Method for rapidly and quantitatively detecting mesenchymal stem cell exosome by capillary electrophoresis

Abstract

The invention discloses a method for rapidly and quantitatively detecting mesenchymal stem cell exosomes by capillary electrophoresis. The method comprises the following steps: taking 50mM BTP-Gly as a background buffer solution, carrying out sample introduction for 5 seconds under the conditions that the pH is 9.0 and the pressure is 50mbar, and carrying out separation under the conditions that the voltage is 25kV and the temperature is 25 DEG C; repeatability verifies that RSD of migration time is equal to 0.30%, and RSD of peak area is equal to 3.62%. A correlation coefficient of a linear regression equation is R2gt; the linear range is 2.62 * 10 < 9 > to 2.1 * 10 < 10 > EV / mL; the optimal detection limit is 9.67 * 10 < 8 > EV / mL; and the recovery rate reaches 90% or above. According to the method, the peak signal of the exosome can be detected in a short time (less than 10 minutes), the operation is simple, derivatization treatment is not needed, the sample consumption is low, the cost is greatly reduced, and rapid and efficient quantitative detection of the mesenchymal stem cell exosome is realized.

Description

Technical Field

[0001] The present invention relates to a method for rapidly and quantitatively detecting mesenchymal stem cell exosomes by using capillary electrophoresis. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of multipotent somatic stem cell that can be derived from a variety of tissues, including bone marrow, umbilical cord blood, placenta, and adipose tissue. They are easy to isolate, culture, and expand, possess self-proliferation capabilities, and possess immunomodulatory properties. They can play a role in tissue repair and treatment for a variety of diseases, and have become the cell type of choice in regenerative medicine. Exosomes (Exo) are lipid-bilayer-encapsulated vesicles secreted by MSCs via a paracrine mechanism, ranging in size from 30 to 150 nm. Exosomes contain a variety of specific proteins, nucleic acids (e.g., DNA, mRNA, miRNA), lipids, and other biomarkers, and are involved in numerous biological processes, including intercellular communication, tumorigenesis, signal transduction, and immune responses.

[0003] Traditional methods for analyzing exosomes include transmission electron microscopy, nanoparticle size tracking analysis, western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. However, these methods face challenges such as large sample volumes, complex procedures, long analysis times, and high costs. Capillary electrophoresis (CE) is an analytical technique that efficiently separates and quantifies components based on their charge ratios. This makes CE applicable to a wide range of analytes and, as a mature analytical and quantitative technique, has been widely used in various fields. This paper utilizes CE to rapidly quantify mesenchymal stem cell exosomes. A novel background buffer is proposed. This buffer has a sufficiently large buffer capacity within a selected pH range, imparts an appropriate charge to the analyte, and exhibits low absorption at the detection wavelength, resulting in minimal interference with the detection of exosome vesicle samples. This buffer allows for the detection of exosome peak signals in a short time (<10 minutes). Furthermore, no derivatization is required, and sample consumption is minimal (approximately 10 nL). This enables rapid and efficient quantitative analysis of mesenchymal stem cell exosomes, simplifying the process and significantly reducing costs.

[0004] Compared with the currently commonly used gel electrophoresis method, the method of the present invention using capillary electrophoresis for rapid quantitative detection of mesenchymal stem cell exosomes has the advantages of higher resolution, smaller sample size, shorter analysis time and lower operating costs. It is also automated and simple to operate, with a low detection limit, high recovery rate, good repeatability and precision, and can accurately detect the content of exosomes in samples.

[0005] Compared with the traditional quantitative method (BCA method), the present invention does not require a complicated sample pretreatment process, can effectively avoid the interference of impurity proteins, obtain more accurate quantitative results, and has short analysis time, small sample consumption, lower cost, easy operation, good repeatability, higher efficiency and good promotion.

[0006] Compared with the quantitative method using transmission electron microscopy, the present invention has lower requirements for sample pretreatment and preparation, simplifies the sample preparation stage, is suitable for large-scale and rapid determination of exosomes, is not affected by the sample separation method and sample properties, and has higher accuracy.

[0007] The present invention creates a more novel and efficient method compared to traditional methods for detecting exosome content. It provides a method for the rapid quantification of mesenchymal stem cell exosomes using capillary electrophoresis, which has many advantages such as high detection efficiency, low cost, good reproducibility, accurate quantitative results, and easy operation for the quality control of exosomes and their preparations. Summary of the Invention

[0008] The present invention provides a method for quantitatively detecting mesenchymal stem cell exosomes using capillary electrophoresis. The method is simple and easy to operate, which not only shortens the detection time and reduces sample consumption, but also solves the shortcomings of existing methods and technologies.

[0009] The present invention discloses a method for quantitatively detecting mesenchymal stem cell exosomes using capillary electrophoresis. The specific technical solution involves adding a mesenchymal stem cell exosome sample to a capillary electrophoresis channel for detection, obtaining a signal peak for the mesenchymal stem cell exosomes, and then quantitatively analyzing the mesenchymal stem cell exosomes based on a sample concentration / peak area standard curve.

[0010] The concentration of the electrophoresis buffer is 50 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane:75 mM glycine mixed in a volume ratio of 1:1, with a pH value of 9.0.

[0011] The capillary electrophoresis instrument was an Agilent 7100 CE, and the detection parameters were as follows: a combined diode array detector (DAD detector) with a detection wavelength of 200 nm; uncoated quartz capillaries with an inner diameter of 75 μm, a total length of 60 cm, and an effective length of 51.5 cm; pressure injection with an injection pressure of 50 mbar; an injection time of 5 s; a capillary column temperature of 25°C; and a separation voltage of 25 kV.

[0012] The method was tested for repeatability, and the results showed that the RSD of migration time was 0.30%, and the RSD of peak area was 3.62%; the linear regression equation was: correlation coefficient R 2 >0.99, linear range is 2.62×10 9 -2.1×1010 EV / mL; optimal detection limit: 9.67×10 8 EV / mL; recovery rate: more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for use in the embodiments. The drawings are provided to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention.

[0014] Figure 1 To investigate the effects of different electrophoresis buffers on the detection of mesenchymal stem cell exosomes; Figure 2 The effect of BTP-Gly electrophoresis buffer with different pH values ​​on the detection of mesenchymal stem cell exosomes; Figure 3 It is a double Y-axis graph between peak area and theoretical plate number under different pH conditions; Figure 4 The effect of electrophoresis buffer with different concentrations of BTP on the detection of mesenchymal stem cell exosomes; Figure 5 It is a double Y-axis graph between migration time and peak area under different BTP concentration conditions; Figure 6 The effect of different separation voltages on the detection of mesenchymal stem cell exosomes; Figure 7 It is a double Y-axis graph between migration time and theoretical plate number under different separation voltage conditions; Figure 8 The effect of different injection times on the detection of mesenchymal stem cell exosomes; Figure 9 It is a double Y-axis graph between peak area and theoretical plate number under different injection time conditions; Figure 10 The linear fitting graph of different concentrations of mesenchymal stem cell exosomes and the total area of ​​the signal peak; Figure 11 This is the electrophoresis result spectrum of the reproducible detection of mesenchymal stem cell exosome samples; Figure 12 The electrophoresis results of the detection of BSA samples, mesenchymal stem cell exosome samples and a mixture of the two samples are shown; Figure 13 is the protein standard curve prepared by BCA method; DETAILED DESCRIPTION

[0015] In order to better understand the essence of the present invention, various exemplary embodiments of the present invention are specifically introduced below, and the characteristics of the present invention are described in detail.

[0016] 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.

[0017] Example 1 Selection of detection conditions for mesenchymal stem cell exosomes Mesenchymal stem cell exosomes were detected using a capillary electrophoresis instrument (7100 CE, Agilent, USA) at a detection wavelength of 200 nm. Uncoated quartz capillaries (Part No. 160-2644-5, Agilent, USA) with an inner diameter of 75 μm, a total length of 60 cm, and an effective length of 51.5 cm were used. Injection was performed using pressure injection at 50 mbar, an injection time of 5 s, a separation voltage of 25 kV, and a capillary column temperature of 25°C. Prior to detection, the capillary channel was cleaned for 3 minutes each with 0.1 M sodium hydroxide solution, deionized water, and electrophoresis buffer.

[0018] The capillary tube is immersed in electrophoresis buffer at both ends and connected to a high-voltage power supply. When the power supply applies a stable high voltage, electroosmotic flow occurs within the capillary, causing the entire liquid within the capillary to migrate toward the negative electrode. Signal fluctuations are generated when the target exosome sample passes through a light detector near the capillary output end. OpenLAB software (Agilent, USA) records the absorbance and dynamically and intuitively displays the exosomes as absorption peaks on the screen, with migration time as the horizontal axis and absorbance as the vertical axis.

[0019] Example 2 Optimization of detection conditions for mesenchymal stem cell exosomes Experiment 1 Selection of electrophoresis buffer system Capillary electrophoresis must be performed in a buffer, so the choice of buffer is crucial. It has a significant impact on signal detection time, peak shape, and sensitivity. In order to obtain the best detection effect, the present invention investigated background buffers with different buffering capacities: acetate-glycine (Acetate-Gly), phosphate-glycine (Phosphate-Gly), borax-glycine (Borax-Gly), 1,3-bis(tris(hydroxymethyl)methylamino]propane-glycine (BTP-Gly). The electrophoretic patterns are shown in Figure 2. Figure 1As shown in Figure 2 , peak signals were detected using both Acetate-Gly and BTP-Gly as background buffers. The BTP-Gly system exhibited a more stable signal baseline and higher reproducibility. This is because the background buffer, composed of organic compounds (BTP and the buffering coion Gly), has a pK value similar to that of BTP, and Gly, as a zwitterion with amino and carboxyl groups, exhibits strong buffering properties. The combination of these two systems results in a high buffer capacity. Using a relatively large buffering counterion such as BTP as a BGE component spatially supports the stability of exosome vesicles during capillary electrophoresis analysis, reducing the likelihood of vesicle aggregation due to particle collisions under the electric field and more effectively suppressing the vesicle relaxation effects and polarization / deformation that occur with inorganic buffer systems. To ensure rapid and efficient signal detection, this system was selected as the electrophoresis buffer for subsequent experiments and further optimized.

[0020] Experiment 2 Selection of pH value of electrophoresis buffer The pH value of the electrophoresis buffer is one of the important factors that determine the migration speed and stability of the test substance, because it affects the size of the electroosmotic flow (EOF) in the microchannel. Most buffers have sufficient buffering capacity only within a limited pH range. In order to obtain the best detection conditions, the present invention explored the detection effect of the electrophoresis buffer in different value ranges (pH 6.0, 7.0, 8.0, 9.0, 10.0). The electrophoretic pattern is shown in Figure 2. Figure 2 As shown in Figure 2, as the pH value increases, the migration time becomes shorter and shorter, while the number of theoretical plates increases, and reaches the optimum at pH = 9 ( Figure 3 Although the peak time is shorter at pH = 10, the stability is not high and the baseline noise is large. Therefore, we finally chose pH = 9 as the optimal value.

[0021] Experiment 3 Selection of BTP concentration in electrophoresis buffer Ion concentration affects electroosmosis by affecting the double layer and its thickness. The higher the concentration, the smaller the double layer thickness and the smaller the electroosmotic flow, which in turn affects the migration time. In order to obtain the best detection conditions, 30 mM, 40 mM, 50 mM, 60 mM, and 70 mM BTP and Gly were mixed in a volume ratio of 1:1. The detection effect of different BTP concentrations was explored. The electrophoretic patterns are shown in the figure below. Figure 4 As shown in Figure 2, with the increase of BTP concentration, the peak area of ​​the peak signal increases sharply, accompanied by the extension of the migration time ( Figure 5 The peak area reaches its maximum at 60 mM. At 70 mM, the BTP concentration is too high, and almost no signal is detected. While 60 mM provides high sensitivity, the signal baseline is unstable. 50 mM provides good sensitivity and a faster migration rate, making it the optimal concentration.

[0022] Test 4: Separation Voltage Selection After the capillary length is fixed, the separation voltage determines the electric field strength, affecting the separation column efficiency and migration time. Increasing the separation voltage can shorten the detection time, but the large amount of Joule heat generated under high pressure will also affect the detection effect. In order to obtain the best detection conditions, the detection effect under different separation voltages (10kV, 15kV, 20kV, 25kV, 30kV) was explored. The electrophoretic patterns are shown in Figure 1. Figure 6 As shown in Figure 2, with the increase of separation voltage, the migration rate increases and the peak time becomes shorter, but the theoretical plate number is lower when the separation voltage is 30 kV ( Figure 7 ). Therefore, the separation voltage of 25 kV was selected as the optimal separation voltage.

[0023] Experiment 5: Injection time selection The injection time determines the injection volume of the analytical sample and affects the peak current and peak shape of the analyte. In theory, increasing the injection time will increase the sensitivity of the detection, but too long an injection time will cause sample overload and peak broadening. In order to obtain the best detection conditions, the detection effect of different injection times (3s, 4s, 5s, 6s, 7s) was explored at a separation voltage of 25kV and a fixed injection pressure of 50 mbar. The electrophoretic patterns are shown in Figure 2. Figure 7 As the injection time increases, the peak area increases, but the theoretical plate number decreases ( Figure 8 After comprehensive consideration, 5s was selected as the optimal injection time.

[0024] Experiment 6 Repeatability Study Under the optimal conditions, in order to explore the feasibility and repeatability of this method, repeatability experiments were carried out on exosome standards. The signal can be quickly detected within 10 minutes, such as Figure 9 As shown in the results, high repeatability was achieved, with the migration time RSD being 0.30%, the peak area RSD being 3.62%, the peak height RSD being 3.46%, and the signal-to-noise ratio (S / N) being greater than 10.

[0025] Experiment 7: Linear Investigation The exosome standard solutions with different concentration gradients were prepared by diluting with electrophoresis buffer. Under the optimal capillary electrophoresis conditions, the standard solutions with different concentrations were tested to obtain the electrophoresis patterns as shown below. Figure 10 Then draw the concentration / peak area standard curve ( Figure 11 ), the peak area is 2.62×10 9 ~2.10×10 10 The EV / mL concentration range showed good linearity (R2=0.9980). According to the peak area standard curve, the optimal detection limit (LOD) of the capillary electrophoresis method was 9.67×10 8EV / ml (calculated by LOD=kSb / m, where k is 3 for a 90% confidence level, Sb is the standard error of the intercept, and m is the sensitivity.

[0026] Experiment 8: Comparison of capillary electrophoresis and BCA protein quantification for exosome detection Capillary electrophoresis (CE) detection of exosomes targets intact exosome vesicles, eliminating the need for lysis. This method effectively detects exosomes of insufficient purity or with the presence of contaminating proteins. To demonstrate its advantages, the advantages and disadvantages of CE and BCA protein quantification for quantitative exosome detection were compared.

[0027] (1) Quantitative detection of exosomes by capillary electrophoresis: Add bovine serum albumin (BSA) as an impurity protein to the exosome sample. Bovine serum is an important component of the culture medium during stem cell culture and expansion. It is the largest natural culture medium used in cell culture. In bovine serum, BSA is the main component of bovine serum, about 30mg-50mg / mL. As a representative of heterologous proteins, it can induce multiple immune responses in the body. Detection structure such as Figure 12 As shown, two signal peaks can be obtained, which can well distinguish the two.

[0028] (2) BCA protein quantification method for quantitative detection of exosomes: The exosome sample was added to the lysis buffer and left on ice for 30 minutes, then centrifuged at 12000 g for 15 minutes at 4°C to remove cell debris, and the supernatant was taken as the lysed exosome sample. The protein standard curve was prepared using the BCA protein quantification method, and the results were as follows: Figure 13 As shown in the figure, concentrations of exosome samples and exosome samples after the addition of BSA were measured. The OD values ​​of the samples measured at a UV wavelength of 562 nm were substituting into the standard curve to obtain the corresponding concentrations. The exosome protein concentration was 1.1233 mg / ml. The exosome protein concentration after the addition of BSA was 1.8455 mg / ml, a significant increase. This result indicates that the presence of impurity proteins can lead to an elevated protein content in exosomes, and the BCA protein quantification method cannot eliminate the interference of such impurities.

[0029] In summary, capillary electrophoresis can separate impurity proteins from exosomes without lysing exosomes, providing a new method for quality control of exosome-containing samples.

[0030] Example 3 Validation of the capillary electrophoresis method for quantitative detection of exosomes Quantitative detection of different batches of samples was performed according to the optimized electrophoresis conditions.

[0031] (1) Sample separation and extraction: Ultracentrifugation was used to extract exosomes derived from mesenchymal stem cells. The pre-treated supernatant was collected into a dedicated ultracentrifuge tube and centrifuged at 120,000 g for 90 min at 4°C. The supernatant was discarded and the pellet was resuspended in PBS. The tube wall was repeatedly blown against the centrifuge tube to avoid loss. The centrifugation cycle was repeated once more. Finally, the pellet containing exosomes was suspended in PBS. The sample was stored at -80°C for long-term use and at 4°C for short-term use for one week.

[0032] (2) NTA detection of exosome concentration: Six batches of samples were tested using NanoSight NS300 (Malvern, UK) to obtain the actual concentration values. The Brownian motion of particles in each sample was recorded in the video five times, each time for 60 seconds.

[0033] (3) Sample pretreatment: Take 100 μl of the sample to be tested and transfer it to a 4°C refrigerator at -80°C for thawing. After 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 by electrophoresis.

[0034] (4) Preparation of electrophoresis buffer: 50 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane: 75 mM Gly (1:1 by volume), pH 9.0. Filter with a disposable filter before use. Before capillary electrophoresis, add the electrophoresis buffer to the instrument's specific electrophoresis sample vial.

[0035] (5) Capillary electrophoresis: Before electrophoresis, clean the capillary with 0.1 M sodium hydroxide and deionized water for 3 minutes each.

[0036] Use a pipette to transfer the sample to be tested to an electrophoresis vial. Set the capillary electrophoresis instrument to the following parameters: injection pressure 50 mbar, injection time 5 seconds, and separation voltage 25 kV. Move the sample to the DAD detector near the output end of the capillary tube. Detect and record the absorbance. Dynamically and visually record each component on the screen as an absorption peak, with migration time as the horizontal axis and absorbance as the vertical axis.

[0037] (6) The peak integrals in the obtained electrophoresis patterns were integrated using OpenLAB software (Agilent, USA) to obtain the peak area, and the measured concentration values ​​were obtained by substituting them into the linear equation of the standard curve.

[0038] Recovery and precision determination: According to the sample treatment method (without adding substrate), samples of different concentrations were taken for spiked recovery experiments, and the recovery and precision were calculated (see Table 1).

[0039] Table 1 Recovery rate and precision of mesenchymal stem cell exosomes

[0040] In summary, the present invention has developed a method for rapid quantification of mesenchymal stem cell exosomes using capillary electrophoresis. This detection method can rapidly identify and quantify exosomes within 10 minutes, with good reproducibility and stability. Within a certain range, a good linear relationship between peak area and particle concentration is observed, effectively enabling quantitative analysis of exosomes.

Claims

1. A method for rapid quantitative detection of mesenchymal stem cell exosomes using capillary electrophoresis.

2. The method according to claim 1, characterized in that The capillary electrophoresis instrument was an Agilent 7100 CE, and the detection parameters were as follows: combined diode array detector (DAD detector), detection wavelength of 200 nm; uncoated quartz capillary, capillary inner diameter of 75 μm, total length of 60 cm, effective length of 51.5 cm; injection pressure of 50 mbar; injection time of 5 s; capillary column temperature of 25 °C; separation voltage of 25 kV.

3. The method according to claim 1, characterized in that The background buffer was 50 mM BTP-Gly, a mixture of 1,3-bis(tris(hydroxymethyl)methylamino)propane and glycine (BTP-Gly) at a mass volume ratio of 1:1, with a pH of 9.0.

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