A method for isolating macrophage migrasomes

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a simple and efficient method for separating macrophage migratory bodies. The present invention adopts a filter to filter and intercept migratory bodies and successfully separates migratory bodies with a diameter range of 0.5-3 μm by reverse filtration and elution. The present invention successfully separates migratory bodies with a vesicle-like structure and wrinkles on the surface, with a diameter of more than 500 nm from RAW264.7 cells. The isolated migratory bodies express characteristic proteins PIGK, EOGT, and TSPAN4, but do not express EVs-specific markers TSG101 and ALIX, and are a unique type of vesicle that is different from EVs. It also does not affect the integrity of the RNA carried by the migratory bodies. The migratory body separation method provided by the present invention has the characteristics of simplicity, high efficiency, good controllability of the separation method, good repeatability, low cost, and no need for large special equipment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for separating macrophage migratory bodies. Background Art

[0002] Extracellular vesicles (EVs), involved in various pathophysiological processes, have rapidly developed as tools for intercellular communication. For example, the proteins, nucleic acids, cytokines, and growth factors contained in EVs mediate immune regulation, angiogenesis, tumor growth, and wound repair. Consequently, EVs have applications in the diagnosis, treatment, and prognosis of a wide range of diseases. Migrasomes are a novel and unique type of vesicle secreted and released by cells during migration. They range in size from 0.5 to 3 μm and have been demonstrated to be present in a variety of cells, tissues, and body fluids. Studies have shown that migrasomes are not only crucial for organ formation during embryonic development but also function as a means of expelling damaged mitochondria to maintain cellular homeostasis. Furthermore, migrasomes are not only involved in the progression of stroke but also serve as non-invasive biomarkers of renal podocyte injury. These findings suggest that, similar to other EVs, migrasomes hold great potential and value in disease diagnosis, treatment, and prognosis. However, our understanding of migrasomes, particularly their functional investigations, remains at a preliminary stage. Efficient isolation of migrasomes is crucial for studying their functions.

[0003] Currently, there are two methods for isolating migrasomes: one is to extract migrasomes by low-speed fractional centrifugation, which only yields a crude extract; the other is to extract migrasomes by low-speed fractional centrifugation, followed by gradient density centrifugation, and finally by ultrahigh-speed centrifugation. This method is currently the most reliable, but its complex procedures, long separation times, and demanding equipment make it difficult to meet the requirements for subsequent large-scale extraction and subsequent applications. Therefore, the development of new methods for isolating migrasomes is urgently needed. This is of great significance for studying the biological functions of these novel EVs and their role as intercellular messengers, which can provide important information for the diagnosis and treatment of diseases. Summary of the Invention

[0004] In view of this, and to solve one of the above technical problems, the present invention provides a simple and efficient method for isolating macrophage migrasomes. The present invention uses a filter to filter and intercept, and then separates migrasomes with a diameter ranging from 0.5 to 3 μm by reverse filtration and elution, thereby achieving rapid isolation of migrasomes in macrophages.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for isolating macrophage migratory bodies, the method comprising the steps of:

[0007] (1) The macrophages to be isolated were cultured in DMEM medium containing fetal bovine serum overnight;

[0008] (2) Place a sterile glass slide in a well plate and inoculate macrophages. Add DMEM culture medium without EV serum to each well and incubate at 37°C in a 50 mL / L CO2 saturated humidity incubator for 24 to 36 hours. Discard the supernatant and add glutaraldehyde (2.5%) to completely cover the cell surface. Place in a refrigerator at 4°C overnight and then discard. Rinse with PBS and fix with pre-cooled osmium phosphate for 1 to 2 hours. Rinse again with PBS and dehydrate with alcohol gradient. Dry in a freeze dryer under vacuum and spray gold coating with an ion sputtering instrument.

[0009] (3) The macrophages obtained in step (2) were inoculated into a cell culture dish, and DMEM culture medium without EV serum was added. The cells were cultured in a 37°C, 50 mL / L CO2 saturated humidity incubator. When the cell confluence reached 40-50%, the cells were digested with trypsin, centrifuged, and the supernatant was collected. After filtering with a filter, the filter direction was reversed for reverse filtration. After elution, ultrafiltration was performed using an ultrafiltration tube with a molecular weight cutoff of 100 KD. The remaining liquid obtained was the migratoria.

[0010] Preferably, the concentration of the alcohol gradient dehydration in step (2) is 50%, 70%, 90%, 95%, or 100%.

[0011] The current of the ion sputtering apparatus in step (2) is 10 mA, and the time is 120 s.

[0012] The filter described in step (3) has filter pores capable of intercepting migratory bodies with a diameter of 0.5-3 μm; preferably, the pore size of the filter is 0.45 μm.

[0013] The reverse filtration described in step (3) is to use a sterile silicone tube to physically connect the filter in the reverse direction to a syringe containing pre-cooled PBS and then perform filtration.

[0014] The diameter of the migration body in step (3) is between 0.5 and 3 μm.

[0015] The present invention uses a filter to capture migratoria and successfully isolates migratoria with diameters ranging from 0.5 to 3 μm through reverse filtration and elution. This method is a simple and efficient method for isolating macrophage migratoria. Specific embodiments of the present invention demonstrate that numerous contractile filaments extend from the tail surface of RAW264.7 cells, with membranous vesicles at their tips or bifurcations, ranging in diameter from 0.5 to 3 μm. The isolated migratoria are round, have a vesicle-like structure, and have wrinkled surfaces. Their diameters are over 500 nm, which is consistent with their morphology and size under scanning electron microscopy. The isolated migratoria express the characteristic proteins PIGK, EOGT, and TSPAN4, but do not express the EV-specific markers TSG101 and ALIX, suggesting that migratoria are a unique type of vesicle distinct from EVs. Furthermore, the integrity of the RNA carried by the migratoria is not compromised. The provided migratoria isolation method is simple, efficient, highly controllable, reproducible, low-cost, and does not require large, specialized equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a morphological feature of macrophages forming migratory bodies under a scanning electron microscope;

[0017] Figure 2 This is a morphological feature of migrasomes isolated from macrophages under transmission electron microscopy;

[0018] Figure 3 is the Western blot expression graph of the characteristic markers of the isolated migrasome;

[0019] Figure 4 is a small RNA profile of mitosomes isolated from macrophages. DETAILED DESCRIPTION

[0020] The present invention is further described by the following examples, however, the scope of the present invention is not limited to these examples. The present invention provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, the reagents, biological materials, etc. used in the following examples were commercially available.

[0021] (1) Macrophage culture

[0022] Mouse RAW264.7 macrophages purchased from the Cell Bank of the Chinese Academy of Sciences were routinely cultured overnight in a 37°C, 50 mL / L saturated humidity CO2 incubator using DMEM medium containing 100 mL / L fetal bovine serum.

[0023] (2) Scanning electron microscopy observation of RAW267.4 cell migratoria formation: Sterile round glass slides were placed in a 24-well plate and 5×10 RAW264.7 cells were inoculated. 4 2 mL of EV serum-depleted DMEM was added to each well and incubated at 37°C in a humidified incubator with 50 mL / L CO2 for 24 h. The supernatant was discarded, and 25 mL / L glutaraldehyde (2.5%) was added to completely cover the cell surface. The cells were refrigerated at 4°C overnight and then aspirated. The cells were rinsed twice with PBS for 10 min each. After discarding the PBS, the cells were fixed with pre-chilled 10 mL / L osmium sulfate for 1 h. The cells were rinsed twice with PBS and dehydrated twice with a gradient of ethanol (50%, 70%, 90%, 95%, 100%) for 15 min each. After drying in a freeze-dryer under vacuum, the cells were sputter-coated with gold using an ion sputtering system (current: 10 mA, time: 120 s). The morphology of RAW267.4 cell migrasomes was observed and images were taken under a scanning electron microscope.

[0024] Figure 1 This is a morphological feature diagram of macrophages forming migratory bodies under a scanning electron microscope; Figure 1 As shown, a large number of contractile filaments extend from the surface of the tail of RAW264.7 cells, and the tips or bifurcations of the filaments have membrane-structured vesicles with a diameter of approximately 0.5-3 μm.

[0025] (3) Isolation of migratory bodies: RAW264.7 cells from step (2) were inoculated into 100 mm cell culture dishes, 6 mL of EV serum-free DMEM culture medium was added, and the cells were cultured in a 37°C, 50 mL / L CO2 saturated humidity incubator; when the cell confluence reached 40-50%, the cells were digested with 2.5 g / L trypsin and centrifuged at 1000 g for 10 min at 4°C; the supernatant was collected and centrifuged at 4000 g for 20 min at 4°C to remove cell debris and impurities; the supernatant was then collected and filtered with a 0.45 μm filter; a sterile silicone tube was then used to physically connect the filter in the reverse direction to a syringe containing 5 mL of pre-cooled PBS, and the filter was then turned in the opposite direction, and reverse filtration was performed with the reverse side of the filter to elute the migratory bodies retained by the filter; finally, a 100KD ultrafiltration tube was used for ultrafiltration, and the remaining liquid obtained was the migratory body and was packaged with EP tubes and stored in a -80°C refrigerator for later use.

[0026] Take 20 μL of the isolated and purified migratoria in Example 1 and mix thoroughly, then add it dropwise to the sample-loaded copper grid and let it stand at room temperature for 1 min. Then, use filter paper to absorb the liquid on the copper grid from the edge of the copper grid. Add 30 g / L phosphotungstic acid solution dropwise to the copper grid and negatively stain it at room temperature for 5 min. After drying under an incandescent lamp, place it in the electron microscope sample chamber, and observe the morphology of the migratoria under a transmission electron microscope and take pictures. Figure 2 is a morphological feature diagram of migratory bodies isolated from macrophages under transmission electron microscopy; Figure 2 As shown in the figure, the migrasomes isolated from RAW264.7 cells were round, had a vesicle-like structure and wrinkled surface, and had a diameter of more than 500 nm; this was basically consistent with the morphology and particle size results under scanning electron microscopy.

[0027] The migrating EVs and macrophages isolated and purified in Example 1 were thoroughly lysed, and total protein was collected and quantified using a BCA protein assay. 60 μg of protein was loaded onto a SDS-PAGE gel and electrotransferred to a PVDF membrane. The membrane was blocked with 50 g / L skim milk powder for 1 hour at room temperature, and antibodies to TSG101, ALIX, PIGK, EOGT, TSPAN4, and β-actin (1:1000) were added at 4°C overnight. After washing with TBST, a secondary antibody (HRP-conjugated goat anti-rabbit antibody) was added (1:2000) and incubated at room temperature for 1 hour. After washing with TBST, a chemiluminescent substrate was added for color development, and the membrane was photographed and analyzed using an ELC chemiluminescence system. Figure 3 It is a Western blot expression diagram of the characteristic markers of the isolated migratory body; Figure 3 As shown in the figure, migrasomes isolated from RAW264.7 cells expressed their characteristic proteins PIGK, EOGT, and TSPAN4, but did not express EVs-specific markers TSG101 and ALIX, suggesting that migrasomes are a unique type of vesicles that are different from EVs.

[0028] RNA was extracted and purified from the isolated misomes and subsequently analyzed using a Labchip bioanalyzer. RNA quantification was performed by Hangzhou Lianchuan Biotechnology Co., Ltd. All procedures were performed according to the kit's recommended procedures. Figure 4 is a small RNA profile of mitosomes isolated from macrophages. Figure 4 As shown, the migratoria produced a better electropherogram of small RNAs with a length of 25-200 nt.

[0029] Thus, it can be seen that the migratoria isolation and extraction method of the present invention can successfully isolate migratoria from macrophages without affecting the integrity of the RNA carried by the migratoria.

[0030] Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples without mutual contradiction.

Claims

1. A method for isolating macrophage migratory bodies, characterized in that: The separation method comprises the following steps: (1) The macrophages to be isolated were cultured in DMEM medium containing fetal bovine serum overnight; (2) Place a sterile glass slide in a well plate and inoculate macrophages. Add DMEM culture medium without EV serum to each well and incubate at 37°C in a 50 mL / L CO2 saturated humidity incubator for 24 to 36 hours. Discard the supernatant and add 2.5% glutaraldehyde to completely cover the cell surface. Place in a 4°C refrigerator overnight and then discard. Rinse with PBS and fix with pre-cooled osmium phosphate for 1 to 2 hours. Rinse again with PBS and dehydrate with alcohol gradient. Dry in a freeze dryer under vacuum and spray gold coating with an ion sputtering instrument. (3) The macrophages obtained in step (2) were inoculated into a cell culture dish, and DMEM culture medium without EV serum was added, and the cells were cultured in a 37°C, 50 mL / L CO2 saturated humidity incubator; when the cell confluence reached 40-50%, the cells were digested with trypsin, centrifuged, and the supernatant was collected; after filtering with a filter with a pore size of 0.45 μm, the filter direction was reversed for reverse filtration, and after elution, ultrafiltration was performed using an ultrafiltration tube with a molecular weight cutoff of 100 KD. The remaining liquid obtained was the migratoria; the reverse filtration described in step (3) was performed by using a sterile silicone tube to physically connect the filter in the reverse direction to a syringe containing pre-cooled PBS and then perform filtration.

2. The separation method according to claim 1, wherein The concentrations of the alcohol gradient dehydration described in step (2) are 50%, 70%, 90%, 95%, and 100%.

3. The separation method according to claim 1, characterized in that The current of the ion sputtering apparatus in step (2) is 10 mA, and the time is 120 s.

4. The separation method according to claim 1, wherein The diameter of the migration body in step (3) is between 0.5 and 3 μm.

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