Efficient purification method of brain tissue small extracellular vesicles
The brain tissue sEVs purification process was optimized by wire mesh cutting and single sucrose pad ultracentrifugation technology, which solved the problems of cumbersome, time-consuming and low-yield brain tissue sEVs purification process in existing technologies, achieved efficient and accurate sEVs purification, and improved purity and repeatability.
Patent Information
- Application Number
- CN202510851549.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-09-12
AI Technical Summary
The existing technology for purifying small extracellular vesicles (sEVs) from brain tissue is cumbersome, time-consuming, low in yield, and poor in purity, which affects experimental efficiency and the accuracy of test results.
A wire mesh cutting device was used to evenly divide brain tissue, and a synergistic enzymatic hydrolysis system of collagenase and papain was combined to simplify the single sucrose cushion ultracentrifugation technology to optimize the purification process of brain tissue sEVs.
The purification efficiency and repeatability of brain tissue sEVs were significantly improved, the damage to vesicles during the enzymatic hydrolysis process was reduced, and the integrity and purity of sEVs were improved to meet the needs of downstream functional analysis.
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Figure CN120624357A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine and clinical medicine, and particularly relates to a method for efficiently purifying small extracellular vesicles of brain tissue. Background Art
[0002] While the technology for purifying small extracellular vesicles (sEVs) from body fluids and cell supernatants is relatively mature, purifying sEVs from tissues remains a significant challenge. sEVs cultured in vitro struggle to mimic the complex pathophysiological changes in vivo, making the purification of sEVs from in vivo tissues of significant scientific value.
[0003] Purification of tissue-derived EVs (sEVs) has been extensively studied in tumors and adipose tissue, but little research has been conducted on brain tissue sEV purification. While research has explored the extraction of sEVs from brain tissue, the mainstream method, ultracentrifugation combined with sucrose gradient centrifugation, is complex, time-consuming, and produces suboptimal purity, severely hindering both basic research and clinical application of brain tissue sEVs.
[0004] The existing brain tissue sEVs purification process mainly includes three core steps: brain tissue homogenization, supernatant crude purification, and sucrose density gradient ultracentrifugation. Each link has obvious technical bottlenecks:
[0005] ① Brain tissue homogenization: The required tissue volume for sEV extraction is unclear; the brain tissue cutting process is time-consuming and prone to contamination by cell debris; uneven cutting results in varying degrees of tissue enzymatic digestion, leading to significant fluctuations in sEV yield and poor experimental reproducibility. From tissue sampling and mechanical dissociation to enzymatic digestion, this step alone can take 1-2 hours.
[0006] ② Supernatant crude purification step: This step usually uses gradient centrifugation to gradually remove cells and cell debris. The specific process is: first, centrifuge at 300g to remove intact cells, then centrifuge at 2000g to remove cell nuclei and larger cell fragments, then centrifuge at more than 5000g (centrifugal force range 5000-16000g) to remove plasma membranes and large vesicles, and finally ultracentrifuge at 100,000-200,000g to precipitate sEVs. This process takes a total of about 4-5 hours.
[0007] Sucrose density gradient ultracentrifugation: Sucrose cushions consisting of five concentrations, 2.0M, 1.5M, 1M, 0.825M, and 0.65M, are prepared. After 16-20 hours of ultracentrifugation (usually overnight), sEVs are collected at concentrations between 0.65-0.825M and 0.825-1.0M. PBS is then added and the pellet is ultracentrifuged again. This step is characterized by cumbersome gradient preparation, lengthy centrifugation cycles (at least 16 hours per centrifugation), and difficulty collecting liquid from the multilayered sucrose cushion, which can easily lead to significant sEV loss.
[0008] In summary, the existing technology takes nearly 30 hours from sample processing to the final acquisition of sEVs precipitation. This not only seriously affects experimental efficiency, but also leads to low sEV yield and poor purity (high impurity residues and impaired vesicle morphological integrity caused by extremely long centrifugation times), resulting in deviations in downstream functional testing, proteomics, RNA analysis and other test results, becoming a key technical obstacle to the study of brain tissue sEVs mechanism and biomarker screening. Summary of the Invention
[0009] Addressing the technical bottlenecks of existing brain tissue sEV purification technologies, such as cumbersome procedures, lengthy processing times, low yields, and poor purity, the present invention aims to provide a highly efficient purification method for small extracellular vesicles from brain tissue. This purification method offers the advantages of high efficiency, precision, and high reproducibility.
[0010] The purpose of the present invention is achieved through the following technical solutions:
[0011] A highly efficient method for purifying small extracellular vesicles from brain tissue comprises the following steps:
[0012] (1) Brain tissue homogenate:
[0013] Mouse brain tissue was cut into uniform small pieces using a wire mesh. A digestion solution containing papain and collagenase, Type III, was then added for dissociation. After dissociation was complete, digestion was terminated, the mixture was filtered, and the filtrate was taken and brought to volume with pre-chilled EBSS buffer to obtain the tissue supernatant.
[0014] (2) Crude purification of supernatant:
[0015] Intact cells, cell debris, plasma membrane, and large vesicles were sequentially removed by gradient centrifugation with increasing centrifugal force. The filtrate was filtered and ultracentrifuged at a centrifugal force of 100,000-200,000 g to obtain sEVs precipitate. The sEVs precipitate was resuspended in PBS buffer to obtain a crude sEVs purification product.
[0016] (3) Single sucrose cushion ultracentrifugation purification:
[0017] A sucrose solution with a concentration of 25-35% (w / v) was injected into the bottom of a centrifuge tube containing PBS buffer. The crude purified sEVs product obtained in step (2) was then added dropwise to the top of the liquid surface of the centrifuge tube. Ultracentrifugation was performed at a centrifugal force of 100,000-200,000 g until a clear boundary line appeared between the sucrose solution and the PBS buffer. The upper layer of PBS buffer was discarded and the PBS buffer was replenished again to mix thoroughly. The ultracentrifugation process was repeated. The resulting precipitate was the purified brain tissue sEVs.
[0018] Furthermore, the step (1) of cutting the mouse brain tissue into uniform small pieces using a wire mesh refers to cutting 0.4-0.5 g (more than half a brain) of mouse brain tissue into small pieces with a size of less than 2*2*2 mm using a wire mesh with a pore size of 1-2 mm.
[0019] The present invention uses a dedicated wire mesh cutting device to evenly divide brain tissue into pieces smaller than 2 mm, ensuring the consistency of tissue block size across different samples. This effectively avoids uneven enzymatic hydrolysis caused by differences in block size and significantly improves the repeatability and stability of experimental operations.
[0020] Furthermore, the digestion solution in step (1) is a Hibernate-E culture medium solution with a papain concentration of 10-30 U / ml and a collagenase III concentration of 0.2-1 mg / ml; the dissociation process is incubated on a shaker at 35-38°C for 10-30 minutes.
[0021] The present invention adopts a composite enzymatic hydrolysis system with the synergistic action of collagenase and papain. While ensuring sufficient digestion of brain tissue, it significantly reduces the amount of collagenase used, reduces the damage to sEVs during the enzymatic hydrolysis process, releases vesicles in a more gentle manner, and improves the integrity and activity of sEVs.
[0022] Furthermore, the digestion in step (1) is stopped by adding Hibernate-E medium containing protease inhibitors and phosphatase inhibitors.
[0023] Furthermore, the filtration in step (1) refers to filtration using a 40 μm filter.
[0024] Furthermore, the gradient centrifugation in step (2) is preferably performed at 300 g for 10 min to remove intact cells, 2000 g for 20 min to remove cell debris, and then 10,000 g for 40 min to remove plasma membranes and large vesicles; the gradient centrifugation process is performed at a temperature of 0-4°C.
[0025] Furthermore, the filtration in step (2) refers to filtration using a 0.22 μm filter.
[0026] Furthermore, the ultracentrifugation in steps (2) and (3) is preferably carried out at a centrifugal force of 160,000 g for 2 to 3 hours.
[0027] Furthermore, the volume ratio of the sucrose solution to the PBS buffer in step (3) is preferably 1:5.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] (1) Solve the problems of uneven tissue cutting and poor repeatability (such as Figure 1 By designing a special wire mesh cutting device, brain tissue can be evenly divided into pieces less than 2 mm, ensuring the consistency of tissue block size between different samples, effectively avoiding the problem of uneven enzymatic hydrolysis caused by differences in block size, and significantly improving the repeatability and stability of experimental operations.
[0030] (2) Optimize the enzymatic hydrolysis system and reduce tissue damage: Innovatively adopt a composite enzymatic hydrolysis system with the synergistic effect of collagenase and papain. While ensuring sufficient digestion of brain tissue, it greatly reduces the amount of collagenase used, reduces the damage to sEVs during the enzymatic hydrolysis process, releases vesicles in a more gentle way, and improves the integrity and activity of sEVs.
[0031] (3) Simplify the gradient centrifugation process to reduce vesicle loss (e.g. Figure 2 As shown in the figure, the complex preparation process of traditional multi-concentration sucrose cushions is abandoned and a single sucrose cushion centrifugation technique is used instead to simplify the centrifugation operation steps. The single sucrose cushion centrifugation technique shortens the centrifugation time to 3 hours (compared to 16 hours of traditional sucrose gradient centrifugation), significantly reducing the loss of sEVs during the centrifugation process, and achieving a dual improvement in purification efficiency and vesicle yield.
[0032] (4) Improving the quality of sEVs purification: Based on the rapid cutting of brain tissue and single sucrose pad centrifugation technology, it can effectively avoid the residual impurities such as foreign proteins and cell fragments. The short-term centrifugation of the single sucrose pad can also minimize the damage to the morphology of sEVs caused by long-term centrifugation. The obtained sEVs particles are clear, abundant and high in purity, which can provide high-quality samples for downstream functional analysis, proteomic analysis, RNA sequencing and other experiments, meeting the strict requirements of brain tissue sEV mechanism research and biomarker screening. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 The figures show the process flow and separation effect of using a special steel wire mesh to cut and separate brain tissue in the present invention (B) and using a scalpel / scissors to cut and separate brain tissue in the conventional method (A).
[0034] Figure 2Schematic diagram of the processing flow and detection results of the traditional sucrose gradient centrifugation method (AC) and the single sucrose cushion ultracentrifugation method (DG) of the present invention. DETAILED DESCRIPTION
[0035] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0036] Example 1
[0037] (1) Brain tissue homogenate:
[0038] 1) After the mouse is perfused with normal saline throughout the heart, the brain is immediately removed and placed on ice. Using a 40 mm steel mesh (1 mm per hole), the brain tissue is cut into small pieces of less than 2 x 2 x 2 mm. At least 0.4-0.5 g of brain tissue (more than half a brain) is required. The above-mentioned process and separation results of brain tissue cutting and separation using a special steel mesh (B) and the conventional method of cutting and separating brain tissue using a scalpel / scissors (A) are shown in the figure. Figure 1 As shown in the figure, the innovative use of wire mesh cutting technology reduces brain tissue cutting time from several minutes with traditional scalpels and scissors to less than 10 seconds, significantly improving sample pre-processing efficiency. By precisely controlling tissue block size to less than 2 mm, differences in enzymatic hydrolysis caused by uneven tissue block size are effectively eliminated, significantly reducing fluctuations in sEV purity and yield between samples.
[0039] 2) Brain tissue enzymatic dissociation: Dissociate the brain tissue using a mild digestive solution. Prepare Hibernate-E medium supplemented with papain (20 U / ml, Worthington) and collagenase III (0.5 mg / ml, Worthington). Add 4 ml of the solution to the brain tissue and incubate on a shaker at 37°C for 20 minutes.
[0040] 3) Stop digestion: Immediately transfer the liquid to ice and add 7 ml of Hibernate-E medium (containing protease inhibitors and phosphatase inhibitors) to the brain tissue. Mix well for 5 minutes to stop digestion.
[0041] 4) Collect the supernatant of the digestion solution: Filter all the liquid from step 3) through a 40 μm filter and collect it. Repeatedly add pre-cooled EBSS buffer to rinse the filter until the final volume is 30 ml.
[0042] (2) Crude purification of supernatant:
[0043] 1) Centrifuge at 300 g for 10 min at 4°C to remove intact cells, 2000 g for 20 min to remove cell debris, and 10,000 g for 40 min to remove plasma membranes and large vesicles.
[0044] 2) The supernatant was filtered through a 0.22 μm filter and then ultracentrifuged at 160,000 g for 3 h at 4°C in a Beckman centrifuge (SW 32 Ti) to obtain the sEV pellet. The sEV pellet was resuspended in 3 ml of PBS to prepare the crude sEV purification sample.
[0045] (3) Single sucrose cushion ultracentrifugation purification:
[0046] 1) Prepare a 4 ml 30% sucrose cushion (w / v, PBS): First add 20 ml PBS, then use an extra-long loading needle to inject 4 ml of 30% sucrose solution into the bottom of the centrifuge tube.
[0047] 2) Gently add the crude purified sEVs sample obtained in step (2) to the top, adjust with PBS, and ultracentrifuge at 160,000 g for 3 h at 4°C in a Beckman centrifuge (SW 32 Ti).
[0048] 3) Once the boundary between the sucrose solution and PBS is visible, gently discard the upper PBS layer without disturbing the solution, retaining the sucrose layer at the bottom. Add PBS again to make up to 31 ml, thoroughly mix the sucrose solution and PBS, and ultracentrifuge again at 160,000 g for 2 hours at 4°C in a Beckman centrifuge (SW32 Ti). The resulting pellet is the sEVs. Resuspend in protein lysis buffer or PBS for the next step.
[0049] The processing flow diagram and test results of the single sucrose cushion ultracentrifugation method (DG) are compared with the traditional sucrose gradient centrifugation method (AC). Figure 2 shown.
[0050] The traditional sucrose gradient centrifugation method is as follows:
[0051] 1) Prepare a sucrose gradient (w / v, PBS): Literature reports indicate that the concentration of sEVs is between 30% and 40%. First, add 20 ml of PBS. Then, use an extra-long loading needle to slowly inject 3 ml of 30% sucrose and 3 ml of 40% sucrose solution into the bottom of the centrifuge tube, avoiding disturbing the sucrose layer.
[0052] 2) Gently add the crude purified sEVs sample obtained in step (2) to the top, adjust with PBS, and ultracentrifuge at 160,000 g for 16 h at 4°C in a Beckman centrifuge (SW 32 Ti).
[0053] 3) Carefully remove 23 ml of PBS supernatant (total volume, approximately 31 ml), leaving 8 ml. Collect the remaining liquid from top to bottom, 2 ml each as a fraction (F1-F4). After collecting all the liquid, add 2 ml of PBS to the bottom of the tube to resuspend the pellet, which is F5.
[0054] 4) Transfer the collected sEVs into new ultracentrifugation tubes, add PBS, mix thoroughly, and ultracentrifuge again at 160,000 g for 3 h at 4°C in a Beckman centrifuge (SW 32 Ti). The resulting precipitate is sEVs.
[0055] 5) The results showed that brain tissue sEVs mainly existed in F2 and F3, and F2 and F3 mainly originated from the 30% sucrose gradient.
[0056] The above results show that the present invention uses a single sucrose cushion centrifugation instead of the traditional multi-concentration sucrose gradient centrifugation, which can significantly reduce the single centrifugation time of sEVs from more than 16 hours to 3 hours, and improve the efficiency by more than 5 times. The simplified gradient preparation process not only reduces the complexity of the operation, but also significantly reduces the physical loss and morphological damage of sEVs during the centrifugation process. Transmission electron microscopy (TEM) observation shows ( Figure 2 Middle E), the purified sEVs have uniform particle morphology and clean background. The purified sEVs are high in purity and quantity, which are significantly improved compared with traditional methods ( Figure 2 As shown in E, the number of sEVs particles in the field of view is relatively large. Figure 2 The C content in the medium increases by 3-5 times and the impurity signal is almost eliminated).
[0057] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A highly efficient method for purifying small extracellular vesicles from brain tissue, characterized in that: The steps include: (1) Brain tissue homogenate: Mouse brain tissue was cut into small uniform pieces using a wire mesh, and then dissociated by adding a digestion solution containing papain and collagenase III. After dissociation, digestion was stopped, the mixture was filtered, and the filtrate was taken and fixed to volume with pre-cooled EBSS buffer to obtain the tissue supernatant; (2) Crude purification of supernatant: Intact cells, cell debris, plasma membrane, and large vesicles were sequentially removed by gradient centrifugation with increasing centrifugal force. The filtrate was filtered and ultracentrifuged at a centrifugal force of 100,000-200,000 g to obtain sEVs precipitate. The sEVs precipitate was resuspended in PBS buffer to obtain a crude sEVs purification product. (3) Single sucrose cushion ultracentrifugation purification: A sucrose solution with a concentration of 25-35% was injected into the bottom of a centrifuge tube containing PBS buffer, and then the crude purified sEVs product obtained in step (2) was added dropwise above the liquid surface of the centrifuge tube. Ultracentrifugation was performed at a centrifugal force of 100,000-200,000 g until a clear boundary line appeared between the sucrose solution and the PBS buffer. The upper layer of PBS buffer was discarded and the PBS buffer was replenished again to mix thoroughly. The ultracentrifugation process was repeated. The resulting precipitate was the purified brain tissue sEVs.
2. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: Cutting the mouse brain tissue into uniform small pieces using a wire mesh in step (1) refers to cutting 0.4-0.5 g of mouse brain tissue into small pieces with a size of less than 2*2*2 mm using a wire mesh with a pore size of 1-2 mm.
3. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: The digestion solution in step (1) is a Hibernate-E culture medium solution with a papain concentration of 10-30 U / ml and a collagenase III concentration of 0.2-1 mg / ml; the dissociation process is incubated on a shaker at 35-38°C for 10-30 minutes.
4. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: The digestion described in step (1) is terminated by adding Hibernate-E medium containing protease inhibitors and phosphatase inhibitors.
5. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: The filtration described in step (1) refers to filtration using a 40 μm filter.
6. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: The gradient centrifugation in step (2) refers to centrifugation at 300g for 10 minutes to remove intact cells, centrifugation at 2000g for 20 minutes to remove cell debris, and then centrifugation at 10000g for 40 minutes to remove plasma membrane and large vesicles; the gradient centrifugation process is carried out at a temperature of 0~4°C.
7. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: The filtration in step (2) refers to filtration using a 0.22 μm filter.
8. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: The ultracentrifugation in steps (2) and (3) refers to ultracentrifugation at a centrifugal force of 160,000 g for 2 to 3 hours.
9. The method for efficiently purifying small extracellular vesicles from brain tissue according to claim 1, characterized in that: The volume ratio of the sucrose solution to the PBS buffer in step (3) is 1:5.