A freeze-dried type of matrigel and its preparation method and application

Abstract

This invention relates to the field of biomedical materials technology, disclosing a lyophilized matrix gel, its preparation method, and its applications. The lyophilized matrix gel is prepared by dissolving a natural basement membrane extract in high-glucose DMEM; the natural basement membrane extract is derived from EHS mouse tumor tissue rich in extracellular matrix proteins. The lyophilized matrix gel provided by this invention uses a natural basement membrane extract as the core active ingredient and is prepared through an optimized freeze-drying process. After reconstitution, it can rapidly restore the three-dimensional fiber network structure, retaining the biological functions of active ingredients such as collagen IV, laminin, and nestin in the basement membrane, and has high storage stability. It also boasts advantages such as good reconstitution effect and good cell compatibility, and can be widely used in cell culture and in vitro disease model construction, solving the technical pain points of traditional liquid matrix gels, such as the need for low-temperature storage, high transportation costs, and easy degradation and inactivation.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a freeze-dried matrix adhesive, its preparation method, and its application. Background Technology

[0002] Matrix is ​​an important component of the extracellular matrix, mainly composed of biomacromolecules such as laminin (approximately 60%), type IV collagen (approximately 30%), nestin (approximately 8%), and heparan sulfate proteoglycans. It also contains small amounts of cytokines and glycoproteins (approximately 2% in total). It plays multiple functions, including supporting cell adhesion, regulating cell proliferation and differentiation, and maintaining tissue morphology and structure, thus playing an irreplaceable role in biomedical research and clinical applications.

[0003] Most commercially available matrix adhesives are in liquid form and require freezing storage below -20°C. They must be used within a short time after thawing, otherwise they are prone to loss of activity due to protease degradation or microbial contamination. These liquid matrix adhesives have stringent requirements for storage and transportation conditions, necessitating a continuous cold chain (-20°C to -80°C). This not only increases logistics costs but also poses a significant quality risk in remote areas or during long-distance transportation, as the cold chain is easily disrupted if the product fails.

[0004] To address the aforementioned issues, existing technologies attempt to improve the stability of matrix gels by adding protective agents (such as trehalose) and then freeze-drying them. However, this method still has some drawbacks, such as incomplete recovery of fiber structure after reconstitution, reduced cell adhesion rate, and low success rate in constructing in vitro models. Therefore, developing a freeze-dried matrix gel that is stable in storage, has good reconstitution properties, and high bioactivity is of significant practical importance. Summary of the Invention

[0005] In order to overcome the problems of high cold chain costs, poor reconstitution structure recovery due to the addition of protective agents in existing freeze-drying technologies, low cell adhesion rate, and high failure rate of in vitro model construction, this invention provides a freeze-dried matrix adhesive, its preparation method, and its application.

[0006] The technical solution of the present invention is as follows: A lyophilized matrix gel is prepared by dissolving a natural basement membrane extract in high-sugar DMEM; the natural basement membrane extract is derived from EHS mouse tumor tissue rich in extracellular matrix proteins.

[0007] Furthermore, an antibiotic combination is added to the high-sugar DMEM, and the final concentration of the antibiotic combination in the solvent system is: gentamicin 50 µg / mL, penicillin 100 U / mL, and streptomycin 0.1 mg / mL.

[0008] Furthermore, the natural basement membrane extract comprises laminin, type IV collagen, nestin, heparan sulfate proteoglycan, and a group of cytokines, wherein the group of cytokines includes at least IGF-1, TGF-β, VEGF, EGF, or bFGF.

[0009] The present invention also provides a method for preparing a lyophilized matrix adhesive, comprising any of the lyophilized matrix adhesives described above, including the following preparation steps: (1) Preparation of matrix gel: Select EHS mouse tumor tissue, add salt solution to thaw at a low temperature of 2–8℃; after thawing, homogenize, centrifuge to remove supernatant, add extraction buffer to dissolve basement membrane protein complex; centrifuge again to remove tissue residue, retain supernatant, and obtain basement membrane extract, i.e. matrix gel, after protein purification. (2) Freeze-drying: Dispense the matrix gel obtained in step (1) into sterile freeze-drying vials, dispensing 1–10 mL into each vial and sealing them for storage at -80°C; remove the aluminum cap before freeze-drying, press the silicone soft stopper halfway into the bottle mouth, place it in a freeze dryer, pre-freeze at -50°C for 1 to 5 hours, then reduce the vacuum to below 10 Pa and maintain at -50°C for 5 hours, then raise the temperature to -25°C and maintain for 5 hours, then raise the temperature to -15°C and maintain for 3 hours, then raise the temperature to -10°C and maintain for 3 hours, and finally raise the temperature to 25°C and maintain for 6 hours to obtain a loose and porous freeze-dried matrix gel; (3) Sealed packaging: The freeze-dried matrix adhesive is sealed in a sterile environment to obtain the finished product.

[0010] Furthermore, step (1) involves extraction with a salt solution containing EDTA, which inhibits the activity of matrix metalloproteinases by chelating calcium ions, thereby reducing protein degradation.

[0011] Furthermore, in step (1), a high-speed tissue homogenizer is used for homogenization, and the homogenizer is homogenized at 8000–10000 rpm for 30 min and then centrifuged at 13000 rpm.

[0012] Furthermore, the heating rate of step (2) throughout the entire heating phase is 1–2 °C / min.

[0013] This invention provides an application of lyophilized matrix gel in cell culture. The lyophilized matrix gel is reconstituted in sterile deionized water pre-cooled to below 4 °C in a clean bench at a reconstitution ratio of 8 mg / mL to 10 mg / mL. After vortexing at 25 °C for 3 minutes, it is transferred to a 4 °C freezer and left to stand overnight. The next day, it is removed and placed on crushed ice. The matrix gel solution is then repeatedly pipetted and mixed in a clean bench. Subsequently, the matrix gel solution is diluted with culture medium to the required concentration or used directly. It is then evenly spread on the surface of a cell culture plate and incubated at 37 °C for 30 minutes to form a gel layer. Finally, tumor cells, stem cells, or epithelial cells are inoculated to promote cell adhesion, proliferation, and differentiation.

[0014] This invention also provides an application of lyophilized matrix gel in the construction of in vitro disease models. The reconstituted matrix gel is used to construct a three-dimensional cell culture model, i.e. an organoid model, to simulate the in vivo microenvironment for studying physiological mechanisms, inflammatory responses, or drug screening.

[0015] The beneficial effects of the present invention include at least the following: The freeze-dried matrix gel provided by this invention uses natural basement membrane extract as the core active ingredient and is prepared through an optimized freeze-drying process. After reconstitution, it can rapidly restore the three-dimensional fiber network structure, retain the biological functions of active components such as collagen IV, laminin, and nestin in the basement membrane, and has high storage stability (more than 12 months at room temperature), eliminating the dependence on cold chain at -20℃ to -80℃, reducing logistics costs by more than 50%. At the same time, it has the advantages of good reconstitution effect (reconstituted with sterile water pre-cooled at 4℃ at a ratio of 8-10mg / mL, completely dissolved within 15 minutes after vortexing at 25℃ for 3 minutes and standing overnight at 4℃) and good cell compatibility (organoid growth rate ≥95%, cell adhesion rate ≥98%, significantly better than the control group containing trehalose). It can be widely used in cell culture and in vitro disease model construction, solving the technical pain points of traditional liquid matrix gels, such as the need for low-temperature storage, high transportation costs, and easy degradation and inactivation. Moreover, the formulation does not contain any sugar protectants, exogenous proteases or microorganisms, and can form a gel after incubation at 37℃ for 30 minutes, which can directly support the efficient adhesion, proliferation and differentiation of tumor cells, stem cells and epithelial cells. Attached Figure Description

[0016] Figure 1 The morphology diagrams of the freeze-dried matrix adhesive obtained in Example 2 of this invention are compared with those of the conventional freeze-dried matrix adhesive.

[0017] Figure 2 The image shows the morphology of the freeze-dried matrix adhesive obtained in Example 2 of this invention after reconstitution and solidification at 37°C.

[0018] Figure 3This is a comparison of the cell compatibility of lyophilized matrix gels under different trehalose concentrations and reconstitution conditions in Example 3.

[0019] Figure 4 This is a morphological diagram of the iPSC supported by the lyophilized matrix gel in Example 3 under a conventional optical microscope.

[0020] Figure 5 This is a morphological diagram of the in vitro angiogenesis obtained in Example 3 under a conventional optical microscope.

[0021] Figure 6 This is a morphological diagram of the human colorectal organoid obtained in Example 5 under a conventional optical microscope.

[0022] Figure 7 The image shows the morphological structure of the lung adenocarcinoma organoids obtained in Example 6 under a conventional optical microscope. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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.

[0024] Example 1 This embodiment provides a lyophilized matrix gel, which is made by dissolving a natural basement membrane extract in high-sugar DMEM; the natural basement membrane extract is derived from EHS mouse tumor tissue rich in extracellular matrix proteins and does not contain exogenous protective agents.

[0025] Furthermore, an antibiotic combination is added to the high-sugar DMEM, and the final concentration of the antibiotic combination in the solvent system is: gentamicin 50 µg / mL, penicillin 100 U / mL, and streptomycin 0.1 mg / mL.

[0026] Furthermore, the natural basement membrane extract comprises laminin, type IV collagen, nestin, heparan sulfate proteoglycan, and a group of cytokines, wherein the group of cytokines includes at least IGF-1, TGF-β, VEGF, EGF, or bFGF.

[0027] The proportions of the components in the natural basement membrane are as follows: laminin 60%, type IV collagen 30%, nestin 8%, and the remainder 2%.

[0028] The lyophilized matrix gel provided in this embodiment is composed of natural basement membrane extract, high-sugar DMEM and the above-mentioned antibiotic combination, and does not contain any exogenous protective agents.

[0029] Example 2 This embodiment provides a method for preparing a lyophilized matrix adhesive, including any of the lyophilized matrix adhesives described above, comprising the following preparation steps: (1) Preparation of matrix gel: Select EHS mouse tumor tissue, add salt solution to thaw at a low temperature of 2–8℃; after thawing, homogenize, centrifuge to remove supernatant, add extraction buffer to dissolve basement membrane protein complex; centrifuge again to remove tissue residue, retain supernatant, and obtain basement membrane extract, i.e. matrix gel, after protein purification. (2) Freeze-drying: Dispense the matrix gel obtained in step (1) into sterile freeze-drying vials, dispensing 1–10 mL into each vial and sealing them for storage at -80°C; remove the aluminum cap before freeze-drying, press the silicone soft stopper halfway into the bottle mouth, place it in a freeze dryer, pre-freeze at -50°C for 1 to 5 hours, then reduce the vacuum to below 10 Pa and maintain at -50°C for 5 hours, then raise the temperature to -25°C and maintain for 5 hours, then raise the temperature to -15°C and maintain for 3 hours, then raise the temperature to -10°C and maintain for 3 hours, and finally raise the temperature to 25°C and maintain for 6 hours to obtain a loose and porous freeze-dried matrix gel; (3) Sealed packaging: The freeze-dried matrix adhesive is sealed in a sterile environment to obtain the finished product.

[0030] Furthermore, step (1) involves extraction with a salt solution containing EDTA, which inhibits the activity of matrix metalloproteinases by chelating calcium ions, thereby reducing protein degradation.

[0031] Furthermore, in step (1), a high-speed tissue homogenizer is used for homogenization, and the homogenizer is homogenized at 8000–10000 rpm for 30 min and then centrifuged at 13000 rpm.

[0032] Furthermore, the heating rate of step (2) throughout the entire heating phase is 1–2 °C / min.

[0033] This embodiment also provides a freeze-dried matrix adhesive of Comparative Example 1, the preparation steps of which are as follows: 1. Prepare the basement membrane extract according to step (1) of Example 1; 2. Preparation of protective agent solution: Weigh 25 g of trehalose, add 100 mL of sterile deionized water to dissolve, filter sterilize through 0.22 μm filtration, and store at 4 ℃; 3. Preparation of the mixture: Mix the matrix adhesive and the protective agent solution at a volume ratio of 4:1, and slowly mix on a shaker at 2–8 ℃ for 30 min to form the matrix mixture; 4. Freeze-drying and sealing packaging: Same as steps (2)-(3) in Example 1.

[0034] The results of comparing the comparative example with those of this embodiment show that the lyophilized powder can form a complete gel, but when it is reconstituted and used for mouse small intestinal crypt culture, organoids cannot be cultured, indicating that the addition of trehalose protectant actually destroys the bioactivity of the matrix gel.

[0035] Therefore, the key to the preparation method in this embodiment lies in the following optimization steps: Optimization of the preparation process of basement membrane extract: Extraction with salt solution containing EDTA is used. EDTA can chelate calcium ions, inhibit the activity of matrix metalloproteinases (MMPs), and reduce protein degradation. At the same time, the purity and homogenity of the extract are ensured by high-speed homogenization at 8000-10000 rpm and centrifugation at 13000 rpm. Optimized freeze-drying process: A multi-stage freeze-drying mode of "pre-freezing - three-stage sublimation - desorption drying" is adopted, wherein: Pre-freezing stage (-50℃, 1~5h): Rapidly freeze the matrix mixture to form small ice crystals, avoiding excessively large ice crystals that could damage the protein structure; Three-stage sublimation process (-50℃, 5h; -25℃, 5h; -10℃, 3h): Gradually increase the temperature to remove free water and bound water in stages to prevent matrix collapse; Drying stage (25℃, 6h): Thoroughly removes residual moisture (moisture content <3%), improving product stability; Optimization of the basement membrane resolution process: Sterile low-temperature pre-cooling water was added, and the mixture was vortexed at 25°C for 3 minutes, then transferred to a 4°C refrigerator and left to stand overnight. The next day, it was removed and placed on crushed ice, and the matrix gel solution was repeatedly mixed by pipetting in a clean bench. This ensured that the proteins in the matrix gel were fully dissolved and kept in a liquid state for easy use.

[0036] The preparation and reconstitution methods are simple to operate, can be mass-produced, and have small batch-to-batch differences.

[0037] like Figure 1 As shown, Figure 1 The left figure shows the morphology of the trehalose-free lyophilized matrix gel obtained in this embodiment. Figure 1 The image on the right shows a freeze-dried matrix gel containing 5% trehalose. Figure 2 The lyophilized matrix adhesive is solidified at 37°C after being reconstituted. The lyophilized matrix adhesive provided in this embodiment exhibits a uniform, loose, and porous morphology after reconstitution, and the three-dimensional fiber network is dense and complete after reconstitution, with significantly better biological activity than traditional methods.

[0038] Example 3 This embodiment provides an application of lyophilized matrix gel in cell culture, including: 1. The lyophilized matrix gel obtained in Example 2 was reconstituted in a clean bench with sterile deionized water pre-cooled to below 4 °C at a reconstitution ratio of 8 mg / mL to 10 mg / mL. After vortexing at 25 °C for 3 minutes, it was transferred to a 4 °C refrigerator and left to stand overnight. The next day, it was taken out, placed on crushed ice, and the matrix gel solution was repeatedly mixed by pipetting in a clean bench. II. Subsequently, the matrix gel solution is diluted with culture medium to the required concentration or used directly, and evenly spread on the surface of the cell culture plate. It is then incubated at 37 °C for 30 minutes to form a gel layer. Finally, tumor cells, stem cells or epithelial cells are inoculated to promote cell adhesion, proliferation and differentiation.

[0039] A comparative example 2 was also provided. The only difference between comparative example 2 and example 3 is that comparative example 2 used phosphate buffer and culture medium for reconstitution. The rest was the same as example 3. Both examples formed complete gels, but comparative example 2 could not culture organoids.

[0040] Example 3 showed significant superiority over Comparative Example 2 in terms of storage stability and cell compatibility. In Example 3, the finished product was stored at 25°C, 4°C, and -20°C for 6 months, and its activity was tested: after storage at 25°C, the organoid culture success rate was 95%; after storage at 4°C, the culture success rate did not change significantly; after storage at -20°C, the performance was consistent with that of the freshly prepared sample; at the same time, the reconstituted matrix was plated on a 6-well plate, and human umbilical vein endothelial cells (HUVECs) were seeded. After 4 hours of culture, the cell adhesion rate was 98%, and obvious tubular structures were formed.

[0041] The gel layer formed after reconstitution in this embodiment can simulate the extracellular matrix microenvironment in vivo, promoting the adhesion and differentiation of tumor cells, stem cells, etc. For example, in the culture of human umbilical vein endothelial cells, this matrix can enable cells to form tubular structures on the matrix surface, which can be used for the study of angiogenesis mechanisms.

[0042] By combining this embodiment with Embodiment 2, the application results of this embodiment in mouse small intestinal organoids, IPSCs, and in vitro angiogenesis models show that: Figure 3 When applied to mouse small intestinal organoids, the organoids in the group without trehalose showed intact organoid structures and vigorous budding, while the group containing 5% trehalose showed almost no organoid formation. Figure 4 The comparison of iPSC clonal morphology on un-lyophilized and lyophilized gels at 4× / 10× shows that iPSCs form dense three-dimensional clones on the gel surface and maintain an undifferentiated state. Figure 5 The formation of capillary networks after HUVECs were cultured on two types of gels indicates that HUVECs spontaneously assembled into interconnected capillary networks within 12 hours on the lyophilized matrix gel, confirming that the matrix perfectly simulates the in vivo microenvironment and supports angiogenesis.

[0043] Example 4 This embodiment provides an application of lyophilized matrix gel in the construction of in vitro disease models. The reconstituted matrix gel is used to construct a three-dimensional cell culture model, namely an organoid model, to simulate the in vivo microenvironment for studying physiological mechanisms, inflammatory responses, or drug screening.

[0044] In this embodiment, during tumor or stem cell culture, cells are induced to form organoid structures. Utilizing their three-dimensional structural characteristics, tumor microenvironment models or inflammation models are constructed for drug screening. For example, in lung cancer drug testing, this three-dimensional model can more accurately reflect the inhibitory effect of drugs on tumor cells and reduce the cost of animal experiments.

[0045] Example 5 This embodiment provides an application of lyophilized matrix gel in intestinal organoids, including: 1. Under aseptic conditions, remove intestinal tissue and place it in DPBS solution containing double antibodies pre-cooled at 4°C. Cut open the intestinal lumen, gently scrape off any surface residue, wash twice, and cut into 2mm wide segments. 2. After washing twice, transfer to pre-cooled DPBS containing 2.5 mmol / L EDTA and digest at 4°C, 60-80 rpm for 60 min; after digestion, wash twice to remove EDTA. 3. Add 30 mL of DPBS containing 0.1% BSA to vortex the tissue fragment, collect the tissue suspension and filter it through a 70 μm filter; centrifuge at 300 g for 3 min to collect the crypts, resuspend in 1 mL of DPBS containing 0.1% BSA, and take 20 μL of the suspension for microscopic examination and crypt counting. 4. After counting, aspirate the suspension containing the required amount of crypts, centrifuge to remove the supernatant, add an appropriate amount of reconstituted lyophilized matrix gel according to the density, mix well on ice, and take 6 μL of the mixed suspension to spot into the center of a 96-well plate; after inoculation, solidify the culture plate at 37°C for about 15 min, and slowly add 100 μL / well of organoid complete culture medium along the wall.

[0046] The results showed that culturable intestinal organoids (e.g., human colon organoids), such as... Figure 6 As shown, the growth is in good condition.

[0047] Example 6 This embodiment provides an application of dry matrix adhesive in tumor organoids, including: 1. Under aseptic conditions, remove the corresponding tissue, wash it 2-3 times with cold epithelial organoid-specific rinsing solution, transfer the tissue to a 1.5mL EP tube, and cut the tissue into fragments of about 0.5 mm3 using sterile tissue scissors; 2. Add 5 mL of tissue digestion solution to resuspend the tissue and digest at 37 ℃ and 100 rpm for 20-60 minutes (depending on the tissue condition). When the tissue blocks are clearly dispersed and the suspension is relatively turbid, take an appropriate amount of the tissue digestion suspension for microscopic examination. When there are many epithelial cells in the suspension, add fetal bovine serum to a final concentration of 1% to 5% to slow down the digestion. At the same time, gently pipette 5 to 10 times. 3. Filter using a 100μm filter, collect 250g of filtrate, centrifuge for 3 minutes, discard the supernatant. If there are a large number of red blood cells, add red blood cell lysis buffer to lyse the red blood cells, then wash the precipitate. Wash the cells twice with basal culture medium to remove residual fetal bovine serum. 4. Resuspend the washed cell pellet in basal culture medium, take a small amount of suspension for viable cell detection and counting, add an appropriate amount of reconstituted lyophilized matrix gel (>70%) and mix on ice, take 6 μL of the mixed suspension and spot it into the center of a 96-well plate, solidify the culture plate at 37°C for about 15 min after seeding, and slowly add 100 μL / well of the complete culture medium of the organoid to be tested along the wall.

[0048] The results showed that tumor organoids (e.g., lung adenocarcinoma organoids) could be cultured, such as... Figure 7 As shown, the growth is in good condition.

[0049] The freeze-dried matrix gel provided by this invention exhibits no activity degradation after being sealed and stored at 25°C for more than 6 months, completely eliminating the need for a cold chain. It can restore a complete three-dimensional fiber network after reconstitution in 15 minutes, with a cell adhesion rate of ≥95%. It can efficiently support cell proliferation, differentiation, and organoid formation. The process parameters are fixed, with batch differences of <5%, and it can be produced on a kilogram-scale basis. It is widely applicable to 2D cell culture, 3D disease model construction, and drug screening, achieving low-cost, high-throughput, and standardized applications.

[0050] The freeze-dried matrix gel provided by this invention achieves long-term storage at room temperature through the synergistic effect of optimized freeze-drying process, and can completely restore the three-dimensional structure and biological activity of the basement membrane after reconstitution.

[0051] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a freeze-dried matrix adhesive, characterized in that, The preparation steps include the following: (1) Preparation of matrix gel: Select tumor tissue from EHS mice, add salt solution to thaw at a low temperature of 2–8℃; after thawing, homogenize, centrifuge to remove supernatant, add extraction buffer to dissolve basement membrane protein complex; Centrifuge again to remove tissue residue, retain the supernatant, and purify the protein to obtain the basement membrane extract, namely matrix gel. (2) Freeze-drying: The matrix gel obtained in step (1) is dispensed into sterile freeze-drying vials, 1–10 mL per vial, and sealed and stored at -80°C. Before freeze-drying, the aluminum cap is removed, the silicone soft stopper is half-pressed into the bottle mouth, and the vial is placed in a freeze dryer. No exogenous protective agent is added during the freeze-drying process. The vial is first pre-frozen at -50°C for 1 to 5 hours, then the vacuum is reduced to below 10 Pa and kept at -50°C for 5 hours. Then the temperature is raised to -25°C and kept for 5 hours, then raised to -15°C and kept for 3 hours, then raised to -10°C and kept for 3 hours, and finally raised to 25°C and kept for 6 hours to obtain a loose and porous freeze-dried matrix gel. (3) Sealed packaging: The freeze-dried matrix adhesive is sealed in a sterile environment to obtain the finished product.

2. The method for preparing the freeze-dried matrix adhesive according to claim 1, characterized in that, Step (1) involves extraction with a salt solution containing EDTA, which inhibits the activity of matrix metalloproteinases by chelating calcium ions, thereby reducing protein degradation.

3. The method for preparing the freeze-dried matrix adhesive according to claim 1, characterized in that, In step (1), a high-speed tissue homogenizer was used for homogenization at 8000–10000 rpm for 30 min, followed by centrifugation at 13000 rpm.

4. The method for preparing the freeze-dried matrix adhesive according to claim 1, characterized in that, The heating rate in step (2) is 1–2 °C / min throughout the entire heating phase.

5. A freeze-dried matrix adhesive prepared by any one of the preparation methods described in claims 1-4, characterized in that, The freeze-dried matrix adhesive is a loose, porous solid without any exogenous protective agents.

6. The application of the lyophilized matrix gel according to claim 5 in cell culture, characterized in that, The lyophilized matrix gel was reconstituted in a clean bench with sterile deionized water pre-cooled to below 4 °C at a reconstitution ratio of 8 mg / mL to 10 mg / mL. After vortexing at 25 °C for 3 minutes, the gel was transferred to a 4 °C freezer and left to stand overnight. The next day, the gel was removed, placed on crushed ice, and repeatedly pipetted to mix the matrix gel solution in a clean bench. Subsequently, the matrix gel solution was diluted with culture medium to the required concentration or used directly. It was then evenly spread on the surface of a cell culture plate and incubated at 37 °C for 30 minutes to form a gel layer. Finally, tumor cells, stem cells, or epithelial cells were inoculated to promote cell adhesion, proliferation, and differentiation.

7. The application of the lyophilized matrix gel according to claim 5 in the construction of in vitro disease models, characterized in that, Three-dimensional cell culture models, or organoid models, can be constructed using reconstituted matrix gel to simulate the in vivo microenvironment for studying physiological mechanisms, inflammatory responses, or drug screening.

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