] Fig. 9 : The molecular weight distribution of the first group of samples of recombinant human matrix gel is shown, with peaks at 1383 and 5322. The GPC results in the table show that the molecular weight distribution of the recombinant matrix gel is uniform and has good polydispersity, which helps it maintain structural stability during degradation.

[0114] Fig.10 :The molecular weight distribution of another group of matrix gel samples is shown, with the main peaks at 1488 and 534, indicating that the molecular weight distribution of samples under different conditions is slightly different. This difference may be related to the use of cross-linking agents or different preparation processes.

[0115] Experimental conclusion: The molecular weight distribution diagram shows that recombinant human matrix gel can maintain good molecular structure integrity during the degradation process and is suitable for short-term implantation materials. Its degradation rate can be adjusted according to the molecular weight distribution characteristics.

[0116] 2. Mechanical properties and material viscoelasticity analysis

[0117] To further evaluate the changes in the mechanical properties of recombinant Matrigel during degradation, we used a rheometer to measure the elastic modulus and viscous modulus of Matrigel, especially to analyze its viscoelastic behavior by tanδ. Fig.11 shown.

[0118] Fig.11 : Analysis of elastic and viscous behavior: Fig.11 The performance of different materials in viscoelastic behavior is shown, and tanδ (loss factor) is used to evaluate the ductility of the material. The analysis shows that when the tanδ value is less than 1, the material exhibits high elasticity and can maintain sufficient mechanical support during the degradation process.

[0119] Result analysis:

[0120] The figure compares porcine collagen and several different recombinant human matrix gel samples. Experiments show that the tanδ value of recombinant matrix gel is less than 1, indicating that its elasticity is better than viscosity and can provide sufficient mechanical support in the early stage of implantation.

[0121] This also indicates that recombinant matrix gel can better support cell adhesion and growth during implantation and is particularly suitable for tissue repair.

[0122] Experimental conclusion: Recombinant human matrix gel has a high elastic modulus under low strain conditions and a moderate viscous modulus, indicating that the material can maintain sufficient mechanical properties and gradually degrade after implantation, and is suitable for soft tissue repair applications.

[0123] 3. In vivo implantation experiment and tissue compatibility assessment

[0124] In order to verify the biocompatibility of recombinant human matrix gel, we conducted an in vivo implantation experiment in a mouse model. The matrix gel was implanted subcutaneously in the back of the mouse, and samples were taken at different time points (1 month, 3 months, and 6 months) to evaluate its degradation and the effect on the surrounding tissues.

[0125] Tissue compatibility observation: Tissue sections taken at different time points showed that the recombinant matrix gel was gradually absorbed and did not cause obvious inflammatory response. Tissue regeneration experiments showed that the degradation process of matrix gel was synchronized with tissue repair, promoting the proliferation of fibroblasts and the formation of new blood vessels.

[0126] Experimental conclusion: After implantation, the matrix gel showed good biocompatibility, gradually degraded and promoted tissue repair. This property makes it have a wide range of application prospects in soft tissue repair and medical aesthetic implants.

[0127] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing recombinant human matrix gel, characterized in that: The following steps are involved: S1. Genetic engineering technology transfection: By genetic engineering technology, the humanized structural protein gene is inserted into an expression vector, and the vector is transfected into a host cell; S2. Cell culture and protein expression: Cultivating host cells under the culture conditions of a bionic human skin model, wherein the host cells produce fully recombinant humanized structural proteins; S3. Recombinant protein purification: Purifying the recombinant humanized structural protein produced by the host cell to obtain a recombinant protein; S4. Preparation of matrix monomer and mixing with cross-linking agent: Using the recombinant protein to prepare a matrix monomer, adding a cross-linking agent to the matrix monomer, and uniformly mixing the two; S5. Matrigel formation: adding a solution to the mixture and subjecting it to appropriate processing to form a matrix gel; S6. Sterile packaging and storage: The formed matrix gel is sterile packaged and stored under specific storage conditions for subsequent use.

2. The method for preparing a recombinant human matrix gel according to claim 1, characterized in that: The host cell in the S1 is a cell strain, and the type of the cell strain is human; the humanized structural proteins in the S1 are human collagen I and human collagen III, and the mass ratio of the two is 1:

5.

3. The method for preparing a recombinant human matrix gel according to claim 1, characterized in that: The culture conditions of the bionic human skin model in S2 include: the culture medium used is DMEM / F12, the culture temperature is a constant temperature of 37° C., the humidity range in the culture dish is 70% to 80%, and the CO2 concentration range is 5% to 10%.

4. The method for preparing a recombinant human matrix gel according to claim 1, characterized in that: The cross-linking agent in S4 is selected from one of glutaminase, oxidase, lipoxygenase, glutamine endopeptidase, peroxidase or horseradish enzyme.

5. The method for preparing a recombinant human matrix gel according to claim 1, characterized in that: The processing in S5 specifically includes filtering operation and sterilization operation, wherein the filtering operation of the solution is performed by filtering through a filter membrane, and the filter pore diameter of the filter membrane is 0.22 μm.

6. The method for preparing a recombinant human matrix gel according to claim 1, characterized in that: The pH value of the solution in S5 is in the range of 6.0-7.0, and the solution includes a physiological saline solution or a phosphate buffer solution of salt and a buffer.

7. The method for preparing a recombinant human matrix gel according to claim 1, characterized in that: The matrix gel in S5 forms a gel state in a water bath, and the water bath temperature ranges from 37°C to 45°C.

8. The method for preparing a recombinant human matrix gel according to claim 1, characterized in that: The storage conditions of the matrix gel in S6 include a storage temperature range of 4°C to 25°C, a storage environment of a sterile environment under light-proof conditions, and a storage period of the matrix gel of 3 to 6 months.

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