A de-nucleated stem cell-based pro-repair microvesicle, and a preparation method and application thereof

Abstract

The application discloses a kind of based on de-nucleated stem cell's promoting repair microvesicle and its preparation method and application, including the following steps: using cytochalasin B to de-nucleate stem cell, using Percoll centrifugation or vortex method separates the stem cell cytoplast of de-nucleated stem cell, using extrusion device with filter membrane repeatedly extruding, namely de-nucleated stem cell microvesicle.The method in the application removes the nucleus of stem cell while retaining the key protein and RNA of stem cell to play the function of promoting repair, reduces the risk of dangerous gene transfer.Moreover, based on extrusion method can effectively control the particle size distribution of microvesicle, and based on the size of filter membrane pore size, the size of the prepared microvesicle is flexibly adjusted, and has significant therapeutic effect in tissue damage and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a repair-promoting microvesicle based on denuclearized stem cells, and a preparation method and application thereof. Background Art

[0002] Due to various physiological and pathological factors, ideal tissue regeneration and repair cannot be achieved in many cases. This is due, firstly, to the fact that cells at the site of tissue damage often age or even undergo abnormalities such as apoptosis and necrosis. Secondly, the microenvironment at the site of injury is severely imbalanced, leading to prolonged inflammation, which further deteriorates the cellular phenotype and disrupts the normal repair process. Therefore, reversing cell aging and improving the microenvironment are key to achieving efficient and rapid tissue repair.

[0003] Stem cells can secrete a variety of bioactive factors that promote repair, thereby improving the microenvironment at the site of injury, activating cells involved in tissue repair, and improving their biological functionality. In addition, stem cells contain a large number of active mitochondria. Studies have shown that stem cells can transport active mitochondria to damaged and mitochondrially dysfunctional cells through nanotubes, vesicles, etc., thereby restoring cell vitality, increasing their energy production, and reducing the negative effects of oxidative stress. Stem cells also have certain immunomodulatory effects. Therefore, stem cells are one of the most commonly used cells in the field of tissue repair. However, in related technologies, studies have found that when stem cells are transplanted into tissue damage in the hope of tissue repair, it is difficult for stem cells to remain in the transplant site for a long time and differentiate in the desired direction, and there is a risk of carcinogenesis and teratogenesis. When other substances are used to promote tissue repair, such as extracellular vesicles secreted by stem cells, there are many other problems. For example, the method of collecting cell culture supernatant and centrifuging to obtain extracellular vesicles requires culturing a large number of stem cells, which is cumbersome, costly, and has low yields. The particle size of the collected extracellular vesicles is uneven and uncontrollable. Therefore, the actual use of stem cells in tissue repair is greatly limited. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a repair-promoting microvesicle based on enucleated stem cells, and its preparation method and application. The method of the present invention can directly utilize stem cells to prepare enucleated stem cell microvesicles based on the extrusion method and the combination of a filter membrane with a specific pore size. Compared with traditional methods, it has more significant technical advantages, simple operation, low cost, high yield, uniform and controllable vesicle particle size, and has extremely high application value.

[0005] The first aspect of the present invention provides a method for preparing enucleated stem cell microvesicles, comprising the following steps:

[0006] The stem cells are enucleated using cytochalasin B, and the denucleated stem cell cytoplasmic bodies are separated using Percoll centrifugation or vortexing. The denucleated stem cell microvesicles are obtained by repeated extrusion using an extrusion device with a filter membrane.

[0007] In some embodiments of the present invention, the denucleated stem cell cytoplasmic bodies are isolated using Percoll centrifugation.

[0008] In this study, the inventors discovered that if intact stem cells are directly extruded, potential risk genes in the cell nucleus cannot be removed from the resulting vesicles. This poses a risk of risk gene transfer when the vesicles are subsequently applied to damaged tissue. To minimize the transfer of risk genes, it is essential to remove the stem cell nucleus from the cell before extrusion to create vesicles.

[0009] In the present invention, the cytoplasm can still maintain its activity after the cell nucleus is removed, and except for the removal of nuclear components, the organelles and other protein macromolecules and RNA components in the stem cells basically do not change, which also provides a material basis for the application of microvesicles in promoting repair and other aspects.

[0010] In some embodiments of the present invention, the stem cells include human stem cells and non-human mammalian stem cells.

[0011] In the present invention, there is no particular limitation on the source of stem cells, including human stem cells and non-human mammalian stem cells. Examples of mammals include mice, rats, rabbits, pigs, dogs, cows, primates (except humans), etc.

[0012] In some embodiments of the present invention, the stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, iPS (induced pluripotent stem cells), etc. according to their functionality or stage.

[0013] In the present invention, the only requirement for stem cells is that they are healthy stem cells, so as to ensure that after the cells undergo the method of the present invention, they can obtain microvesicles containing biologically active mitochondria, proteins, RNA and other contents, and based on this, they can maintain long-term stable activity under storage conditions of 4°C, and can promote cell growth and tissue repair.

[0014] In some embodiments of the present invention, the pore size of the filter membrane is 0.2 μm to 10 μm.

[0015] In some embodiments of the present invention, the pore size of the filter membrane is 3.0 μm to 10 μm.

[0016] In some embodiments of the present invention, the extrusion device is selected from a liposome extruder.

[0017] Of course, those skilled in the art can also select other devices that can be used for cell vesicle extrusion to perform extrusion operations according to actual use requirements, including but not limited to liposome extruders.

[0018] In some embodiments of the present invention, the extrusion times are greater than or equal to 20 times.

[0019] In some embodiments of the present invention, the extrusion times are 20 to 30 times.

[0020] In some embodiments of the present invention, the filter membrane is selected from track-etched filter membranes.

[0021] In some embodiments of the present invention, the extrusion device with a filter membrane is a liposome extruder with a filter membrane. Denucleated stem cell cytoplasms are repeatedly extruded through the liposome extruder with a filter membrane using a syringe or other input device to obtain denucleated stem cell microvesicles.

[0022] In some embodiments of the present invention, the specific extrusion steps are:

[0023] (1) Using a syringe to draw up the stem cell cytoplasm with the cell nucleus removed, the syringe is connected to a liposome extruder having a filter membrane with a pore size of 0.2 μm to 10 μm, and then an empty syringe is connected to the other end of the liposome extruder to receive the substance passing through the filter membrane;

[0024] (2) Push and pull the two syringes back and forth to allow the cytoplasmic suspension to pass through the filter membrane in the middle of the extruder repeatedly. Repeat this process more than 20 times to obtain microvesicles of denuclearized stem cells.

[0025] In the present invention, the method of removing the nucleus of the stem cell and then performing vesicle extrusion has the advantage of uniform and controllable particle size compared to directly using denucleated stem cells. It can be phagocytosed by recipient cells in the tissue in the form of extracellular vesicles, thereby effectively delivering the repair-promoting proteins, RNA, and active mitochondria in the vesicles to the recipient cells, thereby improving the biological activity of the recipient cells.

[0026] The second aspect of the present invention provides denuclearized stem cell microvesicles prepared by the preparation method described in the first aspect of the present invention.

[0027] In some embodiments of the present invention, the enucleated stem cell microvesicles are micro-nanoscale vesicles containing active contents and wrapped by the stem cell plasma membrane.

[0028] In some embodiments of the present invention, the active contents include organelles and / or bioactive macromolecules derived from maternal stem cells.

[0029] In the present invention, the enucleated stem cell microvesicles prepared in the present invention can, on the one hand, be mass-produced, avoiding dangerous gene transfer and low risk of teratogenicity and carcinogenesis; on the other hand, because their content components are highly similar or identical to the active components of the stem cells themselves, and their particle size is uniform and controllable, they can encapsulate organelles and be phagocytosed and utilized by recipient cells, so they can be well absorbed by the body and better promote tissue repair.

[0030] In some embodiments of the present invention, the active contents include organelles and bioactive macromolecules derived from maternal stem cells.

[0031] In some embodiments of the invention, the organelle comprises a mitochondria.

[0032] In some embodiments of the present invention, the bioactive macromolecules include proteins and nucleic acid molecules.

[0033] In some embodiments of the invention, the nucleic acid molecule comprises RNA.

[0034] In some embodiments of the invention, the RNA comprises microRNA.

[0035] In some embodiments of the present invention, the particle size of the enucleated stem cell microvesicles is 0.2 μm to 10 μm.

[0036] In some embodiments of the present invention, the particle size of the enucleated stem cell microvesicles is 1.0 μm to 10 μm.

[0037] The third aspect of the present invention provides the use of the enucleated stem cell microvesicles described in the second aspect of the present invention in the following (1) to (3);

[0038] (1) Preparation of tissue repair or auxiliary repair products;

[0039] (2) preparing products that inhibit cell aging;

[0040] (3) Preparation of products for preventing or treating inflammatory reactions caused by tissue damage.

[0041] In the present invention, the repair-promoting microvesicles prepared based on enucleated stem cells can be phagocytosed by cells at the site of tissue damage, achieving efficient delivery of bioactive factors, thereby improving the biological functions of cells, activating cells to participate in repair, and regulating the microenvironment. They can be used alone or in combination with other carrier materials as repair-promoting substances in the treatment of chronic difficult-to-heal wounds, pressure sores, severe burns, myocardial infarction, bone / cartilage damage and other tissue injuries to promote tissue repair.

[0042] The beneficial effects of the present invention are:

[0043] (1) The preparation method in the application effectively removes the nuclei of stem cells through chemical induction and high-speed centrifugation, while retaining key proteins and RNA of stem cells for playing a promoting repair function, and reducing the risk of dangerous gene transfer.

[0044] (2) The preparation method in the application is based on an extrusion method for preparing microvesicles, through which microvesicles with controllable and uniform particle size distribution can be obtained, and the size of the prepared microvesicles can be flexibly adjusted based on the needs of subsequent experiments and applications by changing the pore size of the track-etched filter membrane, which has excellent use flexibility.

[0045] (3) The application avoids the cumbersome process of extracting natural microvesicles such as exosomes, small extracellular vesicles, etc. from conditioned medium collected after a large number of stem cell culture, has high yield of vesicles, simple and efficient preparation process, and through experimental verification, can be phagocytosed by cells at tissue damage sites, realize efficient delivery of bioactive factors, thereby improving cell biological functions, activating cells to participate in repair and regulate microenvironment, and in the treatment of chronic non-healing wounds, pressure sores, severe burns, myocardial infarction, bone / cartilage damage and other tissue damage, the application can be used as a promoting repair material alone or in combination with other carrier materials to promote tissue repair. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The immunofluorescence staining diagram of the microvesicles of different particle sizes extruded by the method in the embodiment of the application.

[0047] Figure 2 The particle size distribution diagram of the microvesicles prepared by the method in the embodiment of the application, wherein MicroVesicles represents microvesicles, and Microspheres represents flow cytometry particle size calibration microspheres.

[0048] Figure 3 The flow cytometry characterization diagram of the mitochondrial content in the 3 mu microvesicles prepared by the method in the embodiment of the application.

[0049] Figure 4 The effect of the microvesicles prepared by the method in the embodiment of the application on the proliferation of fibroblasts.

[0050] Figure 5 The effect of the microvesicles prepared by the method in the embodiment of the application on the inflammatory factors (TNF-alpha, IL-6) of fibroblasts.

[0051] Figure 6 The cell staining image of HUVEC treated with the microvesicles prepared by the method in the embodiment of the application, wherein the blue cells are senescent HUVEC.

[0052] Figure 7This is a statistical diagram of the aging of HUVECs treated with microvesicles prepared using the method in an example of the present invention. DETAILED DESCRIPTION

[0053] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention.

[0054] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0055] In the present invention, the stem cells used are rat adipose-derived mesenchymal stem cells. It should be understood that this stem cell is merely an example and does not constitute a specific limitation on the type of stem cells used in the present invention. The stem cells that can be used in the present invention include, but are not limited to, rat adipose-derived mesenchymal stem cells.

[0056] Stem cell denucleation

[0057] The specific steps are:

[0058] (1) Rat adipose-derived mesenchymal stem cells (ADMSCs) passaged between P4 and P5 were digested using a cell digestion solution to facilitate their removal from the cell culture flask. The cells were resuspended in a rat adipose-derived mesenchymal stem cell culture medium, and cytochalasin B was added to a final concentration of 10 μg / mL. The cells were incubated in a cell culture incubator for 30 to 60 minutes (30 minutes in this example).

[0059] (2) Take the Percoll separation solution, add 8.5% NaCl at a volume ratio of 9:1 to obtain an isotonic SIP solution, and then dilute it with an equal volume of normal saline to obtain a 50% (v / v) Percoll solution.

[0060] (3) The rat adipose-derived mesenchymal stem cells incubated in (1) were centrifuged at 1200 rpm for 5 min, and the supernatant was removed to obtain a cell pellet. The pellet was resuspended in the 50% (v / v) Percoll solution obtained in (2), and cytochalasin B was added to a final concentration of 20 μg / mL. The pellet was centrifuged at 18,000 × g for 1.5 h at room temperature, and the buffy coat layer between the upper and lower liquids was collected and resuspended in culture medium or PBS buffer (PBS in this embodiment) to obtain an enucleated rat adipose-derived mesenchymal stem cell cytoplasmic suspension.

[0061] Preparation of stem cell microvesicles

[0062] The specific steps are:

[0063] (1) Use a syringe to draw up the enucleated rat adipose-derived mesenchymal stem cell cytoplasms prepared in the above example. Connect the syringe to a liposome extruder containing a filter membrane with a pore size of 0.2 μm to 10 μm (a track-etched filter membrane with a pore size of 3 μm was used in this example). Then, connect an empty syringe to the other end of the liposome extruder to receive the substance that has passed through the filter membrane.

[0064] (2) Place the connected device horizontally on the table, push and pull the two syringes back and forth, so that the cytoplasmic suspension repeatedly passes through the filter membrane in the middle of the extruder. Repeat this for more than 20 times. The residual liquid contains the desired enucleated stem cell microvesicles.

[0065] Characterization of stem cell microvesicles

[0066] (1) Immunofluorescence staining:

[0067] The enucleated stem cell microvesicles prepared based on filter membranes of different pore sizes (0.4 μm, 1.0 μm, and 3.0 μm) were stained using immunofluorescence staining. The dyes used were Dil (cell membrane red fluorescent probe), Hoechst 33342 (cell nucleus stain), and Mito Tracker-Green (mitochondrial green fluorescent probe).

[0068] The specific staining operation is:

[0069] DiI was diluted with PBS at a ratio of 1:200, Hoechst 33342 was diluted at a ratio of 1:100, and MitoTracker-Green was diluted at a ratio of 1:5000 to obtain the corresponding dye working solution. The enucleated cytoplasm was resuspended in the working solution and incubated in an incubator in the dark for 30 minutes. The cytoplasm was centrifuged at 1000×g for 15 minutes to obtain the stained cytoplasm. After washing twice with PBS, the cytoplasm was extruded according to the method in the above example to obtain the stained vesicles.

[0070] The results are as follows Figure 1 As shown in the figure, by observing the immunofluorescence staining of enucleated stem cell microvesicles obtained through filters of different pore sizes (0.4μm, 1.0μm, and 3.0μm), it can be found that all three groups of vesicles do not contain nuclear components. However, only when the enucleated stem cell microvesicles are extruded through a 3μm filter membrane can they effectively encapsulate active mitochondria.

[0071] (2) Particle size distribution:

[0072] The microvesicles of enucleated stem cells prepared with a 3.0 μm pore size filter were compared with flow cytometry particle size calibration microspheres (particle sizes of 1.0 μm, 4.0 μm, 6.0 μm, and 10.0 μm, purchased from ThermoFisher). The particle size differences between the samples were compared using the peak FSC values ​​in the flow cytometry results.

[0073] The results are as follows Figure 2 shown.

[0074] It can be found that when the microvesicles of enucleated stem cells prepared by the 3.0 μm pore size filter membrane are compared with the flow cytometry particle size calibration microspheres, it is found that 70.7% of the vesicles have a diameter distribution between 1 and 4 μm.

[0075] (3) Characterization of the proportion of mitochondria encapsulated in denuclearized stem cell microvesicles:

[0076] According to the method in the above example, mitochondria were labeled with MitoTracker-Green, cell membranes were labeled with DiI, and the proportion of mitochondria encapsulated in microvesicles of enucleated stem cells prepared with a 3.0 μm pore size filter membrane was characterized by flow cytometry.

[0077] The results are as follows Figure 3 shown.

[0078] It was found that about 80.9% of the enucleated stem cell microvesicles prepared by the 3.0 μm pore size filter membrane contained mitochondria.

[0079] Effects of using stem cell microvesicles

[0080] (1) Cell proliferation activity of stem cell microvesicles:

[0081] Rat fibroblasts were cultured in DMEM medium containing 35 mM glucose for at least 5 days, and β-galactosidase staining was used to verify cell senescence. Normal and senescent fibroblasts were seeded in well plates. Normal cells were cultured in low-glucose DMEM, while senescent cells were cultured in high-glucose DMEM. The experimental groups were treated with 20 μg / mL and 40 μg / mL of microvesicles, respectively. Cell proliferation activity was assessed using CCK8 reagent (refer to the user manual for instructions on using the CCK8 reagent).

[0082] The results are as follows Figure 4 shown.

[0083] Fibroblasts are typical cells at wound sites. It was found that, after treating normal and high-sugar senescent fibroblasts with the anucleated stem cell microvesicles prepared by the method in the above examples, by detecting the change in cell proliferation activity, it was found that the anucleated stem cell microvesicles could significantly improve the proliferation activity of high-sugar senescent fibroblasts, thereby benefiting the wound healing at the tissue damage site.

[0084] (2) Inhibition effect of stem cell microvesicles on inflammatory factor TNF-α:

[0085] Normal / senescent fibroblasts (rF) were inoculated in the well plate. Normal cells (Control) were cultured with low-sugar DMEM medium, senescent rF control group (Sen-Control) was cultured with high-sugar DMEM, and senescent cell experimental group was treated with 40 μg / mL microvesicles. The cell culture supernatant was collected on the 3rd day and the 5th day of treatment, and the inflammatory factors TNF-α and IL-6 secreted by the cells were detected by ELISA kit.

[0086] The results are shown in Figure 5 .

[0087] After treating fibroblasts with the anucleated stem cell microvesicles prepared by the method in the above examples, the concentration of inflammatory factors TNF-α and IL-6 secreted by fibroblasts before and after treatment was detected, and it was found that the anucleated stem cell microvesicles could alleviate the inflammatory phenotype of fibroblasts under high-sugar state, thereby benefiting the repair of chronic tissue damage.

[0088] (3) Anti-aging effect of stem cell microvesicles:

[0089] Human umbilical vein endothelial cells (HUVEC) were cultured with DMEM with high glucose concentration (35 mM) for 5 days. The control group was continued to be cultured with high-sugar DMEM, and the two experimental groups were treated with 20 μg / mL and 40 μg / mL microvesicles, respectively. The cells were stained with β-galactosidase on the 3rd, 5th, and 7th days, and the proportion of blue senescent cells in the field of view was counted.

[0090] The results are shown in Figure 6 and Figure 7 .

[0091] After treating human umbilical vein endothelial cells (HUVEC) with the anucleated stem cell microvesicles prepared by the method in the above examples, the aging of HUVEC was characterized by β-galactosidase staining. The results showed that, under the same initial conditions, when HUVEC was treated with a certain concentration of microvesicles for 5 days or more, the aging of HUVEC could be significantly improved.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The use of enucleated stem cell microvesicles in the preparation of products for treating tissue damage or inflammatory responses caused by tissue damage; The denuclearized stem cell microvesicles are prepared by the following method: Stem cells were incubated with cytochalasin B at a final concentration of 10 μg / mL for 30-60 minutes to enucleate. The enucleated stem cell cytoplasms were separated using Percoll centrifugation and cytochalasin B at a final concentration of 20 μg / mL. The enucleated stem cell microvesicles containing mitochondria were obtained by repeated extrusion using an extrusion device with a filter membrane. in, The steps for isolating the denucleated stem cell cytoplasm are: Cytochalasin B was added to separate the stem cell cytoplasmic bodies that had lost their nuclei; The pore size of the filter membrane is 3.0 μm; The extrusion times are greater than or equal to 20 times; The extrusion device is selected from a liposome extruder; The enucleated stem cell microvesicles are micro-nano-scale vesicles wrapped by the stem cell plasma membrane and containing active contents; the active contents include mitochondria derived from maternal stem cells and bioactive macromolecules; The stem cells are adipose-derived mesenchymal stem cells.

2. The use according to claim 1, characterized in that The filter membrane is a track-etched filter membrane.

3. The use according to claim 1, characterized in that The biologically active macromolecules include proteins and nucleic acid molecules.

4. The use according to claim 1, characterized in that The particle size of the denuclearized stem cell microvesicles is 1 μm to 4 μm.

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