Method for separating and culturing primary vascular smooth muscle cells from human abdominal aorta wall

Abstract

The invention discloses a method for separating and culturing primary vascular smooth muscle cells from a human abdominal aorta wall, and relates to the technical field of human cell culture. The method for separating and culturing the primary vascular smooth muscle cells from the human abdominal aorta wall comprises the following steps: obtaining a collagenase A solution; adding a collagenase A solution into the treated human abdominal aortic aneurysm wall tissue specimen, and then cutting into pieces; transferring the cut human abdominal aortic aneurysm wall tissue specimen into a 37-DEG C cell culture box for culturing; after stopping digestion, centrifuging to obtain a cell suspension; discarding the supernate, re-suspending the cell precipitate, and repeating the centrifugation for three times; re-suspending the cell precipitate again, carrying out blowing and beating homogenization, transferring the cell precipitate into a T25 culture bottle, and putting the T25 culture bottle into a 37-DEG C cell culture box for standing culture; when a wave crest and wave trough state appears, carrying out digestion passage by using 0.25% pancreatin, and transferring into a new T25 culture bottle; normal culture and passage to P3 generation are cryopreserved in liquid nitrogen, so that the cells can be prevented from being damaged, and the activity and multiplication capacity of the cells can be maintained.

Description

Technical Field

[0001] The invention relates to the technical field of human cell culture, in particular to a method for separating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta. Background Art

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

[0003] The in vitro culture of arterial smooth muscle cells began in the 1970s, when Ross and Chamley successfully cultured them using the tissue patch method. In vitro cultured vascular smooth muscle cells can be obtained from arteries in different parts of the human body, such as the thoracic aorta, abdominal aorta, pulmonary artery, cerebral artery, mesenteric artery, etc. Human umbilical vein vessels are not only simple to obtain and have an abundant source, but their smooth muscle cells are also highly active and more suitable for culture. There are many methods for culturing vascular smooth muscle cells, the main ones being enzymatic digestion and tissue patch adhesion. Different culture methods have different advantages and uses.

[0004] However, the existing technology is not convenient for preventing cell damage. Timely subculturing can prevent contact inhibition caused by excessive growth and maintain cell activity and proliferation ability. Summary of the invention

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

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for isolating and culturing primary vascular smooth muscle cells from the human abdominal aorta wall, comprising the following steps: obtaining collagenase A solution; adding collagenase A solution to the treated human abdominal aortic aneurysm wall tissue specimen and then cutting it into pieces; transferring the cut human abdominal aortic aneurysm wall tissue specimen to a 37-degree cell culture incubator for culture; terminating digestion and centrifuging to obtain a cell suspension; discarding the supernatant, resuspending the cell pellet, and repeating the centrifugation three times; resuspending the cell pellet again, blowing and homogenizing it, and then transferring it to a T25 culture flask, placing it in a 37-degree cell culture incubator for static culture; when peaks and troughs appear, digesting and passage with 0.25% trypsin, and transferring it to a new T25 culture flask; culturing and passage normally to the P3 generation and freezing it in liquid nitrogen.

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

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

[0009] Furthermore, the processing process of the human abdominal aortic aneurysm wall tissue specimen includes the following steps: taking human abdominal aortic aneurysm wall tissue within 0.5h-1h of in vitro, placing it in a mixture of PBS+triple antibodies and moving it to a clean bench; repeatedly rinsing it with the mixture of PBS+triple antibodies to wash away the blood clots on the human abdominal aortic aneurysm wall tissue to obtain the human abdominal aortic aneurysm wall tissue specimen.

[0010] Furthermore, the processed human abdominal aortic aneurysm wall tissue specimens were up to 35 mm 2 In the culture dish, the human abdominal aortic aneurysm wall tissue specimen was added with collagenase A solution and the volume of the human abdominal aortic aneurysm wall tissue specimen after cutting was 1 mm 3 .

[0011] Further, the cells were transferred to a 37°C cell culture incubator for culture, including the following steps: 2 Transfer the culture dish to a 37-degree cell culture incubator; observe every 30-60 minutes and pipette repeatedly; repeat for 4-6 hours.

[0012] Furthermore, the digestion termination process is as follows: after culturing for 4-6 hours, when the enzyme solution becomes turbid by shaking, SMCM culture medium containing preheated 10% FBS is added to terminate the digestion.

[0013] Furthermore, the method of obtaining a cell suspension by centrifugation after terminating digestion comprises the following steps: using a cell filter to remove incompletely digested tissue fragments; and obtaining a cell suspension by centrifugation at 400 g for 5 minutes.

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

[0015] Furthermore, serum-free medium was used when the cell pellet was resuspended; and 10 ml of 10% FBS SMCM medium was used when the cell pellet was resuspended again.

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

[0017] The method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta uses PBS containing antibiotics to prevent bacterial contamination and maintain the physiological balance of tissues, finely chopped tissue pieces can increase the contact area between enzymes and tissues, collagenase A helps decompose extracellular matrix and promotes cell release, blowing helps disperse cells and prevents cells from aggregating to form clumps, adding serum-containing culture medium can neutralize the activity of digestive enzymes and prevent cell damage, observing the cell growth state, and timely passage can prevent contact inhibition caused by excessive growth and maintain cell activity and proliferation ability.

[0018] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The flowchart of the method is shown in FIG.

[0020] Figure 2 Completed picture for digestion.

[0021] Figure 3 The picture is of day 4 of static culture.

[0022] Figure 4 The picture is on the 7th day of static culture.

[0023] Figure 5 This is a picture of the 10th day of static culture.

[0024] Figure 6 This is a picture of the static culture on the 18th day.

[0025] Figure 7 This is a picture of the P2 generation.

[0026] Figure 8 Immunofluorescence staining of smooth muscle cell cultures for human specimens DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In the description of the present invention, it should be understood that the terms "opening", "upper", "lower", "thickness", "top", "middle", "length", "inside", "all around" and the like indicating orientation or positional relationship are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the components or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0029] See also Figure 1 The embodiment of the present invention provides a technical solution: a method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta, comprising the following steps:

[0030] 1. Take fresh human abdominal aortic aneurysm wall tissue within 0.5h-1h of in vitro separation, put it into a mixture of PBS + three antibodies and move it to a clean bench. Using PBS containing antibiotics can prevent bacterial contamination and maintain the physiological balance of the tissue.

[0031] 2. Rinse repeatedly with a large amount of PBS containing the three antibodies to clean the blood clots. Thorough washing can remove blood and other impurities that may affect cell culture and ensure the purity of the culture environment.

[0032] 3. Transfer the processed specimen to 35mm 2 Add an appropriate amount (not too much to increase viscosity) of collagenase A solution to the culture dish and use ophthalmic scissors to cut the specimen into 1 mm pieces. 3 , add an appropriate amount of collagenase A solution at the right time. Finely chopped tissue pieces can increase the contact area between the enzyme and the tissue. Collagenase A helps decompose the extracellular matrix and promote the release of cells.

[0033] 4. The above 35mm 2 The culture dish was transferred to a 37-degree cell culture incubator and observed every 30-60 minutes, and the cells were gently pipetted repeatedly. The appropriate temperature can accelerate the action of enzymes and promote the digestion of tissues, but it needs to be closely monitored to avoid over-digestion.

[0034] 5. Repeat the above steps for 4-6 hours. When the enzyme solution becomes turbid after shaking, add preheated 10% FBS SMCM culture medium to terminate digestion. Observe the cell digestion under a microscope in time to prevent excessive digestion and cell damage. Puffing helps disperse the cells to prevent cell aggregation and formation of clumps. Adding serum-containing culture medium can neutralize the activity of digestive enzymes and prevent cell damage.

[0035] In order to prevent over-digestion, physical characteristics during the digestion process can be obtained in real time, including cell density, cell morphology, aggregation index, and cell viability index. In addition, the specified cell density, specified cell morphology, specified aggregation index, and specified cell viability index stored in the database when the digestion is completed are obtained.

[0036] The obtained cell density, cell morphology characteristics, aggregation index and cell viability index are compared and analyzed with the reference cell density, reference cell morphology characteristics, 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 status when the reference digestion is completed.

[0037] The method for obtaining the comparison index is 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 feature, Z1 is the aggregation index, H1 is the cell viability index, m2 is the reference cell density, x2 is the reference cell morphology feature, Z2 is the reference aggregation index, H2 is the reference cell viability index, β1 is the density weight factor, β2 is the morphology weight factor, β3 is the aggregation index weight factor, β4 is the viability index 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, then it can be considered that the current state is that the digestion is completed and should be terminated.

[0041] Acquiring real-time data in a quantitative manner (such as cell density, morphological characteristics, etc.) avoids the uncertainty of subjective judgment. Culture conditions can be dynamically adjusted based on real-time data, such as stopping the action of digestive enzymes to avoid over-digestion and cell damage. By comparing with the reference features in the database, a clear standard is provided to determine when digestion is complete, ensuring that each experiment reaches a consistent endpoint. Ensuring the optimal survival state and vitality of cells helps with subsequent experimental operations and data reliability.

[0042] Cell density directly reflects the quantitative level of cell growth and survival. It can indicate whether cells are proliferating normally or whether they are inhibited by certain conditions (such as nutrient deficiency or space limitation). High density may mean that cells are too crowded, which may affect the normal function and morphology of cells. Cell morphological changes are important indicators of cell health and physiological status. Abnormal morphological changes may indicate cell stress or death. Changes in morphological characteristics are often closely related to cell density and vitality indicators, and dense environments may lead to deformed cell morphology.

[0043] The aggregation index describes the degree of cell-cell interaction and aggregation, reflecting the cohesion of the cell population. High aggregation in some cell types indicates a healthy state, while in other types it may indicate a pathological state. High aggregation may be associated with high cell density and morphological changes, which may lead to increased competition for resources in the microenvironment and affect the normal physiological function of the cells. Viability index directly reflects the viability of cells and is a direct method to assess the health status of cells. Low viability indicates that the cells are damaged or dead. Decreased viability may be related to inappropriate density, abnormal morphology or excessive aggregation.

[0044] 6. Use a 100um cell filter to remove incompletely digested tissue fragments. Remove incompletely digested tissue fragments to ensure the purity and consistency of the cells in the resulting suspension.

[0045] 7. Centrifuge the cell suspension at 400g / 5min. Centrifugation can effectively separate cells and supernatant, which helps to remove unwanted small molecules and impurities.

[0046] 8. Aspirate the supernatant, resuspend the cell pellet in serum-free medium, centrifuge (400g / 5min), and repeat three times. Multiple washings with serum-free medium can further remove external contamination and digestive enzymes, and prepare the cells to achieve the ideal culture state.

[0047] 9. Finally, resuspend the cell pellet with 10ml 10% FBS SMCM medium, gently blow and evenly, transfer to a T25 culture flask, and culture in a 37° cell culture incubator. Keep it absolutely still for the first 3-5 days. The growth factors and nutrients in the serum are conducive to the growth and attachment of cells. The T25 culture flask provides sufficient growth area.

[0048] 10. On the 7th to 10th day, you can see the cells growing on the wall, and the growth rate will increase rapidly on the 10th to 15th day. When the peak and trough state appears, use 0.25% trypsin to digest and subculture, and transfer to a new T25 culture bottle. Observe the cell growth state and subculture in time to prevent contact inhibition caused by excessive growth and maintain cell activity and proliferation ability.

[0049] 11. Cryopreservation in liquid nitrogen after normal culture to P3 generation. Cryopreservation can provide a stable cell source for subsequent experiments and is also an effective way to preserve cells for a long time.

[0050] Wherein the preparation method of collagenase A solution is:

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

[0052] 2. Add 0.25% pancreatin (Gibco TrypLE 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.

[0054] 4. The remaining mother liquor was stored in a -80℃ refrigerator for future use.

[0055] Cultured cell identification:

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

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

[0058] Under light microscope:

[0059] Figure 2 Completed picture for digestion. Figure 3 The picture is of the 4th day of static culture. Figure 4 The picture is of the 7th day of static culture. Figure 5 This is a picture of the 10th day of static culture. Figure 6 This is a picture of the static culture on the 18th day. Figure 7 This is a picture of the P2 generation. Figure 8 Immunofluorescence staining of smooth muscle cell culture of human specimens. Blue Dapi indicates cell nuclei, and green SMA indicates vascular smooth muscle cells.

[0060] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0061] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention 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 the wall of human abdominal aorta, comprising the following steps: Obtain collagenase A solution; Add collagenase A solution to the processed human abdominal aortic aneurysm wall tissue specimen and cut into pieces; The minced human abdominal aortic aneurysm wall tissue specimens were transferred to a 37-degree cell culture incubator for culture; After terminating the digestion, the cell suspension was obtained by centrifugation; 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 the peaks and troughs appeared, the cells were digested and passaged with 0.25% trypsin and transferred to new T25 culture flasks; Normal culture was subcultured to P3 and frozen in liquid nitrogen.

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

3. The method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta 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 the wall of human abdominal aorta according to claim 1, characterized in that: The processing of human abdominal aortic aneurysm wall tissue specimens includes the following steps: Take the human abdominal aortic aneurysm wall tissue within 0.5h-1h of in vitro separation, put it into the mixture of PBS + three antibodies and move it to the clean bench; The blood clots on the human abdominal aortic aneurysm wall tissue were washed off repeatedly with a mixture of PBS + three antibodies to obtain a human abdominal aortic aneurysm wall tissue specimen.

5. The method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta according to claim 1, characterized in that: 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 after adding collagenase A solution and cutting into pieces is 1 mm 3 .

6. The method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta according to claim 5, characterized in that: Transfer to a 37°C cell culture incubator for culture, including the following steps: 35mm 2 The culture dish was transferred to a 37°C cell culture incubator; Observe every 30-60 minutes and blow repeatedly with a pipette; Repeat for 4-6 hours.

7. The method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta according to claim 6, characterized in that: The process of terminating digestion is as follows: after culturing for 4-6 hours, when the enzyme solution becomes turbid after shaking, add preheated 10% FBSSMCM culture medium to terminate digestion.

8. The method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta according to claim 1, characterized in that: 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.

9. The method for isolating and culturing primary vascular smooth muscle cells from the wall of human abdominal aorta according to claim 8, characterized in that: The specification of the cell filter is 100 μm.

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

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