A microfluidic chip electrophoresis detection method for extracellular vesicles of mesenchymal stem cells

Abstract

The present invention discloses a microfluidic chip electrophoresis detection method for mesenchymal stem cell extracellular vesicles, belonging to the field of extracellular vesicle detection technology. The method comprises the following steps: adding a mesenchymal stem cell extracellular vesicle sample to a microfluidic chip electrophoresis channel, performing detection, respectively obtaining the signal peaks of the subgroups of exosomes and microvesicles of the mesenchymal stem cell extracellular vesicle sample, and performing qualitative analysis on the mesenchymal stem cell extracellular vesicle subgroups according to the signal peaks; the electrophoresis buffer used in the detection process includes 2-(N-morpholine)-ethanesulfonic acid and L-histidine in a mass ratio of 1:1. The present invention realizes rapid and efficient detection of mesenchymal stem cell extracellular vesicles, can separate and detect the peak signals of two subgroups of exosomes and microvesicles in a short time, and is simple to operate, does not require derivatization treatment, and consumes less sample, which greatly reduces the cost.

Description

Technical Field

[0001] The present invention relates to the technical field of extracellular vesicle detection, and in particular to a microfluidic chip electrophoresis detection method for mesenchymal stem cell extracellular vesicles. Background Art

[0002] Mesenchymal stem cells have the characteristics of strong proliferation ability, low immunogenicity, and multidirectional differentiation potential. They are widely used in regenerative medicine and tissue repair and treatment of various diseases. Mesenchymal stem cells secrete extracellular vesicles (EVs) through a paracrine mechanism, which contain a variety of substances such as proteins, mRNAs, miRNAs, and DNA, and play a role in cell communication. Studies have shown that extracellular vesicles have similar effects to maternal cells, with lower immunogenicity and the advantage of no tumor formation, which has attracted widespread attention from researchers. EVs can be divided into two different subgroups according to particle size: exosomes, with a diameter of 30-150nm; microvesicles, with a diameter of 200-1000nm. They each play an important role in intercellular communication and the regulation of many physiological and pathological processes, but the overlapping characteristics of size, density and biological composition make it difficult to distinguish them completely. Therefore, the individual characteristic information of a single subpopulation may be obscured or lost when studying EVs. In order to learn more about the common and unique physicochemical properties of exosomes and microvesicles, the development of new detection methods is essential.

[0003] Currently, the commonly used methods for detecting extracellular vesicles include transmission electron microscopy, nanoparticle size tracking analysis, protein immunoblotting, enzyme-linked immunosorbent assay and flow cytometry. However, these methods face problems such as complex steps and long analysis time, making them difficult to apply in clinical practice. Capillary electrophoresis separation method is a new type of liquid phase separation technology that enables the analysis of biological molecules from the microliter level to the nanoliter level, and makes single cell analysis and even single molecule analysis possible. Some studies have used capillary electrophoresis combined with ultraviolet detectors or laser-induced fluorescence detectors to achieve the detection and analysis of exosomes, which preliminarily shows the advantages of electrophoresis-based separation analysis, but these methods require complicated derivatization reactions and have problems such as low signal-to-noise ratio and photobleaching. Summary of the invention

[0004] The purpose of the present invention is to provide a microfluidic chip electrophoresis detection method for mesenchymal stem cell extracellular vesicles to solve the problems existing in the above-mentioned prior art. The method can separate and detect two subpopulations of mesenchymal stem cell extracellular vesicles within 1 minute, making up for the deficiency that traditional extracellular vesicle analysis methods cannot separate and analyze subpopulations.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a microfluidic chip electrophoresis detection method for mesenchymal stem cell extracellular vesicles, comprising the following steps:

[0007] The mesenchymal stem cell extracellular vesicle sample is added to the electrophoresis channel of the microfluidic chip for detection, and the signal peaks of the subpopulations of exosomes and microvesicles of the mesenchymal stem cell extracellular vesicle sample are obtained respectively, and the mesenchymal stem cell extracellular vesicle subpopulations are qualitatively analyzed according to the signal peaks;

[0008] The electrophoresis buffer used in the detection process includes 2-(N-morpholino)-ethanesulfonic acid and L-histidine in a mass ratio of 1:1.

[0009] Furthermore, the concentration of the electrophoresis buffer is 40 mM and the pH value is 6.0.

[0010] Furthermore, the detection process is:

[0011] In the injection stage, the sample is transferred from the negative electrode to the positive electrode, the injection voltage is 500V, and the injection time is 40s. In the separation stage, the sample is transferred from the negative electrode to the positive electrode, the separation voltage is 1000V, the operating temperature is 25°C, and the sample conductivity signal in the separation stage is converted into a voltage signal until two obvious signal peaks appear, and the detection is terminated.

[0012] Furthermore, the signal peak that appears first is the extracellular vesicle subpopulation of mesenchymal stem cells, exosomes, and the signal peak that appears later is the extracellular vesicle subpopulation of mesenchymal stem cells, microvesicles.

[0013] Furthermore, the mesenchymal stem cell extracellular vesicle sample is obtained by mixing the mesenchymal stem cell extracellular vesicle with the electrophoresis buffer at a volume ratio of 1:1.

[0014] Furthermore, the concentration of the mesenchymal stem cell extracellular vesicle sample is 10 9 -10 11 Particles / mL.

[0015] Furthermore, before the detection, the electrophoresis channel was cleaned with 0.1 M sodium hydroxide, deionized water and the electrophoresis buffer for 20 minutes respectively.

[0016] Furthermore, the electrophoresis buffer is filtered before use.

[0017] The present invention discloses the following technical effects:

[0018] The present invention realizes the rapid and efficient detection of extracellular vesicles of mesenchymal stem cells, can separate and detect the peak signals of two subpopulations of exosomes and microvesicles in a short time, and is simple to operate, does not require derivatization treatment, consumes less samples, and greatly reduces costs. The present invention provides a useful and easy-to-use solution for the quality control of samples containing extracellular vesicles, and can efficiently and conveniently obtain qualitative information of different subpopulations of extracellular vesicles of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Schematic diagram of the detection scheme of extracellular vesicles of mesenchymal stem cells;

[0021] Figure 2 To investigate the effects of different types of electrophoresis buffer on the separation and detection of extracellular vesicles of mesenchymal stem cells;

[0022] Figure 3 The effect of different concentrations of MES / His electrophoresis buffer on the separation and detection of extracellular vesicles of mesenchymal stem cells;

[0023] Figure 4 The effect of MES / His electrophoresis buffer with different pH values ​​on the separation and detection of extracellular vesicles of mesenchymal stem cells;

[0024] Figure 5 This is the electrophoresis result of mesenchymal stem cell extracellular vesicle sample 1;

[0025] Figure 6 This is the electrophoresis result of mesenchymal stem cell extracellular vesicle sample 2;

[0026] Figure 7 This is the linear fitting graph of the total signal peak area and the extracellular vesicles of mesenchymal stem cells at different concentrations. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Unless otherwise specified, the materials, instruments and reagents used in the present invention can be obtained from commercial channels; the experimental methods used are conventional experimental methods in the art unless otherwise specified.

[0033] 1 Materials and Methods

[0034] 1.1 Experimental methods

[0035] The microchip electrophoresis inorganic ion portable detector (equipment model: MEP-2021A, factory number: 20210001, provided by the Hefei Institutes of Physical Science, Chinese Academy of Sciences, conventional microchip electrophoresis instruments are also applicable) was selected to separate and detect the extracellular vesicle samples of mesenchymal stem cells. The process is as follows:

[0036] Before detection, the microchip channel was cleaned with 0.1M sodium hydroxide solution, deionized water and electrophoresis buffer (BEG) for 20 minutes each. The injection was completed by using the electric cross injection method. During the injection stage, a voltage of 500V was applied to the sample pool (SR) for 40s. Subsequently, the voltage was switched to apply a voltage of 1000V to the BGE pool (BR) for 100s to complete the separation process, and the samples passed through the detection area in turn. The change in solution conductivity was detected by the non-contact conductivity detector and the conductivity signal was converted into a voltage signal output to obtain the signal peaks of the mesenchymal stem cell extracellular vesicle subpopulation exosomes and microvesicles. The schematic diagram of the detection scheme for mesenchymal stem cell extracellular vesicles is shown in Figure 1 .

[0037] 1.2 Optimization of separation conditions

[0038] 1.2.1 Selection of electrophoresis buffer

[0039] Electrophoresis buffer is an important basis for determining whether the sample signal can be stably and efficiently detected. In order to obtain the best detection effect, the present invention tests BEG with different buffering capacities (all at a concentration of 20mM): phosphate, borate, borax, and 2-(N-morpholine)-ethanesulfonic acid / L-histidine (MES / His). The test results are as follows: Figure 2 As shown in the figure, peak signals can be detected when Borax and MES / His are used as background buffers, and electrophoresis patterns with high resolution, better separation, and earlier peak elution time are obtained under MES / His conditions. MES / His, as an amphoteric electrolyte, has the characteristics of low conductivity at high concentrations, and its oxidation reaction is slow, and it is not expected to have a significant impact on biological systems. In order to quickly and efficiently detect signals, it was selected as the electrophoresis buffer for subsequent experiments and was further optimized.

[0040] 1.2.2 Optimization of electrophoresis buffer concentration

[0041] The concentration of electrophoresis buffer has a significant effect on the detection effect. Increasing the concentration of BGE may lead to a decrease in the electroosmotic force (EOF) velocity, an increase in current and Joule heat, and thus a decrease in detection stability and resolution. Parallel experiments were performed using different concentrations of MES / His (10, 20, 40, 80 mM). The results are shown in Figure 2. Figure 3 As shown in the figure, as the concentration increases, the electrophoretic migration speed of EVs increases, the detection time decreases, but the baseline noise increases. Considering that too high a concentration is not conducive to applying a high voltage, a concentration of 80 mM is avoided. In order to achieve fast separation, a flat baseline, high efficiency and resolution, 40 mM is selected as the optimal electrophoresis buffer concentration.

[0042] 1.2.3 Effect of pH value of electrophoresis buffer

[0043] The pH of electrophoresis buffer is one of the important factors that determine the migration speed and stability of the detected substance, because it affects the size of EOF in the microchannel. In order to obtain the best detection conditions, the detection effect of BEG in different pH ranges (pH 3.0, 6.0, 9.0, 12.0) was investigated. The results are shown in Figure 2. Figure 4 As shown in the figure, as the pH increases, the EOF (direction is opposite to the electrophoretic force) increases, the combined force on the particles in the solution decreases, and the migration speed slows down, and the time to reach the detector is prolonged. Under acidic conditions, although the peak time is fast, the separation of the two subpopulations is not high. Under strong alkaline conditions, the stability of EVs is somewhat destructive, resulting in the failure to detect the complete two peak signals. The detection rate is faster when the pH is 6.0, so pH 6.0 is selected as the optimal pH value.

[0044] 1.3 Detection according to optimized separation conditions

[0045] 1.3.1 The ultra-high-speed centrifugation method modified according to the literature (reference: https: / / doi.org / 10.12307 / 2022.383) was used: the supernatant of the human umbilical cord mesenchymal stem cell culture medium was pre-treated and centrifuged at 3,000g, 10,000g for 10 min and 20 min at 4°C to remove cell debris. Then centrifuged at 120,000g for 90 min at 4°C. The supernatant was discarded, the tube wall precipitate was resuspended with PBS, and then centrifuged at 120,000g for 90 min, and then resuspended to obtain the human umbilical cord mesenchymal stem cell extracellular vesicle sample (sample 1). Sample 1 was selected for the following steps:

[0046] (1) Select the buffer system: the electrophoresis buffer is 40 mM 2-(N-morpholine)-ethanesulfonic acid and L-histidine (mass ratio is 1:1), pH 6.0, and is filtered through a 0.22 μm disposable nylon filter before use;

[0047] (2) Sample pretreatment: Take 100 μl of the sample 1 to be tested and transfer it to a 4°C refrigerator for thawing at -80°C. When it is completely thawed, add 100 μl of electrophoresis buffer, shake for 2 minutes to mix, and store at 4°C as the sample to be tested for electrophoresis;

[0048] (3) Electrophoresis: The above electrophoresis samples to be tested are added to a non-contact conductivity microfluidic chip for processing; in the injection stage, the sample is injected from the negative electrode to the positive electrode, the injection voltage is 500 V, and the injection time is 40 s; in the separation stage, the sample is injected from the negative electrode to the positive electrode, the separation voltage is 1000 V, and the operating temperature is 25°C; the two subpopulations of mesenchymal stem cell extracellular vesicles are separated during the movement and pass through the detection area in turn. The non-contact conductivity detector detects the change in solution conductivity and converts the conductivity signal into a voltage signal output, thereby obtaining the signal peaks of the mesenchymal stem cell extracellular vesicle subpopulations of exosomes and microvesicles.

[0049] Before electrophoresis, the channel was cleaned with 0.1 M sodium hydroxide and deionized water for 20 minutes each; before the sample electrophoresis test, pre-electrophoresis was performed using electrophoresis buffer.

[0050] The graphs and data were processed using Microsoft Excel and Origin 2020 software packages. Figure 5 This is the electrophoresis result spectrum of sample 1. As shown in the figure, two peak signals were detected, which are two subpopulations of mesenchymal stem cell extracellular vesicles, exosomes (signal peak 1) and microvesicles (signal peak 2).

[0051] 1.3.2 Prepare the human umbilical cord mesenchymal stem cell extracellular vesicle sample (sample 2) according to the instructions of the cell supernatant exosome extraction kit (catalog number: UR52121, purchased from Shanghai Yumeibo Biotechnology Co., Ltd.), and select sample 2 for detection according to step 1.3.1.

[0052] Test results such as Figure 6 As shown, Figure 5 Compared with the results, two signal peaks were detected in both samples, and they had similar peak migration times, but the total area of ​​the signal peaks was different, which was caused by the difference in the concentration of the two samples. Figure 5-Figure 6 It can be seen that the total signal peak area of ​​the mesenchymal stem cell sample prepared by ultrahigh-speed centrifugation is larger, proving that sample 1 has a higher concentration and less loss than sample 2.

[0053] 1.4 Linear correlation between sample concentration and signal area

[0054] The mesenchymal stem cell extracellular vesicle sample 1 was diluted into different concentration gradients (1.31×10 9 , 2.63×10 9 , 5.25×10 9 , 1.05×10 10 , 2.10×10 10Three samples were taken at each concentration for microfluidic chip electrophoresis detection according to the above steps, and the spectra and data were processed using Microsoft Excel and Origin 2020 software packages. Figure 7 The linear fitting results of the total area of ​​the signal peaks and the extracellular vesicles of mesenchymal stem cells at different concentrations are shown. Figure 7 As shown in the figure, with the increase of concentration, the peak area of ​​the detected signal gradually increases. This is because the increase of extracellular vesicles of mesenchymal stem cells will increase the conductivity of the solution, thereby increasing the conductivity difference between it and the buffer solution. 9 -10 11 Within the particle / mL concentration range, there is a good linear relationship between the concentration of mesenchymal stem cell extracellular vesicles and the signal peak area.

[0055] In summary, the present invention has studied a microfluidic chip electrophoresis detection method for extracellular vesicles of mesenchymal stem cells, which has the potential to distinguish different subpopulations of extracellular vesicles (exosomes and microvesicles). The present invention confirms that there is a linear relationship between electrophoretic mobility and particle size. Smaller particles have a higher charge-to-mass ratio, which makes them migrate faster and have an earlier peak time. Therefore, according to the different particle sizes, two subpopulations of extracellular vesicles can be separated: exosomes (signal peak 1) and microvesicles (signal peak 2). And according to Figure 5-Figure 6 The results show that this detection method can separate and detect two subpopulations of extracellular vesicles of mesenchymal stem cells within 1 minute, making up for the deficiency of traditional extracellular vesicle analysis methods that cannot separate and analyze subpopulations.

[0056] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A microfluidic chip electrophoresis detection method for mesenchymal stem cell extracellular vesicles, characterized in that: The following steps are involved: The mesenchymal stem cell extracellular vesicle sample is added to the electrophoresis channel of the microfluidic chip for detection, and the signal peaks of the subpopulations of exosomes and microvesicles of the mesenchymal stem cell extracellular vesicle sample are obtained respectively, and the mesenchymal stem cell extracellular vesicle subpopulations are qualitatively analyzed according to the signal peaks; The electrophoresis buffer used in the assay consisted of 2-(N-morpholino)-ethanesulfonic acid and L-histidine in a 1:1 mass ratio; The detection process is as follows: In the injection stage, the sample is transferred from the negative electrode to the positive electrode, the injection voltage is 500 V, and the injection time is 40 s. In the separation stage, the sample is transferred from the negative electrode to the positive electrode, the separation voltage is 1000 V, the operating temperature is 25 °C, and the sample conductivity signal in the separation stage is converted into a voltage signal until two obvious signal peaks appear, and the detection is terminated.

2. The microfluidic chip electrophoresis detection method according to claim 1, characterized in that: The concentration of the electrophoresis buffer is 40 mM and the pH value is 6.

0.

3. The microfluidic chip electrophoresis detection method according to claim 1, characterized in that: The first signal peak is the exosomes, a subpopulation of mesenchymal stem cells extracellular vesicles, and the later signal peak is the microvesicles, a subpopulation of mesenchymal stem cells extracellular vesicles.

4. The microfluidic chip electrophoresis detection method according to claim 1, characterized in that: The mesenchymal stem cell extracellular vesicle sample is obtained by mixing the mesenchymal stem cell extracellular vesicle with the electrophoresis buffer at a volume ratio of 1:

1.

5. The microfluidic chip electrophoresis detection method according to claim 4, characterized in that: The concentration of the mesenchymal stem cell extracellular vesicle sample is 10 9 -10 11 Particles / mL.

6. The microfluidic chip electrophoresis detection method according to claim 1, characterized in that: Before detection, the electrophoresis channel was cleaned with 0.1 M sodium hydroxide, deionized water and the electrophoresis buffer for 20 min, respectively.

7. The microfluidic chip electrophoresis detection method according to claim 6, characterized in that: The electrophoresis buffer was filtered before use.

Images