Extracellular vesicle optimized by hydrogel as well as preparation method and application of extracellular vesicle

Abstract

The invention discloses an extracellular vesicle optimized by hydrogel as well as a preparation method and application of the extracellular vesicle. The preparation method comprises the following steps: S1, placing stem cells on fiber membrane hydrogel at the density of 1 * 10 < 5 >-1 * 10 < 6 > / cm < 2 > for culture, and collecting a culture solution; s2, centrifuging the culture solution obtained in the step S1 for 5-10 minutes under 500-1000g, collecting supernate, centrifuging for 30 minutes under the centrifugal force of 5000-10000g, collecting supernate, and removing impurities by using a 0.22 mu m microfiltration membrane, so as to obtain the extracellular vesicles, wherein the fibrous membrane hydrogel in the step S1 contains a gel agent, and the gel agent is a combination of 5 to 20 weight percent of methacrylated gelatin and 0.1 to 5 weight percent of methacrylic acid esterified hyaluronic acid. The specially prepared fibrous membrane hydrogel is used as a matrix for culturing the stem cells, the physiological activity of the stem cells is regulated and controlled through fiber morphology, the function of promoting tissue regeneration of the extracellular vesicles is effectively enhanced, complex gene manipulation or chemical loading is not needed, and the operation process is greatly simplified.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell tissue engineering, and in particular to an extracellular vesicle optimized by hydrogel, and a preparation method and application thereof. Background Art

[0002] Tissue defects are common in clinical practice due to trauma, infection, and tumor resection. Although damaged tissue has a certain ability to regenerate itself, its repair capacity is generally limited. With the aging of the population, the time required for tissue regeneration will be further prolonged, and factors such as obesity may also lead to a decrease in tissue regeneration capacity. Therefore, promoting tissue repair and regeneration has become an important clinical issue. However, the drugs currently used in clinical practice to promote tissue regeneration are often expensive or have significant toxic side effects, limiting their widespread application.

[0003] Studies in recent years have found that the tissue regeneration process is regulated by a variety of cells, especially stem cells with regenerative potential. Stem cells promote tissue regeneration by releasing extracellular vesicles containing biomolecules such as proteins and nucleic acids (such as miRNA). As an important intercellular communication carrier, extracellular vesicles can participate in multiple biological processes such as immune regulation, energy metabolism, tissue repair, and metabolism. Its natural advantages make it a potential drug carrier. Its smaller size and double-layer phospholipid structure help reduce the risk of vascular blockage and are easy for cells to take up. In addition, extracellular vesicles also have low toxicity and immunogenicity. These advantages make it an ideal carrier in the field of tissue regeneration.

[0004] Although extracellular vesicles (EVs) offer numerous advantages as drug carriers, their effectiveness in promoting tissue regeneration is limited by the concentration and type of their contents. Currently, common methods for modifying EVs include genetic engineering and chemical loading, but these methods suffer from complex procedures and low efficiency. Therefore, developing a simple and efficient method for preparing EVs to enhance their ability to promote tissue regeneration has become an important research direction. Summary of the Invention

[0005] In response to the defects in the existing technology, the present invention proposes an extracellular vesicle optimized using hydrogel, and its preparation method and application, aiming to improve the tissue regeneration promoting function of stem cell-derived extracellular vesicles and provide a new idea for clinical application.

[0006] The present invention provides a method for preparing extracellular vesicles, which comprises the following steps:

[0007] (1) Stem cells were collected at a rate of 1×10 5 ~1×10 6 pieces / cm 2The density of the cells was placed on the fiber membrane hydrogel for culture, and the culture fluid was collected;

[0008] (2) The culture medium of (1) is centrifuged at 500-1000 g for 5-10 min, and the supernatant is collected. The culture medium is then centrifuged at 5000-10000 g for 20-40 min, and the supernatant is collected. Impurities are then removed using a 0.22 μm microporous filter membrane to obtain the extracellular vesicles. The centrifugation time at 5000-10000 g is preferably 30 min.

[0009] Wherein, (1) the fiber membrane hydrogel comprises a gelling agent, which is a combination of 5-20 wt% methacrylated gelatin and 0.1-5 wt% methacrylated hyaluronic acid. Gelatin is a degradation product of collagen, and collagen and hyaluronic acid are chemical components of the natural extracellular matrix. Using gelatin and hyaluronic acid as a culture matrix mimics the chemical components of the natural extracellular matrix and imitates the natural living microenvironment of cells, resulting in a good growth effect.

[0010] In some embodiments, (1) the preparation method of the fiber membrane hydrogel is as follows:

[0011] S1: dissolving methacrylated gelatin in hexafluoroisopropanol and methacrylated hyaluronic acid in trifluoroacetic acid, and mixing them to prepare a spinning solution containing 5-20 wt% methacrylated gelatin and 0.1-5 wt% methacrylated hyaluronic acid;

[0012] S2: Spinning the spinning solution described in S1 using an electrospinning device to obtain an electrospinning membrane;

[0013] S3: placing the electrospun membrane described in S2 in a photoinitiator solution, exposing it to ultraviolet light of 200 to 365 nm for 5 to 60 minutes, and then removing excess photoinitiator to obtain the fiber membrane hydrogel.

[0014] Electrospinning can effectively convert polymer solutions into materials with high specific surface area, unique morphology and fiber structure. When hydrogels are combined with electrospinning technology, hydrogels with highly adjustable fiber structures can be prepared, which can provide a larger surface area and more attachment points, promote the binding of cells to the matrix, and enhance cell attachment and proliferation.

[0015] In some embodiments, the photoinitiator solution in S3 contains 0.5 to 5 wt % of 2-hydroxy-2-methylacetone or 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone.

[0016] In some embodiments, the electrospinning parameters of S2 include: a voltage applied between the spinning needle and the stainless steel receiving plate of 10 to 20 kV, a distance between the spinning needle and the stainless steel receiving plate of 10 to 20 cm, and a spinning time of 5 to 30 min.

[0017] In some embodiments, the stem cells are any one of human bone marrow mesenchymal stem cells, human adipose mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human umbilical cord blood mesenchymal stem cells, human placental mesenchymal stem cells, human dental pulp stem cells, human periodontal ligament stem cells, human hair follicle stem cells and human amniotic membrane mesenchymal stem cells, preferably human bone marrow mesenchymal stem cells.

[0018] In some embodiments, the extracellular vesicles are exosomes, microvesicles, or vesicles.

[0019] The present invention also provides an extracellular vesicle obtained by the preparation method.

[0020] The present invention also provides an extracellular vesicle preparation, comprising the extracellular vesicles.

[0021] The present invention also provides a method for improving the tissue regeneration promoting function of extracellular vesicles, and the extracellular vesicles are prepared by culturing stem cells using the fiber membrane hydrogel.

[0022] The present invention also provides application of the preparation method in preparing extracellular vesicles.

[0023] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0024] 1. The present invention uses specially prepared fiber membrane hydrogel as a matrix for culturing stem cells. It regulates the physiological activities of stem cells through fiber morphology, effectively enhancing the function of extracellular vesicles in promoting tissue regeneration. It does not require complex genetic manipulation or chemical loading, greatly simplifying the operation process.

[0025] 2. The preparation of the culture matrix fiber membrane hydrogel of the present invention is simple and convenient, without the introduction of obvious harmful substances. While improving the function of extracellular vesicles, it maintains its natural biocompatibility and low immunogenicity, reduces the risk of potential side effects, and provides a safer and more efficient option for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a scanning electron microscope photograph of the surface morphology of the fiber membrane hydrogel of Example 1 of the present invention; scale: 200 μm;

[0028] Figure 2 This is a fluorescence staining image of mesenchymal stem cells cultured on the surface of the fiber membrane hydrogel in Example 2 of the present invention;

[0029] Figure 3 This is a morphological diagram of mesenchymal stem cells cultured in a culture dish according to Example 2 of the present invention;

[0030] Figure 4 The aspect ratios of mesenchymal stem cells on different culture substrate surfaces in Example 2 of the present invention;

[0031] Figure 5 The scanning electron microscope photograph and particle size distribution diagram of the extracellular vesicles of Example 2 of the present invention are shown;

[0032] Figure 6 This is the RNA sequencing analysis result of Example 2 of the present invention. DETAILED DESCRIPTION

[0033] To help those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used are all commercially available unless otherwise specified.

[0035] Example 1

[0036] This embodiment provides a method for preparing stem cell-derived bone-targeted extracellular vesicles loaded with small molecule drugs, which comprises the following steps:

[0037] (1) Preparation of fiber membrane hydrogel:

[0038] First, 20wt% methacrylated gelatin (Gel-MA) was dissolved in hexafluoroisopropanol, and 4wt% methacrylated hyaluronic acid (HA-MA) was dissolved in trifluoroacetic acid. Then, the Gel-MA solution and the HA-MA solution were mixed in equal volumes to obtain a spinning solution containing 10wt% Gel-MA and 2wt% HA-MA. Subsequently, the spinning solution was loaded into a 5mL syringe and injected through a blunt stainless steel needle (model 23G) at a flow rate of 1.5mL / h. The voltage applied between the spinning needle and the stainless steel receiving plate was set to 20kV, and the distance between the spinning needle and the stainless steel receiving plate was 15cm. After 10min of electrospinning, the Gel-MA / HA-MA electrospun membrane was successfully collected.

[0039] The Gel-MA / HA-MA electrospun membrane after spinning was photocrosslinked under ultraviolet irradiation: First, 0.1g of 2-hydroxy-2-methylacetone (photoinitiator, I 1173) was dissolved in 10mL of ethanol to prepare a photocrosslinking solution. Then, the electrospun membrane was immersed in the photocrosslinking solution for 2h. Subsequently, each side of the electrospun membrane was exposed to 365nm ultraviolet light for 15min. After photocrosslinking, the crosslinked electrospun membrane was immersed in 10mL of ethanol again to remove excess photoinitiator and washed with deionized water. Finally, the crosslinked Gel-MA / HA-MA electrospun membrane was freeze-dried and stored. When in use, the electrospun membrane was immersed in PBS, absorbed water and swelled to form a fiber membrane hydrogel. The scanning electron microscope photo of the fiber membrane hydrogel is shown in the figure. Figure 1 shown.

[0040] (2) Stem cell culture:

[0041] Mesenchymal stem cells were cultured at a rate of 5×10 5 / cm 2 Cells were cultured on the fiber membrane hydrogels at a density of 100 μg / mL. After 2 hours of culture and 72 hours of culture in α-MEM medium supplemented with 10% extracellular vesicle-free serum and 1% double-antibody, the cells were collected. Stem cells in the control group were cultured in the same culture dish under the same conditions.

[0042] (3) Collection of extracellular vesicles:

[0043] The cell culture medium was centrifuged at 1000g for 10 minutes to remove dead cells and cell debris. Larger extracellular vesicles were then removed by centrifugation at 10,000g for 30 minutes. Impurities were further removed using a 0.22μm microporous filter. Finally, extracellular vesicles were collected by centrifugation at 100,000g for one hour. Extracellular vesicles were collected from control stem cells in the same manner.

[0044] Example 2

[0045] Fluorescence staining of mesenchymal stem cells cultured on the surface of fiber membrane hydrogel Figure 2 As shown, the cells are polygonal. The fluorescence staining of mesenchymal stem cells cultured in the control group is shown in Figure 3 As shown, the cells are spindle-shaped, indicating that the mesenchymal stem cells cultured on the fiber membrane hydrogel surface have better adhesion and spreading behavior, and the microfiber morphology is more conducive to cell adhesion and spreading.

[0046] The statistical results are as follows Figure 4 As shown, mesenchymal stem cells cultured on the fiber membrane hydrogel surface have adhesion and spreading behavior in all directions, while mesenchymal stem cells cultured in the control group culture dish adhere to and spread to both ends, indicating that mesenchymal stem cells cultured on the fiber membrane hydrogel surface have better adhesion and spreading behavior, and the microfiber morphology is more conducive to cell adhesion and spreading.

[0047] Particle size distribution diagram Figure 5 As shown, the average particle size of extracellular vesicles is about 250 nm, indicating that extracellular vesicles were successfully collected in this experiment.

[0048] RNA sequencing results Figure 6 As shown, the extracellular vesicles secreted by stem cells cultured on the surface of the fiber membrane hydrogel significantly increased gene expression compared to the extracellular vesicles secreted by stem cells cultured in a culture dish, indicating that culturing stem cells using the fiber membrane hydrogel prepared by the present application can guide the secretion of extracellular vesicles that promote tissue regeneration and enhance function by regulating the physiological activities of stem cells.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing extracellular vesicles, characterized in that: The preparation method comprises the following steps: (1) Stem cells were collected at a rate of 1×10 5 ~1×10 6 Pieces / cm 2 The density of the cells was placed on the fiber membrane hydrogel for culture, and the culture fluid was collected; (2) centrifuging the culture solution of (1) at 500-1000 g for 5-10 min, collecting the supernatant, and then centrifuging at 5000-10000 g for 20-40 min, collecting the supernatant, and removing impurities using a 0.22 μm microporous filter membrane to obtain the extracellular vesicles; Wherein, (1) the fiber membrane hydrogel comprises a gelling agent, and the gelling agent is a combination of 5 to 20 wt % methacrylated gelatin and 0.1 to 5 wt % methacrylated hyaluronic acid.

2. The preparation method according to claim 1, characterized in that: (1) The preparation method of the fiber membrane hydrogel is as follows: S1: dissolving methacrylated gelatin in hexafluoroisopropanol and methacrylated hyaluronic acid in trifluoroacetic acid, and mixing them to prepare a spinning solution containing 5-20 wt % of methacrylated gelatin and 0.1-5 wt % of methacrylated hyaluronic acid; S2: Spinning the spinning solution in S1 using an electrospinning device to obtain an electrospinning membrane; S3: placing the electrospun membrane described in S2 in a photoinitiator solution, exposing it to ultraviolet light of 200 to 365 nm for 5 to 60 minutes, and then removing excess photoinitiator to obtain the fiber membrane hydrogel.

3. The preparation method according to claim 2, characterized in that: S3: The photoinitiator solution contains 0.5-5 wt% of 2-hydroxy-2-methylacetone or 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone.

4. The preparation method according to claim 2, characterized in that: The electrospinning parameters described in S2 include: the voltage applied between the spinning needle and the stainless steel receiving plate is 10 to 20 kV, the distance between the spinning needle and the stainless steel receiving plate is 10 to 20 cm, and the spinning time is 5 to 30 min.

5. The preparation method according to claim 1, characterized in that: The stem cells are any one of human bone marrow mesenchymal stem cells, human adipose mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human umbilical cord blood mesenchymal stem cells, human placental mesenchymal stem cells, human dental pulp stem cells, human periodontal ligament stem cells, human hair follicle stem cells and human amniotic membrane mesenchymal stem cells.

6. The preparation method according to claim 1, characterized in that: The extracellular vesicles are exosomes, microvesicles or cystosomes.

7. An extracellular vesicle, characterized in that The method is obtained by the preparation method according to any one of claims 1 to 6.

8. An extracellular vesicle preparation, characterized in that Comprising the extracellular vesicles of claim 7.

9. A method for improving the tissue regeneration promoting function of extracellular vesicles, characterized in that: The fiber membrane hydrogel described in claim 1 is used to culture stem cells to obtain extracellular vesicles.

10. Use of the preparation method according to any one of claims 1 to 6 in preparing extracellular vesicles.

Images