CN114703120A

CN114703120A - Method for separating animal nervous system blood vessel smooth muscle cell single cell

Info

Publication number: CN114703120A
Application number: CN202210268027.3A
Authority: CN
Inventors: 张倩
Original assignee: Shanghai Newren Biomedical Technology Co ltd
Current assignee: Shanghai Newren Biomedical Technology Co ltd
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2022-07-05
Anticipated expiration: 2042-03-17
Also published as: CN114703120B

Abstract

The invention discloses a method for separating single cells of vascular smooth muscle cells of an animal nervous system, belonging to the technical field of biology. The method comprises the following steps: mixing and culturing the cut fresh neural tissue and cell dissociation digestive juice, centrifuging, adding total cell culture solution, and filtering to obtain a cell mixture A; mixing the cell mixture A with endothelial cell gradient separating medium, purifying and separating to obtain cell mixture B containing vascular smooth muscle cells. And then, the iodixanol solution is further used for separating to obtain pure vascular smooth muscle cells, the method is simple and convenient to operate, low in cost and high in efficiency, a specific instrument is not required, and the method is suitable for industrial production, and the neurovascular smooth muscle cell single cells prepared by the method can be used for single cell sequencing or other analysis, so that the characteristic of the neurovascular smooth muscle cells can be further researched.

Description

Method for separating animal nervous system blood vessel smooth muscle cell single cell

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for separating single cells of vascular smooth muscle cells of an animal nervous system.

Background

Blood vessels play a pivotal role in neural activity. Vascular smooth muscle cells are the main cellular components constituting the tunica media of blood vessels, are one of the important metabolic and endocrine organs of the body, play an important role in various physiological processes, and are closely related to the occurrence and development of diseases. Research shows that in the pathological process, vascular smooth muscle cells participate in the physiological and pathological processes of various vascular diseases such as hypertension, atherosclerosis, transplanted vascular diseases, restenosis after angioplasty, repair after vascular wall injury and the like through self proliferation, migration and extracellular matrix synthesis. Therefore, vascular smooth muscle cells have become an important subject of study in the field of medical care.

Each cell is unique and heterogeneous, even of the same cell type, in the same organ or tissue. The brain, the most important organ of the organism, contains many types of cells, all with their unique characteristics, such as vascular smooth muscle cells, which underlie the enhancement of vasomotor activity. The research on various vascular diseases such as hypertension can find that honest and reliable smooth muscle cells can effectively improve the effect of vascular tension and enable vascular contraction to be more normal, the activity of blood vessels can be effectively improved by means of the smooth muscle cells, the effect is particularly important for curing and preventing the hypertension diseases, the smooth muscle cells can also be found to be important components forming vascular walls in experiments, and the smoothness and the delivery capacity of the blood vessels are undoubtedly improved by means of the smooth muscle cells.

Vascular smooth muscle cells are an important basis for improving disease recovery and therapeutic efficacy. In the current rehabilitation methods of various vascular diseases, better subsequent effects can be ensured only by improving the growth activity of smooth muscle cells, and the application of the smooth muscle cells can be found through smooth muscle cell transaction arrangement, so that the capability of immune tissues can be effectively improved, the subsequent vascular dynamics can be greatly improved, and the resistance of the body can be improved by applying the smooth muscle cells in the disease rehabilitation means to obtain better rehabilitation effects;

to summarize. The vascular smooth muscle cell is an indispensable cell type in the body, and directly influences the growth effect of the vascular wall and the normal function of the blood vessel, so that related scientific researchers are required to know the culture mode and the use method of the vascular smooth muscle cell in the culture of the smooth muscle technology, the growth capacity of the vascular smooth muscle cell is improved in a correct mode, and the vascular wall has stronger power to improve the research and development capacity of the biological science.

The stroke is the first cause of death in China, and the research on the functions of vascular smooth muscle cells can provide a new method and strategy for treating the stroke. However, the kind and function of vascular smooth muscle cells in the central nervous system are not completely clear, and single cell sequencing technology plays an incomparable role in accurately understanding the function and interaction of certain types of cells. However, no method for preparing the vascular smooth muscle cell single cell suspension in the nerve tissue of the mammal specially exists at present.

Disclosure of Invention

Aiming at the defects, the invention provides a preparation method of a single cell suspension of vascular smooth muscle cells of an animal nervous system. The method is simple and convenient to operate, low in cost and high in efficiency, does not need a specific instrument, and is suitable for industrial production, and the single cells of the neural vascular smooth muscle cells prepared by the method can be used for single cell sequencing or other analysis, so that the method is favorable for deeply researching the characteristics of the neural vascular smooth muscle cells.

The invention is realized by the following technical scheme:

the invention provides a method for separating single cells of vascular smooth muscle cells of an animal nervous system, which comprises the following steps:

mixing and culturing the cut fresh neural tissue and cell dissociation digestive juice, centrifuging, adding total cell culture solution, and filtering to obtain a cell mixture A;

mixing the cell mixture A with endothelial cell gradient separation liquid, and purifying and separating to obtain a cell mixture B containing vascular smooth muscle cells and endothelial cells;

mixing the cell mixture B with the gradient separation liquid of the vascular smooth muscle cells, and purifying and separating the single cells of the vascular smooth muscle cells;

the blood vessel smooth muscle cell gradient separation liquid is formed by compounding blood vessel smooth muscle cell separation liquid and total cell culture liquid, and the blood vessel smooth muscle cell separation liquid contains 58-62% of iodixanol by volume percentage.

Further, in a preferred embodiment of the present invention, the vascular smooth muscle cell gradient separation solution is prepared by adding a third solution to the fourth solution in a volume ratio of 1: 0.8-1.2, wherein the volume ratio of the vascular smooth muscle cell gradient separation solution to the total cell culture solution in the third solution is 2-3: 17-18; the volume ratio of the vascular smooth muscle cell separating medium to the total cell culture medium in the fourth solution is 2.5: 17.5.

Further, in a preferred embodiment of the present invention, the endothelial cell gradient separation solution is formed by compounding an endothelial cell separation solution and a total cell culture solution, wherein the endothelial cell separation solution contains silica gel particles with a mass concentration of 1.2-1.4 g/mL, and the silica gel particles are coated with vinylpyrrolidone.

Further, in a preferred embodiment of the present invention, the endothelial cell gradient separation solution is prepared by adding a first solution above a second solution according to a volume ratio of 1: 2-4, wherein the volume ratio of the endothelial cell gradient separation solution to the total cell culture solution in the first solution is 1-3: 37-39; the volume ratio of the endothelial cell separating medium to the total cell culture medium in the second solution is 2: 38.

further, in a preferred embodiment of the present invention, the step of preparing the cell mixture B comprises:

and mixing the cell mixture A with the endothelial cell gradient separation solution to obtain a complete endothelial cell gradient separation system, centrifuging the endothelial cell gradient separation system at the room temperature of 2000-2500 g for 15-20 min, removing the endothelial cell culture medium on the uppermost layer, and other nervous system cells and dead cells on the lowermost layer, retaining the endothelial cell layer containing endothelial cells and vascular smooth muscle cells in the middle, and washing.

Further, in a preferred embodiment of the present invention, the step of washing the endothelial cell layer comprises:

and (3) mixing the endothelial cell layer with the total cell culture solution, centrifuging for 1-3 min at room temperature at 150-250 g, and uniformly mixing the obtained precipitate by using a DMEM (DMEM) culture medium without fetal calf serum to obtain endothelial cell single-cell suspension.

Further, in a preferred embodiment of the present invention, the step of preparing single cells of vascular smooth muscle cells comprises:

and adding the cell mixture B above the gradient separation liquid of the vascular smooth muscle cells to obtain a complete gradient separation system of the vascular smooth muscle cells, centrifuging the gradient separation system of the vascular smooth muscle cells at the room temperature of 700-900 g for 12-17 min, removing the cell culture medium at the uppermost layer and the vascular endothelial cells at the middle layer, retaining the vascular smooth muscle cell layer of which the bottom is 0.5-1.5 mL, and washing.

Further, in a preferred embodiment of the present invention, the step of washing the vascular smooth muscle cell layer comprises:

and (3) mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging for 1-3 min at room temperature at 150-250 g, and uniformly mixing the obtained precipitate with a DMEM culture medium without fetal calf serum to obtain the vascular smooth muscle cell single cell suspension.

Further, in a preferred embodiment of the present invention, the total cell culture solution at least comprises: penicillin/streptomycin double antibody, DMEM medium and fetal bovine serum.

Further, in a preferred embodiment of the present invention, the cell dissociation digestion solution is prepared by adding 1-3 mg/mL of protease E and 22-28U/mL of DNase I to the total cell culture solution.

Compared with the prior art, the invention at least has the following technical effects:

in brain tissue, nerve endothelial cells and vascular smooth muscle cells are precisely connected in structure and function, and the difference between the cell size and the sedimentation coefficient of the nerve endothelial cells and the vascular smooth muscle cells is very small. Furthermore, the number of vascular smooth muscle cells is very small compared to endothelial cells, which makes it difficult to separate single vascular smooth muscle cells from neural endothelial cells using the existing cell separation methods. Therefore, how to effectively remove the nerve endothelial cells, neurons and other nerve cells while maintaining the vitality of the single cells of the vascular smooth muscle cells is very important for successfully separating the pure single cells of the vascular smooth muscle cells.

The separation method provided by the application obtains the neural tissue cell mixture after mixing and digesting fresh neural tissue and cell dissociation digestive juice containing protease and DNase. The cell dissociation digestive juice does not need to be aerated with oxygen in advance, the oxygen content is low, and most of neuron cells die due to oxygen deficiency in the digestion process because the neuron cells are sensitive to oxygen. And then, endothelial cells containing vascular smooth muscle cells are separated by using the endothelial cell gradient separating medium, and as the silica gel particles contained in the endothelial cell gradient separating medium are coated with the vinylpyrrolidone, the osmotic pressure is very low, the viscosity is also very low, the diffusion constant is low, the formed gradient is very stable, the biomembrane is not penetrated, the cells are not toxic, and the settleability of the cell gradient separating medium to different types and dead cells can be specifically adjusted, so that the aim of purifying the endothelial cells is fulfilled. Then, the vascular smooth muscle cells are separated from the vascular endothelial cells by using a vascular smooth muscle cell gradient separating medium, and finally pure vascular smooth muscle cells are obtained. Because the gradient separation liquid of vascular smooth muscle cells contains iodixanol, compared with the conventional separation liquid Ficoll, Percoll, sucrose and cesium chloride (CsCl), the iodixanol separation liquid not only has good separation and purification effects, but also has no influence on the life activities of various cells and organelles.

The preparation method of the animal nervous system blood vessel smooth muscle cell single cell suspension is simple and convenient to operate, low in cost and high in efficiency, does not need to use a specific instrument, is suitable for industrial production, and the nerve vessel smooth muscle cell single cell prepared by the method can be used for single cell sequencing or other analysis, so that the deep research on the characteristics of the nerve vessel smooth muscle cell is facilitated.

Drawings

FIG. 1 is a UMAP map of the vascular smooth muscle cells of the nervous system in the cerebral cortex of the mouse obtained in example 1, and it is understood that almost all the cells are labeled with the marker Acta2 of the vascular smooth muscle cells, indicating that the purity of the obtained cells is high.

FIG. 2 shows the genes expressed specifically for each subtype after the subtype classification using the vascular smooth muscle cells of the nervous system in the cerebral cortex of the mouse obtained in example 1.

FIG. 3 is the result of GO analysis using highly expressed genes in vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained in example 1.

FIG. 4 shows the GO analysis results of highly expressed genes in the vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained in control group 1.

FIG. 5 shows the GO analysis results of highly expressed genes in the vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained in control group 2.

FIG. 6 shows the GO analysis results of highly expressed genes in the vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained in control group 3.

FIG. 7 shows the GO analysis results of highly expressed genes in the vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained in control group 4.

Detailed Description

Embodiments of the present invention will be described in detail with reference to the following examples, but those skilled in the art will understand that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention, and that the specific conditions not specified in the examples are carried out according to conventional conditions or conditions suggested by the manufacturer, and that the reagents or equipment used are not specified by the manufacturer, and are all conventional products available through commercial purchase.

The technical scheme of the invention is as follows:

a method for separating single cells of vascular smooth muscle cells of an animal nervous system comprises the following steps:

step S1: mixing the cut fresh neural tissue with cell dissociation digestive juice, culturing, centrifuging, adding total cell culture solution, and filtering to obtain cell mixture A.

Wherein the fresh nerve tissue is derived from animal tissue, and the fresh nerve tissue is obtained after perfusion treatment. Preferably, the method of obtaining animal tissue is: after the animal is anesthetized by tribromoethanol, the abdominal cavity is cut off rapidly, after the heart is exposed, the injector is inserted into the left ventricle, the right auricle is cut off, precooled Du's phosphate buffer solution (D-PBS) without calcium and magnesium ions is used for heart perfusion until the blood is completely removed, the brain is taken out rapidly on ice, the part of the region of interest is cut off, and the target tissue is cut up by a razor blade at low temperature. D-PBS buffer solution without calcium and magnesium ions is used for perfusion on mammals, so that blood cells are thoroughly removed, and the influence of the D-PBS buffer solution on subsequent sequencing is avoided. If the blood cells are removed separately after digestion, the purity of the blood vessel smooth muscle cells is reduced or the yield of the blood vessel smooth muscle cells is reduced.

After perfusion, the brain or spinal cord of a mammal is taken out quickly on ice, nerve tissues of the region of interest are cut off, the region of interest is cut up quickly on ice by a razor blade, and as nerve cells are sensitive to oxygen, most nerve cells die due to oxygen deficiency in the digestion process, are separated from an endothelial cell layer containing vascular smooth muscle cells during gradient separation, are further purified to obtain endothelial cells, and finally, the vascular smooth muscle cells are further separated and purified by using iodixanol solution.

Further, in the process of preparing the cell mixture, fresh nerve tissues and cell dissociation digestive juice are mixed and then cultured at the constant temperature of 33 ℃ for 30-60 min, and mechanically and uniformly mixed once every 2 minutes. Wherein, the mechanical mixing means that the carrier (such as a centrifuge tube, a test tube and the like) filled with the culture solution is manually shaken or swirled to vibrate in the process of constant-temperature culture, which is beneficial to improving the digestion efficiency and ensuring more complete digestion.

Further, after the constant temperature culture, the centrifugal force in the centrifugation process is 200g, and the centrifugation time is 5 min. During centrifugation, the nerve tissue cells settle at the bottom and are separated from other less massive impurities resulting from digestion. After centrifugation, the supernatant was removed and total cell culture fluid was added and blown to mechanically dissociate. In order to reduce the damage of the pipette tip to cells, the tip of the pipette tip is polished on an alcohol lamp or a Pasteur pipette tip is used for blowing the mammalian tissue.

Further, in the preparation of the cell mixture, filtration was performed using a cell sieve having a pore size of 40 μm to remove bulk tissues and impurities.

Preferably, the total cell culture fluid contains at least: penicillin/streptomycin double antibody, DMEM medium and fetal bovine serum.

Preferably, the cell dissociation digestion solution is prepared by adding 1-3 mg/mL of Pronase E protease and 22-28U/mL of DNase I into the total cell culture solution. The protease Pronase E is mainly used for digesting the protein of the adherent cells, and the DNase I mixture is mainly used for digesting the DNA of the adherent cells and reducing the proportion of double cells or multiple cells, thereby improving the yield of single cells.

The total cell culture solution and the cell dissociation digestive solution do not need to be aerated with oxygen in advance, most nerve cells die due to oxygen deficiency in the digestion process, and are separated from an endothelial cell layer containing vascular smooth muscle cells in gradient separation, so that the aim of purifying the cell mixture A is fulfilled.

Step S2: mixing the cell mixture A with endothelial cell gradient separation liquid, and purifying and separating to obtain a cell mixture B containing vascular smooth muscle cells and endothelial cells;

the endothelial cell gradient separation solution is formed by compounding an endothelial cell separation solution and the total cell culture solution, wherein the endothelial cell separation solution contains silica gel particles with the mass concentration of 1.2-1.4 g/mL, and preferably, the mass concentration is 1.3 g/mL. The silica gel particles are coated with vinylpyrrolidone. The vinylpyrrolidone is used as a synthetic water-soluble high molecular compound, is soluble in water and most of organic solvents, has low toxicity and good physiological compatibility, has excellent physiological inertia, does not participate in human metabolism, has excellent biocompatibility and does not generate toxicity to separated cells.

Further, the endothelial cell gradient separating medium is prepared by adding a first solution above a second solution according to the volume ratio of 1: 2-4, wherein the volume ratio of the endothelial cell separating medium to the total cell culture medium in the first solution is 1-3: 37-39, preferably 1: 18.5-19.5 mixing; the volume ratio of the endothelial cell separation solution to the total cell culture solution in the second solution is 5-7: 33-35, preferably in a volume ratio of 3: 16-18 mixing.

Further, the step of preparing the cell mixture B includes:

and mixing the cell mixture A with the endothelial cell gradient separation solution to obtain a complete endothelial cell gradient separation system, centrifuging the endothelial cell gradient separation system at 2000-2500 g for 15-20 min at room temperature, removing the endothelial cell culture medium at the uppermost layer and other nervous system cells and dead cells at the lowermost layer, retaining an endothelial cell layer containing endothelial cells and vascular smooth muscle cells in the middle, and washing. Preferably, the centrifugal force is 2200-2500 g, and the centrifugal time is 15-17 min.

Preferably, the step of washing the endothelial cell layer comprises:

and (3) mixing the endothelial cell layer with the total cell culture solution, centrifuging for 1-3 min at room temperature at 150-250 g (preferably at 180-230 g for 2min), and uniformly mixing the obtained precipitate with a DMEM (DMEM) culture medium without fetal calf serum to obtain endothelial cell single cell suspension.

The cell mixture B mainly contains vascular smooth muscle cells, and also contains a part of endothelial cells, pericytes and the like, the vascular smooth muscle cells are the main components of the large blood vessels, can be attached to the endothelial cells to provide nutrition or support for the endothelial cells, have similar effects with the pericytes forming the components of the small blood vessels, and are difficult problems in the field of blood vessel research.

More preferably, the centrifugation in the step of purifying and separating endothelial cells is performed using a horizontal rotor centrifuge. In this step, a fixed angle centrifuge is not used, mainly because the fixed angle rotor may cause the cells to gather on one side, resulting in poor separation.

Step S3: and mixing the cell mixture B with the blood vessel smooth muscle cell gradient separating medium, and purifying and separating the blood vessel smooth muscle cell single cells.

The vascular smooth muscle cell gradient separation solution is formed by compounding a vascular smooth muscle cell separation solution and a total cell culture solution, wherein the vascular smooth muscle cell separation solution contains 58-62% by volume of iodixanol, and preferably the volume fraction of the iodixanol is 60%.

Further, the vascular smooth muscle cell gradient separation solution is prepared by adding a third solution above a fourth solution according to the volume ratio of 1: 0.8-1.2, wherein the volume ratio of the vascular smooth muscle cell separation solution to the total cell culture solution in the third solution is 2-3: 17-18, preferably 5: 35; the volume ratio of the vascular smooth muscle cell separation solution to the total cell culture solution in the fourth solution is 6-8: 32-34, and preferably 7: 33.

Further, the step of preparing the vascular smooth muscle cell single cell comprises:

adding the cell mixture B above the blood vessel smooth muscle cell gradient separation liquid to obtain a complete blood vessel smooth muscle cell gradient separation system, centrifuging the blood vessel smooth muscle cell gradient separation system at 700-900 g for 12-17 min (preferably at 750-850 g for 14-16 min), removing the uppermost layer of cell culture medium and the blood vessel endothelial cells in the middle layer, retaining the lowest 0.5-1.5 mL (preferably 0.8-1.2 mL) of blood vessel smooth muscle cell layer, and washing.

Preferably, the step of washing the vascular smooth muscle cell layer comprises:

the step of washing the vascular smooth muscle cell layer comprises:

and (3) mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging for 1-3 min at room temperature at 150-250 g, and uniformly mixing the obtained precipitate with a DMEM culture medium without fetal bovine serum to obtain the vascular smooth muscle cell single cell suspension. Preferably, the centrifugal force is 200g and the centrifugation time is 2 min.

More preferably, the centrifugation in the step of purifying and separating vascular smooth muscle cells is performed by a horizontal rotor centrifuge. In this step, a fixed angle centrifuge is not used, mainly because the fixed angle rotor may cause cells to gather on a certain side, resulting in poor separation.

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Example 1

The embodiment provides a method for separating single cells of vascular smooth muscle cells of an animal nervous system, which comprises the following steps:

(1) preparing a digestion buffer solution special for single cell dissociation: 44.5mL of DMEM medium, 5mL of FBS, 500. mu.L of penicillin/streptomycin double antibody, 2mg/mL of Pronase E protease at a final concentration and 25U/mL of DNase I mixture at a final concentration are sequentially added into a sterile 50mL centrifuge tube, and the mixture is prepared into a digestion buffer solution, 6 mL/tube and subpackaged at-20 ℃ and below for later use.

(2) Preparing a total cell culture solution: 44.5mL of DMEM medium, 5mL of FBS, and 500. mu.L of penicillin/streptomycin diabody were sequentially added to a sterile 50mL centrifuge tube, and stored at 4 ℃.

(3) After the animal is anesthetized by tribromoethanol, the abdominal cavity is cut off rapidly, after the heart is exposed, the injector is inserted into the left ventricle, the right auricle is cut off, precooled D-PBS without calcium and magnesium ions is used for heart perfusion until the blood is completely removed, the brain is taken out rapidly on ice, the part of the interested area is cut, and the target tissue is cut up by a razor blade at low temperature.

(4) Mixing the minced tissue with a digestion buffer solution special for single cell dissociation, incubating for 30min in a constant temperature incubator at 33 ℃, and turning upside down and mixing once every 2 min.

(5) While the above steps were performed, after preparing the first solution and the second solution shown in table 1 in a sterile super clean bench, the first solution was slowly added above the second solution in a 15mL centrifuge tube to obtain an endothelial cell gradient separation solution. The mass concentration of silica gel particles in the cell separation solution in Table 1 was 1.3 g/mL.

TABLE 1 preparation of gradient separating medium for endothelial cells

Solutions of	Endothelial cell isolate (μ L)	Total cell culture fluid (μ L)	Total (μ L)
				First solution	100	1900	2000
Second solution	300	1700	2000

(6) After digestion of nerve tissue, 200g of the tissue is centrifuged for 4min briefly, the supernatant is removed, and 6mL of total cell culture solution is added to blow and beat the tissue mechanically until no obvious tissue block exists.

(7) A20-micron sterile cell sieve previously infiltrated with D-PBS free of calcium and magnesium ions was put into a 50-mL centrifuge tube in a clean bench, and the total cell culture solution was added to a cell sieve having a pore size of 40 μm under a sterile environment by using a 1-mL pipette to filter impurities and undigested tissues, thereby obtaining a cell mixture A.

(8) The cell mixture A is added into the cell gradient separating medium obtained in step (5) very carefully to prepare an integral endothelial cell gradient separating system, and 10mL of solution is counted in the centrifuge tube. The complete endothelial cell gradient separation solution is centrifuged for 15min at room temperature in a horizontal rotor centrifuge of 2500 g.

(9) The upper layer 6mL of the centrifuged endothelial cell gradient separation solution is endothelial cell culture medium containing fragments, the middle layer 2mL is endothelial cell cells, and the lowest layer 2mL is various nervous system cells and dead cells. And (3) sucking and discarding the culture medium on the uppermost layer, transferring 2mL of the solution in the middle layer into a new 15mL centrifuge tube, mixing the solution with the total cell culture solution, centrifuging at room temperature for 2min at 200g, and uniformly mixing the obtained precipitate by using a DMEM culture medium without fetal calf serum to obtain a vascular cell mixture B.

(10) After the third solution and the fourth solution shown in table 2 were prepared in a sterile super clean bench, the third solution was slowly added to the top of the fourth solution in a 15mL centrifuge tube to obtain a vascular smooth muscle cell gradient separation solution. The iodixanol content in the vascular smooth muscle cell isolate in table 2 was 60%.

TABLE 2 preparation of gradient separation liquid for vascular smooth muscle cells

(11) Adding the cell mixture B obtained in the step (9) into the vascular smooth muscle cell gradient separating medium obtained in the step 10 very carefully to prepare an integral vascular smooth muscle cell gradient separating system, wherein the centrifugal tube contains 4mL of solution in total. The complete vascular smooth muscle cell gradient separation system is centrifuged for 15min by a horizontal rotor centrifuge of 800g at room temperature.

(12) 2mL of the upper layer of the centrifuged vascular smooth muscle cell gradient separation system is endothelial cell solution, 1mL of the middle layer is vascular endothelial cells, and 1mL of the lowest layer is vascular smooth muscle cells. The top two layers were aspirated and the lower 1mL of solution was transferred to a new 15mL centrifuge tube. And (3) mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging at room temperature for 2min at 200g, and uniformly mixing the obtained precipitate with a DMEM (DMEM) culture medium without fetal bovine serum to obtain the vascular smooth muscle cell single cell suspension.

(13) 5mL of total cell culture solution is added into the solution, after careful mixing, 200g of total cell culture solution is centrifuged by a horizontal rotor for 2min at room temperature, 1mL of a pipette is used for washing and removing supernatant, the precipitate is mixed uniformly by DMEM without serum, and the survival rate is calculated by staining the platform blue.

Example 2

This example provides a method for separating single cells from vascular smooth muscle cells of the nervous system of an animal, which is substantially the same as example 1, except that the following steps are performed:

(5) while the above steps were performed, after preparing the first solution and the second solution shown in table 3 in a sterile super clean bench, the first solution was slowly added above the second solution in a 15mL centrifuge tube to obtain an endothelial cell gradient separation solution. The mass concentration of silica gel particles in the cell separation solution in Table 3 was 1.4 g/mL.

TABLE 3 preparation of gradient separating medium for endothelial cells

Solutions of	Endothelial cell isolate (μ L)	Total cell culture fluid (μ L)	Total (μ L)
				First solution	150	1850	2000
Second solution	350	1650	2000

(8) The cell mixture A is added into the cell gradient separating medium obtained in step (5) very carefully to prepare an integral endothelial cell gradient separating system, and 10mL of solution is counted in the centrifuge tube. The complete endothelial cell gradient separation solution is centrifuged for 20min at room temperature in a horizontal rotor centrifuge of 2000 g.

(9) The upper layer 6mL of the centrifuged endothelial cell gradient separation solution is endothelial cell culture medium containing fragments, the middle layer 2mL is endothelial cell cells, and the lowest layer 2mL is various nervous system cells and dead cells. And (3) sucking and discarding the culture medium on the uppermost layer, transferring 2mL of the solution in the middle layer into a new 15mL centrifuge tube, mixing the solution with the total cell culture solution, centrifuging the mixture at room temperature for 1min at 250g, and uniformly mixing the obtained precipitate by using a DMEM culture medium without fetal calf serum to obtain a vascular cell mixture B.

(10) After preparing the third solution and the fourth solution shown in table 4 in a sterile super clean bench, the third solution was slowly added to the top of the fourth solution in a 15mL centrifuge tube to obtain a vascular smooth muscle cell gradient separation medium. The iodixanol content in the vascular smooth muscle cell isolate in table 4 was 62%.

TABLE 4 preparation of gradient separation liquid for vascular smooth muscle cells

(11) Carefully adding the cell mixture B obtained in the step (10) into the vascular smooth muscle cell gradient separating medium obtained in the step 10 to prepare an integral vascular smooth muscle cell gradient separating system, wherein the centrifugal tube contains 4mL of solution in total. The complete vascular smooth muscle cell gradient separation system is centrifuged for 12min at room temperature by a horizontal rotor centrifuge of 700 g.

(12) 2mL of the upper layer of the centrifuged vascular smooth muscle cell gradient separation system is endothelial cell solution, 1mL of the middle layer is vascular endothelial cells, and 1mL of the lowest layer is vascular smooth muscle cells. The top two layers were aspirated and the lower layer 0.8mL of solution was transferred to a new 15mL centrifuge tube. And (3) mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging at room temperature for 1min at 250g, and uniformly mixing the obtained precipitate by using a DMEM (DMEM) culture medium without fetal calf serum to obtain a vascular smooth muscle cell single cell suspension.

Example 3

(5) while the above steps were performed, after preparing the first solution and the second solution shown in table 5 in a sterile ultra clean bench, the first solution was slowly added above the second solution in a 15mL centrifuge tube to obtain an endothelial cell gradient separation solution. The mass concentration of silica gel particles in the cell separation solution in Table 5 was 1.2 g/mL.

TABLE 5 preparation of gradient separating medium for endothelial cells

Solutions of	Endothelial cell isolate (μ L)	Total cell culture fluid (μ L)	Total (μ L)
				First solution	50	1950	2000
Second solution	250	1750	2000

(8) The cell mixture A is added into the cell gradient separating medium obtained in step (5) very carefully to prepare an integral endothelial cell gradient separating system, and 10mL of solution is counted in the centrifuge tube. The complete endothelial cell gradient separation solution is centrifuged for 18min at room temperature by a horizontal rotor centrifuge 2300 g.

(9) The upper layer 6mL of the centrifuged endothelial cell gradient separation solution is endothelial cell culture medium containing fragments, the middle layer 2mL is endothelial cell cells, and the lowest layer 2mL is various nervous system cells and dead cells. And (3) sucking and discarding the culture medium on the uppermost layer, transferring 2mL of the solution in the middle layer into a new 15mL centrifuge tube, mixing the solution with the total cell culture solution, centrifuging the mixture at room temperature for 3min at 150g, and uniformly mixing the obtained precipitate by using a DMEM culture medium without fetal calf serum to obtain a vascular cell mixture B.

(10) After preparing the third solution and the fourth solution shown in table 6 in a sterile super clean bench, the third solution was slowly added to the top of the fourth solution in a 15mL centrifuge tube to obtain a vascular smooth muscle cell gradient separation medium. The iodixanol content in the vascular smooth muscle cell isolate in table 6 was 58%. TABLE 6 preparation of gradient separation liquid for vascular smooth muscle cells

(11) Carefully adding the cell mixture B obtained in the step (10) into the vascular smooth muscle cell gradient separating medium obtained in the step 10 to prepare an integral vascular smooth muscle cell gradient separating system, wherein the centrifugal tube contains 4mL of solution in total. The complete vascular smooth muscle cell gradient separation system is centrifuged for 12min at room temperature by a horizontal rotor centrifuge of 900 g.

(12) 2mL of the upper layer of the centrifuged vascular smooth muscle cell gradient separation system is endothelial cell solution, 1mL of the middle layer is vascular endothelial cells, and 1mL of the lowest layer is vascular smooth muscle cells. The top two layers were aspirated and the lower layer 1.2mL of solution was transferred to a new 15mL centrifuge tube. And (3) mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging at room temperature for 3min at 150g, and uniformly mixing the obtained precipitate with a DMEM (DMEM) culture medium without fetal calf serum to obtain the vascular smooth muscle cell single cell suspension.

Experimental example 1

The following tests were performed on the single cells obtained in the present application:

1. and (3) carrying out platform blue staining on the cells obtained in the step, carrying out subsequent operation according to the requirements of a 10X genomics single cell sequencing instrument after the activity reaches 80%, carrying out sequencing and biogenic analysis on the obtained single cells, carrying out dimensionality reduction treatment by using Seurat software to obtain a two-dimensional cell distribution result, marking the obtained cells by using a known vascular smooth muscle cell marker, and observing the proportion of vascular smooth muscle cells.

2. The obtained vascular smooth muscle cells were subjected to subtype classification using the Seurat software, and the results were obtained.

3. The David website is used for carrying out signal path analysis on the gene highly expressed by vascular smooth muscle cells, and observing the main signal paths involved in the signal path analysis.

The results are shown in FIGS. 1 to 3:

FIG. 1 shows the vascular smooth muscle cells of the nervous system in the cerebral cortex of a mouse obtained by the present invention, and it is known that almost all of the obtained single cells can be labeled with the known marker Acta2 of the vascular smooth muscle cells of the nervous system.

FIG. 2 shows the results of subtype classification of the vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained by the present method. Therefore, the vascular smooth muscle cells in the brain are various in types and complex in functions, and have important clinical significance for further research;

FIG. 3 shows the GO analysis result of the highly expressed gene in the neural vascular smooth muscle cells in the cerebral cortex of the mouse obtained by the method, and it can be known from the graph that most of the highly expressed gene in the cells is related to angiogenesis, and the conclusion that the highly expressed gene is the neural vascular smooth muscle cells is proved again.

Experimental example two

In this example, the separation effect of five different cell separation solutions on vascular smooth muscle cells was compared by the method of example 1:

experimental groups: the cell gradient separation medium provided in example 1 of the present application was used.

Control group 1: vascular smooth muscle cells were isolated and purified using only the third solution and the fourth solution shown in Table 7.

TABLE 7 preparation of gradient separation liquid for vascular smooth muscle cells

Control group 2: only the third solution and the fourth solution shown in Table 8 were used to isolate and purify vascular smooth muscle cells.

TABLE 8 preparation of gradient separation of vascular smooth muscle cells

Control group 3: the third solution and the fourth solution shown in table 9 were used to prepare a vascular smooth muscle cell gradient separation medium. Wherein the blood vessel smooth muscle cell separating medium is Ficoll.

TABLE 9 preparation of gradient separation of vascular smooth muscle cells

Control group 4: the third solution and the fourth solution shown in table 10 were used to prepare a vascular smooth muscle cell gradient separation medium. Wherein the blood vessel smooth muscle cell separation liquid is sucralose.

TABLE 10 preparation of gradient separation of vascular smooth muscle cells

The experimental results are as follows:

experimental group (fig. 1): the cells obtained by marking with the marker Acta2 of vascular smooth muscle cells are found to be capable of marking almost all cells by Acta2, which indicates that the single cells obtained by utilizing the conditions have high purity;

control 1 (fig. 4): cells labeled with the marker Acta2 for vascular smooth muscle cells found that Acta2 can label about 47% of the cells;

control 2 (fig. 5): cells labeled with the marker Acta2 for vascular smooth muscle cells found that Acta2 can label about 59% of the cells;

control group 3 (fig. 6): cells labeled with the vascular smooth muscle cell marker Acta2 found that Acta2 labeled about 83% of the cells;

control group 4 (fig. 7): cells labeled with the vascular smooth muscle cell marker Acta2 found that Acta2 labeled about 89% of the cells.

Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating single cells of vascular smooth muscle cells of an animal nervous system is characterized by comprising the following steps:

mixing the cell mixture A with an endothelial cell gradient separation solution, and purifying and separating to obtain a cell mixture B containing vascular smooth muscle cells and endothelial cells;

mixing the cell mixture B with a vascular smooth muscle cell gradient separation solution, and purifying and separating vascular smooth muscle cell single cells;

the blood vessel smooth muscle cell gradient separation solution is formed by compounding a blood vessel smooth muscle cell separation solution and a total cell culture solution, and the blood vessel smooth muscle cell separation solution contains 58-62% of iodixanol by volume percentage.

2. The method for separating single cells of vascular smooth muscle cells in the animal nervous system according to claim 1, wherein the gradient separation solution of vascular smooth muscle cells is prepared by adding a third solution to a fourth solution in a volume ratio of 1: 0.8-1.2, wherein the volume ratio of the vascular smooth muscle cell separation solution to the total cell culture solution in the third solution is 2-3: 17-18; the volume ratio of the vascular smooth muscle cell separation solution to the total cell culture solution in the fourth solution is 6-8: 32-34.

3. The method for separating the vascular smooth muscle cell unicells of the animal nervous system according to claim 1, wherein the endothelial cell gradient separation solution is formed by compounding an endothelial cell separation solution and the total cell culture solution, the endothelial cell separation solution contains silica gel particles with a mass concentration of 1.2-1.4 g/mL, and the silica gel particles are coated with vinylpyrrolidone.

4. The method for separating the smooth muscle cells in the blood vessels of the animal nervous system according to claim 3, wherein the endothelial cell gradient separation solution is prepared by adding a first solution to a second solution in a volume ratio of 1: 2-4, and the volume ratio of the endothelial cell separation solution to the total cell culture solution in the first solution is 1-3: 37 to 39; the volume ratio of the endothelial cell separation solution to the total cell culture solution in the second solution is 5-7: 33 to 35.

5. The method for separating single cells from smooth muscle cells in blood vessels of animal nervous system according to claim 1, wherein the step of preparing the cell mixture B comprises:

and mixing the cell mixture A with the endothelial cell gradient separation solution to obtain a complete endothelial cell gradient separation system, centrifuging the endothelial cell gradient separation system at 2000-2500 g for 15-20 min at room temperature, removing the endothelial cell culture medium at the uppermost layer and other nervous system cells and dead cells at the lowermost layer, retaining an endothelial cell layer containing endothelial cells and vascular smooth muscle cells in the middle, and washing.

6. The method for separating single cells from vascular smooth muscle cells of the animal nervous system according to claim 5, wherein the step of washing the endothelial cell layer comprises:

and (3) mixing the endothelial cell layer with the total cell culture solution, centrifuging for 1-3 min at room temperature at 150-250 g, and uniformly mixing the obtained precipitate by using a DMEM (DMEM) culture medium without fetal calf serum to obtain endothelial cell single cell suspension and the cell mixture B.

7. The method for separating single cells of vascular smooth muscle cells in animal nervous system according to claim 1, wherein the step of preparing the single cells of vascular smooth muscle cells comprises:

and adding the cell mixture B above the vascular smooth muscle cell gradient separation solution to obtain a complete vascular smooth muscle cell gradient separation system, centrifuging the vascular smooth muscle cell gradient separation system at 700-900 g for 12-17 min at room temperature, removing the uppermost layer of cell culture medium and the vascular endothelial cells in the middle layer, retaining the lowest 0.5-1.5 mL of vascular smooth muscle cell layer, and washing.

8. The method for separating single cells from vascular smooth muscle cells in the animal nervous system according to claim 7, wherein the step of washing the vascular smooth muscle cell layer comprises:

and (3) mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging for 1-3 min at room temperature at 150-250 g, and uniformly mixing the obtained precipitate with a DMEM culture medium without fetal bovine serum to obtain the vascular smooth muscle cell single cell suspension.

9. The method for separating single cells from vascular smooth muscle cells of animal nervous system according to claim 1, wherein the total cell culture solution comprises at least: penicillin/streptomycin double antibody, DMEM medium and fetal bovine serum.

10. The method for separating the single cell of the vascular smooth muscle cell in the animal nervous system according to claim 1, wherein the cell dissociation digestion solution is prepared by adding 1-3 mg/mL of Pronase E protease and 22-28U/mL of DNaseI to the total cell culture solution.