CN111172102A - Method for culturing adventitia cells of blood vessels of mammals - Google Patents
Method for culturing adventitia cells of blood vessels of mammals Download PDFInfo
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Abstract
The invention discloses a method for culturing adventitia cells of a mammal blood vessel, which relates to the technical field of cell culture and comprises the following steps: obtaining a tubular vascular adventitia by an oversleeve method; cutting the adventitia into blocks to obtain a plurality of adventitia tissue blocks, wherein the adventitia tissue blocks contain fibroblasts; performing primary culture on fibroblasts in the adventitia tissue block by a tissue adherence method to obtain generated fibroblasts. The method for obtaining the adventitia of the blood vessel by the oversleeve method has the advantages of simple steps, short time, reduction of pollution of fibroblast in the adventitia of the blood vessel, maintenance of the original tissue structure to a great extent by adopting the tissue adherence method for primary culture, closer cell growth under physiological conditions due to no enzyme treatment, and contribution to adaptation of the method to in vitro culture environment.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for culturing adventitia cells of blood vessels of mammals.
Background
At present, the adventitia plays an important role in the occurrence and development processes of vascular remodeling, is the structural basis of the incidence of various cardiovascular diseases, and the research aiming at the adventitia is one of the current cardiovascular field research hotspots. Fibroblasts (AFs) are the major component of the adventitia of blood vessels, and Siow et al used an adenovirus vector expressing B2 galactosidase (Lac 2 z) to label fibroblasts on a rat carotid artery endothelial pull model, and found that the fibroblasts migrated from the adventitia to the intima after arterial injury, which directly proved that the fibroblasts participated in the development and progression of atherosclerosis. Under the action of in vivo vasoactive substances, the collagen participates in the processes of cell phenotype transformation, proliferation, apoptosis, migration and collagen synthesis and secretion by secreting active factors, thereby participating in the processes of vascular function regulation and repair. Fibroblasts (AFs) have become a new target for the treatment of cardiovascular diseases.
Therefore, establishing a simple, efficient and feasible primary fibroblast (AFs) isolation culture method is an essential link for researching the biological characteristics of As under the stimulation action of various factors, selecting a proper method to rapidly generate fibroblast (AFs) is of great significance for developing the research field of vascular remodeling, and a tissue adherence method is commonly used As A Fibroblast (AFs) culture method, but the report of isolating rat primary aorta by the cuff method is rare.
On the other hand, in the prior art, the cost of the traditional primary culture is high, and the period is long, so that the cell culture is usually continued by subculture, but the subcultured cells have certain defects in the cell research process.
Disclosure of Invention
Accordingly, the present invention is directed to a method for culturing adventitia cells in a mammal, which solves at least some of the problems of the related art.
The present application provides a method for culturing adventitial cells in a mammalian blood vessel, comprising: obtaining a tubular vascular adventitia by an oversleeve method; cutting the vascular adventitia into blocks to obtain a plurality of vascular adventitia tissue blocks, wherein the vascular adventitia tissue blocks contain fibroblasts; and performing primary culture on the fibroblasts in the adventitia tissue block by a tissue adherence method to obtain generated fibroblasts.
In some embodiments, the obtaining a tubular adventitia by a cuff method comprises: placing the isolated blood vessel in a culture medium, fixing one end of the blood vessel, and stripping the blood vessel adventitia from the fixed end to the flat laying end to obtain the tubular blood vessel adventitia.
In some embodiments, the adventitia tissue mass has a volumetric range of between 2mm and 3mm castration.
In some embodiments, the primary culturing of fibroblasts in the adventitia tissue mass by tissue apposition comprises: flatly laying and attaching the adventitia tissue block to the wall of a container, and putting the container into an incubator for primary culture; repeating the following steps for a plurality of times on the adventitia tissue block to carry out primary culture: and when the fibroblasts generated on the vascular adventitia tissue block reach a preset density, stripping the generated fibroblasts from the vascular adventitia tissue block to obtain generated fibroblasts, and continuously performing primary culture on the stripped vascular adventitia tissue block by a tissue wall pasting method.
In some embodiments, the method for culturing adventitia cells in a mammal further comprises:
and in the process of repeatedly carrying out primary culture on the adventitia tissue block, when the adventitia tissue block falls off from the wall of the container, re-attaching the adventitia tissue block to the wall, and carrying out primary culture.
In some embodiments, the predetermined density ranges from 80% to 90%.
In some embodiments, the container is a 6-well plate.
In some embodiments, the attaching the adventitia tissue piece to the wall of the container in a flat state includes: at least two vascular adventitia tissue blocks are flatly attached to the well bottom in each well of the 6-well plate.
In some embodiments, the method for culturing adventitia cells in a mammal further comprises: and subculturing the generated fibroblasts.
The invention has the advantages that:
the method for culturing the mammal adventitia cells obtains the adventitia cells through the oversleeve method, has simple steps and short time, reduces the pollution of fibroblast in the adventitia, adopts the tissue adherence method to carry out primary culture, maintains the original tissue structure to a great extent, is more close to the cell growth under the physiological condition because of not being treated by enzyme, and is beneficial to adapting to the in vitro culture environment. In the two processes, the adventitia tissue block is subjected to primary culture in a short time, so that fibroblasts with similar genetic characters and fibroblast cells in the adventitia can be generated, and the method is suitable for cell morphology, function, differentiation and other researches.
In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic flow chart of a method for culturing adventitia cells in a mammal according to an embodiment of the present invention;
FIG. 2 is a schematic view under a microscope after 1 day of a mammalian adventitia cell culture method according to an embodiment of the present invention;
FIG. 3 is a schematic view under a microscope after 5 days of a mammalian adventitia cell culture method according to an embodiment of the present invention;
FIG. 4 is a schematic representation of primary cultured cells of fibroblasts prepared by the method of comparative example 1 under a microscope after 24 hours;
FIG. 5 is an immunofluorescence test chart of cells cultured by a mammalian adventitia cell culture method according to an embodiment of the present invention;
FIG. 6 is a schematic diagram showing immunohistochemical staining of cells cultured by a mammalian adventitia cell culture method according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Fig. 1 is a schematic flow chart of a mammalian adventitia cell culture method according to an embodiment of the present invention, and as shown in fig. 1, the present application provides a mammalian adventitia cell culture method, including the following steps:
step S101, obtaining tubular vascular adventitia through a sleeve method.
In the present embodiment, the isolated blood vessel refers to a blood vessel isolated from a mammal, such as rat, sheep, pig, etc. The sleeve method is used for extracting the adventitia of the blood vessel, the conventional blood vessel dissection is not needed for treatment, the operation is simple, the tissue is easier to adhere to the wall, the treatment time is short, and the pollution is reduced.
In some embodiments, step S101, obtaining the tubular adventitia by the cuff method, may be specifically implemented by:
step S101A, taking the culture of the adventitia cells of SD rat as an example, the method for culturing the adventitia cells of mammal blood vessels includes the following steps: SD rats were sacrificed by amputation after abdominal anesthesia with 2% pentobarbital sodium (35 mg/kg), immediately and completely soaked in 75% alcohol, and rapidly moved to a clean bench after 5 minutes. The thoracic cavity and abdominal cavity of the rat are cut along the median thoracic line, the abdominal viscera are gently turned to the left side, and the left lung lobes are turned.
Step S101B, the aorta is placed in a sterile culture dish and washed by phosphate buffer solution, one end of the blood vessel is fixed by holding the ophthalmology bending forceps with the left hand, and the adventitia of the blood vessel is peeled off by holding the other ophthalmology forceps with the right hand from top to bottom with slight force and is placed in a new culture dish (the intima and the media cells of the blood vessel are removed).
And S102, cutting the vascular adventitia into blocks to obtain a plurality of vascular adventitia tissue blocks, wherein the vascular adventitia tissue blocks contain fibroblasts.
In some embodiments, step S102, cutting the adventitia into pieces to obtain a plurality of adventitia tissue pieces, wherein the adventitia tissue pieces contain fibroblasts, specifically, the method can be implemented by:
step S102A, a small amount of culture fluid containing 30% (volume fraction) serum is added to the new petri dish, and the adventitia is cut into tissue blocks about 2-3mm high-speed cultivation.
In the embodiment of the application, the steps S101A to S102A can be completed within 10-20min, and the effect is better.
Step S103, performing primary culture on the fibroblasts in the adventitia tissue block by a tissue adherence method to obtain generated fibroblasts.
In the embodiment of the application, the tissue adherence method is a common, simple and easy primary culture method with high success rate, which is used for shearing a living tissue into small pieces after the living tissue is taken out and adhering the living tissue to the bottle wall to wait for the cells to be dissociated from the periphery of the tissue. The primary culture refers to the first culture which is carried out by taking out tissues or cells from the body, and is also called as the primary culture, the primary culture has short in vitro time, has similar genetic characters with the cells in the body, and is suitable for the research of cell morphology, function, differentiation and the like.
In some embodiments, step S103, performing primary culture on the fibroblasts in the adventitia tissue mass by tissue adherence to obtain generated fibroblasts, and specifically, may be implemented by the following steps:
step S103A, after the bottom surface of the 6-hole plate is infiltrated with fetal calf serum, serum is sucked away, and the tissue blocks in the culture dish are uniformly laid in the 6-hole plate, wherein each hole is provided with 5-6 tissue blocks.
And step S103B, adhering the tissue blocks of the step S103A to the wall, putting the tissue blocks into an incubator to adhere to the wall, taking out the 6-hole plate after 6-8h, and adding 300 mu L of culture solution containing 30% (volume fraction) of serum along the hole wall. Placing the mixture into an incubator and standing for 24 hours to generate fiber cells.
For the fibroblasts generated in the examples of the present application, the morphology and growth state of the cells were observed under an inverted microscope, fig. 2 is a schematic view of the method for culturing the mammalian adventitia cells under a microscope after 1 day, as shown in fig. 2, after primary culture 1-2d, the fibroblasts start to climb out from the periphery of the tissue mass, and the cells are in a fusiform or polygonal shape.
In the method for culturing the adventitia cells of the mammal blood vessels provided by the embodiment of the application, the tubular adventitia of the blood vessels is obtained by an oversleeve method; cutting the adventitia into blocks to obtain a plurality of adventitia tissue blocks, wherein the adventitia tissue blocks contain fibroblasts; performing primary culture on fibroblasts in the adventitia tissue block by a tissue adherence method to obtain generated fibroblasts. The method for obtaining the adventitia of the blood vessel by the oversleeve method has the advantages of simple steps, short time, reduction of pollution of fibroblast in the adventitia of the blood vessel, maintenance of the original tissue structure to a great extent by adopting the tissue adherence method for primary culture, closer cell growth under physiological conditions due to no enzyme treatment, and contribution to adaptation of the method to in vitro culture environment. In the two processes, the adventitia tissue block is subjected to primary culture in a short time, so that fibroblasts with similar genetic characters and fibroblast cells in the adventitia can be generated, and the method is suitable for cell morphology, function, differentiation and other researches.
In some embodiments, the obtaining a tubular adventitia by a cuff method comprises: placing the isolated blood vessel in a culture medium, fixing one end of the blood vessel, and stripping the blood vessel adventitia from the fixed end to the flat laying end to obtain the tubular blood vessel adventitia.
The blood vessel adventitia cell culture method of mammal that this application embodiment provided peels off the blood vessel adventitia from the stiff end of separation blood vessel to the tiling end slightly hard, and the blood vessel adventitia that acquires is cleaner, pollutes for a short time, does benefit to and cultivates the fibroblast in the blood vessel adventitia.
In some embodiments, the adventitia tissue mass has a volumetric range of between 2mm and 3mm castration.
The tissue block that 2 ~ 3mm volume were carried out to shearing the adventitia through of this application embodiment is favorable to the adherence operation for the adherence is tighter.
In some embodiments, the primary culturing of fibroblasts in the adventitia tissue mass by tissue apposition comprises: flatly laying and attaching the adventitia tissue block to the wall of a container, and putting the container into an incubator for primary culture; repeating the following steps for a plurality of times on the adventitia tissue block to carry out primary culture: and when the fibroblasts generated on the vascular adventitia tissue block reach a preset density, stripping the generated fibroblasts from the vascular adventitia tissue block to obtain generated fibroblasts, and continuously performing primary culture on the stripped vascular adventitia tissue block by a tissue wall pasting method.
In the embodiment of the present application, when fibroblasts generated on the adventitia tissue block reach a preset density, the generated fibroblasts are peeled off from the adventitia tissue block to obtain generated fibroblasts, and the peeled adventitia tissue block is subjected to primary culture by a tissue adherence method, which can be specifically realized by the following steps: when the concentration of the produced fibroblasts reaches a preset concentration, illustratively, when the concentration of the fibroblasts is as long as 80-90% of the confluent state, digestion is performed with pancreatin, wherein the amount of pancreatin is set as required, illustratively, the amount of pancreatin is 500. mu.L. After a large number of cells are exfoliated, carefully blowing the cells, particularly tissue edges, and showing that fibroblasts generated on the tissue edges are completely exfoliated under a microscope, adding 1ml of fetal calf serum culture solution with the volume fraction of 10% to terminate digestion, after passage, retaining tissue blocks, cleaning 6 pore plates by using 1ml of phosphate buffer solution, adding 2ml of fetal calf serum culture solution with the volume fraction of 30% along the pore walls, placing the pore plates into an incubator, continuously culturing the tissue blocks (if the tissue blocks are exfoliated, the tissues can be treated from a new adherent wall), and after 24 hours, the fibroblasts can be seen to be re-germinated from the tissue edges. The tissue can be used for more than 2 months, and the cell state is still good after 7 th digestion.
The application greatly shortens the cell culture time by repeatedly utilizing the digested tissue, and the cells cultured by the common method need to be transferred to the second generation for about 20 days and can only be utilized once, and need to be cultured again if continuing the test. The armhole method needs about 14 days for culturing the cells to be transferred to the second generation, the primary cells can be digested again every 3 to 5 days, and the adventitia tissue of the blood vessel can be used for at least 2 months. The morphology of primary cells generated from adventitia tissue was still good after 7 th extraction. Greatly reducing the culture time, saving the mouse treatment cost, being particularly suitable for extracting primary cells after model building, and being capable of extracting a large amount of cells in a short time after entering the culture of the primary cells of the carotid artery of the mouse.
The method for culturing the mammal adventitia cells, provided by the embodiment of the application, can generate fibroblasts with similar genetic characters to fibroblast cells in the adventitia by reserving the tissue blocks, re-attaching the tissue blocks to the wall and performing primary culture, is suitable for cell morphology, function, differentiation and other researches, can still generate the fibroblast cells in a short time according to the method, and has important significance in cell research.
In some embodiments, the method for culturing adventitia cells in a mammal further comprises:
and in the process of repeatedly carrying out primary culture on the adventitia tissue block, when the adventitia tissue block falls off from the wall of the container, re-attaching the adventitia tissue block to the wall, and carrying out primary culture.
In the embodiment of the application, when the adventitia tissue piece drops from the wall of the container, then it is right the adventitia tissue piece adheres to the wall again, every 2-3 days after adhering to the wall again the culture solution of interchangeable to guarantee that there is sufficient cell growth's culture solution (also can not change) in the container, through adhering to the wall repeatedly, can reach the rational utilization to the tissue piece, need not to acquire the tissue again, thereby reduce the experiment cost, shortened the experimental period.
In some embodiments, the predetermined density is in the range of 80% to 90%, and when the predetermined density of the cells reaches 80% to 90%, the generated fibroblasts are isolated to facilitate the growth of the cells. FIG. 3 is a schematic diagram of a mammalian adventitia cell culture method under a microscope after 5 days according to an embodiment of the present invention, and as shown in FIG. 3, the fibroblast density reaches 80-90% in a primary culture for 5 days.
In some embodiments, the container is a 6-well plate.
In the embodiment of the application, the container can be a culture vessel or a bottle, preferably, the application adopts a 6-well plate, and compared with the culture bottle, the 6-well plate is more beneficial to the operation of the experiment
In some embodiments, the pieces of adventitia tissue are attached flat to the wall of a container, comprising: and in each hole of the 6-hole plate, at least two vascular adventitia tissue blocks are tiled and attached to the hole wall.
In some embodiments, the method for culturing adventitia cells in a mammal further comprises: and subculturing the generated fibroblasts.
In the embodiment of the present application, when the concentration of the generated fibroblasts reaches a predetermined concentration, illustratively, when the concentration of the fibroblasts grows to 80-90% of the fused state, the digestion is performed with pancreatin, wherein the amount of pancreatin is set as required, illustratively, the amount of pancreatin is 500 μ L, after a large amount of cells are exfoliated, 1ml of culture solution containing 10% by volume of fetal calf serum is added to terminate the digestion, the culture solution is aspirated and centrifuged to obtain the generated fibroblasts, and the generated fibroblasts are subcultured.
According to the method for culturing the adventitia cells of the blood vessels of the mammals, the obtained fibroblasts grow dominantly through subculture and run in a radial or reticular interweaving manner, and the cells can be purified after twice passages. Fibroblasts can be generated in a short time by subculture, and the state, shape and similarity of the fibroblasts generated by the method are higher than the similarity of the fibroblasts in the adventitia tissue mass.
In some embodiments, the method for culturing adventitia cells in a mammal further comprises:
after the container is soaked by serum, the adventitia tissue blocks of the blood vessels are uniformly laid in the container and put into an incubator for adherence.
In the embodiment of the application, the tissue blocks are uniformly laid in a vessel soaked by serum, and the uniform laying is used for obtaining a better wall attaching effect; the container is soaked by serum and then is attached to the wall, on one hand, the container is prevented from being overdried, so that the activity and the attachment effect of tissue cells are influenced, on the other hand, the container can also provide nutritional factors for tissues, and can also prevent a tissue block from being soaked in a culture solution due to excessive culture solution; the attachment operation is performed by placing the cells in an incubator to keep the cells in a state simulating an in vivo environment, for example, the cell culture needs a certain gas environment, namely 95% of air (necessary for cell metabolism) and 5% of CO2, to maintain the pH value of the culture solution, and the temperature is set to 37 ℃ to effectively ensure that the cells are in an environment beneficial to growth and effective in maintaining cell activity.
In some embodiments, the tissue apposition methods mentioned herein may also be applied to endothelial cells and smooth muscle cells.
Comparative example 1
Obtaining the adventitia of the blood vessel by a common tissue adherence method: SD rats were sacrificed by amputation after abdominal anesthesia with 2% pentobarbital sodium (35 mg/kg), immediately and completely soaked in 75% alcohol, and rapidly moved to a clean bench after 5 minutes. The thoracic cavity and the abdominal cavity of a rat are cut along the median thoracic line, the abdominal cavity viscera are slightly turned to the left side, the lung lobe at the left side is turned, the aorta is taken to be arranged in a sterile PBS culture dish, the peripheral blood stain of the blood vessel is washed out, the aorta is transferred to a new sterile PBS culture dish to wash the residual blood inside and outside the blood vessel and then is transferred to another sterile PBS culture dish, the blood vessel is longitudinally split by an ophthalmic scissors, the inner membrane is faced upwards, the middle membrane smooth muscle layer close to the inner membrane is carefully torn down after the inner membrane is slightly scraped by an ophthalmic curved forceps from top to bottom, and the residual flocculent milky white tissue is the blood vessel adventitia.
The cell morphology and the growth state of the fibroblasts obtained by the method of comparative example 1 are observed under an inverted microscope, fig. 4 is a schematic diagram of the fibroblasts primarily cultured by the method of comparative example 1 under a microscope after 24 hours, and as shown in fig. 4, the fibroblasts of the adventitia of the blood vessel cultured by the method of comparative example 1 do not climb out in 24 hours and usually climb out after 72 hours.
The results show that:
the fibroblast generated by the culture method of the mammal adventitia cell provided by the embodiment of the application comprises the following steps: the adventitia tissue block is attached to the bottom of the culture bottle, fibroblasts start to climb out from the periphery of the tissue block after primary culture for 1-2 days, the cells are in spindle shapes and polygons (see figure 2), the cells grow for 1-2 days earlier compared with the common tissue wall pasting method (see figure 4) in comparative example 1, then the cells rapidly proliferate, the cells can grow to be in a fusion state after 5-6 days (see figure 3), and multiple layers are formed in the region close to the tissue block after 10-15 days.
Immunofluorescence assay
Vimentin is a characteristic protein of adventitia fibroblast and is used for identifying cells. The steps of immunofluorescence assay: taking cells in logarithmic phase, digesting, blowing and beating the cells into cell suspension, uniformly inoculating the cell suspension into a 96-well plate, taking out phosphate buffer solution for rinsing for 3 minutes multiplied by 3 times the next day; fixing 4% paraformaldehyde for 15 minutes at room temperature; rinsing with phosphate buffer solution for 3 min × 3 times; permeabilize 0.1% Triton X-100 in PBS for 20 minutes at room temperature, rinse with phosphate buffer solution for 3 minutes × 3 times; 5% BSA in PBS for 1 h; the supernatant was aspirated off, primary antibody was added: vimentin (1: 100), putting the wet box into a refrigerator at 4 ℃ overnight, the next day, rewarming at room temperature for 30 min, washing with PBS for 3 times, dripping FITC (1: 100) labeled secondary antibody into a dark room, and incubating at 37 ℃ for 1 h; PBS rinse, 3 min x3 times, care to keep out of light; DAPI staining nuclei, PBS rinsing, 3 minutes multiplied by 3 times, adding a proper amount of PBS, and observing the staining condition under an immunofluorescence microscope. And the smooth muscle cells were excluded by immunofluorescent staining of a-SMA in the same manner. The negative control primary antibody was replaced with PBS.
FIG. 5 is an immunofluorescence test chart of cells cultured by the mammalian adventitia cell culture method according to the embodiment of the present invention, and as shown in FIG. 5, the cells are positive and proved to be fibroblasts by detecting the same cells by both the ER-TR7 method and the vimentin method, and the cells generated in the embodiment of the present application have immunofluorescence and are proved to be fibroblasts, thereby excluding the possibility of smooth muscle cells.
FIG. 6 is a schematic diagram of immunohistochemical staining of cells cultured by a mammalian adventitial cell culture method according to an embodiment of the present invention, as shown in FIG. 6, wherein the cytoplasm of fibroblasts generated in the present application is stained brown-yellow (see the right side of FIG. 6), and the negative control is not stained, indicating that the cells are of mesenchymal origin.
In the application, we culture fibroblast (AFs) by using the oversleeve method, and the Vimentin protein performs immunofluorescence and immunohistochemical staining on purified fibroblast (AFs) respectively, and found that there are uniform Vimentin protein positive particles in the cytoplasm of fibroblast (AFs). Compared with the common tissue adherence method, the immunohistochemical staining judgment cuff method combining cell morphology and immunofluorescent agent is more suitable for primary culture of rat fibroblasts (AFs), the cultured cells are high in purity and good in growth state, and a sufficient and reliable target cell model is provided for researching the biological functions of the rat AFs. The original tissue structure is kept to a great extent by a tissue adherence method, and the cell growth is closer to the cell growth under the physiological condition because of no enzyme treatment, so that the method is beneficial to being adapted to the in vitro culture environment, and the defects of the enzyme digestion method can be avoided.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A method for culturing adventitia cells in a mammalian blood vessel, comprising:
obtaining a tubular vascular adventitia by an oversleeve method;
cutting the vascular adventitia into blocks to obtain a plurality of vascular adventitia tissue blocks, wherein the vascular adventitia tissue blocks contain fibroblasts;
and performing primary culture on the fibroblasts in the adventitia tissue block by a tissue adherence method to obtain generated fibroblasts.
2. The method of claim 1, wherein said obtaining of the tubular adventitia by the cuff method comprises:
placing the isolated blood vessel in a culture medium, fixing one end of the blood vessel, and stripping the blood vessel adventitia from the fixed end to the flat laying end to obtain the tubular blood vessel adventitia.
3. The method of claim 1, wherein the adventitia tissue mass is harvested from 2mm to 3mm by volume.
4. The method for culturing adventitial cells in a mammal according to claim 1, wherein the primary culture of fibroblasts in the adventitial tissue mass by tissue adherence comprises:
flatly laying and attaching the adventitia tissue block to the wall of a container, and putting the container into an incubator for primary culture;
repeating the following steps for a plurality of times on the adventitia tissue block to carry out primary culture:
and when the fibroblasts generated on the vascular adventitia tissue block reach a preset density, stripping the generated fibroblasts from the vascular adventitia tissue block to obtain generated fibroblasts, and continuously performing primary culture on the stripped vascular adventitia tissue block by a tissue wall pasting method.
5. The method of claim 4, further comprising:
and in the process of repeatedly carrying out primary culture on the adventitia tissue block, when the adventitia tissue block falls off from the wall of the container, re-attaching the adventitia tissue block to the wall, and carrying out primary culture.
6. The method of claim 4, wherein the predetermined density is in the range of 80% to 90%.
7. The method of claim 4, wherein the vessel is a 6-well plate.
8. The method of claim 7, wherein the step of laying the perivascular tissue mass on the wall of the container comprises:
at least two vascular adventitia tissue blocks are flatly attached to the well bottom in each well of the 6-well plate.
9. The method for culturing adventitia vasorum cell of a mammal according to any one of claims 1 to 8, further comprising:
and subculturing the generated fibroblasts.
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