A composite hydrogel based on urethral stem cell membrane sheet ECM and exosome and a preparation method and application thereof

Abstract

The application discloses a composite hydrogel based on urethral stem cell membrane sheet ECM and exosomes and a preparation method and application thereof, and relates to the technical field of tissue engineering, and in particular relates to a soluble ECM protein solution obtained by dissolving and enzymatically hydrolyzing urethral stem cell membrane sheet decellularized matrix (ECM) powder and mixing the soluble ECM protein solution with exosomes in a mass ratio of 3:1-10:1, and performing temperature-sensitive crosslinking in DMEM at pH 7.0 and 36-38 DEG C to form a hydrogel. The composite hydrogel based on urethral stem cell membrane sheet decellularized matrix (ECM) and exosomes integrates the cell activity of the urethral stem cell membrane sheet, the structural support of the decellularized matrix (ECM) and the signal regulation of the exosomes, constructs a three-level functional system, forms a synergistic repair effect, and is helpful to promoting angiogenesis in the urethra. Through covalent coupling of the decellularized matrix (ECM) fiber network and the surface protein of the exosomes, the intelligent release of the exosomes in an inflammatory environment is realized, and the composite hydrogel is used for urethral repair and angiogenesis promotion.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering, and particularly relates to a composite hydrogel based on urethral stem cell membrane sheet decellularized matrix (ECM) and exosomes as well as a preparation method and application thereof. BACKGROUND

[0002] In the field of urethral repair, traditional materials such as synthetic polymers have the problems of lacking biological activity and being difficult to promote angiogenesis and tissue regeneration. At the same time, a single exosome delivery system has defects such as easy degradation, poor targeting, and insufficient sustained-release effect. In addition, the natural microstructure is easily damaged in the decellularization process of the decellularized matrix (ECM) material, resulting in the loss of biological active ingredients. Although there are related researches in the existing patents and documents, the active ingredients are not integrated or the problem of rapid release of exosomes is not solved. SUMMARY

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

[0004] The present application is implemented by the following technical solutions:

[0005] A preparation method of a composite hydrogel based on urethral stem cell membrane sheet decellularized matrix (ECM) and exosomes, comprising the following steps: mixing a soluble ECM protein solution obtained by dissolving and enzymatically digesting urethral stem cell membrane sheet decellularized matrix (ECM) powder and exosomes in a mass ratio of 3:1-10:1, and performing temperature-sensitive crosslinking in DMEM at pH 7.0 and 36-38℃ to form a hydrogel.

[0006] Preferably, the concentration of the 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, calculated based on total protein.

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

[0008] 1) Cell acquisition and culture: obtain primary stem cells by collagenase digestion of urethral tissue samples, inoculate in a culture bottle, and culture in a 37℃, 5% CO2 incubator, with liquid replacement every 2 days, and subculture when the cells reach 80% confluence.

[0009] 2) 3D microcarrier amplification: inoculate the cells at 5×10 4 cells / cm 2The cells were seeded at a seeding density of 5 x 104cells / cm2on 3D TableTrix microcarriers in a dynamic culture system with an agitation rate of 15 rpm in an intermittent agitation mode of 5 min on / 30 min off for 7 days, with half of the medium being changed every 2 days.

[0010] 3) Formation of sheets from 2D culture: The cells on microcarriers were collected and seeded in 2D culture dishes and incubated at 37 °C in a 5% CO2incubator, with medium change every 2 days until sheets were formed.

[0011] 4) Decellularization: The cell sheets were freeze-thawed for 3 cycles, then treated in a solution containing 0.5% Triton X-100 and 0.1 M NH4OH at 4 °C overnight, followed by treatment with DNase and RNase at 37 °C for 1 hour, and finally subjected to critical point drying, freeze-drying, and pulverization through a 400-mesh sieve to obtain ECM powder.

[0012] Dissolution and enzymatic digestion of urethral stem cell sheet decellularized matrix (ECM) powder includes the following steps: under ice bath condition, urethral stem cell sheet decellularized matrix (ECM) powder was added into pre-cooled 1 wt.% pepsin (dissolved in 0.01 M hydrochloric acid) solution at a target final concentration of 10 mg / ml, and gently stirred for digestion at 4 °C for 48-72 hours on a rotary mixer, after digestion was completed, the digestion solution was placed on ice, pre-cooled 1 M NaOH solution was added extremely slowly while gentle stirring was continued and pH meter was used to accurately monitor, the pH value of the solution was carefully adjusted to 7.0-7.4 for neutralization, immediately after neutralization, an appropriate amount of 10x or 1x PBS was added to make the solution reach the physiological ion concentration of 1x PBS to terminate the activity of pepsin, then, the viscous solution after neutralization was centrifuged at 4 °C at a speed of 12,000-15,000 g for 20-30 minutes, and the supernatant was carefully aspirated to remove undissolved particles and precipitates. Finally, the total protein concentration in the supernatant was determined by BCA method or Bradford method, and the solution was diluted to a final concentration of 10 mg / mL (based on total protein) with pre-cooled sterile 1x PBS or physiological saline according to the determination results. The obtained 10 mg / mL soluble ECM protein solution can be aliquoted and stored at -20 °C or -80 °C for future use, and repeated freezing and thawing should be avoided.

[0013] The exosome preparation includes the following steps:

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

[0015] 2) Differential centrifugation: the collected supernatant is subjected to 300g centrifugation for 10 minutes, the cell mass is removed; 2,000g centrifugation for 15 minutes, the cell debris is removed; 100,000g centrifugation for 1 hour, the exosome crude extract is collected.

[0016] 3) VesPura density gradient centrifugation: the exosome crude extract is placed in the VesPura density gradient liquid, 100,000g centrifugation for 1 hour, the exosome particle size is 30-150nm, and the TSG101 / CD81 double positive.

[0017] The core of the application is to construct a three-level functional system and a directional slow-release structure, to integrate the cell activity of the urethral stem cell membrane sheet, the structural support of the decellularized matrix (ECM) and the signal regulation of the exosome, to construct a three-level functional system, to form a synergistic repair effect, and to help promote angiogenesis in the urethra.

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

[0019] 2) In the decellularization process, critical point drying is beneficial to the retention of the ECM topological structure.

[0020] The application also protects the use of the composite hydrogel, characterized by being used for urethral repair and promoting angiogenesis.

[0021] The application has the following beneficial effects: based on the urethral stem cell membrane sheet decellularized matrix (ECM) and the exosome, by integrating the cell activity of the urethral stem cell membrane sheet, the structural support of the decellularized matrix (ECM) and the signal regulation of the exosome, a three-level functional system is constructed, a synergistic repair effect is formed, and it is helpful to promote angiogenesis in the urethra; by covalent coupling of the decellularized matrix (ECM) fiber network and the exosome surface protein, the intelligent release of the exosome in the inflammatory environment is realized, and it is used for urethral repair and promoting angiogenesis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1These are images showing the state of urethral stem cells in the microcarrier dynamic culture system after 12 hours and 48 hours of culture in Example 1. A: Morphology of urethral stem cells (PUMSLCs) after 12 hours (left image) and 48 hours (right image) of culture in the microcarrier dynamic culture system; B: Immunofluorescence staining (cytoskeleton / green cell nucleus / blue) performed on urethral stem cells after 48 hours of culture in the microcarrier dynamic culture system, where the left image shows nuclear staining and the right image shows cytoskeleton staining; C: Live-dead cell staining performed on cells after 48 hours of culture in the urethral stem cell microcarrier dynamic culture system (where the left image shows live cell staining and the right image shows dead cell staining).

[0023] Figure 2 This is a schematic diagram of the cell membrane preparation process in Example 1; where A: Microscopic images of urethral stem cells transferred into 2D culture for 1 day (left image) and 14 days (right image) to form a membrane; B: Cell membrane after 14 days of culture (top: cell nucleus staining, bottom: collagen staining) undergoing fluorescence-free staining, expressing a large amount of extracellular matrix component collagen; C: Morphological image of cell membrane after 14 days of culture (top: morphology in culture plate, bottom: morphology after elution).

[0024] Figure 3 The image shows the characterization of urethral stem cell exosomes prepared in Example 2; where A is the exosome particle size analysis image, B is the exosome transmission electron microscopy image, and C is the marker protein detected by Western blotting.

[0025] Figure 4 The results of the in vitro angiogenesis experiment in Example 4 (comparison of tube formation experiments) are shown. In this example, A is the composite hydrogel formed in Example 3 of the present invention at pH 7.0 and 37°C, and B is the commercially available matrix gel, matrigel. Detailed implementation method:

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

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

[0028] 1) Cell acquisition and culture: Refer to Zhang, S., Li, J., Li, C., Xumin Xie, 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. Take the pediatric urethral tissue sample, collagenase (1 mg / mL) 37°C digestion for 2 hours, resuspended with mesenchymal stem cell professional culture medium, obtain primary stem cells, inoculate in culture flask, 37°C, 5% CO2 incubator, change liquid every 2 days, when the cells reach 80% confluence, passaged.

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

[0030] 3) Trans-2D culture to form a membrane sheet: The cells on the microcarriers in step 2) were collected and seeded in a 2D culture dish in a medium containing 10 ng / mL VEGF and 50 μg / mL vitamin C, 37°C, 5% CO2 culture, liquid change every 2 days, continue to culture for 14d to form a complete membrane sheet.

[0031] 4) Decellularization treatment: The cell membrane sheet was frozen at -80°C for 2 hours, then thawed at 4°C for 2 hours, and the freeze-thaw cycle was repeated 3 times, then the freeze-thawed cell membrane sheet was treated in a solution containing 0.5% Triton X-100 and 0.1M NH4OH at 4°C overnight, after thorough rinsing, 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 preserve the natural microstructure of ECM, after freeze-drying and crushing, pass through a 400 mesh sieve (pore size about 38 μm) to obtain ECM powder.

[0032] Example 2: Exosome preparation

[0033] Cell culture supernatant collection: The cell culture supernatant was collected from the 3D microcarrier culture system of urethral stem cells in Example 1.

[0034] Differential centrifugation: The collected supernatant was sequentially centrifuged at 300g for 10 min to remove cell pellets, 2,000g for 15 min to remove cell debris, and 100,000g for 1 h to collect the crude exosome extract.

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

[0036] Example 3: Preparation of composite hydrogel

[0037] 1) ECM powder dissolution and enzymatic digestion: The ECM powder obtained in Example 1 was dissolved and enzymatically digested by the following steps: under ice bath condition, the ECM powder was added into pre-cooled 1 wt.% pepsin (dissolved in 0.01 M hydrochloric acid) solution at a target final concentration of 10 mg / ml, and the mixture was gently stirred on a rotary mixer at 4°C for 48-72 hours. After digestion, the digestion solution was placed on ice, and pre-cooled 1 M NaOH solution was added dropwise very slowly while continuously gently stirring and using a pH meter to accurately monitor the solution pH, and the solution pH was carefully adjusted to 7.0-7.4 for neutralization (this step needs to be extremely cautious to avoid local over-alkalization leading to protein precipitation). Immediately after neutralization, an appropriate amount of 10x or 1x PBS was added to the solution to reach the physiological ion concentration of 1x PBS to terminate the pepsin activity. Subsequently, the neutralized viscous solution was centrifuged at 12,000-15,000g at 4°C for 20-30 minutes, and the supernatant was carefully aspirated to remove undissolved particles and precipitates. Finally, the total protein concentration in the supernatant was determined by BCA method or Bradford method, and the solution was diluted to a final concentration of 10 mg / mL (based on total protein) using pre-cooled sterile 1x PBS or physiological saline according to the determination results. The obtained 10 mg / mL soluble ECM protein solution can be aliquoted and stored at -20°C or -80°C for future use, and repeated freezing and thawing should be avoided.

[0038] 2) Thermo-sensitive crosslinking: The soluble ECM protein solution (10 mg / mL) obtained by ECM powder dissolution and enzymatic digestion in step 1) was mixed with the exosomes (1 mg / mL) obtained in Example 2 at a ratio of 3:1 (w / w) in DMEM: The mixture was subjected to thermo-sensitive crosslinking at pH 7.0 and 37°C to form a hydrogel.

[0039] Example 4: In vitro tube formation function verification

[0040] The composite hydrogel obtained in Example 3 at pH 7.0 and 37°C (as the experimental group) and Matrigel matrix gel (as the control group) were melted according to the instructions and spread into 96-well plates, 50 μL per well, and cured at 37°C for 30 minutes. In the experimental group, HUVEC cells were introduced at a concentration of 1 × 10⁻⁶ cells / well. 4 HUVEC cells were seeded at a density of 1 × 10⁶ cells / well in the composite hydrogel, while in the control group, HUVEC cells were seeded at a density of 1 × 10⁶ cells / well. 4 Cells were seeded at a density of 100 μL / well on Matrigel cells, with 100 μL of complete endothelial cell culture medium added to each well. The cells were incubated at 37°C in a 5% CO2 incubator. Tubular structures were imaged under a microscope (100×) after 8 hours. Results are as follows: Figure 4 The composite hydrogel of this invention is superior to Matrigel matrix gel.

Claims

1. A method for preparing a composite hydrogel based on urethral stem cell sheet decellularized matrix and exosomes, characterized in that, The method comprises the following steps: The soluble ECM protein solution obtained by dissolving and enzymatic hydrolysis of the urethral stem cell membrane sheet acellular matrix powder is mixed with the exosomes at a mass ratio of 3:1-10:1, and a hydrogel is formed by warm crosslinking in DMEM at 36-38℃ and pH 7.0; the preparation of the urethral stem cell membrane sheet acellular matrix powder comprises the following steps: 1) Cell acquisition and culture: urethral tissue samples are taken, primary stem cells are obtained by collagenase digestion, and are inoculated into culture bottles, which are cultured in a 37℃, 5% CO2 incubator, the liquid is changed every 2 days, and the cells are subcultured when the cell confluence reaches 80%; 2) 3D microcarrier culture: the cells on the microcarriers are collected and inoculated into 3D culture bottles, which are cultured in a 37℃, 5% CO2 incubator, the liquid is changed every 2 days, and the cells are subcultured when the cell confluence reaches 80%; 3) 2D culture to form a membrane sheet: the cells on the microcarriers are collected and inoculated into 2D culture dishes, which are cultured in a 37℃, 5% CO2 incubator, the liquid is changed every 2 days, and the cells are subcultured when the cell confluence reaches 80%; and 4) decellularization treatment: the cell membrane sheet is subjected to freeze-thaw cycles for 3 times, then is treated in a solution containing 0.5% Triton X-100 and 0.1M NH4OH at 4℃ overnight, is treated with DNase and RNase at 37℃ for 1 hour, and finally is subjected to critical point drying, is freeze-dried, is crushed, and is sieved through a 400-mesh sieve to obtain the ECM powder. 2) 3D microcarrier expansion: Cells were seeded at a seeding density of 5 x 10 4 cells / cm2on 3D TableTrix microcarriers in a dynamic culture system at a stirring rate of 15 rpm with an intermittent stirring pattern of 5 min on / 30 min off for 7 days with half medium exchange every 2 days; The concentration of the exosomes is 0.5-1.5 mg / mL. The concentration of the acellular matrix in the soluble ECM protein solution is 10 mg / ml in terms of total protein.

2. The method of claim 1, wherein, The dissolving and enzymatic hydrolysis of the urethral stem cell membrane sheet acellular matrix powder comprises the following steps: under ice bath conditions, the urethral stem cell membrane sheet acellular matrix powder is added to a pre-cooled 1wt.% pepsin solution dissolved in 0.01M hydrochloric acid at a target final concentration of 10 mg / ml, and is gently stirred and digested at 4℃ for 48-72 hours on a rotary mixer, after the digestion is completed, the digestion solution is placed on ice, pre-cooled 1M NaOH solution is slowly added dropwise, gentle stirring is continuously performed, and a pH meter is used to accurately monitor the pH value, the pH value of the solution is adjusted to 7.0-7.4 for neutralization, immediately after neutralization, an appropriate amount of 10x or 1x PBS is added to make the solution reach the physiological ion concentration of 1x PBS to terminate the activity of pepsin, then, the viscous solution after neutralization is centrifuged at 12,000-15,000 g at 4℃ for 20-30 minutes, the supernatant is aspirated, and finally, the solution is diluted to a final concentration of 10 mg / mL with pre-cooled sterile 1x PBS or physiological saline.

3. The method of claim 1, wherein, The preparation of the exosomes comprises the following steps:

4. The method of claim 1, wherein, 1) The cell culture supernatant is collected from the urethral stem cell 3D microcarrier culture system in step 2) of the preparation of the urethral stem cell membrane sheet acellular matrix powder; 5. The method of claim 1, wherein, 2) The collected supernatant is sequentially subjected to 300g centrifugation for 10 minutes, 2,000g centrifugation for 15 minutes, and 100,000g centrifugation for 1 hour, and the exosome crude extract is collected; 3) VesPura density gradient centrifugation: the exosome crude extract is placed in a VesPura density gradient liquid, and is centrifuged at 100,000g for 1 hour to obtain the exosomes. ​ ​

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