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CN-121991883-A - Extraction and culture method for central nervous system vascular wall cells

CN121991883ACN 121991883 ACN121991883 ACN 121991883ACN-121991883-A

Abstract

The application discloses an extraction and culture method of central nervous system vascular wall cells, and relates to the technical field of cell culture. The microvascular cluster is separated by combining one-step enzymolysis with two-time centrifugation and four-time natural sedimentation, and a large number of high-purity and high-activity vascular wall cells are obtained through in-vitro culture and five passages, so that the method has the advantages of simplicity and convenience in operation, low cost, high efficiency, good cell activity and high purity. The method comprises the following steps of S1, separating the obtained brain and spinal cord tissues by papain, S2, beating the brain and spinal cord tissues to obtain brain and spinal cord tissue homogenate, S3, adding bovine serum albumin into the brain and spinal cord tissue homogenate to perform centrifugation twice to obtain a low-purity microvascular cluster, S4, performing four natural sedimentation on the low-purity microvascular cluster to obtain a high-purity microvascular cluster, S5, inoculating the high-purity microvascular cluster into a culture plate, S6, performing in-vitro culture, and climbing out cells from the microvascular cluster to obtain vascular wall cells, and S7, performing five passages to obtain the high-purity vascular wall cells.

Inventors

  • ZHOU TIAN
  • ZHENG YIMING

Assignees

  • 南方医科大学

Dates

Publication Date
20260508
Application Date
20260214

Claims (9)

  1. 1. A method for extracting and culturing central nervous system vascular wall cells, which is characterized by comprising the following steps: s1, separating the obtained brain and spinal cord tissues by using papain; s2, using syringe needles of different types to blow and beat dissociated brain and spinal cord tissues to obtain brain and spinal cord tissue homogenate; S3, adding bovine serum albumin into the brain and spinal cord tissue homogenate, and centrifuging for two times to remove myelin sheath fragments, dead cells and foreign cells, thereby obtaining a low-purity microvascular plexus; s4, carrying out four times of natural sedimentation on the low-purity microvascular cluster, and obtaining the high-purity microvascular cluster 5min each time; s5, inoculating the high-purity microvascular cluster into a culture plate coated with gelatin; S6, in-vitro culture is carried out by using a selective culture medium, and cells climb out of the high-purity microvascular plexus to form vascular wall cells; S7, carrying out five passages to obtain the high-purity vascular wall cells.
  2. 2. The method for the extraction and culture of cells of the vascular wall of the central nervous system according to claim 1, wherein the concentration of papain is 1 mg/ml.
  3. 3. The method for the extraction and culture of cells on a vessel wall of the central nervous system according to claim 1, wherein the gelatin concentration of the culture plate is 4.8 mg/ml.
  4. 4. The method for the extraction and culture of cells of the vascular wall of the central nervous system according to claim 1, wherein the syringe needles are 18G and 30G in size.
  5. 5. The method according to claim 1, wherein the concentration of bovine serum albumin is 22%.
  6. 6. The method according to claim 1, wherein the conditions for the two centrifugation are 4℃and 2600 rpm, and the centrifugation is 10 min.
  7. 7. The method according to claim 1, wherein the four natural sedimentation steps comprise adding 500ul of wall cell culture medium to the low-purity microvascular cluster obtained by the two centrifugation steps for the first time to resuspend the vascular cluster, allowing the vascular cluster to naturally sediment for 5 minutes, removing 400ul of supernatant, adding 400ul of wall cell culture medium to resuspend the vascular cluster for the second time, naturally sediment for 5 minutes, removing 400ul of supernatant, and repeating the second natural sedimentation step for two times until the fourth natural sedimentation is completed, thereby obtaining the high-purity microvascular.
  8. 8. The method for extracting and culturing cells of a vessel wall of a central nervous system according to claim 1, wherein the selective medium is a medium specific for cells of a vessel wall.
  9. 9. The method according to claim 1, wherein the brain and spinal cord tissue is brain and spinal cord tissue of a young mouse.

Description

Extraction and culture method for central nervous system vascular wall cells Technical Field The application relates to the technical field of cell culture, in particular to an extraction culture method of central nervous system vascular wall cells. Background The vascular wall cells (or perivascular cells) of the central nervous system are cells located on the basement membrane of microvascular endothelial cells, are closely adjacent to endothelial cells, and interact with endothelial cells through physical contact and molecular signals, playing an important role in maintaining the integrity of the blood brain barrier, regulating vascular function, supporting neuronal activity, and the like. In addition, the vascular wall cells may have characteristics of stem cells or progenitor cells, be capable of differentiating into various cell types, and participate in regeneration and repair of tissues, and thus, the isolated culture of the vascular wall cells has a central nervous system critical role. Currently, there are multi-step enzymolysis, flow cell sorting, and magnetic bead sorting for the separation of cells from the vessel wall. The multi-step enzymolysis method for separating and extracting the blood vessel wall cells can cause the cells to be in contact with digestive enzymes for too long, so that part of the cells are digested in a transitional way, and the cell activity is inhibited. The flow cytometry is one application, and the specific antigen on the cell surface of the sample is marked with fluorescein conjugated antibody or the cell has fluorescent report protein, and through high voltage electric field, after laser irradiation, the fluorescent signal emitted by each cell is collected, and the sample cell is treated and analyzed to express some antigen molecule, so as to separate the blood vessel wall cell. The disadvantages of flow cytometry are 1, the high price of flow cytometry and sorting equipment, high maintenance and operation costs, 2, the need for specialized technical knowledge and operation experience, which may lead to low sorting efficiency or inaccurate results, 3, the possibility of mechanical damage to the cells during sorting, which may affect the viability and function of the cells especially during high-speed sorting, 4, the need for single cell suspension for samples, the preparation process may be complicated for some cells or tissues which are difficult to disperse, 5, the sorting often depends on specific cell surface markers, which may lead to missed or erroneous sorting of some rare cell subsets, 6, some cell types may not be easily sorted due to their specific physical characteristics (e.g. size, shape). 7. The amount of data generated by flow cytometry is large, requiring specialized software and expertise to analyze the data. The magnetic bead sorting method is to connect magnetic beads with antibodies first and then to separate target cells by means of the principle of combining antibodies with cell surface antigen and the magnetic beads to adsorb the cells expressing corresponding antigen through the action of magnetic field. However, since no specific antibody can label the vessel wall cells, this method can result in the extracted vessel wall cells being mixed with other cell types, which are not of high purity and cannot be used for important studies related to such cells. Disclosure of Invention The embodiment of the application provides an extraction culture method of central nervous system vascular wall cells, which combines one-step enzymolysis with twice centrifugation and four times natural sedimentation to separate microvascular clusters, cultures outside a gelatin-coated culture plate and a selective culture matrix, and obtains a large number of high-purity and high-activity vascular wall cells through five passages. The embodiment of the application provides an extraction culture method of central nervous system vascular wall cells, which comprises the following steps of S1, using papain to carry out enzymolysis to obtain brain and spinal cord tissues, S2, using different types of syringe needles to blow and beat the dissociated brain and spinal cord tissues to obtain brain and spinal cord tissue homogenate, S3, adding bovine serum albumin into the brain and spinal cord tissue homogenate, carrying out centrifugation twice to remove myelin sheath fragments, dead cells and mixed cells to obtain low-purity microvascular clusters, S4, carrying out four times of natural sedimentation on the low-purity microvascular clusters, obtaining high-purity microvascular clusters each time for 5min, S5, inoculating the high-purity microvascular clusters into a gelatin-coated culture plate, S6, using a selective culture medium to carry out in vitro culture, climbing out cells from the high-purity microvascular clusters to form vascular wall cells, and S7, carrying out five passages to obtain the high-purity microvascular wall cells. Further, the concentration of papain