Extraction method of brain tissue vascular wall cells

Abstract

The invention provides a brain tissue vascular wall cell extraction method, which comprises: 1, carrying out dissociation and myelin sheath removal treatment on a brain tissue to obtain a cell precipitate; 2, re-suspending the cell precipitate obtained in the step 1 in a buffer solution B, and sorting by adopting CD45 immunomagnetic beads to obtain CD45-cells; step 3, suspending the CD45-cells obtained in the step 2 in a buffer solution B again, adding a biotinylated CD140b antibody, and incubating; 4, centrifugally washing a product obtained in the step 3 by adopting a buffer solution B, and removing supernate to obtain a cell precipitate; and 5, re-suspending the cell precipitate obtained in the step 4 in a buffer solution B, adding antibiotin microspheres, incubating, and sorting to obtain the CD45- / CD140b + cells. The method disclosed by the invention can be used for efficiently extracting single cells of vascular wall cells in human brain tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell extraction, and particularly relates to a method for extracting cerebrovascular wall cells from brain tissue. Background Art

[0002] During brain development, the generation of new blood vessels is very active, while in the healthy adult brain, this process is almost in a static state. However, in vascular-dependent encephalopathies (such as cerebrovascular malformations and brain tumors), the generation of blood vessels will be reactivated, indicating that blood vessels are closely related to brain diseases and brain development. There is a special physiological barrier between cerebrovascular and brain tissue - the blood-brain barrier (BBB). The blood-brain barrier plays a crucial "gatekeeper" role in protecting the brain from the invasion of harmful substances, and its dysfunction is often the starting factor of the pathophysiological processes of various diseases.

[0003] Mural cells (MCs) are an important part of the blood-brain barrier, including vascular smooth muscle cells (VSMCs) and pericytes (PC), and undertake the core responsibility of maintaining the function of the blood-brain barrier, playing a key role in blood vessel development and stability, and participating in the formation of the normal vasculature. Studying the single-cell characteristics of these cells will help to deeply understand the mechanism of action of nervous system wall cells. However, there is still a lack of effective methods for preparing single-cell suspensions of vascular wall cells in human brain tissue, resulting in the inability to obtain single vascular wall cells in human brain tissue. Summary of the Invention

[0004] In order to solve the above problems of the prior art, the present invention provides a method for extracting cerebrovascular wall cells, which can efficiently extract single cerebrovascular wall cells from human brain tissue.

[0005] The present invention is realized through the following technical solutions: A method for extracting cerebrovascular wall cells from brain tissue, comprising the following steps: Step 1, dissociating and demyelinating the brain tissue to obtain cell precipitate; Step 2, resuspending the cell precipitate obtained in Step 1 in buffer B, and sorting with CD45 immunomagnetic beads to obtain CD45 - cells; Step 3, resuspending the CD45 - cells obtained in Step 2 in buffer B, adding biotinylated CD140b antibody, and incubating; Step 4, centrifuging and washing the product obtained in Step 3 with buffer B, removing the supernatant, and obtaining cell precipitate; Step 5: Resuspend the cell pellet obtained in Step 4 in Buffer B, add avidin microbeads, incubate, and then perform sorting to obtain CD45 - / CD140b + cells.

[0006] Preferably, the specific process of dissociation in Step 1 is as follows: Add the brain tissue to the enzyme mixture, repeatedly suspend it with a needle until the liquid flows smoothly; then, incubate it using a rotator, and separate the cells with a needle every preset time during incubation; finally, add cold D-PBS, filter, centrifuge, and remove the supernatant to obtain a cell pellet.

[0007] Further, the specific process of demyelination in Step 1 is as follows: Resuspend the cell pellet obtained by dissociation with D-PBS, add the demyelination solution, mix, cover the obtained cell suspension with cold D-PBS, centrifuge to form three phases, remove the upper two phases, then add cold D-PBS, centrifuge, and remove the supernatant to obtain a cell pellet.

[0008] Preferably, the specific process of Step 2 is as follows: Resuspend the cell pellet obtained in Step 1 in Buffer B, add CD45 immunomagnetic beads, incubate, add them to a sorting column, place the sorting column in a sorter, wash the sorting column with Buffer B, collect the washing solution, centrifuge, and remove the supernatant to obtain CD45 - cells.

[0009] Preferably, in Step 2, add 10 7 μL of CD45 immunomagnetic beads to every 10 cells.

[0010] Preferably, in Step 3, add 2 6 μL of biotinylated CD140b antibody to every 10 cells.

[0011] Preferably, in Step 3, the incubation temperature is 2 - 8 °C and the incubation time is 10 minutes.

[0012] Preferably, in Step 5, add 20 7 μL of avidin microbeads to every 10 cells.

[0013] Preferably, in Step 5, the incubation temperature is 2 - 8 °C and the incubation time is 15 minutes.

[0014] Preferably, step 5 specifically includes: resuspending the cell pellet obtained in step 4 in buffer B, adding avidin microbeads to the obtained cell suspension, incubating, then diluting the cell suspension with buffer B, adding the diluted cell suspension into a sorting column, placing the sorting column in a sorter, flushing the sorting column with buffer B, then removing the sorting column from the sorter, adding a culture medium into the sorting column, flushing out the magnetically labeled cells, centrifuging, and removing the supernatant; resuspending the obtained cell pellet in D-PBS, adding trypsin, incubating, then centrifuging and removing the supernatant to obtain CD45 - / CD140b + Parietal cells.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The method for extracting brain tissue vascular wall cells of the present invention first dissociates brain tissue into single cells, and performs preliminary purification to remove myelin; then uses CD45 immunomagnetic beads to sort out CD45 - cells, removes CD45 + cells, then uses biotinylated CD140b antibody to act on the cells, and finally uses avidin microbeads to sort out the cells bound by the biotinylated CD140b antibody, thereby obtaining CD45 - / CD140b + cells, that is, brain tissue vascular wall cells. This method is simple to operate, low in cost and high in efficiency, does not require special equipment, and can efficiently extract vascular wall cells with high purity, high survival rate and suitable for long-term preservation from brain tissue samples. The brain tissue vascular wall cells sorted by the method of the present invention can be used for subsequent single-cell sequencing and other analysis and research, providing an effective way to deeply explore the characteristics of brain tissue vascular wall cells, and will provide valuable experimental materials for the research on the pathogenesis and treatment methods of cerebrovascular diseases and other nervous system diseases. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 It is a microscopic examination diagram of vascular wall cells after dissociation and sorting in Example 1 of the present invention; Figure 2 It is a Q-PCR data analysis diagram of Example 2 of the present invention; Figure 3Schematic diagram of the experiment for sorting vascular wall cells of normal human brain tissue and single-cell RNA sequencing in Example 3 of the present invention. Detailed implementation manners

[0018] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0019] It should be noted that the process equipment or devices not specifically noted in the following examples all adopt conventional equipment or devices in the art.

[0020] It should be noted that the terms "include" and "have" and any of their variations are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, rather than limiting the arrangement order of each method step or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in technical content, should also be regarded as the scope in which the present invention can be implemented.

[0021] The present invention realizes the extraction of vascular wall cells of brain tissue through an immunomagnetic bead sorting technique, including the following steps: Step 1: Dissociate and demyelinate the brain tissue to obtain cell precipitates; Step 2: Resuspend the cell precipitates obtained in Step 1 in buffer B, and perform sorting using CD45 immunomagnetic beads to obtain CD45 - cells; Step 3: Resuspend the CD45 - cells obtained in Step 2 in buffer B, add biotinylated CD140b antibody, and incubate; Step 4: Centrifuge and wash the product obtained in Step 3 with buffer B, remove the supernatant to obtain cell precipitates; Step 5: Resuspend the cell precipitates obtained in Step 4 in buffer B, add anti-biotin microbeads, incubate and then perform sorting to obtain CD45 - / CD140b + wall cells.

[0022] The present invention uses CD45 immunomagnetic beads to sort out CD45 - cells and remove CD45+ cells. CD45 + Cells refer to cells that express the CD45 antigen. In the brain tissue, these cells are mainly immune cells such as lymphocytes (T cells and B cells), monocytes, macrophages, and certain activated glial cells, etc., which participate in the immune response and inflammatory reaction of the brain tissue. CD45 - Cells in the brain tissue mainly include neurons, unactivated glial cells (such as resting astrocytes and microglia), and other non-immune cells. In the brain tissue, CD140b + Cells mainly refer to mural cells that express CD140b (also known as PDGFRβ). The present invention uses biotinylated CD140b antibody to act on CD45 - cells, and finally uses biotin microbeads to sort out the cells that will be bound by the biotinylated CD140b antibody, so as to obtain CD45 - / CD140b + cells, that is, to obtain brain tissue vascular wall cells.

[0023] As some preferred embodiments of the present invention, the specific process of dissociation in step 1 is as follows: add the brain tissue into the enzyme mixture, repeatedly suspend it with a needle until the liquid is smooth; then, incubate it using a rotator, and separate the cells with a needle every preset time during the incubation; finally, add cold D-PBS, filter, centrifuge, remove the supernatant, and obtain cell precipitate.

[0024] In some specific embodiments of the present invention, the enzyme mixture is obtained by mixing neutral protease II solution, collagenase I solution, collagenase II solution, and collagenase IV solution. The solvent of the neutral protease II solution is HEPES, the collagenase I solution is DMEM medium prepared with Hanks' balanced salt solution (HBSS), the solvent of the collagenase II solution is HBSS, and the solvent of the collagenase IV solution is HBSS.

[0025] As some preferred embodiments of the present invention, the specific process of demyelination in step 1 is as follows: resuspend the cell precipitate obtained by dissociation with D-PBS, add the demyelination solution, mix, cover cold D-PBS on the upper layer of the obtained cell suspension, centrifuge to form three phases, remove the upper two phases, then add cold D-PBS, centrifuge, remove the supernatant, and obtain cell precipitate.

[0026] Among them, D-PBS is Dulbecco's phosphate buffered saline containing HBSS.

[0027] As some preferred embodiments of the present invention, the specific process of step 2 is as follows: The cell pellet obtained in step 1 is resuspended in buffer B, CD45 immunomagnetic beads are added, incubated, and then added into a sorting column. The sorting column is placed in a sorter, and the sorting column is rinsed with buffer B. The rinsing solution is collected, centrifuged, and the supernatant is removed to obtain CD45 - cells.

[0028] In some specific embodiments of the present invention, buffer B is prepared by the following method: 0.1% bovine serum albumin (BSA) and 2 millimoles per liter of ethylenediaminetetraacetic acid (EDTA) are added to phosphate buffered saline (PBS) to make the pH = 7.4.

[0029] In some specific embodiments of the present invention, in step 2, 80 microliters of buffer B are added per 10 7 cells, and 10 microliters of CD45 immunomagnetic beads are added per 10 7 cells.

[0030] In some specific embodiments of the present invention, in step 3, 98 microliters of buffer B are used to suspend per 10 6 cells, and 2 microliters of biotinylated CD140b antibody are added per 10 6 cells. If there are 2 × 10 6 cells, it is scaled up proportionally, and 4 microliters of antibody and 196 microliters of buffer B are used.

[0031] In some specific embodiments of the present invention, in step 3, the incubation temperature is 2 - 8 °C, and the incubation time is 10 minutes (note that high temperature and long incubation time will both lead to non-specific cell labeling).

[0032] In some specific embodiments of the present invention, in step 5, 80 microliters of buffer B are used to suspend per 10 7 cells, and 20 microliters of antibiotin microbeads are added per 10 7 cells. If there are 5 × 10 6 cells, then 10 microliters of antibiotin microbeads are needed.

[0033] In some specific embodiments of the present invention, in step 5, the incubation temperature is 2 - 8 °C, and the incubation time is 15 minutes.

[0034] In some specific embodiments of the present invention, step 5 specifically includes: resuspending the cell pellet obtained in step 4 in buffer B, adding avidin microbeads to the obtained cell suspension, incubating, then diluting the cell suspension with buffer B, adding the diluted cell suspension into a sorting column, placing the sorting column in a sorter, flushing the sorting column with buffer B, then taking out the sorting column from the sorter, adding a culture medium into the sorting column, flushing out the magnetically labeled cells, centrifuging, and removing the supernatant; resuspending the obtained cell pellet in D-PBS, adding trypsin, incubating, then centrifuging and removing the supernatant to obtain CD45 - / CD140b + Parietal cells.

[0035] Example 1: 1. Dissociation of human brain tissue samples (1) Prepare the following solutions: Solutions required for 1 gram of human brain tissue: Neutral protease II solution: Dissolve 20 mg of neutral protease II in 200 μL of HEPES.

[0036] Collagenase I solution: Dissolve 20 mg of collagenase I in 200 μL of DMEM medium prepared with Hank's balanced salt solution (HBSS).

[0037] Collagenase II solution: Dissolve 20 mg of collagenase II in 200 μL of HBSS.

[0038] Collagenase IV solution: Dissolve 20 mg of collagenase IV in 200 μL of HBSS.

[0039] (2) Operating steps 1) Prepare an enzyme mixture using the solutions in step (1) above.

[0040] 2) Take 1 gram of surgically resected normal brain tissue, cut it into 3-mm pieces, place them in a 50-mL centrifuge tube, add the enzyme mixture for dissociation, and repeatedly resuspend using an 18-gauge pink needle and a 21-gauge yellow needle until the liquid flows smoothly.

[0041] 3) Incubate using a rotator for 1 hour, and separate the cells with a 21-gauge yellow needle every 10 minutes during incubation to check whether the single-cell state is achieved.

[0042] 4) Add cold D-PBS, filter through a 70-μm sieve, centrifuge at 4°C and 300×g for 10 minutes, and remove the supernatant.

[0043] 2. Demyelination (1) Prepare cold demyelination solution and cold D-PBS.

[0044] 1 gram of human brain tissue requires 1.8 ml of myelin removal solution, 6.2 ml of D-PBS, and 4 ml of D-PBS added secondarily.

[0045] (2)Operation steps 1) Gently suspend the cell pellet obtained in step 1 with D-PBS and add the myelin removal solution and mix well.

[0046] 2) Cover the cell suspension with an appropriate amount of cold D-PBS.

[0047] 3) Centrifuge at 3000×g for 10 minutes at 4°C to form three phases. Remove the upper two phases and only retain the bottom phase.

[0048] 4) Add cold D-PBS and gently invert the tube 3 times, avoiding shaking during the inversion process.

[0049] 5) Centrifuge at 1000×g for 10 minutes at 4°C and completely aspirate the supernatant to obtain the cell pellet.

[0050] 3. Isolate CD45 - Cells (1)Preparation of buffer B: Add 0.1% BSA and 2 mmol / L EDTA to PBS buffer to make the pH = 7.4.

[0051] (2)Operation steps: 1) Resuspend the cell pellet obtained in step 2 in buffer B, adding 80 μl of buffer B per 10 7 cells.

[0052] 2) Add 10 μl of CD45 immunomagnetic beads per 10 7 cells and mix well. Incubate at 4°C for 15 minutes, then add 500 μl of buffer B to dilute the cell suspension.

[0053] 3) Add the cell suspension to an LS separation column (from MACS Miltenyi Biotec, USA) and place it in a MACS separator (MidiMACS™ separator).

[0054] 4) Wash the separation column 3 times with 3 ml of buffer B and collect the unlabeled cells (CD45 + cells).

[0055] 5) Remove the LS separation column from the MACS separator and place it at the mouth of a newly taken 15 ml centrifuge tube. Add 5 ml of buffer B to the LS separation column and use a push rod to wash out the magnetically labeled cells and collect the cell suspension.

[0056] 6) Centrifuge CD45 at 300×g for 10 min at 4°C -The cell suspension was centrifuged to discard the supernatant, and the cell pellet was obtained.

[0057] 7) The cell pellet was resuspended in 100 μl of Buffer B to obtain CD45 - cells.

[0058] 4. Isolation of CD45 - / CD140b + parietal cells Procedure: 1) No more than 10 6 CD45 - cells were suspended in 98 μl of Buffer B, and 2 μl of biotinylated CD140b antibody was added.

[0059] 2) After thorough mixing, the mixture was incubated at 4 °C in the dark for 10 minutes.

[0060] 3) 2 ml of Buffer B was added, and the cells were washed by centrifugation at 350 × g for 5 minutes. The supernatant was completely aspirated. This step was repeated, 1 ml of Buffer B was added and centrifuged at 350 × g for 5 minutes to remove the supernatant, and the cell pellet was obtained.

[0061] 4) The cell pellet (per 10 7 cells) was suspended in 80 μl of Buffer B. For every 10 7 cells, 20 μl of antibiotin microbeads was added. After thorough mixing, the mixture was incubated at 4 °C in the dark for 15 minutes and diluted to 500 μl with Buffer B.

[0062] 5) The LS separation column was placed in the magnetic field of a suitable MACS separator and prepared by flushing the column with 3 ml of Buffer B. The cell suspension was applied to the LS separation column, and the LS separation column was flushed 3 times with 3 ml of Buffer B. The unlabeled cells (CD45 - / CD140b - cells) were collected.

[0063] 6) The LS separation column was removed from the separator and placed on a 15-ml centrifuge tube. 5 ml of culture medium was added to the LS separation column, and the magnetically labeled cells were flushed out immediately by pushing the plunger forcefully into the LS separation column. The magnetically labeled cells (CD45 - / CD140b + parietal cells) were collected.

[0064] 7) The suspension of CD45 - / CD140b + parietal cells was centrifuged at 350 × g for 5 minutes, and the supernatant was completely aspirated. In a 15-ml centrifuge tube, CD45 - / CD140b +Parietal cells were suspended in approximately 90 μL of D-PBS containing 0.04% BSA, and 300 μL of trypsin was added to the cell suspension.

[0065] 8) Incubate at 37 °C for 7 minutes and shake for 5 seconds, then incubate at 37 °C for 5 minutes and shake for 10 seconds; pipette thoroughly, and take 10 μL of the cell suspension and mix it with trypan blue, and check the single-cell state under a microscope.

[0066] 9) Add 600 μL of D-PBS containing 0.04% BSA, centrifuge at 300×g for 10 minutes, completely aspirate the supernatant, and resuspend the obtained cell pellet in 30 μL of D-PBS containing 0.04% BSA, count the cells, and check the cell number and viability rate (dilution factor is 1:2) The results are as Figure 1 shown. It can be seen that vascular wall cells were indeed sorted by the method of the present invention. Among them, the white bright spots are live vascular wall cells, and the blue dim spots are dead cells.

[0067] Example 2: The CD45 + cells, CD45 - / CD140b - cells, CD45 - / CD140b + cells obtained in Example 1 were lysed, RNA was extracted, and Q-PCR identification was performed to detect the markers of various cells: P2RY13, CD163, P2RY13, CD163. Among them, the P2RY13 gene encodes a G protein-coupled receptor protein, which participates in regulating immune cell migration, regulating the differentiation of macrophages into anti-inflammatory or pro-inflammatory phenotypes, and maintaining the homeostasis of immune cells in CD45 + cells. CD163 is a highly glycosylated single-chain transmembrane protein that participates in regulating the inflammatory response and is related to the differentiation and phagocytic ability of macrophages. PDGFRB is platelet-derived growth factor receptor β, which is crucial for angiogenesis. NOTCH3 is a transmembrane receptor protein belonging to the Notch family members, which is related to the differentiation of vascular smooth muscle cells and can also regulate the support of vascular wall cells for the vascular wall.

[0068] Through Q-PCR analysis, it was found that the expression of CD163 was very high in the screened CD45 + cell population and the expression of P2RY13 was relatively high in the screened CD45 + cell population, and the expression was relatively low in the screened CD45 - / CD140b - cell population, indicating that the method of the present invention has separated most of the CD45 +The cells were screened out. The mural cell markers PDGFRB and NOTCH3 were highly expressed in the CD45 - / CD140b + cell population and were lowly expressed in the CD45 - / CD140b - cell population, indicating that through the magnetic bead sorting technology, the present invention effectively screened out mural cells. ( Figure 2 ).

[0069] Example 3: The CD45 + cells, CD45 - / CD140b - cells, and CD45 - / CD140b + cells obtained in Example 1 were subjected to single-cell RNA sequencing technology, and then UMAP analysis was performed on the transcriptome data of CD45 - / CD140b + cells. The results showed that CD45 - / CD140b + cells could be clustered into 6 subpopulations, named pericyte 1 (PC 1) subpopulation, pericyte 2 (PC 2) subpopulation, venous vascular smooth muscle cell (vSMC) subpopulation, arterial vascular smooth muscle cell 1 (aSMC 1) subpopulation, arterial vascular smooth muscle cell 2 (aSMC 2) subpopulation, and unknown vascular smooth muscle cell (SMC-unknown) subpopulation ( Figure 3 ).

[0070] In summary, through the application of magnetic bead sorting technology, the present invention effectively separated various different types of mural cells, providing an effective separation and extraction method for in-depth exploration of the characteristics of vascular wall cells in human brain tissue.

Claims

1. A method for extracting cerebrovascular wall cells, characterized in that, It includes the following steps: Step 1: Dissociate and demyelinate the brain tissue to obtain cell precipitates; Step 2: Resuspend the cell pellet obtained in Step 1 in Buffer B and perform sorting using CD45 immunomagnetic beads to obtain CD45 - cells; Step 3: Resuspend the CD45 cells obtained in Step 2 in Buffer B, add biotinylated CD140b antibody, and incubate. - ​ Step 4: Centrifuge and wash the product obtained in Step 3 with Buffer B, remove the supernatant to obtain cell precipitates; Step 5: Resuspend the cell pellet obtained in Step 4 in Buffer B, add anti-biotin microbeads, incubate, and then perform sorting to obtain CD45 - / CD140b + cells.

2. The method for extracting brain tissue vascular wall cells according to claim 1, wherein, The specific dissociation process in Step 1 is as follows: Add the brain tissue into the enzyme mixture, repeatedly suspend it with a needle until the liquid flows smoothly; then, incubate it using a rotator, and separate the cells with a needle every preset time during the incubation; finally, add cold D-PBS, filter, centrifuge, and remove the supernatant to obtain cell precipitates.

3. The method for extracting brain tissue vascular wall cells according to claim 2, wherein, The specific demyelination process in Step 1 is as follows: Resuspend the cell precipitates obtained by dissociation with D-PBS, add the demyelination solution, mix, cover the upper layer of the resulting cell suspension with cold D-PBS, centrifuge to form three phases, remove the upper two phases, then add cold D-PBS, centrifuge, and remove the supernatant to obtain cell precipitates.

4. The method for extracting brain tissue vascular wall cells according to claim 1, wherein The specific process of Step 2 is as follows: Resuspend the cell pellet obtained in Step 1 in Buffer B, add CD45 immunomagnetic beads, incubate, add to the sorting column, place the sorting column in the sorter, wash the sorting column with Buffer B, collect the wash solution, centrifuge, and remove the supernatant to obtain CD45 - cells.

5. The method for extracting brain tissue vascular wall cells according to claim 1, characterized in that, In step 2, add 10 microliters of CD45 immunomagnetic beads to every 10 7 cells.

6. The method for extracting brain tissue vascular wall cells according to claim 1, wherein In step 3, add 2 μL of biotinylated CD140b antibody to every 10 6 cells.

7. The method for extracting brain tissue vascular wall cells according to claim 1, characterized in that, In Step 3, the incubation temperature is 2 - 8 °C and the incubation time is 10 minutes.

8. The method for extracting brain tissue vascular wall cells according to claim 1, wherein In step 5, 20 μL of avidin beads are added to every 10 7 cells.

9. The method for extracting brain tissue vascular wall cells according to claim 1, characterized in that, In Step 5, the incubation temperature is 2 - 8 °C and the incubation time is 15 minutes.

10. The method for extracting brain tissue vascular wall cells according to claim 1, wherein Step 5 specifically includes: resuspending the cell pellet obtained in Step 4 in buffer B, adding anti-biotin microbeads to the obtained cell suspension, incubating, then diluting the cell suspension with buffer B, adding the diluted cell suspension into a sorting column, placing the sorting column in a sorter, rinsing the sorting column with buffer B, then taking out the sorting column from the sorter, adding a culture medium into the sorting column, flushing out the magnetically labeled cells, centrifuging, and removing the supernatant; resuspending the obtained cell pellet in D-PBS, adding trypsin, incubating, then centrifuging and removing the supernatant to obtain CD45 - / CD140b + parietal cells.

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