Extracellular matrix for three-dimensional cell culture and preparation method thereof

Abstract

The invention relates to the field of cell culture, in particular to an epimatrix for three-dimensional cell culture and a preparation method of the epimatrix. The external matrix comprises xanthan gum, polyvinyl alcohol methyl acrylate, serum protein, arginine, trehalose, mannitol, dimethyl sulfoxide, ethylene glycol and a bacteriostatic agent. During preparation, dimethyl sulfoxide and ethylene glycol are mixed firstly, serum protein and an aqueous solution of xanthan gum are sequentially added to the surface layer of the mixture, oxygen is introduced from the bottom layer of the mixture, and a first precursor solution is obtained; introducing carbon dioxide into the bottom layer of a mixed aqueous solution of trehalose, mannitol and arginine to obtain a second precursor solution; after the first precursor solution and the second precursor solution are uniformly mixed, the polyvinyl alcohol methyl acrylate and the bacteriostatic agent are added and uniformly mixed again to obtain the epimatrix, and the epimatrix highly simulates the epimatrix in the human liver tissue and is beneficial to construction of cell spheres or complex tissue structures closer to the in-vivo physiological state.

Description

Technical Field

[0001] This invention relates to the field of cell culture, and in particular to an exogenous matrix for three-dimensional cell culture and a method for its preparation. Background Technology

[0002] Conventional cell culture typically employs adherent two-dimensional culture. However, due to limited communication between cells, cell populations cannot fully mimic the structural and physiological / biochemical characteristics found in vivo. In contrast, three-dimensional cell culture can more effectively simulate the in vivo environment, thus aiding in its simulation.

[0003] Liver cancer (HCC) is a type of cancer with few or no early symptoms. In HCC research, simulating the physiological environment of hepatocytes is crucial for evaluating anti-HCC drugs. By constructing an exomatrix that closely resembles the physiological environment of hepatocytes, the three-dimensional structure and microenvironment of hepatocytes can be more realistically reproduced. This exomatrix is ​​typically composed of collagen, fibronectin, matrix metalloproteinases, etc., and supports normal hepatocyte functions, such as albumin secretion and urea synthesis. Through this physiological environment simulation, cells can not only better maintain their biological characteristics, but also effectively evaluate the efficacy and toxicity of anti-HCC drugs. For example, hepatocytes exhibit biological responses more similar to those in vivo under this exomatrix, accurately reflecting the inhibitory effect of drugs on liver cancer cells, metastatic behavior, and drug resistance mechanisms.

[0004] However, there is currently a lack of three-dimensional cell matrix that closely resembles the physiological environment of hepatocytes. Summary of the Invention

[0005] The purpose of this invention is to provide an exogenous matrix for three-dimensional cell culture that closely resembles the physiological environment of hepatocytes and a method for its preparation.

[0006] To achieve the above objectives, in a first aspect, the present invention provides the following technical solution:

[0007] An exomatrix for three-dimensional cell culture includes xanthan gum, polyvinyl methyl acrylate, serum protein, arginine, trehalose, mannitol, dimethyl sulfoxide, ethylene glycol, and an antibacterial agent.

[0008] Optionally, the concentration of xanthan gum is any value between 6% and 10%, and the concentration of polyvinyl methyl acrylate is any value between 3% and 5%.

[0009] Optionally, the concentration of the serum protein is any value between 13% and 15%, the concentration of the arginine is any value between 3% and 6%, and the concentration of the antibacterial agent is less than or equal to 1%.

[0010] Optionally, the concentration of trehalose is any value between 19% and 25%, and the concentration of mannitol is any value between 2% and 4%.

[0011] Optionally, the concentration of the dimethyl sulfoxide is any value between 1.3% and 2%, and the concentration of the ethylene glycol is any value between 1% and 2.5%.

[0012] Secondly, the present invention also provides a method for preparing the above-mentioned exogenous matrix for three-dimensional cell culture, comprising:

[0013] The first mixture obtained by mixing the dimethyl sulfoxide and the ethylene glycol;

[0014] An aqueous solution of the serum protein and an aqueous solution of the xanthan gum were sequentially added to the surface of the first mixture, and oxygen was introduced from the bottom layer to obtain the first precursor solution.

[0015] The trehalose, mannitol and arginine are dissolved in water to obtain a second mixture. Carbon dioxide is then introduced into the bottom of the second mixture to obtain a second precursor solution.

[0016] After mixing the first precursor solution and the second precursor solution, a precursor gel is obtained. The polyvinyl methyl acrylate and the antibacterial agent are added to the precursor gel and stirred to obtain an exogenous matrix for three-dimensional cell culture.

[0017] Optionally, the dimethyl sulfoxide and the ethylene glycol are shaken in a constant temperature water bath at any temperature between 35°C and 45°C for 8 min to 15 min, and then allowed to stand for 10 min to 20 min to obtain the first mixture.

[0018] After the trehalose, mannitol and arginine are added to water, they are shaken in a constant temperature water bath at any temperature between 30°C and 40°C for 15 min to 18 min to obtain the second mixture.

[0019] Optionally, the flow rate of the oxygen introduced is any value from 0.8 L / min to 1.5 L / min, and the flow rate of the carbon dioxide introduced is any value from 1 L / min to 1.2 L / min.

[0020] Optionally, the first precursor liquid and the second precursor liquid are stirred in a mixer at a speed of 200 r / min to 300 r / min for 15 min to 30 min to obtain the precursor gel.

[0021] Optionally, the precursor gel, polyvinyl methyl acrylate, and the antibacterial agent are stirred at a speed of 50 r / min to 200 r / min for 5 min to 15 min.

[0022] According to a first aspect of the present invention, xanthan gum has strong viscosity and good compatibility with substances such as collagen and sugars. It can form a network structure that provides three-dimensional support to cells and maintains strong structural stability under different environmental conditions, preventing it from being decomposed by various enzymes secreted by hepatocytes. Compared with other colloidal graft copolymers, such as gelatin and natural guar gum, the structural scaffold formed by xanthan gum has greater elasticity and support capacity, which helps to simulate the strong elasticity of liver tissue. In addition, when simulating the extracellular matrix, the polysaccharide properties of xanthan gum make it closer to the components in natural tissues, and can better simulate the microenvironment of the liver in vivo. Polyvinyl alcohol methyl acrylate (PVA-MA) is readily soluble in water and can form a stable colloidal structure in water. This structure has high strength and toughness, which can strengthen the structure and thus simulate the strong strength and toughness of liver tissue. PVA-MA is chemically stable and has a certain water absorption capacity, which can maintain a certain humidity in a humid environment and effectively adsorb water, inhibiting the denaturation or dehydration of the extracellular matrix during culture. Xanthan gum, a natural polysaccharide, is rich in hydroxyl groups and other hydrophilic groups. Trehalose, also a polysaccharide, contains multiple hydroxyl groups in its molecule. The hydroxyl groups in PVA-MA and the ester groups of methyl acrylate can form hydrogen bonds with hydrophilic groups, as well as achieve physical cross-linking through van der Waals interactions. Serum proteins have abundant amino acid groups, including amino and carboxyl functional groups. These groups can interact with xanthan gum and PVA-MA through hydrogen bonds, electrostatic interactions, or partial covalent cross-linking. The hydrophobic regions in serum proteins may interact with the glycan chains of xanthan gum through hydrophobic interactions, forming relatively stable physical connections. Because the three-dimensional structural framework formed by xanthan gum, PVA-MA, serum proteins, and trehalose is mainly physically cross-linked through hydrogen bonds, it exhibits a certain degree of dynamism, mimicking the highly dynamic characteristics of liver tissue, thus adapting to the needs of hepatocyte regeneration and metabolism. As a major nutrient source, serum proteins can provide hepatocytes with necessary bioactive factors, supporting cell growth and functional expression, especially promoting cell attachment and differentiation. Arginine, as an amino acid, not only plays a crucial role in cell metabolism but also promotes cell proliferation and metabolic function, helping hepatocytes maintain a physiological state similar to that in vivo in vitro. Trehalose and mannitol, as osmotic pressure regulators, effectively regulate the osmotic pressure of the culture medium, maintain intracellular and extracellular water balance, reduce cell damage caused by osmotic pressure changes, and mimic the natural osmotic environment of liver tissue. DMSO and ethylene glycol, as cryoprotectants, effectively prevent the formation of intracellular ice crystals during low-temperature storage, reducing cell damage and thus improving cell survival rate and activity. Antibacterial agents inhibit bacterial infection. The synergistic effect of these components allows the culture medium to mimic the complex microenvironment of liver tissue, providing hepatocytes with a stable condition that supports their function and growth.It should be noted that although the exomatrix in this invention has high adaptability to hepatocytes, it can also be used for the culture of other cells.

[0023] According to a second aspect of the invention, after the xanthan gum aqueous solution is added to the surface of a first mixture containing dimethyl sulfoxide and ethylene glycol, it does not quickly dissolve into a homogeneous phase but floats on the upper layer. At this point, oxygen is introduced from the bottom layer. Because the upper layer is covered with a highly viscous xanthan gum aqueous solution, the oxygen is less likely to escape, providing a slight pressurizing effect and facilitating the full dissolution of poorly soluble oxygen in the liquid. The resulting highly fluid second precursor solution is then mixed with the less fluid first precursor solution, which helps to uniformly disperse the components. The introduction of oxygen and carbon dioxide helps to better simulate the in vivo environment. Introducing oxygen and carbon dioxide into the first and second precursor solutions respectively prevents the excessive dissolution of easily soluble carbon dioxide from hindering the dissolution of poorly soluble oxygen. PVA-MA is added to the precursor gel after the remaining components are fully mixed to prevent the structural framework from being difficult to incorporate other substances after PVA-MA reinforcement. This method of mixing and preparing the components helps to achieve uniform dispersion of the components, thereby simulating the liver tissue environment.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0025] Figure 1 This is a flowchart of a method for preparing an exogenous matrix for three-dimensional cell culture, as shown in Embodiment 1 of the present invention. Detailed Implementation

[0026] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0028] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0029] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] This invention application protects an exomatrix for three-dimensional cell culture, comprising xanthan gum, polyvinyl methyl acrylate, serum protein, arginine, trehalose, mannitol, dimethyl sulfoxide, ethylene glycol, and an antibacterial agent.

[0031] Xanthan gum possesses strong viscosity and good compatibility with collagen, sugars, and other substances. It can form a three-dimensional support network structure for cells and maintains strong structural stability under various environmental conditions, preventing its degradation by various enzymes secreted by hepatocytes. Compared to other colloidal graft copolymers, such as gelatin and natural guar gum, the structural scaffold formed by xanthan gum has greater elasticity and support capacity, helping to simulate the highly elastic characteristics of liver tissue. Furthermore, when simulating the extracellular matrix, the polysaccharide properties of xanthan gum make it closer to the components found in natural tissues, effectively mimicking the in vivo liver microenvironment. Polyvinyl alcohol methyl acrylate (PVA-MA) is readily soluble in water and can form a stable colloidal structure in water. This structure has high strength and toughness, reinforcing the structure and thus simulating the high strength and toughness of liver tissue. PVA-MA is chemically stable and has a certain water absorption capacity, maintaining a certain level of humidity in humid environments and effectively absorbing water, inhibiting denaturation or dehydration of the extracellular matrix during culture. Xanthan gum, a natural polysaccharide, is rich in hydroxyl groups and other hydrophilic groups. Trehalose, also a polysaccharide, contains multiple hydroxyl groups in its molecule. The hydroxyl groups in PVA-MA and the ester groups of methyl acrylate can form hydrogen bonds with hydrophilic groups, as well as achieve physical cross-linking through van der Waals interactions. Serum proteins have abundant amino acid groups, including amino and carboxyl functional groups. These groups can interact with xanthan gum and PVA-MA through hydrogen bonds, electrostatic interactions, or partial covalent cross-linking. The hydrophobic regions in serum proteins may interact with the glycan chains of xanthan gum through hydrophobic interactions, forming relatively stable physical connections. Because the three-dimensional structural framework formed by xanthan gum, PVA-MA, serum proteins, and trehalose is mainly physically cross-linked through hydrogen bonds, it exhibits a certain degree of dynamism, mimicking the highly dynamic characteristics of liver tissue, thus adapting to the needs of hepatocyte regeneration and metabolism. As a major nutrient source, serum proteins can provide hepatocytes with necessary bioactive factors, supporting cell growth and functional expression, especially promoting cell attachment and differentiation. Arginine, as an amino acid, not only plays a crucial role in cell metabolism but also promotes cell proliferation and metabolic function, helping hepatocytes maintain a physiological state similar to that in vivo in vitro. Trehalose and mannitol, as osmotic pressure regulators, effectively regulate the osmotic pressure of the culture medium, maintain intracellular and extracellular water balance, reduce cell damage caused by osmotic pressure changes, and mimic the natural osmotic environment of liver tissue. DMSO and ethylene glycol, as cryoprotectants, effectively prevent the formation of intracellular ice crystals during low-temperature storage, reducing cell damage and thus improving cell survival rate and activity. Antibacterial agents inhibit bacterial infection. The synergistic effect of these components allows the culture medium to mimic the complex microenvironment of liver tissue, providing hepatocytes with a stable condition that supports their function and growth.It should be noted that although the exomatrix in this invention has high adaptability to hepatocytes, it can also be used for the culture of other cells.

[0032] In some embodiments, the concentration of xanthan gum is any value from 6% to 10%, for example, any value from 6%, 7%, 8%, 9%, and 10%, and the concentration of polyvinyl methyl acrylate is any value from 3% to 5%, for example, any value from 3%, 3.5%, 4%, 4.5%, and 5%. Adjusting the concentrations of xanthan gum and polyvinyl methyl acrylate by constraint helps to adjust the elasticity and rigidity of the excipient. All percentage concentrations in this invention are mass fractions.

[0033] In some embodiments, the concentration of serum protein is any value between 13% and 15%, for example, any value between 13%, 13.5%, 14%, 14.5%, and 15%; the concentration of arginine is any value between 3% and 6%, for example, any value between 3%, 4%, 5%, and 6%; and the concentration of the antibacterial agent is less than or equal to 1%, for example, any value between 0.001%, 0.01%, 0.1%, and 1%.

[0034] In some embodiments, the concentration of trehalose is any value between 19% and 25%, for example, any value between 19%, 20%, 21%, 22%, 23%, 24%, and 25%, and the concentration of mannitol is any value between 2% and 4%, for example, any value between 2%, 2.5%, 3%, 3.5%, and 4%.

[0035] In some embodiments, the concentration of dimethyl sulfoxide is any value from 1.3% to 2%, for example, any value from 1.3%, 1.5%, 1.7%, 1.9%, and 2%, and the concentration of ethylene glycol is any value from 1% to 2.5%, for example, any value from 1%, 1.3%, 1.6%, 1.9%, 2.2%, and 2.5%.

[0036] This invention also claims protection for the above-mentioned method for preparing the exogenous matrix for three-dimensional cell culture, comprising:

[0037] S1, the first mixture obtained by mixing dimethyl sulfoxide and ethylene glycol.

[0038] S2. Add an aqueous solution of serum protein and an aqueous solution of xanthan gum to the surface of the first mixture in sequence, and introduce oxygen from the bottom layer to obtain the first precursor solution.

[0039] S3. Dissolve trehalose, mannitol and arginine in water to obtain a second mixture. Pass carbon dioxide through the bottom of the second mixture to obtain a second precursor solution.

[0040] S4. After mixing the first and second precursor solutions, a precursor gel is obtained. Polyvinyl methyl acrylate and an antibacterial agent are added to the precursor gel and stirred to obtain an exomatrix for three-dimensional cell culture.

[0041] In some embodiments, dimethyl sulfoxide and ethylene glycol are shaken in a constant temperature water bath at any temperature between 35°C and 45°C for 8 min to 15 min, and then allowed to stand for 10 min to 20 min to obtain a first mixture.

[0042] After adding trehalose, mannitol, and arginine to water, the mixture is shaken in a constant temperature water bath at any temperature between 30°C and 40°C for 15 min to 18 min to obtain a second mixture.

[0043] When xanthan gum aqueous solution is added to the surface of a first mixture containing dimethyl sulfoxide and ethylene glycol, it does not quickly dissolve into a homogeneous phase but floats on the upper layer. At this point, oxygen is introduced from the bottom layer. Because the upper layer is covered with a viscous xanthan gum aqueous solution, the oxygen is less likely to escape, creating a slight pressurizing effect that helps the poorly soluble oxygen dissolve fully in the liquid. The resulting more fluid second precursor solution is then mixed with the less fluid first precursor solution, which helps to evenly disperse the components. The introduction of oxygen and carbon dioxide helps to better simulate the in vivo environment. Introducing oxygen and carbon dioxide into the first and second precursor solutions respectively prevents the excessive dissolution of easily soluble carbon dioxide from hindering the dissolution of poorly soluble oxygen. Furthermore, oxygen helps to oxygenate and form the first precursor solution, while carbon dioxide helps to carbonize and form the second precursor solution. PVA-MA is added to the thoroughly mixed precursor gel to prevent the structural framework from being difficult to incorporate other substances. This method of mixing and preparing the components helps to evenly disperse the components, thereby simulating the liver tissue environment.

[0044] In some embodiments, the flow rate of oxygen is any value from 0.8 L / min to 1.5 L / min, and the flow rate of carbon dioxide is any value from 1 L / min to 1.2 L / min, for example, any value from 1 L / min, 1.1 L / min and 1.2 L / min.

[0045] In some embodiments, the first precursor solution and the second precursor solution are stirred in a mixer at a speed of any value between 200 r / min and 300 r / min for 15 min to 30 min to obtain a precursor gel. The stirring speed can be, for example, any value between 200 r / min, 220 r / min, 240 r / min, 260 r / min, 280 r / min, and 300 r / min, and the stirring time can be, for example, any value between 15 min, 20 min, 25 min, and 30 min.

[0046] In some embodiments, the precursor gel, polyvinyl methyl acrylate, and antibacterial agent are stirred at a speed of 50 r / min to 200 r / min for 5 min to 15 min. The stirring speed can be, for example, any value among 50 r / min, 100 r / min, 150 r / min, and 200 r / min, and the stirring time can be, for example, any value among 5 min, 7 min, 9 min, 11 min, 13 min, and 15 min.

[0047] Please refer to the following examples for details.

[0048] Example 1:

[0049] Please see Figure 1 The method for preparing the exogenous matrix for three-dimensional cell culture shown in a preferred embodiment of this application includes:

[0050] S1, the first mixture obtained by mixing dimethyl sulfoxide and ethylene glycol.

[0051] S2. Add an aqueous solution of serum protein and an aqueous solution of xanthan gum to the surface of the first mixture in sequence, and introduce oxygen from the bottom layer to obtain the first precursor solution.

[0052] S3. Dissolve trehalose, mannitol and arginine in water to obtain a second mixture. Pass carbon dioxide through the bottom of the second mixture to obtain a second precursor solution.

[0053] S4. After mixing the first and second precursor solutions, a precursor gel is obtained. Polyvinyl methyl acrylate and an antibacterial agent are added to the precursor gel and stirred to obtain an exomatrix for three-dimensional cell culture.

[0054] In this embodiment, all experimental operations were carried out in a sterile environment, and all components were sterilized.

[0055] In step S1, 1.8g of dimethyl sulfoxide and 2g of ethylene glycol are weighed, shaken in a constant temperature water bath at 40°C for 15 minutes, and then allowed to stand for 15 minutes to obtain a first mixture without bubbles.

[0056] Step S2 includes:

[0057] S201. Weigh 8g xanthan gum and 15g serum protein. Add water slowly to the xanthan gum several times while stirring until the xanthan gum is completely dissolved. Dissolve the serum protein in the same way to obtain aqueous solutions of xanthan gum and serum protein, respectively.

[0058] S202. Guided by a glass rod, the aqueous solutions of serum protein and xanthan gum are added sequentially to the surface of the first mixture. Then, a sterile gas delivery tube is inserted into the bottom layer of the first mixture, and oxygen is introduced through the gas delivery tube at a flow rate of 1.2 L / min for about 5 minutes to obtain the first precursor solution.

[0059] In step S3, weigh 22g of trehalose, 3g of mannitol and 6g of arginine, add about 26ml of water, shake in a constant temperature water bath at 35℃ for 15min to obtain a second mixture, and then introduce carbon dioxide into the bottom of the second mixture through a gas delivery tube at a flow rate of 1L / min for about 5min to obtain a second precursor solution.

[0060] Step S4 includes:

[0061] S401. Stir the first and second precursor solutions in a mixer at a speed of 250 r / min for 25 min to obtain the precursor gel.

[0062] S402. Weigh 0.3g kanamycin, 0.2g penicillin, 0.1g nystatin and 4g polyvinyl methyl acrylate, add water and stir thoroughly. Then add the mixture to the precursor gel and stir at 50r / min for 10min. Alternatively, you can stir manually.

[0063] S403. The mixture obtained in step S402 is dehydrated until its total mass is 100g, yielding an exogenous matrix for three-dimensional cell culture. In this embodiment, gradient dehydration is performed in a constant temperature and humidity chamber to prevent over-dehydration; other methods can also be used for dehydration.

[0064] The obtained exomatrix was cut into a shape suitable for culture dishes and placed inside. Cell suspension was added for MIHA cell line culture. After one week of continuous culture, the cell viability was found to be 95.7% and the cell activity was 91.2%.

[0065] Comparative Example 1:

[0066] The only difference between this comparative example and Example 1 is that xanthan gum in step S2 is replaced with gelatin, and polyvinyl alcohol methyl acrylate is not added in step S4. After continuous culture of the MIHA cell line for one week, the cell survival rate was measured to be 93.3% and the cell activity was 88.4%.

[0067] Comparative Example 2:

[0068] The only difference between this comparative example and Example 1 is that the MIHA cell line was cultured in the existing Dulbecco's Modified Eagle Medium (DMEM) basal medium. After one week of continuous culture, the cell viability was measured to be 91.5% and the cell activity was 85.1%.

[0069] The beneficial effects of this invention lie in providing an exomatrix with high compatibility with human hepatocytes and its preparation method, which helps to simulate the human liver environment, thereby constructing cell spheres or complex tissue structures that more closely resemble the physiological state in vivo. Under three-dimensional culture conditions, this exomatrix exhibits excellent temperature control stability and long-term activity maintenance capabilities, making it of great application potential and promotional value in biomedical research, regenerative medicine, and the field of biological cells.

[0070] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An excipient matrix for three-dimensional cell culture, characterized in that, It includes xanthan gum, polyvinyl methyl acrylate, serum protein, arginine, trehalose, mannitol, dimethyl sulfoxide, ethylene glycol, and antibacterial agents.

2. The exomammary matrix for three-dimensional cell culture as described in claim 1, characterized in that, The concentration of xanthan gum is any value between 6% and 10%, and the concentration of polyvinyl methyl acrylate is any value between 3% and 5%.

3. The exomammary matrix for three-dimensional cell culture as described in claim 2, characterized in that, The concentration of the serum protein is any value between 13% and 15%, the concentration of the arginine is any value between 3% and 6%, and the concentration of the antibacterial agent is less than or equal to 1%.

4. The exomammary matrix for three-dimensional cell culture as described in claim 1, characterized in that, The concentration of trehalose is any value between 19% and 25%, and the concentration of mannitol is any value between 2% and 4%.

5. The exomammary matrix for three-dimensional cell culture as described in claim 1, characterized in that, The concentration of the dimethyl sulfoxide is any value between 1.3% and 2%, and the concentration of the ethylene glycol is any value between 1% and 2.5%.

6. The method for preparing an exogenous matrix for three-dimensional cell culture as described in any one of claims 1 to 5, characterized in that, include: The first mixture obtained by mixing the dimethyl sulfoxide and the ethylene glycol; An aqueous solution of the serum protein and an aqueous solution of the xanthan gum were sequentially added to the surface of the first mixture, and oxygen was introduced from the bottom layer to obtain the first precursor solution. The trehalose, mannitol and arginine are dissolved in water to obtain a second mixture. Carbon dioxide is then introduced into the bottom of the second mixture to obtain a second precursor solution. After mixing the first precursor solution and the second precursor solution, a precursor gel is obtained. The polyvinyl methyl acrylate and the antibacterial agent are added to the precursor gel and stirred to obtain an exogenous matrix for three-dimensional cell culture.

7. The method for preparing the exogenous matrix for three-dimensional cell culture as described in claim 6, characterized in that, The dimethyl sulfoxide and the ethylene glycol are shaken in a constant temperature water bath at any temperature between 35°C and 45°C for 8 min to 15 min, and then allowed to stand for 10 min to 20 min to obtain the first mixture. After the trehalose, mannitol and arginine are added to water, they are shaken in a constant temperature water bath at any temperature between 30°C and 40°C for 15 min to 18 min to obtain the second mixture.

8. The method for preparing the exogenous matrix for three-dimensional cell culture as described in claim 6, characterized in that, The oxygen flow rate is any value between 0.8 L / min and 1.5 L / min, and the carbon dioxide flow rate is any value between 1 L / min and 1.2 L / min.

9. The method for preparing the exogenous matrix for three-dimensional cell culture as described in claim 6, characterized in that, The first precursor solution and the second precursor solution are stirred in a mixer at a speed of 200 r / min to 300 r / min for 15 min to 30 min to obtain the precursor gel.

10. The method for preparing the exogenous matrix for three-dimensional cell culture as described in claim 6, characterized in that, The precursor gel, polyvinyl methyl acrylate, and the antibacterial agent are stirred at a speed of 50 r / min to 200 r / min for 5 min to 15 min.

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