Composite hydrogel based on urethral stem cell membrane ECM and exosome as well as preparation method and application of composite hydrogel

Abstract

The invention discloses a composite hydrogel based on urethral stem cell membrane ECM and exosome and a preparation method and application thereof.The preparation method comprises the steps that urethral stem cell membrane ECM powder is dissolved and subjected to enzymolysis to obtain a soluble ECM protein solution, the soluble ECM protein solution and the exosome are mixed according to the mass ratio of 3: 1-10: 1, temperature-sensitive crosslinking is conducted in a DMEM at the pH of 7.0 and the temperature of 36-38 DEG C, the hydrogel is formed, and the composite hydrogel is prepared. Based on a urethral stem cell membrane ECM (acellular matrix) and an exosome, a three-level functional system is constructed by integrating the cell viability of the urethral stem cell membrane, the structural support effect of the ECM and the signal regulation effect of the exosome, a synergistic repair effect is formed, and angiogenesis in the urethra is promoted; through covalent coupling of an acellular matrix (ECM) fiber network and exosome surface protein, intelligent release of the exosome in an inflammation environment is realized, and the exosome is used for urethra repair and angiogenesis promotion.

Description

Technical field:

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a composite hydrogel based on a urethral stem cell membrane decellularized matrix (ECM) and exosomes, and a preparation method and application thereof. Background technology:

[0002] Currently, in the field of urinary tract repair, traditional materials such as synthetic polymers lack bioactivity and are difficult to promote angiogenesis and tissue regeneration. Furthermore, single-exosome delivery systems suffer from drawbacks such as easy degradation, poor targeting, and insufficient sustained-release effects. Furthermore, the decellularization process of the acellular matrix (ECM) material easily destroys the natural microstructure, leading to the loss of bioactive components. While relevant research is available in existing patents and literature, none of these incorporate active ingredients or address the rapid release of exosomes. Summary of the invention:

[0003] The purpose of the present invention is to provide a composite hydrogel based on urethral stem cell membrane acellular matrix (ECM) and exosomes, as well as a preparation method and application thereof.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for preparing a composite hydrogel based on urethral stem cell membrane decellularized matrix (ECM) and exosomes comprises the following steps: dissolving and enzymatically hydrolyzing urethral stem cell membrane decellularized matrix (ECM) powder to obtain a soluble ECM protein solution and mixing it with exosomes in a mass ratio of 3:1-10:1, performing thermosensitive crosslinking in DMEM at pH 7.0 and 36-38°C to form a hydrogel.

[0006] Preferably, the concentration of exosomes is 0.5-1.5 mg / mL. The concentration of the decellularized matrix (ECM) in the soluble ECM protein solution is 10 mg / ml based on total protein.

[0007] The preparation of the urethral stem cell membrane decellularized matrix (ECM) powder comprises the following steps:

[0008] 1) Cell Acquisition and Culture: Urethral tissue samples were obtained and digested with collagenase to obtain primary stem cells. The cells were seeded into culture flasks and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 2 days and the cells were passaged after reaching 80% confluency.

[0009] 2) 3D microcarrier expansion: cells were cultured at a rate of 5×10 4 cells / cm 2The cells were inoculated at a density of 100 μg / mL on 3D TableTrix microcarriers and placed in a dynamic culture system with a stirring rate of 15 rpm and an intermittent stirring mode of 5 min on / 30 min off. The culture was carried out for 7 days, during which half of the culture medium was replaced every 2 days.

[0010] 3) Transfer to 2D culture to form membrane sheets: The cells on the microcarriers were collected and inoculated into 2D culture dishes. The cells were cultured in a 37°C, 5% CO2 incubator, with the medium changed every 2 days until the cells formed membrane sheets.

[0011] 4) Decellularization: The cell membrane was freeze-thawed three times, then treated in a solution containing 0.5% Triton X-100 and 0.1 M NH4OH at 4°C overnight, and then treated with DNAse and RNAse at 37°C for 1 hour. Finally, critical point drying was performed, freeze-dried, crushed, and passed through a 400-mesh sieve to obtain ECM powder.

[0012] The dissolution and enzymatic hydrolysis of the urethral stem cell membrane decellularized matrix (ECM) powder includes the following steps: under ice bath conditions, the urethral stem cell membrane decellularized matrix (ECM) powder is added to a pre-cooled 1 wt.% pepsin (dissolved in 0.01 M hydrochloric acid) solution at a target final concentration of 10 mg / ml, and digested on a rotating mixer at 4°C with gentle stirring for 48-72 hours. After digestion is complete, the digestion solution is placed on ice, and pre-cooled 1 M NaOH solution is added very slowly dropwise while continuously stirring gently and accurately monitored using a pH meter. The pH value of the solution is carefully adjusted to 7.0-7.4 for neutralization. After neutralization, an appropriate amount of 10x or 1x PBS is immediately added to make the solution reach the physiological ion concentration of 1x PBS to terminate the activity of pepsin. Subsequently, the neutralized viscous solution is centrifuged at 12,000-15,000g at 4°C for 20-30 minutes, and the supernatant is carefully aspirated to remove undissolved particles and precipitates. Finally, determine the total protein concentration in the supernatant using the BCA or Bradford assay. Based on the assay results, dilute the solution with pre-chilled sterile 1x PBS or saline to a final concentration of 10 mg / mL (based on total protein). The resulting 10 mg / mL soluble ECM protein solution can be aliquoted and frozen at -20°C or -80°C until use. Avoid repeated freeze-thaw cycles.

[0013] The exosome preparation comprises the following steps:

[0014] 1) Collection of cell culture supernatant: Collect cell culture supernatant from the urethral stem cell 3D microcarrier culture system in step 2) of the preparation of the urethral stem cell membrane decellularized matrix (ECM) powder.

[0015] 2) Differential centrifugation: The collected supernatant was centrifuged at 300 g for 10 minutes to remove cell clumps; 2,000 g for 15 minutes to remove cell debris; and 100,000 g for 1 hour to collect the crude exosome extract.

[0016] 3) VesPura density gradient centrifugation: The crude exosome extract was placed in VesPura density gradient medium and centrifuged at 100,000 g for 1 hour to obtain exosomes with a particle size of 30-150 nm and TSG101 / CD81 double positive.

[0017] The core invention of this invention lies in the construction of a three-level functional system and a directional sustained-release structure. By integrating the cell activity of the urethral stem cell membrane, the structural support function of the acellular matrix (ECM), and the signal regulation function of exosomes, a three-level functional system is constructed to form a synergistic repair effect, which helps to promote angiogenesis in the urethra; through the covalent coupling of the amino groups of the acellular matrix (ECM) collagen fiber network with the carboxyl groups of the exosome surface protein, the intelligent release of exosomes in an inflammatory environment is achieved.

[0018] The key technical points in the preparation of the urethral stem cell membrane decellularized matrix (ECM) powder of the present invention are as follows: 1) In the 3D microcarrier expansion culture, an intermittent stirring mode of 5 minutes on and 30 minutes off is adopted to maintain cell activity.

[0019] 2) During decellularization, critical point drying is beneficial to preserving the ECM topology.

[0020] The present invention also protects the application of the composite hydrogel, which is characterized in that it is used for urethra repair and promoting angiogenesis.

[0021] The beneficial effects of the present invention are as follows: Based on the urethral stem cell membrane decellularized matrix (ECM) and exosomes, the present invention integrates the cell activity of the urethral stem cell membrane, the structural support function of the decellularized matrix (ECM) and the signal regulation function of the exosomes to construct a three-level functional system, forming a synergistic repair effect, which helps to promote angiogenesis in the urethra; through the covalent coupling of the decellularized matrix (ECM) fiber network and the exosome surface protein, the intelligent release of exosomes in an inflammatory environment is realized, which is used for urethral repair and promotes angiogenesis. Description of the drawings:

[0022] Figure 1Figures 1 and 2 show the morphology of urethral stem cells after 12 and 48 hours of culture in the microcarrier dynamic culture system. A: Morphology of urethral stem cells (PUMSLCs) after 12 hours (left) and 48 hours (right) of culture in the microcarrier dynamic culture system. B: Immunofluorescence staining (cytoskeleton / green nucleus / blue) of urethral stem cells after 48 hours of culture in the microcarrier dynamic culture system, with the left image showing nuclear staining and the right image showing cytoskeleton staining. C: Live-dead staining of urethral stem cells after 48 hours of culture in the microcarrier dynamic culture system (left image showing live cell staining and right image showing dead cell staining).

[0023] Figure 2 Schematic diagram of the preparation process of the cell membrane in Example 1; wherein, A: Microscope images of the membrane formed by urethral stem cells after 1 day (left) and 14 days (right) of 2D culture; B: Fluorescence-free staining of the cell membrane after 14 days of culture (top: cell nucleus staining, bottom: collagen staining), expressing a large amount of extracellular matrix component collagen; C: Morphology of the cell membrane after 14 days of culture (top: morphology in the culture plate, bottom: morphology after elution).

[0024] Figure 3 : These are characterization images of the urethral stem cell exosomes prepared in Example 2; wherein, A is an exosome particle size analysis image, B is a transmission electron microscopy image of the exosomes, and C is a WB detection marker protein.

[0025] Figure 4 This is the result of the in vitro angiogenesis experiment in Example 4 (comparison diagram of tube formation experiment), wherein A is the composite hydrogel formed at pH 7.0 and 37° C. in Example 3 of the present invention, and B is commercially available matrigel. Specific implementation method:

[0026] The following is a further description of the present invention, but not a limitation of the present invention.

[0027] Example 1: Preparation of urethral stem cell membrane ECM

[0028] 1) Cell acquisition and culture: Refer to Zhang, S., Li, J., Li, C., XuminXie, He, J., Ling, F., & Liu, G. (2022). Isolation and identification of a mesenchymal stem / stromal cell-like population from pediatric urethral tissue. In vitro cellular & developmental biology. Animal, 58(6), 503–511. Obtain urinary urethral tissue samples, digest with collagenase (1 mg / mL) at 37°C for 2 hours, resuspend in mesenchymal stem cell culture medium to obtain primary stem cells, inoculate into culture flasks, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2 days, and passage the cells after reaching 80% confluence.

[0029] 2) 3D microcarrier expansion: cells were cultured at a rate of 5×10 4 cells / cm 2 The cells were inoculated at a density of 100 μg / mL on 3D TableTrix microcarriers and placed in a culture medium containing 10 ng / mL VEGF and 50 μg / mL vitamin C. The cells were cultured in a dynamic culture system (15 rpm intermittent stirring, 5 min on / 30 min off) for 7 days, during which half of the culture medium was replaced every 2 days.

[0030] 3) Transfer to 2D culture to form membrane sheets: Collect the cells on the microcarriers in step 2) and inoculate them into 2D culture dishes. Incubate in a medium containing 10 ng / mL VEGF and 50 μg / mL vitamin C at 37°C and 5% CO2. Change the medium every 2 days and continue culturing for 14 days until the cells form a complete membrane sheet.

[0031] 4) Decellularization: The cell membrane sheets were frozen at -80°C for 2 hours, then thawed at 4°C for 2 hours, and the freeze-thaw cycle was repeated three times. The cell membrane sheets after the freeze-thaw cycle were then treated in a solution containing 0.5% Triton X-100 and 0.1 M NH4OH at 4°C overnight. After thorough rinsing, the cells were treated with DNase (50 μg / mL) and RNase (100 μg / mL) at 37°C for 1 hour to remove residual nucleic acids. Finally, critical point freeze-drying was performed to retain the natural microstructure of the ECM. After freeze-drying and grinding, the membranes were passed through a 400-mesh sieve (pore size approximately 38 μm) to obtain ECM powder.

[0032] Example 2: Exosome Preparation

[0033] Collection of cell culture supernatant: Collect cell culture supernatant from the urethral stem cell 3D microcarrier culture system of Example 1.

[0034] Differential centrifugation: The collected supernatant was centrifuged at 300 g for 10 minutes to remove cell clumps; 2,000 g for 15 minutes to remove cell debris; and 100,000 g for 1 hour to collect crude exosome extracts.

[0035] VesPura density gradient centrifugation: The crude exosome extract was placed in VesPura density gradient medium and centrifuged at 100,000g for 1 hour. The exosome particle size was 30-150nm and TSG101 / CD81 double positive. Figure 3 .

[0036] Example 3: Preparation of composite hydrogel

[0037] 1) The ECM powder obtained in Example 1 was dissolved and enzymatically hydrolyzed, comprising the following steps: The ECM powder was added to a pre-chilled 1 wt.% pepsin solution (dissolved in 0.01 M hydrochloric acid) at a target final concentration of 10 mg / ml in an ice bath. The mixture was then gently stirred on a rotary mixer at 4°C for 48-72 hours. After digestion, the digestion solution was placed on ice and very slowly, dropwise added with pre-chilled 1 M NaOH solution while stirring gently and accurately monitored using a pH meter. The pH of the solution was carefully adjusted to 7.0-7.4 for neutralization (this step required extreme caution to avoid localized over-alkalinity that could lead to protein precipitation). Following neutralization, an appropriate amount of 10x or 1x PBS was immediately added to bring the solution to the physiological ionic concentration of 1x PBS to terminate pepsin activity. The neutralized viscous solution was then centrifuged at 12,000-15,000 g for 20-30 minutes at 4°C. The supernatant was carefully aspirated to remove any undissolved particles and precipitate. Finally, determine the total protein concentration in the supernatant using the BCA or Bradford assay. Based on the assay results, dilute the solution with pre-chilled sterile 1x PBS or saline to a final concentration of 10 mg / mL (based on total protein). The resulting 10 mg / mL soluble ECM protein solution can be aliquoted and frozen at -20°C or -80°C until use. Avoid repeated freeze-thaw cycles.

[0038] 2) Thermosensitive crosslinking: Step 1) ECM powder was dissolved and enzymatically hydrolyzed to obtain a soluble ECM protein solution (10 mg / mL) and the exosomes (1 mg / mL) obtained in Example 2 were mixed in DMEM at a ratio of 3:1 (w / w): the mixture was thermosensitively crosslinked at pH 7.0 and 37°C to form a hydrogel.

[0039] Example 4: Verification of in vitro tube formation function

[0040] The composite hydrogel (as the experimental group) and Matrigel (as the control group) obtained in Example 3 at pH 7.0 and 37°C were melted according to the instructions and plated in a 96-well plate, 50 μL per well, and solidified at 37°C for 30 minutes. 4 The density of cells / well was inoculated into the composite hydrogel. In the control group, HUVEC cells were seeded at a density of 1×10 4 The cells were seeded on Matrigel at a density of 100 cells / well and 100 μL of complete endothelial cell culture medium was added to each well. The cells were cultured in a 37°C, 5% CO2 incubator. Images of the tube structures were taken using a microscope (100×) at 8 hours. Figure 4 The composite hydrogel of the present invention is superior to Matrigel.

Claims

1. A method for preparing a composite hydrogel based on a urethral stem cell membrane decellularized matrix and exosomes, characterized in that: The following steps are involved: The soluble ECM protein solution obtained by dissolving and enzymatically hydrolyzing the urethral stem cell membrane decellularized matrix powder was mixed with exosomes at a mass ratio of 3:1-10:1, and thermosensitive cross-linking was performed in DMEM at pH 7.0 and 36-38°C to form a hydrogel.

2. The method according to claim 1, characterized in that The concentration of exosomes was 0.5-1.5 mg / mL.

3. The method according to claim 1, characterized in that The concentration of the decellularized matrix in the soluble ECM protein solution was 10 mg / ml based on total protein.

4. The method according to claim 1, wherein The preparation of the urethral stem cell membrane acellular matrix powder comprises the following steps: 1) Cell acquisition and culture: Urethral tissue samples were obtained and digested with collagenase to obtain primary stem cells. The cells were seeded into culture flasks and cultured in a 37°C, 5% CO2 incubator with medium replacement every 2 days. Cells were passaged after reaching 80% confluency. 2) 3D microcarrier expansion: cells were cultured at a rate of 5×10 4 cells / cm 2 The cells were inoculated at a density of 100 μg / mL onto 3D TableTrix microcarriers and placed in a dynamic culture system with a stirring rate of 15 rpm and an intermittent stirring mode of 5 min on / 30 min off for 7 days, during which half of the culture medium was replaced every 2 days. 3) Transformation to 2D culture to form membrane sheets: The cells on the microcarriers were collected and inoculated into 2D culture dishes. The culture dishes were cultured in a 37°C, 5% CO2 incubator, with the medium changed every 2 days until the cells formed membrane sheets. 4) Decellularization: The cell membrane was freeze-thawed three times, then treated in a solution containing 0.5% Triton X-100 and 0.1 M NH4OH at 4°C overnight, and then treated with DNAse and RNAse at 37°C for 1 hour. Finally, critical point drying was performed, freeze-dried, crushed, and passed through a 400-mesh sieve to obtain ECM powder.

5. The method according to claim 1, wherein The urethral stem cell membrane decellularized matrix powder dissolution and enzymatic hydrolysis comprises the following steps: under ice bath conditions, the urethral stem cell membrane decellularized matrix powder is added to a pre-cooled 1 wt.% pepsin solution dissolved in 0.01 M hydrochloric acid at a target final concentration of 10 mg / ml, and digested on a rotary mixer with gentle stirring at 4°C for 48-72 hours. After digestion is complete, the digestion solution is placed on ice, and pre-cooled 1 M NaOH solution is slowly added dropwise while continuously stirring gently and accurately monitored using a pH meter to adjust the pH value of the solution to 7.0-7.4 for neutralization. After neutralization, an appropriate amount of 10x or 1x PBS is immediately added to make the solution reach the physiological ion concentration of 1x PBS to terminate the activity of pepsin. Subsequently, the neutralized viscous solution is centrifuged at 12,000-15,000g at 4°C for 20-30 minutes, and the supernatant is aspirated. Finally, the solution is diluted with pre-cooled sterile 1x PBS or physiological saline to a final concentration of 10 mg / mL.

6. The method according to claim 1, characterized in that The exosome preparation comprises the following steps: 1) collecting cell culture supernatant from the urethral stem cell 3D microcarrier culture system in step 2) of preparing the urethral stem cell membrane decellularized matrix powder according to claim 4; 2) The collected supernatant was centrifuged at 300 g for 10 minutes, 2,000 g for 15 minutes, and 100,000 g for 1 hour to collect the crude exosome extract. 3) VesPura density gradient centrifugation: The crude exosome extract was placed in VesPura density gradient medium and centrifuged at 100,000 g for 1 hour to obtain exosomes.

7. Use of the composite hydrogel obtained by the preparation method according to claim 1, characterized in that: Used for urethral repair and promoting angiogenesis.

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