A method for isolating and culturing primary vascular smooth muscle cells from human abdominal aortic wall

Abstract

The application discloses a method for separating and culturing primary vascular smooth muscle cells from human abdominal aortic wall, and relates to the technical field of human cell culture. The method comprises the following steps: obtaining a collagenase A solution; adding the collagenase A solution to a treated human abdominal aortic aneurysm wall tissue sample and then cutting the sample; transferring the cut human abdominal aortic aneurysm wall tissue sample to a 37-degree cell culture box for culture; centrifuging a cell suspension obtained after termination of digestion; discarding supernatant, resuspending cell precipitate, and repeating centrifugation for three times; resuspending the cell precipitate again, uniformly transferring the cell precipitate to a T25 culture bottle after blowing, and placing the cell precipitate in a 37-degree cell culture box for culture; when a wave peak and wave trough state appears, the cell precipitate is digested and subcultured by using 0.25% trypsin, and then the cell precipitate is transferred to a new T25 culture bottle; and the cell precipitate is subcultured normally to the P3 generation and then stored in liquid nitrogen, so that the cell damage is prevented and the activity and proliferation capacity of the cell are maintained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of human cell culture, in particular to a method for isolating and culturing primary vascular smooth muscle cells from human abdominal aortic wall. BACKGROUND

[0002] Vascular smooth muscle cells (VSMCs) are the main cell components of the middle layer of blood vessels, and their contraction and relaxation, proliferation, migration and calcification are closely related to the occurrence of cardiovascular diseases. In vitro culture of human umbilical vascular smooth muscle cells is the basis and important means for studying the pathogenesis and prevention and treatment of cardiovascular diseases.

[0003] In vitro culture of arterial vascular smooth muscle cells began in the 1970s, and was successfully cultured by Ross and Chamley using tissue patch method; the vascular smooth muscle cells cultured in vitro can be taken from different parts of the human body, such as thoracic aorta, abdominal aorta, pulmonary artery, cerebral artery, mesenteric artery, etc.; human umbilical vein blood vessels not only have simple material taking, abundant source, but also have strong smooth muscle cell activity, which are more suitable for culture; there are many methods for culturing vascular smooth muscle cells, among which the main methods are enzyme digestion and tissue block adhesion, and different culture methods have different advantages and uses.

[0004] However, the prior art is inconvenient to prevent cell damage, and timely subculture can prevent contact inhibition caused by overgrowth, maintain the activity and proliferation ability of the cells. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application provides a method for isolating and culturing primary vascular smooth muscle cells from human abdominal aortic wall, which solves the problem that the prior art is inconvenient to prevent cell damage and timely subculture can prevent contact inhibition caused by overgrowth, maintain the activity and proliferation ability of the cells.

[0006] To achieve the above purpose, the present application realizes the following technical scheme: a method for isolating and culturing primary vascular smooth muscle cells from human abdominal aortic wall, comprising the following steps: obtaining a collagenase A solution; cutting the human abdominal aortic aneurysm wall tissue specimen after adding the collagenase A solution; transferring the cut human abdominal aortic aneurysm wall tissue specimen to a 37-degree cell culture box for culture; centrifuging to obtain a cell suspension after termination of digestion; discarding the supernatant, resuspending the cell precipitate, and repeating centrifugation three times; resuspending the cell precipitate again, homogenizing by blowing, and transferring to a T25 culture bottle, and placing in a 37-degree cell culture box for culture; when the wave peak and wave trough state appears, subculture with 0.25% trypsin, and transfer to a new T25 culture bottle; normally subculture to P3 generation and freeze in liquid nitrogen.

[0007] Further, the preparation method of the collagenase A solution comprises the following steps: dissolving collagenase A in SMCM special medium; adding 0.25% trypsin; filtering the solution by using a syringe and a sterile filter screen to obtain the collagenase A solution.

[0008] Further, the specification of the sterile filter screen is 0.22 μm.

[0009] Further, the processing procedure of the human abdominal aortic aneurysm wall tissue sample comprises the following steps: taking the human abdominal aortic aneurysm wall tissue within 0.5-1 h after being removed from the body, putting the human abdominal aortic aneurysm wall tissue into a mixed solution of PBS+triple antibody and moving to a clean bench; repeatedly washing the human abdominal aortic aneurysm wall tissue by using the mixed solution of PBS+triple antibody to clean the blood clots on the human abdominal aortic aneurysm wall tissue to obtain the human abdominal aortic aneurysm wall tissue sample.

[0010] Further, the processed human abdominal aortic aneurysm wall tissue sample is placed in a 35 mm 2 culture dish; the volume of the human abdominal aortic aneurysm wall tissue sample after being cut into pieces by adding the collagenase A solution is 1 mm 3 .

[0011] Further, the culture in the 37-degree cell culture box comprises the following steps: transferring the 35 mm 2 culture dish into the 37-degree cell culture box; observing once every 30-60 minutes and repeatedly blowing and beating by using a pipette; repeating for 4-6 h.

[0012] Further, the processing procedure of terminating the digestion comprises the following steps: after culturing for 4-6 h, shaking to see that the enzyme solution is turbid, and adding preheated 10% FBS SMCM culture medium to terminate the digestion.

[0013] Further, the centrifugation after terminating the digestion to obtain the cell suspension comprises the following steps: using a cell filter screen to remove the tissue fragments which are not completely digested; centrifuging at 400 g for 5 min to obtain the cell suspension.

[0014] Further, the specification of the cell filter screen is 100 μm.

[0015] Further, the serum-free culture medium is used when resuspending the cell precipitate; and 10 ml of 10% FBS SMCM culture medium is used when resuspending the cell precipitate again.

[0016] The present application has the following beneficial effects:

[0017] The method for isolating and culturing primary vascular smooth muscle cells from human abdominal aortic wall uses PBS containing antibiotics to prevent bacterial contamination and maintain the physiological balance of the tissue, finely chopped tissue pieces increase the contact area between enzymes and tissue, collagenase A helps to break down the extracellular matrix and promote cell release, blowing helps to disperse the cells and prevent them from forming clumps, adding serum-containing medium can neutralize the activity of digestive enzymes and prevent cell damage, observing cell growth status and timely subculture can prevent contact inhibition caused by overgrowth and maintain cell activity and proliferation ability.

[0018] Of course, implementing any product of the present application does not necessarily require all the advantages described above to be achieved simultaneously. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The method flow chart.

[0020] Figure 2 Picture of digestion completion.

[0021] Figure 3 Picture of static culture on the 4th day.

[0022] Figure 4 Picture of static culture on the 7th day.

[0023] Figure 5 Picture of static culture on the 10th day.

[0024] Figure 6 Picture of static culture on the 18th day.

[0025] Figure 7 Picture of P2 generation.

[0026] Figure 8 Human specimen smooth muscle cell culture immunofluorescence staining DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0028] In the description of the present application, it should be understood that the terms "opening", "upper", "lower", "thickness", "top", "middle", "length", "inner", "periphery" and the like indicate the orientation or positional relationship, only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the components or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore cannot be understood as a limitation of the present application.

[0029] Please refer to Figure 1 The embodiment of the present application provides a technical scheme: a method for isolating and culturing primary vascular smooth muscle cells from human abdominal aortic wall, comprising the following steps:

[0030] 1. Take fresh human abdominal aortic aneurysm wall tissue within 0.5h-1h after excision, and put it into a mixed solution of PBS+triple antibody and move to a clean bench. Using PBS containing antibiotics can prevent bacterial contamination and maintain the physiological balance of the tissue.

[0031] 2. Wash the blood clots repeatedly with a large amount of PBS containing triple antibody. Thorough cleaning can remove blood and other impurities that may affect cell culture, ensuring the purity of the culture environment.

[0032] 3. Transfer the treated specimen to a 35mm 2 culture dish, add an appropriate amount (not too much, increase the viscosity) of collagenase A solution, and use ophthalmic scissors to cut the specimen into 1mm 3 pieces, and add an appropriate amount of collagenase A solution in a timely manner. The finely chopped tissue pieces can increase the contact area between the enzyme and the tissue, and collagenase A can help break down the extracellular matrix to promote cell release.

[0033] 4. Transfer the above 35mm 2 culture dish to a 37-degree cell culture incubator, and observe every 30-60 minutes and repeatedly and gently blow with a pipette. The appropriate temperature can accelerate the action of the enzyme and promote the digestion of the tissue, but close monitoring is required to avoid excessive digestion.

[0034] 5. Repeat the above operation, 4-6h, shake the enzyme solution to see turbidity, and add preheated 10% FBS SMCM medium to terminate digestion. Observe the cell digestion under a microscope in a timely manner to prevent excessive digestion and cause cell damage. Blowing helps to disperse cells and avoid cell aggregation to form clumps. Adding serum-containing medium can neutralize the activity of the digestive enzyme to prevent cell damage.

[0035] In order to prevent excessive digestion, the physical characteristics during digestion can be obtained in real time, including cell density, cell morphological characteristics, aggregation index and cell viability index. In addition, the reference cell density, reference cell morphological characteristics, reference aggregation index and reference cell viability index when reaching the digestion completion state are obtained from the database.

[0036] The acquired cell density, cell morphology characteristics, aggregation index, and cell viability indicators are compared with the reference cell density, reference cell morphology characteristics, reference aggregation index, and reference cell viability indicators to obtain a comparison index, which represents the similarity between the current state of digestion and the state of the reference complete digestion.

[0037] The comparison index is obtained as follows:

[0038] Bd = β1 * |m1 - m2| + β2 * σ(x1, x2) + β3 * |Z1 - Z2| + β4 * |H1 - H2|;

[0039] In the formula, Bd is the comparison coefficient, m1 is the cell density, x1 is the cell morphology characteristics, Z1 is the aggregation index, H1 is the cell viability indicator, m2 is the reference cell density, x2 is the reference cell morphology characteristics, Z2 is the reference aggregation index, H2 is the reference cell viability indicator, β1 is the density weight factor, β2 is the morphology weight factor, β3 is the aggregation index weight factor, β4 is the viability indicator weight factor, and σ(x1, x2) is the similarity function of x1 and x2.

[0040] If the comparison index is less than or equal to the comparison threshold stored in the database, it can be considered that the current state is complete digestion and should be terminated.

[0041] By obtaining real-time data in a quantitative manner (such as cell density, morphology characteristics, etc.), the uncertainty of subjective judgment is avoided. The culture conditions can be dynamically adjusted based on the real-time data, such as stopping the action of digestive enzymes, to avoid excessive digestion and cell damage. By comparing with the reference characteristics in the database, a clear standard is provided to determine when the digestion is complete, ensuring that each experiment can reach a consistent endpoint. Ensuring the best survival state and viability of cells helps subsequent experimental operations and the reliability of data.

[0042] Cell density directly reflects the quantitative level of cell growth and survival. It can indicate whether the cells are in normal proliferation or are inhibited by certain conditions (such as insufficient nutrition or space limitation). High density may mean that cells are too crowded, which can affect the normal function and morphology of cells. Changes in cell morphology are important indicators of cell health and physiological state. Abnormal morphological changes may indicate cell stress or death. Changes in morphological characteristics are often closely related to cell density and viability indicators, and a dense environment can lead to cell morphological abnormalities.

[0043] Aggregation index describes the degree of interaction and aggregation between cells, reflecting the cohesiveness of the cell population. High aggregation in certain cell types indicates a healthy state, while in others it may indicate a pathological state. High aggregation can be associated with high cell density and morphological changes, which can lead to increased competition for resources in the microenvironment, affecting the normal physiological function of cells. Viability indicators directly reflect the survival ability of cells, which is a direct method to assess the health status of cells. Low viability indicates that cells are damaged or dead. Decreased viability can be related to inappropriate density, abnormal morphology, or excessive aggregation.

[0044] 6. Use a 100-um cell strainer to remove incompletely digested tissue fragments. Removing incompletely digested tissue fragments ensures the purity and consistency of the cells in the resulting suspension.

[0045] 7. Centrifuge the cell suspension at 400g for 5 minutes. Centrifugation can effectively separate cells and supernatant, helping to remove unwanted small molecules and impurities.

[0046] 8. Remove the supernatant and resuspend the cell pellet in serum-free medium, centrifuge (400g / 5min), and repeat three times. Multiple washes with serum-free medium can further remove external contaminants and digestive enzymes, preparing the cells for ideal culture conditions.

[0047] 9. Finally, resuspend the cell pellet in 10 ml of 10% FBS SMCM medium, gently blow to uniformity, and transfer to a T25 culture flask. Place in a 37°C cell culture incubator for culture. The first 3-5 days must be absolutely stationary and not moved. The growth factors and nutrient components in the serum are beneficial to cell growth and adhesion. The T25 culture flask provides sufficient growth area.

[0048] 10. Cell attachment and growth can be observed on the 7th-10th day, and the growth rate increases from the 10th-15th day. When the wave peak and trough state appears, use 0.25% trypsin to digest and passage, and transfer to a new T25 culture flask. Observe the cell growth state and passage in time to prevent contact inhibition caused by excessive growth, maintaining the activity and proliferation ability of the cells.

[0049] 11. Normal culture and passage to P3 generation are stored in liquid nitrogen. Cryopreservation can provide a stable source of cells for subsequent experiments, and is also an effective way to store cells for a long time.

[0050] The preparation method of collagenase A solution is as follows:

[0051] 1. Collagenase A (Sigma / Roche 11088793001, 0.23 U / mg) is dissolved in SMCM special medium (Sciencell 23906 1101). The final working concentration used in the experiment is 0.5-50 mg / ml.

[0052] 2. Add 0.25% Trypsin (Gibco TrypLE Select® TM Express Enzyme (1X) with phenol red).

[0053] 3. After collagenase A is prepared, filter the solution using a syringe and a 0.22um sterile filter screen.

[0054] 4. The remaining mother liquor is stored at -80°C refrigerator for later use.

[0055] Culture cell identification:

[0056] 1. Observe cell morphology using inverted phase contrast microscope

[0057] 2. Cell immunofluorescence identification (use anti-a-sm-actin, ab5694, use working solution concentration is 1:50)

[0058] Under light microscope:

[0059] Figure 2 Digestion completed picture. Figure 3 Picture of static culture for 4 days. Figure 4 Picture of static culture for 7 days. Figure 5 Picture of static culture for 10 days. Figure 6 Picture of static culture for 18 days. Figure 7 Picture of P2 generation. Figure 8 Picture of human specimen smooth muscle cell culture immunofluorescence staining. Blue Dapi represents cell nucleus, green SMA represents vascular smooth muscle cell.

[0060] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It is to be understood that where the application is specifically recited as comprising, having or being, any specific characteristics, but also any of these terms is meant to also encompass the respective opposite phrasing. For example, a device including a component A or having a component A also can be interpreted to mean that the device does not include a component A or does not have a component A. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising", or "includes" and / or "including" when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0061] The preferred embodiments of the application disclosed above are only used to help explain the application. The preferred embodiments do not describe all the details of the application, and the application is not limited to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the specification. The specification selects and specifically describes these embodiments in order to better explain the principles and practical application of the application, so that those skilled in the art can well understand and utilize the application. The application is limited only by the claims and their full scope and equivalents.

Claims

1. A method for isolating and culturing primary vascular smooth muscle cells from human abdominal aorta wall specimens, comprising the following steps: Obtain collagenase A solution; Collagenase A solution was added to the processed human abdominal aortic aneurysm wall tissue specimens and then minced; The processing of human abdominal aortic aneurysm wall tissue specimens includes the following steps: Human abdominal aortic aneurysm wall tissue was obtained within 0.5h-1h of in vitro isolation, placed in a mixture of PBS and three antibodies, and moved to a clean bench; The human abdominal aortic aneurysm wall tissue specimens were obtained by repeatedly washing with a mixture of PBS and three antibodies to remove the blood clots on the human abdominal aortic aneurysm wall tissue; The processed human abdominal aortic aneurysm wall tissue specimens were up to 35mm 2 In a petri dish; The volume of the human abdominal aortic aneurysm wall tissue specimen was 1 mm after adding collagenase A solution and cutting. 3 The minced human abdominal aortic aneurysm wall tissue specimens were transferred to a 37-degree cell culture incubator for culture; 35mm 2 The culture dish was transferred to a 37°C cell culture incubator; Observe every 30-60 minutes and pipette repeatedly; Repeat for 4-6 hours; After 4-6 hours of incubation, shake until the enzyme solution becomes turbid, and add preheated 10% FBS SMCM medium to terminate the digestion; After terminating the digestion, the cell suspension was obtained by centrifugation; The process of determining whether to terminate digestion is as follows: Real-time acquisition of physical characteristics during digestion, including cell density, cell morphology, aggregation index, and cell viability indicators; Obtaining the specified cell density, specified cell morphological characteristics, specified aggregation index, and specified cell viability index stored in the database when the digestion is completed; Compare and analyze the obtained cell density, cell morphology, aggregation index and cell viability index with the reference cell density, reference cell morphology, reference aggregation index and reference cell viability index to obtain a comparison index, which is used to indicate the similarity between the current digestion status and the reference digestion completion status; The method for obtaining the comparison index is as follows: ； Where, is the comparison coefficient, is the cell density, Cell morphological characteristics, is the clustering index, is an indicator of cell viability, To determine the cell density, To determine the cell morphological characteristics, is the parameter aggregation index, To determine the cell viability index, is the density weight factor, is the morphological weight factor, is the aggregation index weight factor, is the vitality index weight factor, for and Similarity function of If the comparison index is less than or equal to the comparison threshold stored in the database, the current state is considered complete digestion, and the digestion is terminated; The supernatant was discarded, the cell pellet was resuspended, and the centrifugation was repeated three times; Resuspend the cell pellet again, pipette and homogenize it, then transfer it to a T25 culture flask and place it in a 37-degree cell culture incubator for static culture. When peaks and troughs appeared, cells were digested and passaged with 0.25% trypsin and transferred to new T25 culture flasks; The cells were cultured normally and passaged to P3 and then frozen in liquid nitrogen.

2. The method for isolating and culturing primary vascular smooth muscle cells from human abdominal aorta wall specimens according to claim 1, characterized in that: The preparation method of collagenase A solution comprises the following steps: Dissolve collagenase A in SMCM special culture medium; Add 0.25% pancreatin; Filter the solution using a syringe and a sterile filter to obtain a collagenase A solution.

3. The method for isolating and culturing primary vascular smooth muscle cells from human abdominal aorta wall specimens according to claim 2, characterized in that: The specification of the sterile filter screen is 0.22 μm.

4. The method for isolating and culturing primary vascular smooth muscle cells from human abdominal aorta wall specimens according to claim 1, wherein: The step of terminating the digestion and then centrifuging to obtain a cell suspension comprises the following steps: Use a cell strainer to remove incompletely digested tissue fragments; The cell suspension was obtained by centrifugation at 400 g for 5 min.

5. The method for isolating and culturing primary vascular smooth muscle cells from human abdominal aorta wall specimens according to claim 4, characterized in that: The specification of the cell filter is 100 μm.

6. The method for isolating and culturing primary vascular smooth muscle cells from human abdominal aorta wall specimens according to claim 1, characterized in that: Use serum-free medium when resuspending the cell pellet; Resuspend the cell pellet in 10 ml of 10% FBS SMCM medium.

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