Method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology

Abstract

The invention provides a method for separating cerebrovascular endothelial cells based on a magnetic bead sorting technology, which comprises the following steps: S1, digesting brain tissues, and then removing myelin sheaths and red blood cells to obtain a cell precipitate; s2, sorting the cell precipitate by using CD45 magnetic beads, so as to obtain CD45 negative cells; s3, the CD45 negative cells are sorted through CD31 magnetic beads, and CD45 negative cells and CD31 positive cells are obtained. According to the method, the high-purity cerebrovascular endothelial cells can be obtained through separation, the method is suitable for single cell sequencing and other analysis, and the characteristics of the cerebrovascular endothelial cells can be deeply researched.

Description

Technical Field

[0001] The present invention belongs to the field of isolation of vascular endothelial cells, and particularly relates to a method for isolating cerebrovascular endothelial cells based on magnetic bead sorting technology. Background Art

[0002] The blood-brain barrier (BBB) is a crucial vascular specialization structure that can both protect and nourish brain neurons and glial cells, and also hinder the delivery of drugs in the blood. It strictly controls the homeostasis of the central nervous system, and many blood-derived drugs have poor therapeutic effects because they cannot cross the cerebrovascular wall.

[0003] Vascular endothelial cells (ECs) are the key cellular components that constitute the BBB. They form a continuous complex through tight junctions and adherens junctions between EC-ECs, and together constitute the blood-brain barrier. Existing studies have shown that molecular alterations of cerebrovascular endothelial cells have been observed in patients and animal models of various brain diseases (such as stroke, multiple sclerosis, traumatic brain injury, and glioblastoma). Therefore, isolating cerebrovascular endothelial cells for disease research is of great significance.

[0004] However, the traditional method for isolating vascular endothelial cells is density gradient centrifugation, which has difficulties in separating capillary clusters, resulting in low separation purity and cell recovery rate. Although some studies have used CD31 immunomagnetic bead sorting to isolate lymphatic endothelial cells or intestinal mucosal microvascular endothelial cells, due to the complex types of cerebrovascular endothelial cells, using CD31 immunomagnetic bead sorting will mix a large number of immune cells, and high-purity vascular endothelial cells cannot be obtained, making it still difficult to accurately analyze the molecular characteristic changes of cerebrovascular endothelial cells in different diseases. Summary of the Invention

[0005] To solve the above problems of the prior art, the present invention provides a method for isolating cerebrovascular endothelial cells based on magnetic bead sorting technology, which can isolate cerebrovascular endothelial cells with high purity, is suitable for single-cell sequencing and other analyses, and helps to deeply study the characteristics of cerebrovascular endothelial cells.

[0006] The present invention is achieved through the following technical solutions: A method for isolating cerebrovascular endothelial cells based on magnetic bead sorting technology, comprising: S1, digesting the brain tissue, and then removing myelin and red blood cells to obtain a cell pellet; S2, sorting the cell pellet with CD45 magnetic beads to obtain CD45-negative cells; S3. Sort the CD45-negative cells using CD31 magnetic beads to obtain CD45-negative and CD31-positive cells.

[0007] Preferably, in S1, the digestion of the brain tissue is specifically as follows: Digest the brain tissue with an enzyme mixture of neutral protease II, collagenase I, collagenase II, and collagenase IV, add cold D-PBS, filter, centrifuge, and remove the supernatant to obtain a cell pellet.

[0008] Preferably, in S1, the removal of myelin sheaths is specifically as follows: Resuspend the cell pellet obtained after digestion with cold D-PBS, add cold myelin sheath removal solution, mix, cover with cold D-PBS on the upper layer, then centrifuge, remove the upper two phases, add cold D-PBS again, centrifuge, and remove the supernatant to obtain a cell pellet.

[0009] Preferably, in S1, the removal of red blood cells is specifically as follows: Resuspend the cell pellet obtained after removing myelin sheaths with red blood cell lysate, incubate, centrifuge, and remove the supernatant to obtain a cell pellet.

[0010] Preferably, S2 is specifically as follows: Resuspend the cell pellet in buffer B, add CD45 magnetic beads, mix and incubate at 2-8 °C, then perform magnetic separation to collect CD45-negative cells.

[0011] Preferably, in S2, the addition amount of CD45 magnetic beads is: add 10 μL of CD45 magnetic beads per 10 7 cells.

[0012] Preferably, S3 is specifically as follows: Resuspend the CD45-negative cells in buffer B, vortex and then add CD31 magnetic beads, incubate at 2-8 °C, then perform magnetic separation to collect CD45-negative and CD31-positive cells.

[0013] Preferably, the addition amount of CD31 magnetic beads is: add 10 μL of CD31 magnetic beads per 10 7 CD45-negative cells.

[0014] Preferably, if the collected CD45-negative and CD31-positive cells show a clumping phenomenon, resuspend the CD45-negative and CD31-positive cells in DPBS solution, add trypsin, incubate at 37 °C, and then vortex; check the cell status under a microscope. If the CD45-negative and CD31-positive cells dissociate into single-cell state, add DPBS solution containing BSA, centrifuge, and remove the supernatant to obtain CD45-negative and CD31-positive cells.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for isolating cerebrovascular endothelial cells. First, CD45-negative cells (CD45 - cells) are screened out using CD45 sorting magnetic beads, and this process can remove immune cells (CD45 + cells). Then, CD45-negative and CD31-positive cells (CD31 + / CD45 - cells) are screened out through CD31 sorting magnetic beads, which are cerebrovascular endothelial cells. This method can remove pericytes, smooth muscle cells, and immune cells from cerebrovascular cells and screen out cerebrovascular endothelial cells. Compared with the traditional density gradient centrifugation method, when isolating cerebrovascular endothelial cells, the method of the present invention can overcome the difficulty of separating capillary clusters in traditional techniques, greatly improving the separation purity and cell recovery rate; using magnetic bead sorting technology, the operation is simple, the cost is low, the efficiency is high, and no special equipment is required. This method can quickly and accurately extract cerebrovascular endothelial cells with high purity, high yield, and long survival time from surgically resected human brain tissue, and can perform single-cell RNA sequencing (scRNA-seq), thereby constructing a molecular map of human brain ECs, revealing the heterogeneity of the BBB and its molecular changes in diseases, helping to deeply study the characteristics of human cerebrovascular endothelial cells, and providing important information for formulating effective treatment plans and optimizing drug delivery strategies. BRIEF 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, without creative efforts, other drawings can be obtained based on these drawings.

[0017] Figure 1 Single human cerebrovascular endothelial cells isolated by the magnetic bead sorting technology in Example 1 of the present invention; Figure 2 The qPCR analysis results of cell surface markers of three different sorting populations in Example 2 of the present invention. a is CD45 + cells, b is CD31 + / CD45 - cells, c is CD45 - / CD31 - cells; Figure 3The BBB model constructed in Example 3 of the present invention and the transport efficiency of TMZ across the blood-brain barrier; a shows the BBB model constructed using a Transwell insert, where Endothelial cells are human cerebral vascular endothelial cells, Luminal is the upper chamber, and Abluminal is the basolateral chamber; b shows the permeability of temozolomide (TMZ) across the blood-brain barrier in the presence of LGK974. 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 the 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 use 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 specified, 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 relationships, without substantial change in technical content, should also be regarded as the scope in which the present invention can be implemented.

[0021] The method for separating cerebral vascular endothelial cells based on magnetic bead sorting technology of the present invention includes the following steps: S1, digest the brain tissue, and then remove myelin and red blood cells to obtain cell precipitates; S2, sort the cell precipitates using CD45 magnetic beads to obtain CD45 - cells; S3, sort the CD45 - cells using CD31 magnetic beads to obtain CD31 + / CD45 - cells.

[0022] The cell pellet obtained by the present invention through digestion, myelin removal, and red blood cell removal mainly contains pericytes, endothelial cells, smooth muscle cells, immune cells, etc. The cerebral blood vessels are mainly composed of endothelial cells and pericytes / smooth muscle cells. Pericytes represent a specific type of interstitial cell closely related to smooth muscle cells and have finger-like protrusions that wind around the lumen of endothelial cells. In normal tissues, pericytes provide paracrine support signals to endothelial cells. Since the sedimentation coefficients of pericytes, endothelial cells, smooth muscle cells, and immune cells are very similar, it is difficult to separate them by ordinary gradient centrifugation methods, and there is no mature method for separating these several types of cells in the industry. The present invention uses immunomagnetic bead sorting to purify and separate these cells. CD45 magnetic beads can be used to remove the mixed immune cells, and further CD31 magnetic beads can be used to remove smooth muscle cells, etc., to obtain pure endothelial cell single cells.

[0023] In the method for separating cerebral vascular endothelial cells based on magnetic bead sorting technology of the present invention, the digestion of the brain tissue in S1 is specifically as follows: The brain tissue is digested with an enzyme mixture of neutral protease II, collagenase I, collagenase II, and collagenase IV, cold D-PBS is added, filtered, centrifuged, and the supernatant is removed to obtain a cell pellet.

[0024] When the present invention is implemented, the specific process of digesting the brain tissue includes: (1) First, prepare the following reagents: N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) is an amphoteric ion organic chemical buffer; Hank's Balanced Salt Solution (HBSS) is one of the commonly used phosphate buffer solutions in cell separation or culture, and its main components include NaCl, KCl, KH2PO4, Na2HPO4, NaHCO3, CaCl2, MgCl2, MgSO4, and glucose; DMEM (dulbecco's modified eagle medium) is a medium containing various amino acids and glucose. 20 mg of neutral protease II is dissolved in 200 μL of HEPES, 20 mg of collagenase I is dissolved in 200 μL of DMEM, 20 mg of collagenase II is dissolved in 200 μL of DMEM, and 20 mg of collagenase IV is dissolved in 200 μL of DMEM.

[0025] (2) Prepare an enzyme mixture (40 μL of neutral protease II solution + 40 μL of collagenase I solution, 40 μL of collagenase II solution, 100 μL of collagenase IV solution, 40 μL of DNAse solution, 2.28 mL of DMEM).

[0026] (3) Cut the brain tissue into small pieces of 2 - 4 mm and place them in 50 - ml tubes. Each tube can digest up to 1 g, with a total of no more than 3 g of brain tissue.

[0027] (4) Transfer the brain tissue to the enzyme mixture and suspend and digest it using a syringe and 18 - gauge pink needle and 21 - gauge yellow needle until the aspiration is smooth.

[0028] (5) Incubate the digested sample on a rotator for 1 hour. Separate the cells with a yellow needle every 10 minutes until the aspiration is smooth and check the single - cell status under a microscope.

[0029] (6) Add cold D - PBS to the centrifuge tube, then filter the cell suspension through a 70 - micron sieve, and centrifuge at 300 g for 10 minutes at 4°C, and then remove the supernatant.

[0030] In the method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology of the present invention, the removal of myelin sheath in S1 is specifically as follows: Resuspend the cell pellet obtained after digestion with cold D - PBS, add cold myelin - sheath removal solution, mix, cover with cold D - PBS on the upper layer, then centrifuge, remove the upper two phases, add cold D - PBS again, centrifuge, and remove the supernatant to obtain a cell pellet.

[0031] When the present invention is implemented, the specific process of removing the myelin sheath includes: (1) Prepare 1.8 ml of cold myelin - sheath removal solution, a 15 - ml centrifuge tube, 6.2 ml of cold D - PBS, and add 4 ml of cold D - PBS again (for 1 g of tissue).

[0032] (2) Gently resuspend the digested cell pellet with an appropriate amount of cold D - PBS, and then add an appropriate amount of cold myelin - sheath removal solution and mix well.

[0033] (3) Very gently cover with an appropriate amount of cold D - PBS, and slowly aspirate to ensure that the D - PBS layer covers the cell suspension and the two layers do not mix.

[0034] (4) Centrifuge at 4°C and 3000 g for 10 minutes, with an acceleration rate of 9 and a deceleration rate of 3. After centrifugation, three phases are formed, and completely aspirate the upper two phases.

[0035] (5) Supplement with cold D - PBS and gently invert the tube three times.

[0036] (6) Centrifuge at 4°C and 1000 g for 10 minutes, and then aspirate the supernatant.

[0037] In the method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology of the present invention, the removal of red blood cells in S1 is specifically as follows: Resuspend the cell precipitate obtained after removing myelin with a red blood cell lysate, incubate, centrifuge, remove the supernatant, and obtain a cell precipitate.

[0038] When the present invention is implemented, the specific process of removing red blood cells includes: (1) Prepare PB buffer (DPBS containing 0.5% BSA); buffer B (PBS containing 0.1% BSA and 2 mmol EDTA, pH 7.4); red blood cell lysate.

[0039] (2) Resuspend the cell precipitate in 1 ml of cold red blood cell lysate, vortex for 5 seconds, and then incubate at room temperature for 2 minutes.

[0040] (3) Add 10 ml of cold PB buffer to terminate the incubation, centrifuge at 1000 rpm for 10 minutes at room temperature (15 ml tube), then completely aspirate the supernatant, and carefully resuspend the cells with 100 - 150 μl of buffer B by slowly pipetting up and down.

[0041] (4) Count the cells and check the cell number and viability.

[0042] In the method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology of the present invention, S2 is specifically as follows: Resuspend the cell precipitate in buffer B, add CD45 magnetic beads, mix and incubate at 2 - 8 °C, then perform magnetic separation to collect CD45-negative cells.

[0043] When the present invention is implemented, the specific process of S2 includes: (1) Resuspend the cell precipitate in 80 μl of buffer B.

[0044] (2) Add 10 μl of CD45 magnetic beads to every 10 7 cells, mix and incubate in a refrigerator at 2 - 8 °C for 15 minutes.

[0045] (3) Add the cell suspension diluted with 500 μl of buffer B to the sorting column.

[0046] (4) Place the sorting column in a magnetic separator, wash the sorting column with 3 ml of buffer B, collect the unlabeled CD45 - cells, and wash the sorting column with an appropriate amount of buffer B (3 times, 3 ml each time).

[0047] (5) Remove the sorting column from the magnetic separator, place it on a 15 ml centrifuge tube, wash the sorting column with 5 ml of medium, and immediately wash out the magnetically labeled cells with a push rod.

[0048] (6) Resuspend the CD45 - cells in 100 - 120 μL with Buffer B, count the cells, and check the cell number and viability rate.

[0049] (7) While the tube containing CD45 - cells is still in the magnetic cell sorter, transfer the supernatant to a new tube, centrifuge at 300 g for 10 minutes, and completely aspirate the supernatant to obtain CD45 - cells.

[0050] In the method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology of the present invention, S3 specifically is: Resuspend the CD45 - cells in Buffer B, vortex and then add CD31 magnetic beads, incubate at 2 - 8 °C, and then perform magnetic separation to collect CD31 + / CD45 - cells.

[0051] When implementing the present invention, the specific process of S3 includes: (1) Prepare a DPBS solution containing 0.04% BSA.

[0052] (2) Resuspend the CD45 - cells in Buffer B to a concentration lower than 1×10 7 cells per 90 μL of Buffer B, vortex and then add 10 μL of CD31 magnetic beads, and incubate in the refrigerator (2 - 8 °C) for 15 minutes.

[0053] (3) Add 500 μL of Buffer B to dilute the cell suspension; place the sorting column in the magnetic cell sorter and rinse the sorting column with 3 mL of Buffer B to prepare; add the cell suspension to the sorting column and remove the unlabeled cells.

[0054] (4) Remove the sorting column from the magnetic cell sorter and place it on a 15 mL centrifuge tube. Add 5 mL of culture medium dropwise to the sorting column and rinse the CD31 + / CD45 - cells through the push rod, centrifuge at 300 g for 10 minutes, and completely remove the supernatant.

[0055] (5) If the cells show aggregation, resuspend the CD31 + / CD45 - cells in 90 μL of DPBS solution; subsequently, add 300 μL of trypsin to the cell suspension, incubate at 37 °C for 7 minutes, and vortex for 5 seconds.

[0056] (6) Check the cell status under the microscope, such as CD31 + / CD45 -Once the cells are dissociated into a single-cell state, 600 μl of DPBS solution containing BSA is added. Subsequently, the cells are centrifuged at 300 g for 10 minutes, and the supernatant is completely removed. The cells are resuspended in 50 μl of DPBS solution containing 0.04% BSA.

[0057] (7)Count the cells and check the cell number and viability.

[0058] The volumes of the reagents given above are applicable to 1 g of brain tissue. Adjust the reagent volumes proportionally according to the weight of the brain tissue.

[0059] Example 1 Isolate vascular endothelial cells from human brain tissue, which specifically includes the following steps: 1. Dissociation of human brain tissue samples (1)First, prepare the following reagents: N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) is an amphoteric organic chemical buffer; Hank's Balanced Salt Solution (HBSS) is one of the commonly used phosphate buffer solutions in cell separation or culture, and its main components include NaCl, KCl, KH2PO4, Na2HPO4, NaHCO3, CaCl2, MgCl2, MgSO4, and glucose; DMEM (dulbecco's modified eagle medium) is a medium containing various amino acids and glucose. 20 mg of neutral protease II is dissolved in 200 μl of HEPES, 20 mg of collagenase I is dissolved in 200 μl of DMEM, 20 mg of collagenase II is dissolved in 200 μl of DMEM, and 20 mg of collagenase IV is dissolved in 200 μl of DMEM.

[0060] (2)Prepare an enzyme mixture (40 μl of neutral protease II solution + 40 μl of collagenase I solution, 40 μl of collagenase II solution, 100 μl of collagenase IV solution, 40 μl of DNase solution, 2.28 ml of DMEM).

[0061] (3)Cut 1 g of human brain tissue into 3-mm pieces, place them in a 50-ml centrifuge tube, add the enzyme mixture, and use a syringe and 18-gauge pink needle and 21-gauge yellow needle to suspend and digest until the aspiration is smooth.

[0062] (4)Incubate the human brain tissue sample using a rotator for 1 hour. Separate the cells with a yellow needle every 10 minutes until the aspiration is smooth, and check the single-cell state under a microscope.

[0063] (5) Add cold D-PBS to the centrifuge tube, then filter the cell suspension through a 70-μm sieve, remove the supernatant after centrifugation at 300 g for 10 minutes at 4 °C to obtain cell pellets.

[0064] 2. Demyelination (1) Prepare 1.8 mL of cold demyelination solution, a 15-mL centrifuge tube, 6.2 mL of cold D-PBS, and add 4 mL of cold D-PBS again (for 1 g of human brain tissue).

[0065] (2) Gently resuspend the cell pellet with 6.2 mL of cold D-PBS. Then add 1.8 mL of cold demyelination solution and mix well.

[0066] (3) Very gently cover with 4 mL of cold D-PBS, slowly aspirate to ensure that the D-PBS layer covers the cell suspension and the two layers do not mix.

[0067] (4) Centrifuge at 4 °C and 3000 g for 10 minutes with an acceleration rate of 9 and a deceleration rate of 3. After centrifugation, three phases are formed, and completely aspirate the upper two phases.

[0068] (5) Supplement with 12 mL of cold D-PBS and gently invert the tube three times.

[0069] (6) Centrifuge at 4 °C and 1000 g for 10 minutes, then aspirate the supernatant to obtain cell pellets.

[0070] 3. Removal of red blood cells (1) Prepare PB buffer (DPBS containing 0.5% BSA); buffer B (PBS containing 0.1% BSA and 2 mmol EDTA, pH 7.4); red blood cell lysate.

[0071] (2) Resuspend the cell pellet in 1 mL of cold red blood cell lysate, vortex for 5 seconds, and then incubate at room temperature for 2 minutes.

[0072] (3) Add 10 mL of cold PB buffer to terminate the incubation, centrifuge at 1000 rpm for 10 minutes at room temperature (15-mL tube), then completely aspirate the supernatant to obtain cell pellets; carefully resuspend the cells with 120 μL of buffer B by slowly pipetting up and down.

[0073] (4) Count the cells and check the cell number and viability.

[0074] 4. Isolation of CD45 - Cells (1) Resuspend the cell pellet obtained in step 3 in 80 μL of buffer B.

[0075] (2) Every 107 Add 10 μL of CD45 magnetic beads to the cells, mix, and incubate in a refrigerator at 4 °C for 15 minutes.

[0076] (3) Add 500 μL of buffer B to dilute the cell suspension, and then add it to the sorting column.

[0077] (4) Place the sorting column in a magnetic separator, and rinse the sorting column with 3 mL of buffer B; collect the unlabeled CD45 - cells, and rinse the sorting column with an appropriate amount of buffer B (3 times, 3 mL each time).

[0078] (5) Remove the sorting column from the magnetic separator, place it on a 15 mL centrifuge tube, rinse the sorting column with 5 mL of culture medium, and immediately rinse out the magnetically labeled cells (CD45 + cells) with a push rod.

[0079] (6) Resuspend the CD45 - cells in 110 μL with buffer B, and count the cells to check the cell number and viability rate.

[0080] (7) When the centrifuge tube is still in the magnetic separator, transfer the supernatant to a new tube, centrifuge at 300 g for 10 minutes, and completely aspirate the supernatant to obtain CD45 - cells.

[0081] 5. Isolate CD31 + cells (1) Prepare a DPBS solution containing 0.04% BSA.

[0082] (2) Resuspend the CD45 - cells in buffer B to a concentration of less than 1×10 7 cells per 90 μL of buffer B. After vortexing, add 10 μL of CD31 magnetic beads and incubate in the refrigerator (4 °C) for 15 minutes.

[0083] (3) Add 500 μL of buffer B to the CD45 ﹣ cells, and then add it to the sorting column. Place the sorting column in a magnetic separator and rinse the activated sorting column with 3 mL of buffer B. Add the cell suspension to the sorting column and remove the unlabeled cells (CD45 - / CD31 - cells).

[0084] (4) Remove the sorting column from the magnetic separator and place it on a 15 mL centrifuge tube. Add 5 mL of culture medium dropwise to the column and rinse the labeled cells (CD31 + / CD45 -Cells), centrifuge at 300 g for 10 minutes, and completely remove the supernatant.

[0085] (5) Resuspend the CD31 + / CD45 - cells in 90 μL of DPBS solution. Subsequently, add 300 μL of trypsin solution to the cell suspension, incubate at 37 °C for 7 minutes, and vortex for 5 seconds.

[0086] (6) Examine the cell status under a microscope. The CD31 + / CD45 - cells are completely dissociated into single-cell state ( Figure 1 ), add 600 μL of DPBS solution containing BSA. Subsequently, centrifuge at 300 g for 10 minutes and completely remove the supernatant. Resuspend the cells in 50 μL of DPBS solution containing 0.04% BSA.

[0087] (7) Count the cells and check the cell number and viability.

[0088] Example 2: Identification of human brain vascular endothelial cell surface markers by qPCR To explore the changes in surface markers of different types of cells, qPCR analysis was performed on the cells of three different sorted populations obtained in Example 1. The specific cell populations include: R1 population (CD31 + / CD45 - cells), R2 population (CD45 - / CD31 - cells) and R3 population (CD45 + cells). The surface markers of cells in each population were detected by qPCR to compare the differences in surface markers of different types of cells. The cell populations obtained by qPCR analysis include CD45 + , CD45 - / CD31 - and CD31 + / CD45 - three categories, and the expression levels of genes P2RY13, CD163, CDH5, PECAM1, CD34 were detected.

[0089] The results are as Figure 2 shown. It can be seen from Figure 2 that the expression of the immune cell marker CD163 in the CD45 + cell population is significantly higher than that in other populations, while the expression is lower in the CD31 + / CD45 - cell population, indicating that immune cells have been effectively screened out. Further analysis found that CD31 + / CD45- The cells in the cell population exhibited typical cerebrovascular endothelial cell characteristics and were able to highly express multiple endothelial cell and blood-brain barrier-related markers, such as CDH5, PECAM1, and CD34, indicating that these cells were an endothelial cell population. The finally screened CD31 + / CD45 - cells can be used for subsequent in vitro blood-brain barrier construction and single-cell transcriptomics research.

[0090] Example 3: Using cerebrovascular endothelial cells to construct a BBB model, it was found that the Wnt inhibitor LGK974 affected the permeability of the chemotherapeutic drug temozolomide across the blood-brain barrier.

[0091] Human cerebrovascular endothelial cells are a cell line derived from surgically resected brain tissue. Through immunomagnetic bead sorting technology, the present invention can efficiently and selectively isolate single cerebrovascular endothelial cells, which can be cultured in vitro and applied to pathological research of the central nervous system, exploration of drug transport mechanisms, neurotoxicity assessment, and research at the cellular and molecular levels of the nervous system. Due to the characteristics of the blood-brain barrier of human cerebrovascular endothelial cells, it can simulate the barrier function of cerebrovascular endothelium in vitro.

[0092] The human cerebrovascular endothelial cells screened by the above method were used to construct an in vitro model of the BBB, as shown in Figure 3 a. The human cerebrovascular endothelial cells were seeded in Transwell chambers (pore size 8.0 μm, diameter 6.5 mm, product of Corning), and the seeding amount was 1×10 6 cells per well, and the cell density per well was 5×10 4 cells. The cells were cultured for 7 - 12 days until a blood-brain barrier (BBB) was formed. After the blood-brain barrier was treated with 100 μl of LGK974 for three days, the medium in the luminal chamber was replaced with a 40 μg / ml temozolomide (TMZ) solution, and 200 μl of the medium in the luminal chamber was replaced with freshly prepared aliquot temozolomide solution every 30 minutes. 50 μl of samples were collected from the abluminal chamber every 30 minutes. The group without LGK974 treatment was used as the Control group. The results showed that the permeability of temozolomide in the group treated with LCK974 was significantly higher than that in the Control group ( Figure 3 b), confirming that inhibiting the Wnt signaling pathway can increase the transport efficiency of TMZ across the blood-brain barrier.

[0093] The above results indicate that the cerebrovascular endothelial cells sorted by immunomagnetic beads can be used to construct a blood-brain barrier in vitro, providing a good cell model for studying changes in blood-brain barrier permeability, primary cell culture, etc.

Claims

1. A method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology, characterized in that, Comprising: S1, digest the brain tissue, then remove myelin and red blood cells to obtain cell precipitate; S2, sort the cell precipitate with CD45 magnetic beads to obtain CD45-negative cells; S3, sort the CD45-negative cells with CD31 magnetic beads to obtain CD45-negative and CD31-positive cells.

2. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 1, wherein In S1, the digestion of the brain tissue is specifically as follows: digest the brain tissue with an enzyme mixture of neutral protease II, collagenase I, collagenase II and collagenase IV, add cold D-PBS, filter, centrifuge, and remove the supernatant to obtain cell precipitate.

3. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 1, wherein In S1, the removal of myelin is specifically as follows: resuspend the cell precipitate obtained after digestion with cold D-PBS, add cold myelin removal solution, mix, cover with cold D-PBS on the upper layer, then centrifuge, remove the upper two phases, add cold D-PBS again, centrifuge, and remove the supernatant to obtain cell precipitate.

4. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 1, wherein In S1, the removal of red blood cells is specifically as follows: resuspend the cell precipitate obtained after removing myelin with red blood cell lysate, incubate, centrifuge, and remove the supernatant to obtain cell precipitate.

5. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 1, wherein S2 is specifically as follows: resuspend the cell precipitate in buffer B, add CD45 magnetic beads, mix and incubate at 2-8°C, then perform magnetic separation to collect CD45-negative cells.

6. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 5, wherein, In S2, the addition amount of CD45 magnetic beads is: 10 microliters of CD45 magnetic beads are added per 10 7 cells.

7. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 1, characterized in that, S3 is specifically as follows: resuspend the CD45-negative cells in buffer B, vortex and add CD31 magnetic beads, incubate at 2-8°C, then perform magnetic separation to collect CD45-negative and CD31-positive cells.

8. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 3, wherein, The addition amount of CD31 magnetic beads is: for every 10 7 CD45-negative cells, 10 microliters of CD31 magnetic beads are added.

9. The method for separating cerebrovascular endothelial cells based on magnetic bead sorting technology according to claim 1, characterized in that, If the collected CD45-negative and CD31-positive cells show a clumping phenomenon, resuspend the CD45-negative and CD31-positive cells in DPBS solution, add trypsin, incubate at 37°C, then vortex; check the cell state under a microscope. If the CD45-negative and CD31-positive cells dissociate into a single-cell state, add DPBS solution containing BSA, centrifuge, and remove the supernatant to obtain CD45-negative and CD31-positive cells.

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