Exosome transmission electron microscope sample preparation method

Abstract

The application relates to the field of transmission electron microscopes, in particular to an exosome transmission electron microscope sample preparation method, which comprises the following steps: 1) arranging exosomes in an iodixanol-containing buffer system to prepare a sample solution; 2) adsorbing the sample solution in step 1) on a carrier to prepare a pre-electron microscope sample; and 3) removing the solution on the surface of the pre-electron microscope sample in step 2), performing rinsing and negative staining, and then drying to prepare a transmission electron microscope sample. The exosome electron microscope sample prepared by the method does not collapse, presents a negative staining effect, a clean background, a clear and complete exosome contour imaging effect, can obtain an exosome image with a complete structure and without collapse, can be automatically counted by using image processing software, and makes exosome particle size counting more accurate and convenient.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of transmission electron microscopy, in particular to a method for preparing an exosome transmission electron microscopy sample. BACKGROUND

[0002] Exosomes are nanometer-sized (30-150 nm) extracellular vesicles secreted by cells, which play a role in intercellular communication and are involved in many important physiological and pathological processes, such as immune response, tumor metastasis, etc. Exosomes have natural molecular transport properties, are non-immunogenic compared to synthetic nanoparticles, have long and stable circulation in the body, and have great application potential in the field of drug carriers.

[0003] Transmission electron microscopy is an important characterization method for the nanostructure morphology and particle size of exosomes. However, due to the liquid state of the contents of these vesicle structures, which are not substantial nanostructures, the ion concentration difference in the process of negative staining with uranium salt can cause the vesicles to rupture or collapse, resulting in poor electron microscopy imaging effects, which affects the statistical results of morphology and particle size. In some methods, glutaraldehyde and other fixing solutions are used to fix exosomes to maintain their morphology. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, to solve the technical problem of vesicle rupture or collapse in the process of preparing an electron microscopy sample of exosomes in the prior art, which affects the electron microscopy imaging effect, the purpose of the present application is to provide a method for preparing an exosome transmission electron microscopy sample, which solves the problems in the prior art.

[0005] To achieve the above-mentioned and other related purposes, one aspect of the present application provides a method for preparing an exosome transmission electron microscopy sample, comprising the following steps:

[0006] 1) Exosomes are disposed in a buffer system containing iodixanol to prepare a sample solution;

[0007] 2) The sample solution of step 1) is adsorbed on a carrier to prepare a pre-electron microscopy sample;

[0008] 3) The solution on the surface of the pre-electron microscopy sample of step 2) is removed, and after rinsing and negative staining, the transmission electron microscopy sample is prepared by air drying.

[0009] In any embodiment of the present application, in step 1), the concentration of iodixanol is 3% to 45% (w / v).

[0010] In any embodiment of the present application, in step 1), the buffer system comprises a combination of one or more of a phosphate buffer solution, a tris-hydroxymethyl aminomethane buffer solution, a morpholine propyl sulfonic acid buffer solution, and a hydroxyethyl piperazine ethanesulfonic acid buffer solution.

[0011] In any embodiment of the present application, in step 2), the adsorption time is 1-10 min.

[0012] In any embodiment of the present application, in step 2), the carrier is selected from one or more of an ultrathin carbon copper grid, a conventional carbon film copper grid, and an enhanced carbon film copper grid.

[0013] In any embodiment of the present application, in step 3), the rinsing solution is selected from one or more of physiological saline, phosphate buffer, and uranium solution; preferably, the rinsing solution is a uranyl acetate solution; more preferably, the rinsing solution is a 1-2% (m / v) uranyl acetate solution. The rinsing time is 10-30 s.

[0014] In any embodiment of the present application, in step 3), the negative staining solution is selected from one or more of sodium phosphotungstate, potassium phosphotungstate, and uranium solution; preferably, the negative staining solution is a uranyl acetate solution; more preferably, the negative staining solution is a 1-2% (m / v) uranyl acetate solution. The negative staining time is 1-10 min.

[0015] The second aspect of the present application provides a transmission electron microscope sample prepared by the exosome transmission electron microscope sample preparation method.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. The sample preparation method has low requirements for exosomes, and is not limited by the source, preparation method, purification method, purity, functional loading, solution environment, and density of the exosomes, so that the exosome particles can be maximally settled on the carrier grid.

[0018] 2. The exosome transmission electron microscope sample prepared by the present application does not collapse, and has uniform negative staining, clean background, clear and complete imaging effect of exosome outline.

[0019] 3. Compared with the traditional sample preparation method, the exosome image obtained without adding glutaraldehyde fixation is complete and does not collapse, and image processing software can be used for automatic statistics, so that the exosome particle size statistics are more accurate and convenient. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure shows the electron microscope image obtained by photographing the sample prepared by the control group without fixation and direct negative staining electron microscope sample preparation.

[0021] Figure 2 The figure shows the electron microscope image obtained by photographing the sample prepared by the electron microscope sample preparation method using 2.5% glutaraldehyde fixation.

[0022] Figure 3The electron micrograph shown was taken of a sample prepared using the electron microscope sample preparation method with 5% iodixanol buffered solution.

[0023] Figure 4 The electron micrograph shown was taken of a sample prepared using the electron microscope sample preparation method with 25% iodixanol buffered solution. DETAILED DESCRIPTION

[0024] In order to make the purposes, technical solutions and beneficial effects of the present application clearer, the present application will be further described below in conjunction with examples. It should be understood that the examples are only used to explain the present application and not intended to limit the scope of the application. The test methods used in the following examples are conventional methods, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure.

[0025] The "range" disclosed in the present application is defined in the form of lower limit and upper limit, a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, i.e. any lower limit can be combined with any upper limit to form a range. For example, if the ranges of 60-120 and 80-110 are listed for a particular parameter, it is understood that the ranges of 60-110 and 80-120 are also contemplated. In addition, if the minimum range values of 1 and 2 are listed, and if the maximum range values of 3, 4 and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present application, unless otherwise stated, the numerical range "a-b" represents a shorthand notation for any real combination of integers between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed herein, and "0-5" is only a shorthand notation for these numerical combinations. In addition, when a parameter is stated to be ≥2 integers, it is equivalent to disclose that the parameter is, for example, integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0026] Unless otherwise specified, all steps of the present application can be performed sequentially or randomly, and preferably sequentially. For example, the method comprises steps 1) and 2), which means that the method can comprise sequentially performed steps 1) and 2), or sequentially performed steps 2) and 1).

[0027] The inventors of the present application have found, through a large number of exploratory researches, an exosome transmission electron microscope sample preparation method, which replaces the conventional preparation method, thereby solving the problem of easy rupture and collapse of exosomes in the existing method. On this basis, the present application is completed.

[0028] The application provides an exosome transmission electron microscope sample preparation method, comprising the following steps:

[0029] 1) The exosome is arranged in an iodixanol-containing buffer system to prepare a sample solution;

[0030] 2) The sample solution in step 1) is adsorbed on a carrier to prepare a pre-electron microscope sample;

[0031] 3) The solution on the surface of the pre-electron microscope sample in step 2) is removed, and after rinsing and negative staining, the transmission electron microscope sample is prepared by air drying.

[0032] In the exosome transmission electron microscope sample preparation method provided by the application, in step 1), the exosome includes exosomes of unlimited sources, unlimited purification methods and purity. The exosome may be from cell culture solution, plasma, urine, cerebrospinal fluid, emulsion, lymph or saliva, etc. It should be understood that the above is only used as an example and is not used for limitation, and in fact, any exosome is suitable for the application. In a specific embodiment of the application, the exosome includes natural exosome and functionalized exosome loaded with nucleic acid, chemical small molecule, polypeptide, etc., and hybrid exosome fused with liposome. It should be understood that the above exosome is only used as an example and is not used for limitation, and in fact, the exosome loaded with any substance is suitable for the application.

[0033] In the exosome transmission electron microscope sample preparation method provided by the application, in step 1), the concentration of iodixanol is 3% to 45% (m / v), preferably, the concentration of iodixanol is 20% to 40% (m / v). In a specific embodiment of the application, the concentration of iodixanol is 25%. The iodixanol, also known as iodixanol, iodoxanol, iodixanol, etc., has a molecular weight of 1550.18 and a purity of 2.295 g / cm 3 , which is commercially available (StemCell, OptiPrep TM #07820). Iodixanol is a new type of non-ionic, isotonic, double-body iodine-containing contrast agent, which has a unique isotonicity advantage, good water solubility, can enhance the stability of the phospholipid bilayer of the exosome, thereby reducing the rupture phenomenon of the exosome during the negative staining and drying process of the exosome transmission electron microscope sample preparation process, improving the electron microscope imaging effect of the exosome, and optimizing the evaluation results of the sample morphology and particle size.

[0034] In the exosome transmission electron microscope sample preparation method provided by the application, in step 1), the buffer system includes one or more combinations of phosphate buffer solution, Tris buffer solution, MOPS buffer solution and HEPES buffer solution.

[0035] In the exosome transmission electron microscope sample preparation method provided in the present application, in step 2), the adsorption time is 1-10 min. In a specific embodiment of the present application, the adsorption may, for example, be that the sample solution is dropped on the carrier and left to adsorb. The standing adsorption time is 1-3 min, and the sample solution is dropped in an amount of 5 μL.

[0036] In the exosome transmission electron microscope sample preparation method provided in the present application, in step 2), the carrier is selected from one or more of an ultrathin carbon copper grid, a regular carbon film copper grid, and an enhanced carbon film copper grid. In a specific embodiment of the present application, the carrier is an enhanced carbon film copper grid. The carrier is used to load the biological sample in electron microscope observation.

[0037] In the exosome transmission electron microscope sample preparation method provided in the present application, in step 3), the solution for removing the pre-electron microscope sample surface may, for example, be that the solution is absorbed with filter paper. This method can directly rinse and negatively stain without glutaraldehyde fixation, and can also obtain exosome images with complete structure and without collapse. The rinsing solution is selected from one or more of physiological saline, phosphate buffer, and uranium solution. Preferably, the rinsing solution is an uranyl acetate solution. More preferably, the rinsing solution is a 1-2% (m / v) uranyl acetate solution. The rinsing time is 10-30 s. Preferably, the rinsing time is 10-15 s. In a specific embodiment of the present application, the rinsing is performed once. The rinsing is used to wash away the excess exosome sample.

[0038] In the exosome transmission electron microscope sample preparation method provided in the present application, the negative staining solution is selected from one or more of sodium phosphotungstate, potassium phosphotungstate, and uranium solution. Preferably, the negative staining solution is an uranyl acetate solution. More preferably, the negative staining solution is a 1-2% (m / v) uranyl acetate solution. The negative staining time is 1-10 min. Preferably, the negative staining time is 1-3 min. In a specific embodiment of the present application, the negative staining is performed once. The negative staining is used to contrast the exosome sample by using the difference in density between the exosome sample and the negative staining reagent.

[0039] Another aspect of the present application provides a transmission electron microscope sample prepared by the exosome transmission electron microscope sample preparation method as described. The electron microscope sample does not collapse, presents a uniform negative staining, a clean background, a clear and complete exosome outline imaging effect, and can be automatically counted using image processing software, so that the exosome particle size counting is more accurate and convenient.

[0040] The present application is further illustrated by the following examples, but the scope of the present application is not limited thereby.

[0041] Example 1

[0042] Step 1, extraction and purification of exosomes: macrophages (RAW 264.7 cells) were cultured in vitro. When the cells were cultured to 80% density in complete nutrient medium (Gibco RPMI Medium 1640 containing 10% fetal bovine serum (Gibco, FBS)), they were washed 3 times with PBS and then replaced with medium without 10% fetal bovine serum / vessel-free serum. After 24 hours of culture, the supernatant was collected. Exosomes were extracted and purified using ultracentrifugation, and the specific steps were as follows:

[0043] 1) The cell supernatant was centrifuged at 300g for 10 min at 4°C to remove cells and dead cells, and the supernatant was taken;

[0044] 2) The supernatant obtained in step 1) was centrifuged at 2000g for 10 min at 4°C to remove cell debris, and the supernatant was taken;

[0045] 3) The supernatant obtained in step 2) was centrifuged at 10,000g for 30 min at 4°C to remove large-sized extracellular vesicles, and the supernatant was taken;

[0046] 4) After centrifugation of the supernatant obtained in step 3) at 12,000g for 70 min at 4°C, the supernatant was discarded, and the exosome precipitate was collected and resuspended with 1xHB (25mM HEPES, 100mM KCl, 10mM MgCl2, pH 7);

[0047] 5) After centrifugation of the exosome resuspension obtained in step 4) at 12,000g for 70 min at 4°C, the supernatant was discarded, and the exosome precipitate was collected and resuspended with 1xHB to obtain an exosome suspension with a concentration of 100μg / mL.

[0048] Step 2, preparation of electron microscope samples

[0049] 5μl of exosome suspension with a concentration of about 100μg / ml was diluted to 5% with iodixanol-containing 1xHB (25mM HEPES, 100mM KCl, 10mM MgCl2, pH 7) solution, respectively; the enhanced carbon film copper mesh was treated with a glow discharge instrument at 25mA for 1min, then the enhanced carbon film copper mesh was clamped with a self-locking forceps, 5μL of exosome diluent was added to the front surface of the copper mesh, and after standing at room temperature for 1min, the excess liquid was absorbed with filter paper after the exosomes were adsorbed on the surface of the copper mesh. Subsequently, 10μL of 0.2% uranyl acetate solution was added to the surface of the copper mesh, and after rinsing for 10s, the excess uranium solution was absorbed with filter paper. Finally, 10μL of 0.2% uranyl acetate solution was added to the surface of the copper mesh for negative staining, and the time was 1min. After negative staining, the excess uranium solution was absorbed with filter paper, and the copper mesh was dried to obtain the experimental group exosome transmission electron microscope sample. The experimental group exosomes were characterized by transmission electron microscopy, and the following photographs were taken Figure 3 .

[0050] Example 2

[0051] Step 1 and Step 2 were the same as Example 1, except that the concentration of iodixanol 1x HB solution was 25%, and the photographs were taken at Figure 4 .

[0052] Comparative Example 1

[0053] Step 1 was the same as Example 1.

[0054] Step 2: Take 5 μl of exosome suspension with a concentration of about 100 μg / mL, and dilute 20 times with 1x HB solution; the enhanced carbon film copper mesh is treated with a glow discharge instrument at 25 mA for 1 min, then the enhanced carbon film copper mesh is clamped with a self-locking forceps, 5 μL of exosome diluent is added to the front of the copper mesh, and after standing at room temperature for 1 min, the excess liquid is absorbed with filter paper after the exosomes are adsorbed on the surface of the copper mesh. Then, 10 μL of 0.2% uranyl acetate solution is added to the surface of the copper mesh, and after rinsing for 10 s, the excess uranium solution is absorbed with filter paper. Finally, 10 μL of 0.2% uranyl acetate solution is added to the surface of the copper mesh for negative staining, and the time is 1 min. After negative staining, the excess uranium solution is absorbed with filter paper, and after the copper mesh is dried, the exosome transmission electron microscope sample of the control group is obtained. The exosomes of the control group are characterized by transmission electron microscopy, and the photographs are taken at Figure 1 .

[0055] Comparative Example 2

[0056] Take 5 μl of exosome suspension with a concentration of about 100 μg / mL, and dilute 20 times with 1x HB solution; the enhanced carbon film copper mesh is treated with a glow discharge instrument at 25 mA for 1 min, then the enhanced carbon film copper mesh is clamped with a self-locking forceps, 5 μL of exosome diluent is added to the front of the copper mesh, and after standing at room temperature for 1 min, the excess liquid is absorbed with filter paper after the exosomes are adsorbed on the surface of the copper mesh. Then, 10 μL of 2.5% glutaraldehyde is added to the surface of the copper mesh, and after incubation for 5 min, it is absorbed with filter paper. Subsequently, 10 μL of 0.2% uranyl acetate solution is added to the surface of the copper mesh, and after rinsing for 10 s, the excess uranium solution is absorbed with filter paper. Finally, 10 μL of 0.2% uranyl acetate solution is added to the surface of the copper mesh for negative staining, and the time is 1 min. After negative staining, the excess uranium solution is absorbed with filter paper, and after the copper mesh is dried, the exosome transmission electron microscope sample of the control group is obtained. The exosomes of the control group are characterized by transmission electron microscopy, and the photographs are taken at Figure 2 .

[0057] Comparison Figure 1 and Figure 2It can be observed that the exosomes are not treated and directly added on the copper mesh for negative staining sample preparation. The exosomes cannot be effectively settled on the copper mesh, and the morphology of the exosomes cannot be observed in the electron microscope. After the exosomes are fixed by 2.5% glutaraldehyde, negative staining sample preparation is performed again, which can increase the deposition of the exosomes and has a negative staining effect. The morphology and particle size of the exosomes can be collected by the electron microscope, but the number of the exosomes is small, and multiple electron microscope pictures need to be taken to meet the requirement of the particle size of the exosomes.

[0058] Comparison Figure 2 and Figure 3 It can be observed that after the exosomes are diluted by 5% iodixanol buffer solution, no 2.5% glutaraldehyde fixation is needed, and direct negative staining can observe a large number of exosomes, and the exosomes maintain a certain morphology, and only part of the exosomes are broken.

[0059] Further comparison Figure 3 and Figure 4 It can be observed that compared with 5% iodixanol buffer solution, after the exosomes are diluted by 25% iodixanol buffer solution, the exosome morphology is best, no rupture phenomenon, and the exosomes are retained on the copper mesh, and the particle size of the exosomes can be accurately measured and counted.

[0060] In summary, the exosomes diluted by 25% iodixanol buffer solution have the best electron microscope sample preparation effect, have excellent negative staining effect, and the exosome morphology is full, no rupture phenomenon, and enough number of exosomes in the electron microscope pictures can be used for counting the particle size.

[0061] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought of the present application should be covered by the claims of the present application.

Claims

1. An exosome transmission electron microscopy sample preparation method, characterized by, The method comprises the following steps: 1) disposing exosomes in a buffer system containing iodixanol to prepare a sample solution; 2) adsorbing the sample solution of step 1) on a carrier to prepare a pre-TEM sample; 3) removing the solution on the surface of the pre-TEM sample of step 2), and after rinsing and negative staining, drying to prepare a TEM sample.

2. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 1), the concentration of iodixanol is 3% to 45% (w / v).

3. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 1), the buffer system comprises a combination of one or more of a phosphate buffer solution, a tris buffer solution, a morpholinepropanesulfonic acid buffer solution, and a hydroxyethylpiperazineethanesulfonic acid buffer solution.

4. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 2), the adsorption time is 1 to 10 minutes.

5. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 2), the carrier is selected from one or more of an ultrathin carbon copper grid, a regular carbon film copper grid, and an enhanced carbon film copper grid.

6. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 3), the rinsing solution is selected from one or more of normal saline, a phosphate buffer, and uranium solution.

7. The exosome transmission electron microscopy sample preparation method of claim 6, wherein, The rinsing solution is an uranyl acetate solution.

8. The exosome transmission electron microscopy sample preparation method of claim 6, wherein, The rinsing solution is a 1 to 2% (m / v) uranyl acetate solution.

9. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 3), the rinsing time is 10 to 30 seconds.

10. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 3), the negative staining solution is selected from one or more of sodium phosphotungstate, potassium phosphotungstate, and uranium solution.

11. The exosome transmission electron microscopy sample preparation method of claim 10, wherein, The negative staining solution is an uranyl acetate solution.

12. The exosome transmission electron microscopy sample preparation method of claim 10, wherein, The negative staining solution is a 1 to 2% (m / v) uranyl acetate solution.

13. The exosome transmission electron microscopy sample preparation method of claim 1, wherein, In step 3), the negative staining time is 1 to 10 minutes.

14. A TEM sample prepared by the exosome TEM sample preparation method according to any one of claims 1 to 13.

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