Extraction and culture method of central nervous system vascular wall cells
By separating microvascular plexuses through one-step enzymatic hydrolysis combined with centrifugation and natural sedimentation, and then using gelatin-coated culture plates and selective culture media, high-purity and highly active vascular wall cells were obtained through five passages. This method solves the problems of expensive equipment, complex operation, severe cell damage, and low purity in existing technologies, and achieves simple, low-cost, and efficient cell extraction.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTHERN MEDICAL UNIVERSITY
- Filing Date
- 2026-02-14
- Publication Date
- 2026-05-08
AI Technical Summary
Existing technologies for isolating vascular wall cells from the central nervous system suffer from problems such as expensive equipment, complex operation, severe cell damage, low purity, and high cost, making it difficult to obtain highly active and pure vascular wall cells.
Microvascular plexuses were separated by a one-step enzymatic digestion combined with two centrifugations and four natural sedimentation processes. The cells were then cultured in vitro in gelatin-coated culture plates and selective culture medium. After five passages, high-purity and highly active vascular wall cells were obtained.
It achieves simple operation, low cost, high efficiency, good cell activity, and high purity, shortens the preparation cycle of scientific research experimental materials, and improves the input-output ratio.
Smart Images
Figure CN121991883A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of cell culture technology, and in particular to a method for extracting and culturing vascular wall cells of the central nervous system. Background Technology
[0002] Vascular wall cells (or perivascular cells) in the central nervous system are cells located on the basement membrane of microvascular endothelial cells. They are closely adjacent to endothelial cells and interact with them through physical contact and molecular signals, playing a crucial role in maintaining the integrity of the blood-brain barrier, regulating vascular function, and supporting neuronal activity. Furthermore, vascular wall cells may possess stem cell or progenitor cell characteristics, capable of differentiating into various cell types and participating in tissue regeneration and repair. Therefore, in vitro culture of vascular wall cells plays a vital role in the central nervous system.
[0003] Currently, the separation of vascular wall cells involves multiple enzymatic digestion methods, flow cytometry sorting, and magnetic bead sorting. Multi-step enzymatic digestion can lead to prolonged contact between cells and digestive enzymes, resulting in over-digestion of some cells and inhibition of cell viability. Flow cytometry sorting, an application of flow cytometry, involves labeling sample cells with specific antigens on their surface using fluorescein-conjugated antibodies, or using cells with their own fluorescent reporter proteins. After laser irradiation under a high-voltage electric field, the fluorescence signal emitted by each cell is collected, and the data is processed and analyzed to determine the strength of antigen expression in the sample cells, thus separating the vascular wall cells. The disadvantages of flow cytometry sorting are: 1. Flow cytometers and sorting equipment are expensive, and maintenance and operating costs are also high; 2. It requires professional technical knowledge and operating experience, otherwise it may lead to low sorting efficiency or inaccurate results; 3. Cells may be mechanically damaged during the sorting process, especially at high speeds, which may affect cell viability and function; 4. Samples need to be single-cell suspensions, and the preparation process may be more complex for some cells or tissues that are difficult to disperse; 5. Sorting usually relies on specific cell surface markers, which may lead to the missed detection or missorting of some rare cell subpopulations; 6. Some cell types may be difficult to sort due to their special physical characteristics (such as size and shape); 7. Flow cytometry generates a large amount of data, requiring specialized software and professional knowledge to analyze the data. Magnetic bead sorting involves first connecting magnetic beads to antibodies, and then utilizing the principle of antibody binding to cell surface antigens, the magnetic beads adsorb cells expressing the corresponding antigens through the action of a magnetic field, thereby separating the target cells. However, since there are no specific antibodies to label blood vessel wall cells, this method results in the extracted blood vessel wall cells being mixed with other cell types, with low purity, making them unsuitable for important research related to this type of cell. Summary of the Invention
[0004] The embodiments of this application provide a method for extracting and culturing vascular wall cells of the central nervous system. The method involves separating microvascular plexuses through one-step enzymatic hydrolysis combined with two centrifugations and four natural sedimentation processes. The cells are then cultured in vitro on gelatin-coated culture plates and selective culture medium, and after five passages, a large number of high-purity and highly active vascular wall cells are obtained. This method has the advantages of simple operation, low cost, high efficiency, good cell activity, and high purity.
[0005] To achieve the above objectives, embodiments of this application provide a method for extracting and culturing vascular wall cells of the central nervous system, comprising the following steps: S1. Dissociating brain and spinal cord tissues using papain; S2. Homogenizing the dissociated brain and spinal cord tissues using syringe needles of different sizes to obtain a brain and spinal cord tissue homogenate; S3. Adding bovine serum albumin to the brain and spinal cord tissue homogenate and centrifuging twice to remove myelin fragments, dead cells, and contaminating cells, resulting in a low-purity microvascular plexus; S4. Allowing the low-purity microvascular plexus to settle naturally four times, 5 minutes each time, to obtain a high-purity microvascular plexus; S5. Seeding the high-purity microvascular plexus into a gelatin-coated culture plate; S6. Culturing in vitro using a selective culture medium, allowing cells to emerge from the high-purity microvascular plexus and form vascular wall cells; S7. Obtaining high-purity vascular wall cells after five passages.
[0006] Furthermore, the concentration of the papainase is 1 mg / ml.
[0007] Furthermore, the gelatin concentration of the culture plate is 4.8 mg / ml.
[0008] Furthermore, the syringe needles are of models 18G and 30G.
[0009] Furthermore, the concentration of the bovine serum albumin is 22%.
[0010] Furthermore, the conditions for both centrifugations were 4°C, 2600 rpm, and 10 min.
[0011] Furthermore, the four natural sedimentation steps specifically include: first, adding 500 μL of parietal cell culture medium to the low-purity microvascular plexus obtained after two centrifugations to resuspend the vascular plexus, allowing it to settle naturally for 5 minutes, removing 400 μL of supernatant, then adding another 400 μL of parietal cell culture medium to resuspend the vascular plexus, allowing it to settle naturally for 5 minutes, removing 400 μL of supernatant, and repeating the second natural sedimentation step twice, until the fourth natural sedimentation is completed, resulting in high-purity microvessels. Furthermore, the selective culture medium is a culture medium specifically for vascular wall cells.
[0012] Furthermore, the brain and spinal cord tissue is brain and spinal cord tissue from young mice.
[0013] This application has the following advantages over the prior art: 1. The method for extracting and culturing vascular wall cells of the central nervous system in this application involves one-step enzymatic hydrolysis combined with two centrifugation separations of microvascular plexuses, followed by in vitro culture in gelatin-coated culture plates and selective culture medium, and then five passages to obtain a large number of high-purity, high-activity vascular wall cells. This method has the advantages of simple operation, low cost, high efficiency, good cell activity, and high purity.
[0014] 2. In the extraction and culture method of central nervous system vascular wall cells in this application embodiment, the concentration of papainase used is 1 mg / ml, which is low and can greatly reduce cell damage by digestive enzymes.
[0015] 3. In the extraction and culture method of vascular wall cells of the central nervous system in this application embodiment, the gelatin concentration used to coat the six-well plate is 4.8 mg / ml, which can coat the well plate in a short time.
[0016] 4. In the extraction and culture method of vascular wall cells of the central nervous system in this application embodiment, the two centrifugations are carried out at 2600 rpm for 10 minutes at 4 degrees Celsius, which is conducive to the removal of residual myelin fragments or cell fragments. The four natural sedimentation processes further reduce the interference of impurities to the greatest extent.
[0017] 5. The method for extracting and culturing vascular wall cells of the central nervous system in the embodiments of this application can shorten the preparation cycle of scientific research experimental materials and improve the input-output ratio of scientific research. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0019] Figure 1 This is a flowchart for extracting vascular plexuses from the brain or spinal cord tissue of young mice.
[0020] Figure 2 This image shows the result of extracting wall cells from a vascular plexus in a cell culture dish.
[0021] Figure 3 This is a graph showing the immunofluorescence identification results of parietal cells after passage. Detailed Implementation
[0022] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0023] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.
[0024] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation", "connection" and "joining" should be interpreted broadly, for example, they can refer to fixed connections, detachable connections, or integral connections; those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.
[0025] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" can explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.
[0026] This application discloses a method for extracting and culturing vascular wall cells from the central nervous system. This method isolates vascular plexuses from the brain and spinal cord of young mice, grows large quantities of vascular wall cells (pericytes) in vitro using these plexuses, and obtains high-purity vascular wall cells through conditioned medium culture. The vascular wall cells obtained by this method are suitable for various life science research, including in vitro cell characterization studies, transcriptomics, proteomics, and lipidomics applications, and can also be used for cell and molecular biology research. It should be noted that this method is applicable to mouse brain and spinal cord, as well as human brain, demonstrating its cross-species and multi-tissue applicability.
[0027] Reference Figures 1 to 3 The method for extracting and culturing vascular wall cells of the central nervous system in this application includes the following steps: S1. Brain and spinal cord tissues of juvenile mice were obtained by dissociation using papain at a concentration of 1 mg / ml. This low concentration of papain significantly reduces cell damage caused by digestive enzymes.
[0028] S2. Using 18G and 30G syringe needles, the dissected brain and spinal cord tissues were aspirated to obtain homogenates. Different syringe sizes help protect the integrity of the extracted vascular plexus.
[0029] S3. Add 22% bovine serum albumin (BSA) to the homogenate of brain and spinal cord tissue, and centrifuge twice to remove myelin fragments, dead cells and other cells to obtain microvascular plexus.
[0030] S31. Add 22% bovine serum albumin (BSA) to the homogenate of brain and spinal cord tissue and perform a first centrifugation to remove myelin fragments, dead cells, and other contaminating cells from the supernatant. The first centrifugation was performed at 4°C, 2600 rpm, for 10 min.
[0031] S32. Perform a second centrifugation, adding 22% bovine serum albumin (BSA) to the brain and spinal cord tissue homogenate after the first centrifugation to remove myelin fragments, dead cells, and other contaminating cells from the upper layer, resulting in a low-purity microvascular plexus. The conditions for the second centrifugation are the same as the first. Two centrifugations can remove some myelin fragments, dead cells, and other contaminating cells from the brain and spinal cord tissue homogenate.
[0032] S4. The low-purity microvascular plexus is subjected to four natural sedimentation processes to further remove myelin fragments, dead cells, and impurities from the upper layer, resulting in a high-purity microvascular plexus.
[0033] The four-stage natural sedimentation process involves: first, adding 500 μL of parietal cell culture medium to the low-purity microvascular plexus obtained after two centrifugations to resuspend the vascular plexus, allowing it to settle naturally for 5 minutes, and then removing 400 μL of supernatant. Then, adding another 400 μL of parietal cell culture medium to resuspend the vascular plexus, allowing it to settle naturally for 5 minutes, and then removing 400 μL of supernatant. This second natural sedimentation process is repeated twice until the fourth natural sedimentation is completed, yielding high-purity microvessels. S5. High-purity microvascular plexuses were seeded into gelatin-coated culture plates. The gelatin concentration of the culture plates was 4.8 mg / m², which was sufficient to coat the wells of the plates quickly.
[0034] S6. Cells are cultured in vitro using a selective medium, allowing them to emerge from the microvascular plexus and yield a large number of vascular wall cells. The selective medium is a special medium for vascular wall cells.
[0035] S7. After five passages, a large number of high-purity vascular wall cells were obtained. It should be noted that high-purity vascular wall cells refer to cells that are positive for wall cell marker proteins, exhibit good colocalization with the expressed protein markers PDGFRβ, α-SMA, Desmin, and NG2, and do not express endothelial cell marker CD31, astrocyte marker GFAP, or microglia marker IBA-1.
[0036] Figure 2 This image shows the result of extracting wall cell growth from a vascular plexus in a cell culture dish. Figure 2 It can be seen that the extracted vascular plexus adheres well to the wall, and a large number of cells crawl out of the vascular plexus. Figure 3 This image shows the immunofluorescence identification results of parietal cells after passage. Figure 3 As can be seen from the immunofluorescence identification results, all isolated and cultured cells showed positive results for wall cell marker proteins. The vascular wall cells had good co-localization with the expressed protein markers PDGFRβ, α-SMA, Desmin and NG2, and did not express endothelial cell marker CD31, astrocyte marker GFAP, or microglia marker IBA-1. In other words, the immunofluorescence identification results of vascular wall cells after 5 passages showed good co-localization with multiple vascular wall cell markers.
[0037] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.
Claims
1. A method for extracting and culturing vascular wall cells of the central nervous system, characterized in that, Includes the following steps: S1. Brain and spinal cord tissues obtained by dissociation using papain; S2. Using syringe needles of different sizes, the dissociated brain and spinal cord tissues were blown and aerated to obtain brain and spinal cord tissue homogenates; S3. Add bovine serum albumin to the homogenate of brain and spinal cord tissue and centrifuge twice to remove myelin fragments, dead cells and impurities to obtain a low-purity microvascular plexus. S4. The low-purity microvascular plexus was subjected to four natural sedimentation cycles, each lasting 5 minutes, to obtain a high-purity microvascular plexus. S5. Inoculate high-purity microvascular plexuses into gelatin-coated culture plates; S6. Using selective culture medium for in vitro culture, cells crawl out from high-purity microvascular plexuses to form vascular wall cells; S7. After five passages, high-purity vascular wall cells were obtained.
2. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, The concentration of the papainase was 1 mg / ml.
3. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, The gelatin concentration in the culture plate was 4.8 mg / ml.
4. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, The syringe needles are of models 18G and 30G.
5. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, The concentration of bovine serum albumin was 22%.
6. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, Both centrifugation conditions were 4℃, 2600 rpm, and 10 min.
7. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, The four natural sedimentation steps specifically include: first, adding 500 μL of parietal cell culture medium to the low-purity microvascular plexus obtained after two centrifugations to resuspend the vascular plexus, allowing it to settle naturally for 5 minutes, and removing 400 μL of supernatant; second, adding another 400 μL of parietal cell culture medium to resuspend the vascular plexus, allowing it to settle naturally for 5 minutes, removing 400 μL of supernatant, and repeating the second natural sedimentation step twice, until the fourth natural sedimentation is completed, resulting in high-purity microvessels.
8. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, The selective culture medium is a culture medium specifically for vascular wall cells.
9. The method for extracting and culturing vascular wall cells of the central nervous system according to claim 1, characterized in that, The brain and spinal cord tissues are from young mice.