CN114703120B - Separation method of animal nervous system vascular smooth muscle cell single cells - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses a method for separating animal nervous system vascular smooth muscle cells single cells, and belongs to the technical field of biology. The method comprises the following steps: mixing and culturing the chopped fresh nerve tissue and cell dissociation digestive juice, centrifuging, adding the total cell culture solution, and filtering to obtain a cell mixture A; the cell mixture A is firstly mixed with endothelial cell gradient separating liquid, and the cell mixture B containing vascular smooth muscle cells is obtained through purification and separation. Then the iodixanol solution is used for further separation to obtain pure vascular smooth muscle cells, the method has simple operation, low cost and high efficiency, does not need to use a specific instrument, is suitable for industrial production, the single cell of the neurovascular smooth muscle cell prepared by the method can be used for single cell sequencing or other analysis, and is beneficial to deep research on the characteristics of the neurovascular smooth muscle cell.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a separation method of animal nervous system vascular smooth muscle cells.
Background
The blood vessels play a central role in neural activity. Vascular smooth muscle cells are the main cellular components constituting the vascular media, are one of the important metabolic and endocrine organs of the body, play an important role in various physiological processes, and vascular smooth muscle cell dysfunction is closely related to the occurrence and development of diseases. Research shows that vascular smooth muscle cells participate in physiological and pathological processes of various vascular diseases such as hypertension, atherosclerosis, graft vascular diseases, restenosis after angioplasty, repair after vascular wall injury and the like through self proliferation, migration and synthesis of extracellular matrixes in the pathological process. Vascular smooth muscle cells have therefore become an important subject of investigation in the field of medical care.
Each cell is unique and has heterogeneity even with cells of the same type in the same organ or tissue. The brain, which is the most important organ of the organism, contains many types of cells, all of which have their unique characteristics, such as vascular smooth muscle cells, which are the basis for increasing vasomotor activity. The research on various vascular diseases such as hypertension at present can find that the reliable smooth muscle cells can effectively improve the vascular tension effect, so that the vascular contraction is more normal, the smooth muscle cells can effectively improve the vitality of blood vessels, the smooth muscle cells have an important role in curing and preventing the hypertension diseases, the smooth muscle cells can be found to be important components forming the vascular wall in experiments, and the smoothness and the conveying capability of the blood vessels are definitely improved by the smooth muscle cells.
Vascular smooth muscle cells are an important basis for improving disease recovery and therapeutic effects. In the current rehabilitation methods for various vascular diseases, better subsequent effects can be ensured only by improving the growth vigor of smooth muscle cells, and the smooth muscle cells can be used for effectively improving the immunity of immune tissues to greatly improve the subsequent vascular power, and the smooth muscle cells can be used for improving the body resistance in the rehabilitation means to obtain better rehabilitation effects;
in summary. Vascular smooth muscle cells are an indispensable cell type in the body, and directly influence the growth effect of the vascular wall and the normal function of the blood vessel, so that related scientific researchers are more required to know the culture mode and the use method in the culture of smooth muscle technology, the growth capacity of the smooth muscle cells is improved in a correct way, and the vascular wall has stronger power to improve the research and development capacity of bioscience.
As the first cause of death in China, research on the function of vascular smooth muscle cells can provide a new method and strategy for treating stroke. However, the type of vascular smooth muscle cells in the central nervous system and their function are not completely understood, and single cell sequencing technology plays an incomparable role in precisely knowing the functions of certain types of cells and interactions between them. However, no method is currently available for preparing a single cell suspension of vascular smooth muscle cells specifically in mammalian neural tissue.
Disclosure of Invention
The invention provides a preparation method of a single cell suspension of vascular smooth muscle cells of an animal nervous system aiming at the defects. The method 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 can be used for single-cell sequencing or other analysis, thereby being beneficial to deep research on the characteristics of the vascular smooth muscle cells of the nervous system.
The invention is realized by the following technical scheme:
the invention provides a method for separating animal nervous system vascular smooth muscle cells, which comprises the following steps:
mixing and culturing the chopped fresh nerve tissue and cell dissociation digestive juice, centrifuging, adding the total cell culture solution, and filtering to obtain a cell mixture A;
mixing the cell mixture A with endothelial cell gradient separating liquid, purifying and separating to obtain a cell mixture B containing vascular smooth muscle cells and endothelial cells;
mixing the cell mixture B with vascular smooth muscle cell gradient separating liquid, and purifying and separating vascular smooth muscle cell single cells;
wherein the vascular smooth muscle cell gradient separating liquid is formed by compounding vascular smooth muscle cell separating liquid and total cell culture liquid, and the vascular smooth muscle cell separating liquid contains 58-62% iodixanol by volume percent.
Further, in a preferred embodiment of the present invention, the gradient separation solution for vascular smooth muscle cells is prepared by adding a third solution to the top of the fourth solution according to a volume ratio of 1:0.8-1.2, wherein the volume ratio between the vascular smooth muscle cell separation solution and the total cell culture solution in the third solution is 2-3: 17-18; the volume ratio between vascular smooth muscle cell separation solution and total cell culture solution 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 with 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 the preferred embodiment of the present invention, the endothelial cell gradient separating solution is prepared by adding the first solution above the second solution according to a volume ratio of 1:2-4, wherein the volume ratio between the endothelial cell separating solution and the total cell culture solution in the first solution is 1-3:37-39; the volume ratio between the endothelial cell separating liquid and the total cell culture liquid 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 endothelial cell gradient separating liquid to obtain a complete endothelial cell gradient separating system, centrifuging 2000-2500 g of the endothelial cell gradient separating system at room temperature for 15-20 min, removing the uppermost endothelial cell culture medium, the lowermost other nervous system cells and dead cells, retaining an 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:
after mixing the endothelial cell layer with the total cell culture solution, centrifuging 150-250 g for 1-3 min at room temperature, and uniformly mixing the obtained precipitate by using a DMEM culture medium without fetal calf serum to obtain the endothelial cell single-cell suspension.
Further, in a preferred embodiment of the present invention, the step of preparing vascular smooth muscle cells comprises:
adding the cell mixture B to the upper part of the vascular smooth muscle cell gradient separating liquid to obtain a complete vascular smooth muscle cell gradient separating system, centrifuging 700-900 g of the vascular smooth muscle cell gradient separating system at room temperature for 12-17 min, removing the uppermost cell culture medium and the vascular endothelial cells of the middle layer, retaining the vascular smooth muscle cell layer with the lowest volume of 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 includes:
mixing vascular smooth muscle cell layer with total cell culture liquid, centrifuging at room temperature of 150-250 g for 1-3 min, the resulting pellet was mixed well with DMEM medium without fetal bovine serum to obtain vascular smooth muscle cell single cell suspension.
Further, in a preferred embodiment of the present invention, the total cell culture solution at least contains: penicillin/streptomycin diabodies, DMEM medium and fetal bovine serum.
Further, in a preferred embodiment of the present invention, the cell dissociation digestate is prepared by adding 1-3 mg/mL of Pronase E protease and 22-28U/mL of DNase I to the total cell culture broth.
Compared with the prior art, the invention has at least the following technical effects:
in brain tissue, nerve endothelial cells and vascular smooth muscle cells are connected precisely in structure and function, and the difference of cell size and sedimentation coefficient between the two is very small. Also, the number of vascular smooth muscle cells is very small relative to endothelial cells, which makes it difficult to separate vascular smooth muscle cell single cells from neural endothelial cells using existing cell separation methods. Therefore, how to effectively remove the nerve endothelial cells, neurons and other nerve cells while maintaining the single cell viability of the vascular smooth muscle cells is important to whether the vascular smooth muscle cells can be successfully isolated.
The separation method provided by the application obtains the nerve tissue cell mixture by mixing and digesting fresh nerve tissue with cell dissociation digestive juice containing protease and DNase. The cell dissociation digestive juice does not need to be pre-aerated, the oxygen content is small, and most of the neuron cells die due to hypoxia in the digestion process because the neuron cells are sensitive to oxygen. Then, endothelial cells containing vascular smooth muscle cells are firstly separated by using an endothelial cell gradient separating liquid, and as the silica gel particles contained in the endothelial cell gradient separating liquid are coated with the vinylpyrrolidone, the osmotic pressure is very low, the viscosity is very low, the diffusion constant is low, the formed gradient is very stable, the formed gradient does not penetrate through a biomembrane, and is non-toxic to cells, the settleability of the cell gradient separating liquid to different types and dead cells can be specifically regulated, so that the aim of purifying the endothelial cells is fulfilled. And then separating vascular smooth muscle cells from vascular endothelial cells by using vascular smooth muscle cell gradient separating liquid, and finally obtaining pure vascular smooth muscle cells. As 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 has good separation and purification effects and has no influence on the life activities of various cells and organelles.
The preparation method of the single cell suspension of the vascular smooth muscle cells of the animal nervous system has the advantages of simple operation, low cost and high efficiency, does not need to use a specific instrument, is suitable for industrial production, can be used for single cell sequencing or other analysis, and is favorable for deeply researching the characteristics of the vascular smooth muscle cells.
Drawings
FIG. 1 is a UMAP diagram of vascular smooth muscle cells of the nervous system in the cerebral cortex of the mouse obtained in example 1, and it is understood from the figure that almost all cells can be labeled with the vascular smooth muscle cell marker Acta2, indicating that the obtained cells are high in purity.
FIG. 2 shows the specific expression genes of the subtypes after 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 shows the results of GO analysis using the genes highly expressed in the vascular smooth muscle cells of the nervous system in the cerebral cortex of the mice obtained in example 1.
FIG. 4 shows the results of GO analysis of genes highly expressed in vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained in control group 1.
Fig. 5 is the result of GO analysis of genes highly expressed in vascular smooth muscle cells of nervous system in the cerebral cortex of mice obtained in control group 2.
FIG. 6 shows the results of GO analysis of genes highly expressed in vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained in control group 3.
FIG. 7 shows the results of GO analysis of genes highly expressed in 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 below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
The technical scheme of the invention is as follows:
a method for isolating single cells of vascular smooth muscle cells of an animal nervous system, comprising:
step S1: fresh nerve tissue after cutting after the cell dissociation digestive juice is mixed and cultured, centrifuging, adding the total cell culture solution, and filtering to obtain a cell mixture A.
Wherein, the fresh neural tissue is derived from animal tissue, and the fresh nerve tissue is fresh animal tissue obtained after perfusion treatment. Preferably, the method of obtaining animal tissue is: after the animals were anesthetized with tribromoethanol, the abdominal cavity was rapidly cut, after exposing the heart, the syringe was inserted into the left ventricle, the right auricle was cut, pre-cooled, magnesium-free Du's phosphate buffer (D-PBS) was used to perfuse the heart to completely remove blood, the brain was rapidly removed on ice, the area of interest was cut, and the target tissue was minced with a razor blade at low temperature. And the D-PBS buffer solution without calcium and magnesium ions is used for perfusion of mammals, so that blood cells are thoroughly removed, and the influence of the blood cells on subsequent sequencing is avoided. Removal of blood cells alone after digestion may result in reduced vascular smooth muscle cell purity or reduced vascular smooth muscle cell yield.
After perfusion, firstly, the brain or spinal cord of mammal should be rapidly taken out on ice, the nerve tissue of the region of interest is cut off, and then the region of interest is rapidly cut off by a razor blade on ice, most nerve cells die due to hypoxia in the digestion process because the nerve cells are sensitive to oxygen, and the nerve cells are separated from an endothelial cell layer containing vascular smooth muscle cells during gradient separation, so that the endothelial cells are purified, and finally, the vascular smooth muscle cells are further separated and purified by iodixanol solution.
Further, in the process of preparing the cell mixture, fresh nerve tissue and cell dissociation digestive juice are mixed and then cultured for 30-60 min at a constant temperature of 33 ℃, and mechanically mixed uniformly every 2min. Wherein mechanical mixing means that a carrier (such as a centrifuge tube, a test tube and the like) filled with culture solution is manually rocked or vibrated by vortex in the constant temperature culture process, which is beneficial to improving digestion efficiency and ensuring complete digestion.
Further, after the constant temperature culture, the centrifugal force in the centrifugation process was 200g, and the centrifugation time was 5min. During centrifugation, the neural tissue cells settle to the bottom and separate from other impurities of lesser mass produced by digestion. After centrifugation, the supernatant was removed and the total cell culture medium was added for mechanical dissociation by pipetting. In order to reduce the damage of the pipette tip to cells, the tip of the tip should be polished on an alcohol lamp or a Pasteur tip should be used when the tip is used to blow mammalian tissues.
Further, in the process of preparing the cell mixture, a cell sieve having a pore size of 40 μm is used for filtration to remove bulk tissues and impurities.
Preferably, the total cell culture medium contains at least: penicillin/streptomycin diabodies, DMEM medium and fetal bovine serum.
Preferably, the cell dissociation digests are prepared by adding 1-3 mg/mL of Pronase E protease and 22-28U/mL of DNase I to the total cell culture broth. The Pronase E protease is mainly used for digesting the protein of the adhesion cells, the DNase I mixture is mainly used for digesting the DNA of the adhesion cells, and the proportion of double cells or multiple cells is reduced, so that the yield of single cells is improved.
The total cell culture solution and the cell dissociation digestive solution do not need to be introduced with oxygen in advance, most nerve cells die due to hypoxia in the digestion process, and the nerve cells are separated from an endothelial cell layer containing vascular smooth muscle cells in gradient separation, so that the purpose of purifying the cell mixture A is achieved.
Step S2: mixing the cell mixture A with endothelial cell gradient separating liquid, purifying and separating to obtain a cell mixture B containing vascular smooth muscle cells and endothelial cells;
the endothelial cell gradient separating liquid is formed by compounding an endothelial cell separating liquid and the total cell culture liquid, and the endothelial cell separating liquid contains silica gel particles with the mass concentration of 1.2-1.4 g/mL, preferably 1.3g/mL. The silica gel particles are coated with vinyl pyrrolidone. The vinyl pyrrolidone 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 metabolism of a human body, has excellent biocompatibility and does not generate toxicity to separated cells.
Further, the endothelial cell gradient separating liquid is prepared by adding a first solution to the second solution according to a volume ratio of 1:2-4, wherein the volume ratio between the endothelial cell separating liquid and the total cell culture liquid in the first solution is 1-3:37-39, preferably according to a volume ratio of 1:18.5 to 19.5; the volume ratio between the endothelial cell separating liquid and the total cell culture liquid in the second solution is 5-7: 33 to 35, preferably according to a volume ratio of 3: 16-18.
Further, the step of preparing the cell mixture B includes:
and mixing the cell mixture A with the endothelial cell gradient separating liquid to obtain a complete endothelial cell gradient separating system, centrifuging 2000-2500 g of the endothelial cell gradient separating system at room temperature for 15-20 min, removing the uppermost endothelial cell culture medium, the lowermost other nervous system cells and dead cells, 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:
after mixing the endothelial cell layer with the total cell culture solution, centrifuging at room temperature for 1-3 min (preferably at 180-230 g for 2 min) at 150-250 g, and uniformly mixing the obtained precipitate with DMEM culture medium without fetal bovine serum to obtain endothelial cell single-cell suspension.
The cell mixture B mainly contains vascular smooth muscle cells, and also contains partial endothelial cells, pericytes and the like, wherein the vascular smooth muscle cells are main components of large blood vessels, can be attached to endothelial cells to provide nutrition or support for the endothelial cells, have similar actions with pericytes which form small blood vessel components, are always difficult problems of separation in the field of vascular research, have no mature established technology at present, and are required to be further purified and separated due to small difference of sedimentation coefficients of the three types of cells so as to remove the endothelial cells and the pericytes mixed in the vascular smooth muscle cells, thus obtaining pure vascular smooth muscle cells.
More preferably, the centrifugation in the step of purifying and separating endothelial cells employs 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 accumulate on one side, resulting in poor separation.
Step S3: and mixing the cell mixture B with a vascular smooth muscle cell gradient separating liquid, and purifying and separating vascular smooth muscle cell single cells.
The vascular smooth muscle cell gradient separating liquid is formed by compounding vascular smooth muscle cell separating liquid and total cell culture liquid, wherein the vascular smooth muscle cell separating liquid contains 58-62% by volume of iodixanol, and preferably the volume fraction of iodixanol is 60%.
Further, the vascular smooth muscle cell gradient separating liquid 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 between the vascular smooth muscle cell separating liquid and the total cell culture liquid in the third solution is 2-3: 17 to 18, preferably 5:35; the volume ratio between the vascular smooth muscle cell separation liquid and the total cell culture liquid in the fourth solution is 6-8:32-34, preferably 7:33.
Further, the step of preparing the vascular smooth muscle cell single cell comprises:
adding the cell mixture B above the vascular smooth muscle cell gradient separation liquid to obtain a complete vascular smooth muscle cell gradient separation system, centrifuging 700-900 g of the vascular smooth muscle cell gradient separation system for 12-17 min (preferably, centrifuging 750-850 g for 14-16 min) at room temperature, removing the uppermost cell culture medium and vascular endothelial cells in the middle layer, retaining the vascular smooth muscle cell layer of 0.5-1.5 mL (preferably, 0.8-1.2 mL) at the bottom, 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 the room temperature by 150-250 g, and uniformly mixing the obtained precipitate by using a DMEM (DMEM medium) without fetal calf serum to obtain vascular smooth muscle cell single-cell suspension. Preferably, the centrifugal force is 200g and the centrifugation time is 2min.
More preferably, the centrifugation in the step of purifying and separating vascular smooth muscle cells employs 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 accumulate on one side, resulting in poor separation.
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Example 1
The embodiment provides a method for separating single cells of smooth muscle cells of blood vessels of an animal nervous system, which comprises the following steps:
(1) Preparing a digestion buffer special for single cell dissociation: 44.5mL of DMEM culture medium, 5mL of FBS,500 mu L of penicillin/streptomycin double antibody, 2mg/mL of Pronase E protease with final concentration and 25U/mL of DNase I mixture with final concentration are sequentially and respectively added into a sterile 50mL centrifuge tube, 6 mL/tube of digestion buffer is prepared for split charging, and the digestion buffer is preserved at the temperature of minus 20 ℃ and below for standby.
(2) Preparing a total cell culture solution: 44.5mL of DMEM medium, 5mL of FBS, 500. Mu.L of penicillin/streptomycin diabodies were sequentially added to a sterile 50mL centrifuge tube, and stored at 4 ℃.
(3) After the animals were anesthetized with tribromoethanol, the abdominal cavity was rapidly cut, after exposing the heart, the syringe was inserted into the left ventricle, the right auricle was cut, pre-cooled, calcium-magnesium ion free D-PBS was perfused through the heart to completely remove blood, the brain was rapidly removed on ice, the region of interest was cut, and the target tissue was minced with a razor blade at low temperature.
(4) The minced tissue is mixed with a digestion buffer solution special for single cell dissociation, incubated for 30min in a constant temperature incubator at 33 ℃, and mixed up and down at intervals of 2min.
(5) While the above steps were being carried out, 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 over the second solution in a 15mL centrifuge tube to obtain an endothelial cell gradient separating solution. The mass concentration of silica gel particles in the cell separation liquid in Table 1 was 1.3g/mL.
TABLE 1 preparation of endothelial cell gradient separation solution
| Solution | Endothelial cell separating liquid (mu L) | Total cell culture fluid (μL) | Aggregate (mu L) |
| First solution | 100 | 1900 | 2000 |
| Second solution | 300 | 1700 | 2000 |
(6) After digestion of the nerve tissue 200g was centrifuged briefly for 4min, the supernatant was removed and 6mL of total cell culture medium was added for mechanical dissociation by pipetting until no distinct tissue mass was present.
(7) A20 μm sterile cell sieve, which had been previously infiltrated with calcium-magnesium ion-free D-PBS, was placed in a 50mL centrifuge tube in an ultra-clean bench, and the above total cell culture solution was added to a cell sieve having a pore size of 40 μm under sterile conditions using a 1mL pipette to filter impurities and undigested tissues, thereby obtaining a cell mixture A.
(8) The cell mixture A was carefully added to the cell gradient separation liquid of step (5) to prepare a complete endothelial cell gradient separation system, at which time 10mL of the solution was counted in a centrifuge tube. The whole endothelial cell gradient isolate was centrifuged at 2500g for 15min in a horizontal rotor centrifuge at room temperature.
(9) The upper layer 6mL of the centrifuged endothelial cell gradient separating liquid is endothelial cell culture medium containing fragments, the middle layer 2mL is endothelial cell, and the lowest layer 2mL is various nervous system cells and dead cells. The uppermost medium was aspirated, 2mL of the intermediate layer solution was transferred to a new 15mL centrifuge tube, mixed with the total cell culture broth, centrifuged at 200g for 2min at room temperature, and the resulting pellet was uniformly mixed with DMEM medium without fetal bovine serum to obtain vascular cell mixture B.
(10) After the third solution and the fourth solution shown in table 2 were placed in a sterile super clean bench, the third solution was slowly added over the fourth solution in a 15mL centrifuge tube to obtain a vascular smooth muscle cell gradient separation solution. The content of iodixanol in the vascular smooth muscle cell separation solution in table 2 was 60%.
TABLE 2 preparation of vascular smooth muscle cell gradient separation liquid
(11) The cell mixture B obtained in (9) was carefully added to the vascular smooth muscle cell gradient separation solution obtained in step 10 to prepare a complete vascular smooth muscle cell gradient separation system, and a total of 4mL of the solution was added to the centrifuge tube. The whole vascular smooth muscle cell gradient separation system was centrifuged at 800g for 15min at room temperature in a horizontal rotor centrifuge.
(12) The upper layer 2mL of the centrifuged vascular smooth muscle cell gradient separation system is endothelial cell solution, the middle layer 1mL is vascular endothelial cells, and the lowest layer 1mL is vascular smooth muscle cells. The top two layers were pipetted off and the lower 1mL solution was transferred to a new 15mL centrifuge tube. After mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging for 2min at room temperature at 200g, and uniformly mixing the obtained precipitate with DMEM medium without fetal calf serum to obtain vascular smooth muscle cell single-cell suspension.
(13) After adding 5mL of the total cell culture solution to the above solution and carefully mixing, 200g of the solution was centrifuged with a horizontal rotor at room temperature for 2min, the supernatant was washed with a 1mL pipette, the precipitate was mixed well with serum-free DMEM, and the viability was calculated by staining with a blue dish.
Example 2
The present example provides a method for separating single cells of vascular smooth muscle cells of an animal nervous system, which has the steps substantially identical to those of example 1, except for the steps of:
(5) While the above steps were being carried out, 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 over the second solution in a 15mL centrifuge tube to obtain an endothelial cell gradient separating solution. The mass concentration of silica gel particles in the cell separation liquid in Table 3 was 1.4g/mL.
TABLE 3 preparation of endothelial cell gradient separation solution
| Solution | Endothelial cell separating liquid (mu L) | Total cell culture fluid (μL) | Aggregate (mu L) |
| First solution | 150 | 1850 | 2000 |
| Second solution | 350 | 1650 | 2000 |
(8) The cell mixture A was carefully added to the cell gradient separation liquid of step (5) to prepare a complete endothelial cell gradient separation system, at which time 10mL of the solution was counted in a centrifuge tube. The whole endothelial cell gradient separation liquid was centrifuged at 2000g for 20min in a horizontal rotor centrifuge at room temperature.
(9) The upper layer 6mL of the centrifuged endothelial cell gradient separating liquid is endothelial cell culture medium containing fragments, the middle layer 2mL is endothelial cell, and the lowest layer 2mL is various nervous system cells and dead cells. The uppermost medium was aspirated, 2mL of the intermediate layer solution was transferred to a new 15mL centrifuge tube, mixed with the total cell culture broth, centrifuged at 250g for 1min at room temperature, and the resulting pellet was uniformly mixed with DMEM medium without fetal bovine serum to obtain vascular cell mixture B.
(10) After the third solution and the fourth solution shown in table 4 were placed in a sterile super clean bench, the third solution was slowly added over the fourth solution in a 15mL centrifuge tube to obtain a vascular smooth muscle cell gradient separation solution. The content of iodixanol in the vascular smooth muscle cell separation solution in table 4 was 62%.
TABLE 4 preparation of vascular smooth muscle cell gradient separation solution
(11) The cell mixture B obtained in step (10) was carefully added to the vascular smooth muscle cell gradient separation solution obtained in step 10 to prepare a complete vascular smooth muscle cell gradient separation system, and a total of 4mL of the solution was placed in a centrifuge tube. The whole vascular smooth muscle cell gradient separation system was centrifuged at 700g for 12min at room temperature in a horizontal rotor centrifuge.
(12) The upper layer 2mL of the centrifuged vascular smooth muscle cell gradient separation system is endothelial cell solution, the middle layer 1mL is vascular endothelial cells, and the lowest layer 1mL is vascular smooth muscle cells. The top two layers were pipetted off and the lower 0.8mL solution was transferred to a new 15mL centrifuge tube. After mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging at 250g for 1min at room temperature, and uniformly mixing the obtained precipitate with DMEM medium without fetal calf serum to obtain vascular smooth muscle cell single-cell suspension.
Example 3
The embodiment provides a method for separating animal nervous system vascular smooth muscle cells single cells, the procedure was substantially as in example 1, except for the following steps:
(5) While the above steps were being carried out, after preparing the first solution and the second solution shown in table 5 in a sterile super clean bench, the first solution was slowly added over the second solution in a 15mL centrifuge tube to obtain an endothelial cell gradient separating solution. The mass concentration of silica gel particles in the cell separation liquid in Table 5 was 1.2g/mL.
TABLE 5 preparation of endothelial cell gradient separation solution
| Solution | Endothelial cell separating liquid (mu L) | Total cell culture fluid (μL) | Aggregate (mu L) |
| First solution | 50 | 1950 | 2000 |
| Second solution | 250 | 1750 | 2000 |
(8) The cell mixture A was carefully added to the cell gradient separation liquid of step (5) to prepare a complete endothelial cell gradient separation system, at which time 10mL of the solution was counted in a centrifuge tube. The whole endothelial cell gradient isolate was centrifuged at 2300g for 18min at room temperature in a horizontal rotor centrifuge.
(9) The upper layer 6mL of the centrifuged endothelial cell gradient separating liquid is endothelial cell culture medium containing fragments, the middle layer 2mL is endothelial cell, and the lowest layer 2mL is various nervous system cells and dead cells. The uppermost medium was aspirated, 2mL of the intermediate layer solution was transferred to a new 15mL centrifuge tube, mixed with the total cell culture broth, centrifuged at 150g for 3min at room temperature, and the resulting pellet was uniformly mixed with DMEM medium without fetal bovine serum to obtain vascular cell mixture B.
(10) After the third solution and the fourth solution shown in table 6 were placed in a sterile super clean bench, the third solution was slowly added over the fourth solution in a 15mL centrifuge tube to obtain a vascular smooth muscle cell gradient separation solution. The content of iodixanol in the vascular smooth muscle cell separation solution in Table 6 was 58%. TABLE 6 preparation of vascular smooth muscle cell gradient separation solution
(11) The cell mixture B obtained in step (10) was carefully added to the vascular smooth muscle cell gradient separation solution obtained in step 10 to prepare a complete vascular smooth muscle cell gradient separation system, and a total of 4mL of the solution was placed in a centrifuge tube. Gradient separation of the intact vascular smooth muscle cells the system was centrifuged at 900g for 12min at room temperature in a horizontal rotor centrifuge.
(12) The upper layer 2mL of the centrifuged vascular smooth muscle cell gradient separation system is endothelial cell solution, the middle layer 1mL is vascular endothelial cells, and the lowest layer 1mL is vascular smooth muscle cells. The top two layers were pipetted off and the lower 1.2mL solution was transferred to a new 15mL centrifuge tube. After mixing the vascular smooth muscle cell layer with the total cell culture solution, centrifuging at 150g for 3min at room temperature, and uniformly mixing the obtained precipitate with DMEM medium without fetal calf serum to obtain vascular smooth muscle cell single-cell suspension.
Experimental example 1
The following test was performed on the smooth muscle endothelial single cells of the nervous system obtained in the present application:
1. and (3) carrying out blue-desk staining on the cells obtained in the steps, carrying out subsequent operation according to the requirement of a 10X genemics single-cell sequencing instrument after the activity reaches 80%, sequencing the obtained single cells, carrying out belief analysis, carrying out dimension reduction treatment by using the SEurat software to obtain a two-dimensional cell distribution result, marking the obtained cells by using known vascular smooth muscle cell markers, and observing the proportion of vascular smooth muscle cells.
2. Subtype classification was performed on the obtained vascular smooth muscle cells using the semat software, and the obtained results were obtained.
3. The David website is used for carrying out signal path analysis on genes with high expression of vascular smooth muscle cells, and observing which main signal paths are involved in the genes.
The results are shown in FIGS. 1 to 3:
FIG. 1 shows that almost all single cells obtained by using the known marker Acta2 for vascular smooth muscle cells of the nervous system in the cerebral cortex of a mouse obtained by the present invention can be labeled.
FIG. 2 is a graph showing the results of subtype classification of vascular smooth muscle cells of the nervous system in the cerebral cortex of mice using the present method. Therefore, the vascular smooth muscle cells in the brain are multiple in variety and complex in function, and have important clinical significance for further research;
FIG. 3 shows the results of GO analysis of highly expressed genes in vascular smooth muscle cells of the nervous system in the cerebral cortex of mice obtained by the present method, and it is understood from the figure that most of the highly expressed genes in the cells are angiogenesis-related pathways, and it is again confirmed that the obtained genes are vascular smooth muscle cells of the nervous system.
Experimental example two
The experimental example compares the separation effect of five different cell separation liquids on vascular smooth muscle cells by the method of example 1:
experimental group: the cell gradient separation liquid 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 and fourth solutions shown in Table 7.
TABLE 7 preparation of vascular smooth muscle cell gradient separation solution
Control group 2: vascular smooth muscle cells were isolated and purified using only the third and fourth solutions shown in table 8.
TABLE 8 preparation of vascular smooth muscle cell gradient separation solution
Control group 3: the vascular smooth muscle cell gradient separation solution was prepared using the third solution and the fourth solution as shown in table 9. Wherein the vascular smooth muscle cell separation liquid is Ficoll.
TABLE 9 preparation of vascular smooth muscle cell gradient separation solution
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Control group 4: the vascular smooth muscle cell gradient separation solution was prepared using the third solution and the fourth solution as shown in table 10. Wherein the vascular smooth muscle cell separation liquid is cross.
TABLE 10 preparation of vascular smooth muscle cell gradient separation solution
The experimental results are:
experimental group (fig. 1): cells marked by using the vascular smooth muscle cell marker Acta2 find that Acta2 can mark almost all cells, which indicates that single cells obtained by using the conditions have high purity;
control group 1 (fig. 4): labeling the resulting cells with Acta2, a marker for vascular smooth muscle cells, found that Acta2 labeled about 47% of the cells;
control group 2 (fig. 5): labeling the resulting cells with Acta2, a marker for vascular smooth muscle cells, found that Acta2 labeled about 59% of the cells;
control group 3 (fig. 6): labeling the resulting cells with Acta2, a marker for vascular smooth muscle cells, found that Acta2 labeled about 83% of the cells;
control group 4 (fig. 7): labeling the resulting cells with Acta2, a marker for vascular smooth muscle cells, found that Acta2 labeled about 89% of the cells.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for isolating single cells of vascular smooth muscle cells of an animal nervous system, comprising:
mixing and culturing the chopped fresh nerve tissue and cell dissociation digestive juice, centrifuging, adding the total cell culture solution, and filtering to obtain a cell mixture A; the cell dissociation digestive juice 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;
mixing the cell mixture A with endothelial cell gradient separating liquid, purifying and separating to obtain a cell mixture B containing vascular smooth muscle cells and endothelial cells;
mixing the cell mixture B with vascular smooth muscle cell gradient separating liquid, and purifying and separating vascular smooth muscle cell single cells;
the vascular smooth muscle cell gradient separating liquid is formed by compounding vascular smooth muscle cell separating liquid and total cell culture liquid, and the vascular smooth muscle cell separating liquid contains 58-62% iodixanol by volume percent;
the vascular smooth muscle cell gradient separating liquid is prepared by adding a third solution above a fourth solution according to a volume ratio of 1:0.8-1.2, wherein the volume ratio between the vascular smooth muscle cell separating liquid and the total cell culture liquid in the third solution is 2: (14-18); the volume ratio of the vascular smooth muscle cell separation liquid to the total cell culture liquid in the fourth solution is 6-8:32-34;
the endothelial cell gradient separating liquid is formed by compounding an endothelial cell separating liquid and the total cell culture liquid, the endothelial cell separating liquid contains silica gel particles with the mass concentration of 1.2-1.4 g/mL, and the silica gel particles are coated with vinylpyrrolidone;
the endothelial cell gradient separating liquid is prepared by adding a first solution above a second solution according to a volume ratio of 1:2-4, wherein the volume ratio between the endothelial cell separating liquid and the total cell culture liquid in the first solution is 1-3: 37-39; the volume ratio between the endothelial cell separating liquid and the total cell culture liquid in the second solution is 5-7: 33-35.
2. The method for isolating single cells of vascular smooth muscle cells of the nervous system of animals according to claim 1, wherein the step of preparing said cell mixture B comprises:
and mixing the cell mixture A with the endothelial cell gradient separation liquid to obtain a complete endothelial cell gradient separation system, centrifuging 2000-2500 g of the endothelial cell gradient separation system at room temperature for 15-20 min, removing the uppermost endothelial cell culture medium, the lowermost other nervous system cells and dead cells, retaining an endothelial cell layer containing endothelial cells and vascular smooth muscle cells in the middle, and washing.
3. The method of isolating single cells of vascular smooth muscle cells of the nervous system of an animal of claim 2, 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 the room temperature at 150-250 g, and uniformly mixing the obtained precipitate by using a DMEM (DMEM medium) without fetal bovine serum to obtain the cell mixture B.
4. The method for isolating vascular smooth muscle cells of the nervous system of an animal according to claim 1, wherein the step of preparing vascular smooth muscle cells comprises:
and adding the cell mixture B to the upper part of the vascular smooth muscle cell gradient separation liquid to obtain a complete vascular smooth muscle cell gradient separation system, centrifuging 700-900 g of the vascular smooth muscle cell gradient separation system at room temperature for 12-17 min, removing the uppermost cell culture medium and vascular endothelial cells in the middle layer, retaining the vascular smooth muscle cell layer with the lowest surface of 0.5-1.5 mL, and washing.
5. The method of isolating vascular smooth muscle cells of the nervous system of an animal as set forth in claim 4, 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 the room temperature of 150-250 g, and uniformly mixing the obtained precipitate by using a DMEM (DMEM medium) without fetal calf serum to obtain vascular smooth muscle cell single-cell suspension.
6. The method for isolating single cells of vascular smooth muscle cells of the nervous system of animals according to claim 1, wherein the total cell culture solution comprises at least: penicillin/streptomycin diabodies, DMEM medium and fetal bovine serum.
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